Subepithelial Tissues Research Articles

Role of the epithelium in human papillomavirus and human immunodeficiency virus infections in the female genital tract.

Two-thirds of people living with human immunodeficiency virus type 1 (HIV-1) infection reside in Sub-Saharan Africa, where there are the highest prevalence and incidence rates of human papillomavirus (HPV) infection. Both infections are sexually transmitted and enter the body via the epithelium. This review describes the extent of involvement of the epithelium in each infection in the female genital tract. A narrative review was conducted on the role of the epithelium in HPV and HIV-1 infections. An intact epithelial barrier is the predominant form of protection against viral entry and infection, including from HIV-1 and HPV. HPV is an intraepithelial pathogen, and thus, its growth and amplification, which are dependent on squamous cell differentiation, occur in the epithelium. It gains entry to the basal cells of the stratified squamous epithelium via micro-abrasions or other epithelial injuries that expose the basement membrane. HIV-1, conversely, passes through the epithelium to infect subepithelial tissues. Following deposition of the HIV-1-containing inoculum into the lumen, the virus enters the mucosa through breaks in the epithelial barrier within hours of infection. Further, HIV-1 penetrates the epithelium via various mechanisms, including paracellular passage or across epithelial cells through transcytosis. The capture of the virus from the mucosal surface by intraepithelial and/or subepithelial target cells has also been documented. Epithelial disruption is the major pathogenetic pathway in HIV-1 and HPV infections. Therefore, biochemical compounds that strengthen the epithelial barrier must be prioritized to prevent these infections.

Dietary and circadian cues regulate gut barrier integrity by synchronizing epithelial cell-macrophage crosstalk

Abstract Small intestinal epithelial cells (sIECs) form a critical barrier essential for nutrient absorption and host defense. sIECs undergo continuous renewal to maintain their integrity. Phagocytes, such as macrophages, facilitate the clearance of apoptotic cells that enter subepithelial tissues. However, little is known about how sIEC-phagocyte crosstalk maintains barrier integrity. Our study reveals synchrony between sIEC death and the accumulation of a specific subset of macrophages engaged in engulfing dying cells. Notably, we observed a diurnal rhythmicity in the death of sIECs during homeostasis. A fraction of these dying cells enter the subepithelial space, where Tim4+ CD4+ macrophages rhythmically eliminate them. Our findings demonstrate that Tim4+ CD4+ macrophage maintenance depends on dietary vitamin A via macrophage-intrinsic retinoic acid receptor (RAR) signaling. We show that rhythmic Tim4+ CD4+ macrophage accumulation relies on light cues and the macrophage circadian clock. Impaired macrophage RAR signaling disrupts the clearance of dying sIECs, leading to compromised gut barrier integrity and increased bacterial translocation to distant organs. Our work reveals an intricate relationship between sIECs and phagocytes and identifies a crucial role for Tim4+ CD4+ macrophages in maintaining intestinal barrier integrity. Understanding these dynamics could suggest new strategies for maintaining intestinal barrier function during infection and inflammatory disease.

Comparison of temperature and renal tissue thermal damage by holmium laser with different energy parameters during lithotripsy: in vitro porcine kidney model.

Holmium laser percutaneous nephrolithotripsy was simulated by porcine kidney calculus model in vitro to investigate thermal damage of renal tissue by different energy parameters of the holmium laser. We placed human kidney calculus specimen in fresh vitro porcine kidney, then insert thermocouple temperature probes into the submucosa of the renal pelvis and reheated in a 37°C water bath. A percutaneous nephrological sheath was used to penetrate the renal parenchyma with a moderate irrigation rate of 30ml/min at 18℃. The Holmium laser was used to fragment the stones under a nephroscope, and the temperature was recorded. The four independent models were lithotripsy with 30W and 60W laser for 5 and 10min, respectively; the mean temperature of 30W vs. 60W within 5min was 36.06°C vs. 39.21°C (t = 5.36, P < 0.01) and the highest temperature was 43.60°C vs. 46.60°C; the mean temperature of 30W vs. 60W within 10min was 37.91°C vs. 40.13℃ (t = 5.28, P < 0.01), maximum temperature 46.80℃ vs. 49.20℃. Pathologically, each kidney was observed to have different degrees of thermal damage lesions, and the higher power and longer time the more severe the injury, but the injury was mainly limited to the uroepithelial and subepithelial tissues, with rare damage to renal tubules. The higher laser excitation power and longer duration raised the intrarenal temperature significantly and caused a certain degree of thermal damage to the kidney tissue, but overall it was found to be safe and reliable. Urologists can avoid further side effects through surgical expertise.

Residual nasal polyp tissue following dupilumab therapy is associated with periostin-associated fibrosis.

Dupilumab, an anti-interleukin-4 receptor alpha monoclonal antibody, is a new treatment for severe uncontrolled chronic rhinosinusitis with nasal polyps. However, data on the effect of dupilumab on histological changes in nasal polyp tissue are lacking. We aimed to investigate the effect of dupilumab on real-life clinical conditions and nasal polyp tissues from patients with eosinophilic chronic rhinosinusitis (ECRS), which is a refractory subtype. We conducted an open-label, prospective, observational, single-centre study on 63 patients with refractory ECRS on the basis of the criteria of the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis Study. These patients had a history of surgery and received dupilumab for 24weeks. Patient-reported sinonasal symptoms, T&T olfactometry and nasal polyp scores were prospectively evaluated. In 23 patients with residual nasal polyps following dupilumab treatment, changes in systemic and local periostin expression, and total collagen deposition in nasal polyp tissues were investigated before and after dupilumab administration. Dupilumab rapidly improved sinonasal symptoms and reduced the nasal polyp score 24weeks after initiation. 40 (63.5%) patients had resolution of nasal polyps, but the reduction was limited in the remaining 23 (36.5%) patients. Periostin expression in serum and nasal lavage fluid was decreased, whereas periostin and the total collagen deposition area in subepithelial tissues in residual nasal polyps were enhanced after dupilumab administration. Dupilumab improves sinonasal symptoms and reduces the nasal polyp score in refractory ECRS. Periostin-associated tissue fibrosis may be involved in the differential effect of dupilumab on nasal polyp reduction.

