Cultures Of Human Epithelial Cells Research Articles

BPA-induced prostatic hyperplasia in vitro is correlated with the unbalanced gene expression of AR and ER in the epithelium and stroma

As a typical environmental endocrine disruptor (EED), bisphenol A (BPA) can induce pathological hyperplasia of the prostatic epithelium and stroma. This study concentrates mainly on the effect and underlying mechanisms of BPA on prostatic hyperplasia, which is based on the culture of primary human prostate epithelial cells (HPEpiC) and human prostate fibroblasts (HPrF). In an effect to screen the optimal pro-survival BPA levels, HPEpiC and HPrF were, respectively, exposed to concentration gradients of BPA (10-12M-10-4M) solution diluted with two corresponding medium and incubated for 72h at 37°C. CCK-8 assay showed that 10-9 M-10-5M BPA could facilitate the proliferation of HPEpiC, while similar proliferative effect of HPrF only needed 10-11 M-10-7M BPA. HPrF were more sensitive to BPA than HPEpiC. The qualification of PCNA gene expression measured using quantitative real-time polymerase chain reaction (qRT-PCR) also mirrored the BPA-induced cell proliferation. Additionally, our results considered that androgen receptor (AR), estrogen receptor (ERα, ERβ), and NFKB1 gene expressions exhibited up-regulation in HPEpiC treated with 10-9M BPA for 72h. However, in HPrF, the identical BPA treatment could activate ERα, ERβ, and NFKB1 gene expressions and down-regulated the expression of AR levels. It is further confirmed that low-dose BPA can indeed promote the proliferation of human prostate cells in vitro, and the mechanisms of BPA for prostatic epithelial and stromal hyperplasia may not be consistent.

Kakkonto Inhibits Cytokine Production Induced by Rhinovirus Infection in Primary Cultures of Human Nasal Epithelial Cells.

Rhinovirus (RV) is a primary etiologic agent of common cold that can subsequently acutely exacerbate bronchial asthma or chronic obstructive pulmonary disease. Kakkonto (Ge-gen-tang in Chinese), one of the most frequently prescribed traditional Japanese (Kampo) medicines, is used for treating common cold, shoulder stiffness, or inflammatory diseases of the upper body. Previous experimental studies have indicated that kakkonto exerts antiviral and anti-inflammatory effects on the influenza virus and the human respiratory syncytial virus. However, there is a lack of reports investigating the efficacy of kakkonto in RV infection. Hence, the aim of the current study was to investigate the effects of kakkonto on RV infection of human nasal epithelial (HNE) cells. HNE cells obtained via endoscopic sinus surgery were cultured and infected with RV14, with or without kakkonto treatment. The supernatants from the cells were collected, and the RV14 titer and cytokine levels were assessed. Reverse transcription-polymerase chain reaction was performed to determine the amount of viral RNA, while the level of nuclear factor kappa B (NF-κB) subunits in the nucleus was assessed by enzyme-linked immunosorbent assay. Although kakkonto treatment did not reduce RV14 titer or RNA levels, indicating that it did not inhibit RV14 proliferation, it was found to reduce the production of specific pro-inflammatory cytokines, including interleukin (IL)-8, tumor necrosis factor (TNF)-α, and monocyte chemotactic protein-1 (MCP-1). Unlike that observed with the kakkonto extract, none of the crude drugs contained in kakkonto reduced IL-8 level. Furthermore, though kakkonto treatment significantly reduced p50 levels, it did not impact the p65 subunit of NF-κB. These results indicated that kakkonto can inhibit inflammation caused by RV infection and may exert an immunomodulatory effect on HNE cells. This is the first report to elucidate the effects of kakkonto extract on RV infection in primary cultures of HNE cells, providing evidence that kakkonto may act as an effective therapy for RV infection and subsequent airway inflammation.

Epithelial–stromal cell interactions and extracellular matrix mechanics drive the formation of airway-mimetic tubular morphology in lung organoids

SummaryComplex human airway cellular organization where extracellular matrix (ECM) and epithelial and stromal lineages interact present challenges for organ study in vitro. Current in vitro lung models that focus on the lung epithelium do not represent complex airway morphology and cell-ECM interactions seen in vivo. Models including stromal populations often separate them via a semipermeable barrier precluding cell–cell interaction or the effect of ECM mechanics. We investigated the effect of stromal cells on basal epithelial cell-derived bronchosphere structure and function through a triple culture of human bronchial epithelial, lung fibroblast, and airway smooth muscle cells. Epithelial–stromal cross-talk resulted in epithelial cell-driven branching tubules with stromal cells surrounding epithelial cells termed bronchotubules. Agarose– Matrigel scaffold (Agrigel) formed a mechanically tuneable ECM, with adjustable viscoelasticity and stiffness enabling long-term tubule survival. Bronchotubule models may enable research into how epithelial–stromal cell and cell–ECM communication drive tissue patterning, repair, and development of disease.

The proton ATPase inhibitor bafilomycin A1 reduces the release of rhinovirus C and cytokines from primary cultures of human nasal epithelial cells

Rhinovirus species C (RV-C) causes more severe asthma attacks than other rhinovirus species. However, the modulation of RV-C replication by drugs has not been well studied. Primary human nasal epithelial (HNE) cells cultured on filter membranes with air-liquid interface methods were infected with RV-C03, and the levels of RV-C03 RNA collected from the airway surface liquid (ASL) of HNE cells were measured with a SYBR Green assay. Pretreatment of HNE cells with the specific vacuolar H+-ATPase inhibitor bafilomycin A1 reduced the RV-C03 RNA levels in the ASL; inflammatory cytokines, including interleukin (IL)-1β, IL-6 and IL-8, in the supernatant; the mRNA expression of the RV-C receptor cadherin-related family member 3 (CDHR3) in the cells; and the number of acidic endosomes where RV-B RNA enters the cytoplasm. The levels of RV-C03 RNA in the ASL obtained from HNE cells with the CDHR3 rs6967,330 G/A genotype tended to be higher than those obtained from HNE cells with the G/G genotype. Pretreatment with the Na+/H+ exchanger inhibitor ethyl-isopropyl amiloride or either of the macrolides clarithromycin or EM900 also reduced RV-C03 RNA levels in the ASL and the number of acidic endosomes in HNE cells. In addition, significant levels of RV-A16, RV-B14 and RV-C25 RNA were detected in the ASL, and bafilomycin A1 also decreased the RV-C25 RNA levels. These findings suggest that bafilomycin A1 may reduce the release of RV-Cs and inflammatory cytokines from human airway epithelial cells. RV-Cs may be sensitive to drugs, including bafilomycin A1, that increase endosomal pH, and CDHR3 may mediate virus entry through receptor-mediated endocytosis in human airway epithelial cells.

Improvement of human embryonic stem cell-derived retinal pigment epithelium cell adhesion, maturation, and function through coating with truncated recombinant human vitronectin.

To explore an xeno-free and defined coating substrate suitable for the culture of H9 human embryonic stem cell-derived retinal pigment epithelial (hES-RPE) cells in vitro, and compare the behaviors and functions of hES-RPE cells on two culture substrates, laminin521 (LN-521) and truncated recombinant human vitronectin (VTN-N). hES-RPE cells were used in the experiment. The abilities of LN-521 and VTN-N at different concentrations to adhere to hES-RPE cells were compared with a high-content imaging system. Quantitative real-time polymerase chain reaction was used to evaluate RPE-specific gene expression levels midway (day 10) and at the end (day 20) of the time course. Cell polarity was observed by immunofluorescent staining for apical and basal markers of the RPE. The phagocytic ability of hES-RPE cells was identified by flow cytometry and immunofluorescence. The cell adhesion assay showed that the ability of LN-521 to adhere to hES-RPE cells was dose-dependent. With increasing coating concentration, an increasing number of cells attached to the surface of LN-521-coated wells. In contrast, VTN-N presented a strong adhesive ability even at a low concentration. The optimal concentration of LN-521 and VTN-N required to coat and adhesion to hES-RPE cells were 2 and 0.25 µg/cm2, respectively. Furthermore, both LN-521 and VTN-N could facilitate adoption of the desired cobblestone cellular morphology with tight junction and showed polarity by the hES-RPE cells. However, hES-RPE cells cultivated in VTN-N had a greater phagocytic ability, and it took less time for these hES-RPE cells to mature. VTN-N is a more suitable coating substrate for cultivating hES-RPE cells.

P53 regulation by MDM2 contributes to self-renewal and differentiation of basal stem cells in mouse and human airway epithelium.

Proper physiological function of mammalian airways requires the differentiation of basal stem cells into secretory or multiciliated cells, among others. In addition, the self-renewal ability of these basal stem cells is crucial for developing a quick response to toxic agents in order to re-establish the epithelial barrier function of the airways. Although these epithelial missions are vital, little is known about those mechanism controlling airway epithelial regeneration in health and disease. p53 has been recently proposed as the guardian of homeostasis, promoting differentiation programs, and antagonizing a de-differentiation program. Here, we exploit mouse and human tracheal epithelial cell culture models to study the role of MDM2-p53 signaling in self-renewal and differentiation in the airway epithelium. We show that p53 protein regulation by MDM2 is crucial for basal stem cell differentiation and to keep proper cell proliferation. Therefore, we suggest that MDM2/p53 interaction modulation is a potential target to control regeneration of the mammalian airway epithelia without massively affecting the epithelium integrity and differentiation potential.

Measles virus exits human airway epithelia within dislodged metabolically active infectious centers.

Measles virus (MeV) is the most contagious human virus. Unlike most respiratory viruses, MeV does not directly infect epithelial cells upon entry in a new host. MeV traverses the epithelium within immune cells that carry it to lymphatic organs where amplification occurs. Infected immune cells then synchronously deliver large amounts of virus to the airways. However, our understanding of MeV replication in airway epithelia is limited. To model it, we use well-differentiated primary cultures of human airway epithelial cells (HAE) from lung donors. In HAE, MeV spreads directly cell-to-cell forming infectious centers that grow for ~3–5 days, are stable for a few days, and then disappear. Transepithelial electrical resistance remains intact during the entire course of HAE infection, thus we hypothesized that MeV infectious centers may dislodge while epithelial function is preserved. After documenting by confocal microscopy that infectious centers progressively detach from HAE, we recovered apical washes and separated cell-associated from cell-free virus by centrifugation. Virus titers were about 10 times higher in the cell-associated fraction than in the supernatant. In dislodged infectious centers, ciliary beating persisted, and apoptotic markers were not readily detected, suggesting that they retain functional metabolism. Cell-associated MeV infected primary human monocyte-derived macrophages, which models the first stage of infection in a new host. Single-cell RNA sequencing identified wound healing, cell growth, and cell differentiation as biological processes relevant for infectious center dislodging. 5-ethynyl-2’-deoxyuridine (EdU) staining located proliferating cells underneath infectious centers. Thus, cells located below infectious centers divide and differentiate to repair the dislodged infected epithelial patch. As an extension of these studies, we postulate that expulsion of infectious centers through coughing and sneezing could contribute to MeV’s strikingly high reproductive number by allowing the virus to survive longer in the environment and by delivering a high infectious dose to the next host.

PAR2-Induced Tissue Factor Synthesis by Primary Cultures of Human Kidney Tubular Epithelial Cells Is Modified by Glucose Availability.

Coagulopathies common to patients with diabetes and chronic kidney disease (CKD) are not fully understood. Fibrin deposits in the kidney suggest the local presence of clotting factors including tissue factor (TF). In this study, we investigated the effect of glucose availability on the synthesis of TF by cultured human kidney tubular epithelial cells (HTECs) in response to activation of protease-activated receptor 2 (PAR2). PAR2 activation by peptide 2f-LIGRLO-NH2 (2F, 2 µM) enhanced the synthesis and secretion of active TF (~45 kDa) which was blocked by a PAR2 antagonist (I-191). Treatment with 2F also significantly increased the consumption of glucose from the cell medium and lactate secretion. Culturing HTECs in 25 mM glucose enhanced TF synthesis and secretion over 5 mM glucose, while addition of 5 mM 2-deoxyglucose (2DOG) significantly decreased TF synthesis and reduced its molecular weight (~40 kDa). Blocking glycosylation with tunicamycin also reduced 2F-induced TF synthesis while reducing its molecular weight (~36 kDa). In conclusion, PAR2-induced TF synthesis in HTECs is enhanced by culture in high concentrations of glucose and suppressed by inhibiting either PAR2 activation (I-191), glycolysis (2DOG) or glycosylation (tunicamycin). These results may help explain how elevated concentrations of glucose promote clotting abnormities in diabetic kidney disease. The application of PAR2 antagonists to treat CKD should be investigated further.

Retracted: Primary Culture and Characterization of Human Renal Inner Medullary Collecting Duct Epithelial Cells.

Retracted: Primary Culture and Characterization of Human Renal Inner Medullary Collecting Duct Epithelial Cells.

Differential Effect of Proinflammatory Cytokines on Corneal and Conjunctival Epithelial Cell Mucins and Glycocalyx.

PurposeOcular surface mucins and glycocalyx are critical for providing ocular hydration as well lubrication and repelling pathogens or allergens. Elevated levels of tear proinflammatory cytokines in dry eye may have detrimental effect on mucins and glycocalyx. The present study tested the effect of proinflammatory cytokines IL-6, TNF-α, and IFN-γ on membrane-tethered mucins expression, glycocalyx, and viability of ocular surface epithelial cells.MethodsStratified cultures of human corneal and conjunctival epithelial cells were exposed to different concentrations of IL-6, TNF-α, and IFN-γ for 24 hours. The mucins gene and protein expressions were quantified by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The glycocalyx was imaged using confocal microscopy after staining with Alexa 488-conjugated wheat germ agglutinin lectin. Apoptotic and necrotic cell death was quantified using flow cytometry.ResultsIL-6, TNF-α, and IFN-γ treatment resulted in a significant increase in mucins (MUC)1 and MUC4 gene and protein expression in human corneal epithelial cells but caused no significant changes in the levels of these mucins in conjunctival epithelial cells. Further, these cytokines decreased MUC16 expression in both corneal and conjunctival epithelial cells. Moreover, no notable change in glycocalyx or apoptotic cell death in corneal and conjunctival epithelial cells was noted with any of the tested cytokines, but IL-6 and TNF-α exposure increased necrotic cell death in corneal and conjunctival epithelial cells, respectively.ConclusionsOur results demonstrate that proinflammatory cytokines have differential effects on human corneal and conjunctival epithelial cell mucins expression, but do not cause any damage to ocular surface epithelial cell glycocalyx.

Association between mucosal barrier disruption by Pseudomonas aeruginosa exoproteins and asthma in patients with chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is a common chronic respiratory condition, frequently associated with asthma and affecting the majority of cystic fibrosis (CF) patients. Pseudomonas aeruginosa infections and biofilms have been implicated in recalcitrant CRS. One of the mechanisms of action for bacteria in CRS and CF is mucosal barrier disruption by secreted products that contribute to the inflammation. However, the role of biofilm and planktonic forms of P. aeruginosa in this process is not known. The aim is to determine the effect of P. aeruginosa exoproteins isolated from CF and non-CF CRS patients on the mucosal barrier. Exoproteins from 40 P. aeruginosa isolates were collected in planktonic and biofilm forms and applied to air-liquid interface (ALI) cultures of primary human nasal epithelial cells (HNECs). Mucosal barrier integrity was evaluated by transepithelial electrical resistance (TEER), passage of FITC-dextrans and immunofluorescence of tight junction proteins. Cytotoxicity assays were performed to measure cell viability, and IL-6 ELISA was carried out to evaluate pro-inflammatory effects. Planktonic exoproteins from 20/40 (50%) clinical isolates had a significant detrimental effect on the barrier and significantly increased IL-6 production. Barrier disruption was characterized by a reduced TEER, increased permeability of FITC-dextrans and discontinuous immunolocalization of tight junction proteins and was significantly more prevalent in isolates harvested from patients with comorbid asthma (P<.05). Exoproteins from planktonic P. aeruginosa clinical isolates from asthmatic CRS patients have detrimental effects on the mucosal barrier and induce IL-6 production potentially contributing to the mucosal inflammation in CRS patients.

Cells feel the strain

Cell Biology Cells respond to mechanical strain by stiffening their actin cytoskeleton, a process that is energetically costly. Salvi et al. show that cells anticipate this cost by increasing glucose transport when subjected to mechanical strain. In canine or human epithelial cells in culture, exposure to strain caused accumulation of the glucose transporter GLUT1 at cell-cell junctions. The increase in transporters increased the uptake of glucose required for cytoskeletal stiffening. The transporters were tethered by the ankyrin G protein to E-cadherin–containing adhesion complexes at the cell junctions. Cells expressing a mutant form of E-cadherin that did not interact with ankyrin G failed to accumulate GLUT1 and were unable to form an effective epithelial barrier. Nat Cell Biol. 23 , 457 (2021).

Long-term modulation of airway remodelling in severe asthma following bronchial thermoplasty.

Bronchial thermoplasty is a mechanical therapeutic intervention that has been advocated as an effective treatment option for severe asthma. The mechanism is promoted as being related to the attenuation of airway smooth muscle which has been shown to occur in the short-term. However, long-term studies of the effects of bronchial thermoplasty on airway remodeling are few with only limited assessment of airway remodeling indices. To evaluate the effect of bronchial thermoplasty on (a) airway epithelial and smooth muscle cells in culture, and (b), airway remodeling in patients with severe asthma who have been prescribed bronchial thermoplasty up to 12-months post-treatment. The distribution of heat within the airway by bronchial thermoplasty was assessed in a porcine model. Culture of human airway smooth muscle cells and bronchial epithelial cells evaluated the impact of thermal injury. Histological evaluation and morphometric assessment were performed on bronchial biopsies obtained from severe asthma patients at baseline, 6-weeks, and 12-months following bronchial thermoplasty. Bronchial thermoplasty resulted in heterogenous heating of the airway wall. Airway smooth muscle cell cultures sustained thermal injury, whilst bronchial epithelial cells were relatively resistant to heat. Airway smooth muscle and neural bundles were significantly reduced at 6-weeks and 12-months post-treatment. At 6-weeks post treatment, submucosal collagen was reduced, and vessel density increased, with both indices returning to baseline at 12-months. Goblet cell numbers, submucosal gland area and subbasement membrane thickness, were not significantly altered at any timepoint examined. Bronchial thermoplasty primarily affects airway smooth muscle and nerves with the effects still present at 12-months post-treatment.

P082 Assessment of anti-inflammatory effect of high acetate administration in UC patient-derived epithelial monolayer cultures

Abstract Background Human organoid-based intestinal monolayer cultures provide an interesting tool to preclinically assess new therapeutic properties in inflammatory bowel disease (IBD). Recently, short-chain fatty acids (SCFA) such as acetate and butyrate have gained interest for their potential beneficial effects in IBD therapy. Most studies have focused on butyrate, given its beneficial effects on gut microbiome composition, intestinal barrier function and the immune system1. Effects of acetate are less well known, despite its lower toxicity to epithelial cells and its potential to support growth of butyrate-producing bacteria by metabolic cross-feeding2. In fact, a recent report suggested that the probiotic potential of Saccharomyces cerevisiae var. boulardii might be related to its high acetic acid production3. We here studied the effect of acetate on organoid-derived epithelial monolayer cultures from ulcerative colitis (UC) patients. Methods Colonic biopsies were obtained from non-inflamed regions in 3 patients with UC. Crypts were isolated, cultured and expanded as 3D-organoids. These organoids were then dissociated and transferred to transwell inserts as monolayer cultures. Upon confluency of the monolayer, evaluated by Transepithelial electrical resistance (TEER), cells were basolaterally stimulated with control medium (CTRL) or an inflammatory mix (INFL) containing 100 ng/ml TNF-α, 20 ng/ml IL-1β and 1 µg/ml Flagellin (Fig. 1). After 24h, cells were apically stimulated with control medium or high acetate (HA) (100mM). TEER was measured at 0, 24 and 48h stimulation. After 48h, cells were subjected to RNA extraction followed by reverse transcriptase qPCR targeting a selection of key diagnostic marker genes. The apical and basolateral media were collected for cytokine determination by Mesoscale (Proinflammatory panel 1). Data were analyzed using GraphPad Prism 9 (D’Agostino &amp; Pearson test for normality followed by a Friedman test). Results A protective effect (Fig. 2) of high acetate administration on TEER values (HA vs INFL: p=0˒017) was observed, together with a trend towards decrease in IL8 (p=0˒11), TNFA (INFL vs INFL+HA: p=0˒68) and CLDN2 (CTRL vs HA: p=0˒03). Moreover, this was confirmed by a decrease of most proinflammatory cytokines (Fig. 3) in the apical and basolateral media upon HA stimulation e.g., IFN-γ (INFL vs INFL+HA: p=0˒25) and IL 10 (CTRL vs HA: p=0˒11). Conclusion In this patient-derived human epithelial cell culture model, a protective effect of high acetate administration on TEER-values, gene expression and cytokine production was observed. Ongoing experiments are expanding the number of patients to 10. References

A toolbox for studying respiratory viral infections using air-liquid interface cultures of human airway epithelial cells

Submerged cultures of primary human airway epithelial cells or human airway epithelial cell lines have been a mainstay of airway epithelial biology research for decades due to their robust in vitro proliferative capacity, relatively low maintenance culture conditions, and clinically translatable results to nasal or bronchial brushings. With the development and improvement of air-liquid interface (ALI) cultures of human airway epithelial cells, such cultures have been considered superior to immortalized cell lines and primary cell monolayers, as such cultures effectively recapitulate in vivo epithelial architecture and cell types. Although ALI culture growth protocols are well-established and widely available, many researchers have avoided their use, as ALI cultures not only take longer to grow but also present technical challenges and limitations that make in vitro intracellular and structural assays taxing. Challenges arise relating to their complex structure, requirements for air exposure, the constraints of transwell growth apparatus, and interference in assays caused by mucus secretion. Although few publications briefly describe technical adaptations for some assays, there is still considerable trial and error required for researchers to establish consistent and reliable assay adaptations, often becoming a deterrent for pursuing mechanistic investigation. We have created a user-friendly toolbox detailing comprehensive protocols for numerous techniques and assay adaptations, particularly focusing on respiratory virus infections. By expanding the repertoire of ALI culture-adapted in vitro assays, we hope to facilitate the widespread adoption of this valuable culture system for mechanistic investigations of respiratory viral infections or other epithelial-pathogen models.

Potential of Pharmaceutical Intervention in Platelets and Cancer Positive Feedback Loop

Significance High platelet counts and advanced stage of ovarian cancer go hand-in-hand in promoting each other in a feed-forward loop that results in blood coagulation and chemotherapy resistance. This results in a high incidence of death due to thrombosis in ovarian cancer patients, and especially in patients with the ovarian clear cell carcinoma histologic type. Objective and Hypothesis We sought to develop an experimental model of the positive interactions between platelets and cancer cells and test the hypothesis that interference with platelet clotting will inhibit this interaction. Approach The effects of platelets on spheroid formation by cancer or healthy epithelial cells were evaluated using a magnetic 3D cancer spheroids assay. The ES2 and MESOV cell lines, which represent clear cell carcinoma and high grade serous histologies, respectively were used for the ovarian cancer cells. Primary human fallopian tube secretory epithelial cell cultures were used to represent healthy cells. The spheroids were imaged and measured using the Optronix GelCount colony counter and evaluated using metabolic viability (MTT) and protein concentration (SRB) assays. The shear-free platelet aggregation assay was performed in the presence of healthy or cancer cells or their conditioned media. Furthermore, we evaluated possible interruption of this feed-forward loop using antiplatelet agents: aspirin—a cyclooxygenase (COX)-1 and -2 inhibitor, celecoxib—a selective COX-2 inhibitor, clopidogrel—an ADP binding inhibitor, dipyridamole—an ADP uptake inhibitor, eptifibatide—a platelet's GP IIb/IIIa inhibitor, and prostacyclin—a platelet aggregation inhibitor. Results Incubation of platelets with cancer spheroids as they are forming decreased the size and density of the spheres in less than 15 minutes of exposure. The MTT assay indicated that condensed spheres were just as live and viable as the spheres that formed in the absence of platelets. Incubation of cancer cells or cancer cells’ conditioned media with platelets caused clumping of platelets in a cancer cells number-dependent manner. Healthy cells’ conditioned media did not cause platelets aggregation. Pre-treating platelets with up to a 1 mM of aspirin, clopidogrel, dipyridamole, and prostacyclin did not prevent cancer cell-induced aggregation, unlike celecoxib, which prevented aggregation at high concentrations, and eptifibatide, which was able to prevent aggregation at low concentrations as 0.1 µM. Conclusions The positive interaction between platelets and cancer cells can be mimicked in co-culture conditions. This interaction appears to involve platelets GPIIb/IIIa binding.

IL-17A or IL-17F is sufficient to maintain innate response and control of H. pylori immunopathogenesis

Abstract Interleukin-17 receptor (IL-17R) signaling is required for control of many extracellular pathogens due to its impact on neutrophil recruitment and antimicrobial responses. Infection with the extracellular pathogen Helicobacter pylori results in Th1 and Th17 cell activation and a chronic inflammatory process which can lead to adverse outcomes such as gastric cancer. Previously, we identified IL-17RA as a requirement for the recruitment of neutrophils. Surprisingly, H. pylori infected IL-17RA−/− mice had significantly more chronic inflammation than H. pylori infected WT mice. In the current study, in vitro human epithelial cell cultures and in vivo mouse models were used to investigate differential roles for IL-17A, IL-17F and IL-17A/F during H. pylori infection. The data indicate generally that epithelial cells responded to IL-17A or IL-17A/F better than IL-17F. Increased gene expression of some factors, such as Cxcl8, required a co-stimulus such as H. pylori, TNF or IL-22 in epithelial cells, but expression of Pigr and Nox1 were induced by IL-17A alone. In vivo deficiencies of IL-17A or IL-17F alone did not significantly change the immunopathological response to H. pylori, but if both cytokines were absent, a hyperinflammatory lymphocytic response developed similar to what was observed in IL-17RA−/− mice. These data imply that IL-17A and IL-17F may have some overlapping role or compensatory roles in maintenance of the gastric mucosal response to infection which is required for preventing immunopathology.

Down-regulation of STAT3 enhanced chemokine expression and neutrophil recruitment in biliary atresia.

Biliary atresia (BA) is an immune-related disorder and signal transducer and activator of transcription 3 (STAT3) is a key signalling molecule in inflammation. The present study was designed to clarify the function of STAT3 in BA. STAT3 expression was examined in patients and a mouse BA model in which STAT3 levels were further altered with a specific inhibitor or activator. Neutrophil accumulation and the levels of the neutrophil chemoattractants (C–X–C motif) ligand 1 (CXCL1) and IL-8 were determined. The effects of STAT3 inhibition on IL-8 expression were examined in human biliary epithelial cell (BEC) cultures. Functional changes in liver STAT3+ neutrophils in the mouse model were analysed with 10× single cell RNA-seq methods. Results showed STAT3 and p-STAT3 expression was reduced in BA liver tissue compared with control samples. Administration of a STAT3 inhibitor increased jaundice and mortality and reduced body weight in BA mice. In contrast, the STAT3 activator ameliorated BA symptoms. Extensive neutrophil accumulation together with CXCL1 up-regulation, both of which were suppressed by an anti-CXCL1 antibody, were observed in the STAT3 inhibitor-treated group. Recombinant IL-8 administration increased disease severity in BA mice, and the STAT3 activator had the reverse effect. Inhibiting STAT3 increased apoptosis of human BECs together with up-regulated IL-8 expression. RNA-seq analysis revealed reduced the numbers of STAT3 expressing neutrophil in BA which was accompanied by marked enhanced interferon-related antiviral activities. In conclusion, STAT3 reduction, enhanced IL-8 and CXCL1 expression and promoted the accumulation of interferon-responsive neutrophils resulting in BEC damage in BA.

Inhibition of Bitter Taste from Oral Tenofovir Alafenamide.

Children have difficulty swallowing capsules. Yet, when presented with liquid formulations, children often reject oral medications due to their intense bitterness. Presently, effective strategies to identify methods, reagents, and tools to block bitterness remain elusive. For a specific bitter-tasting drug, identification of the responsible bitter receptors and discovery of antagonists for those receptors can provide a method to block perceived bitterness. We have identified a compound (6-methylflavone) that can block responses to an intensely bitter-tasting anti-human immunodeficiency virus (HIV) drug, tenofovir alafenamide (TAF), using a primary human taste bud epithelial cell culture as a screening platform. Specifically, TAS2R39 and TAS2R1 are the main type 2 taste receptors responding to TAF observed via heterologously expressing specific TAS2R receptors into HEK293 cells. In this assay, 6-methylflavone blocked the responses of TAS2R39 to TAF. In human sensory testing, 8 of 16 subjects showed reduction in perceived bitterness of TAF after pretreating (or "prerinsing") with 6-methylflavone and mixing 6-methylflavone with TAF. Bitterness was completely and reliably blocked in two of these subjects. These data demonstrate that a combined approach of human taste cell culture-based screening, receptor-specific assays, and human psychophysical testing can successfully discover molecules for blocking perceived bitterness of pharmaceuticals, such as the HIV therapeutic TAF. Our hope is to use bitter taste blockers to increase medical compliance with these vital medicines. SIGNIFICANCE STATEMENT: Identification of a small molecule that inhibits bitter taste from tenofovir alafenamide may increase the compliance in treating children with human immunodeficiency virus infections.

Production of HPV16 capsid proteins in suspension cultures of human epithelial cells

Human papillomavirus (HPV) infection is a leading cause of morbidity and mortality in women worldwide. The virus is associated with benign warts and a broad spectrum of malignancies, including cervical cancer, considered a disease of high clinical relevance, especially in developing countries. In this study we developed the production of recombinant proteins HPV16 L1 and HPV16 L2 in human cells in suspension (293-F), which were transiently co-transfected with the pUF3L1h and pUF3L2h vectors. Expressions of recombinant HPV16 L1 and L2 capsid proteins was detected by laser scanning confocal microscopy and flow cytometry. Both proteins were identified intracellularly in the nucleus and cytoplasm of cells. The presence of these heterologous proteins and VLPs formation were detected by transmission electron microscopy (TEM) through colloidal gold immunolabeling and negative staining. Cell extracts containing recombinant proteins were purified by affinity chromatography and immunization of Balb/c mice with the formulation HPV16 L1/L2 VLPs containing adjuvant was able to induce higher titer of anti-HPV16 L1, when compared to HPV16 L2 antibodies by indirect ELISA assay. These data indicate that transient expression in 293-F cells was efficiently established. The results are promising for obtain recombinant proteins of the HPV capsid for future studies involving human papillomavirus, as well as to contribute for the development of other vaccine strategies for prevention against HPV.