Cigarette smoke extract impairs gingival epithelial barrier function

We previously showed that junctional adhesion molecule 1 (JAM1) and coxsackievirus and adenovirus receptor (CXADR), tight junction-associated proteins, have important roles to maintain epithelial barrier function in gingival tissues. Smoking is considered to be a significant risk factor for periodontal disease. The present study was conducted to examine the effects of cigarette smoke extract (CSE) on JAM1 and CXADR in human gingival epithelial cells. CSE was found to cause translocation of JAM1 from the cellular surface to EGFR-positive endosomes, whereas CXADR did not. Using a three-dimensional multilayered gingival epithelial tissue model, CSE administration was found to increase permeability to lipopolysaccharide and peptidoglycan, whereas overexpression of JAM1 in the tissue model prevented penetration by those substrates. Furthermore, vitamin C increased JAM1 expression, and inhibited penetration of LPS and PGN induced by CSE. These findings strongly suggest that CSE disrupts gingival barrier function via dislocation of JAM1, thus allowing bacterial virulence factors to penetrate into subepithelial tissues. Furthermore, they indicate that vitamin C increases JAM1 expression and prevents disruption of gingival barrier function by CSE.

Evaluation of grade and invasiveness of bladder urothelial carcinoma using infrared imaging and machine learning.

Urothelial bladder carcinoma (BC) is primarily diagnosed with a subjective examination of biopsies by histopathologists, but accurate diagnosis remains time-consuming and of low diagnostic accuracy, especially for low grade non-invasive BC. We propose a novel approach for high-throughput BC evaluation by combining infrared (IR) microscopy of bladder sections with machine learning (partial least squares-discriminant analysis) to provide an automated prediction of the presence of cancer, invasiveness and grade. Cystoscopic biopsies from 50 patients with clinical suspicion of BC were histologically examined to assign grades and stages. Adjacent tissue cross-sections were IR imaged to provide hyperspectral datasets and cluster analysis segregated IR images to extract the average spectra of epithelial and subepithelial tissues. Discriminant models, which were validated using repeated random sampling double cross-validation, showed sensitivities (AUROC) ca. 85% (0.85) for the identification of cancer in epithelium and subepithelium. The diagnosis of non-invasive and invasive cases showed sensitivity values around 80% (0.84-0.85) and 76% (0.73-0.80), respectively, while the identification of low and high grade BC showed higher sensitivity values 87-88% (0.91-0.92). Finally, models for the discrimination between cancers with different invasiveness and grades showed more modest AUROC values (0.67-0.72). This proves the high potential of IR imaging in the development of ancillary platforms to screen bladder biopsies.

Efficient and simple genetic engineering of enteroids using mouse isolated crypts for investigating intestinal functions

Intestinal epithelial cells separate subepithelial tissues from luminal environment formed with food, incoming pathogens, and resident intestinal microbiota, etc., and elicit various intestinal function. Enteroid, a three-dimensional culture system of small intestinal epithelial cells, has been widely used for analyzing the intestinal function, further a transgenic enteroid was developed to investigate the molecular mechanisms. However, conventional transgenic enteroid production method, which transfer gene into single stem cells, has limitations including low efficiency and time-consuming. Here we show that by gene transfer into small intestinal isolated crypts maintaining stem cell niche, a transgenic enteroid was obtained quickly and efficiently. Isolated crypts were transfected by lentiviral vector without separating into single cells, and transgenic enteroid composed of all lineages of intestinal epithelial cells was generated at day 7 with yield of 56%, maintaining the intestinal function in drug transport and innate immunity. Our efficient and simple transgenic enteroid generation method enables high-throughput investigation of intestinal epithelial cells and contributes to understanding intestinal function.

Lactococcus lactis subsp lactis CRL1655 and Schleiferilactobacillus perolens CRL1724 inhibit the adherence of common bovine mastitis pathogens to mammary gland cells, without causing histological changes in the mammary gland.

The present work assessed the ability of two selected lactic acid bacteria (LAB) strains (Schleiferilactobacillus perolens CRL1724 and Lactococcus lactis subsp. lactis CRL1655) to inhibit the adherence of bovine mastitis pathogens to mammary epithelial cells (MAC-T) and their effects (if any) on the structure of the gland after intramammary inoculation at dry-off. Established bovine mammary epithelial cells (MAC-T) were used to assess the LAB strains' ability to inhibit the adherence of bovine mastitis pathogens. Monolayers of MAC-T cells were co-cultured with the LABs and then individual pathogen was added. Both strains prevented the adherence of S. aureus RC108, S. chromogenes, S. uberis UT102 and E. coli ATCC 35218. Adherence of the latter two pathogens was inhibited most strongly in vitro. To evaluate the effect of the LAB on the structure of the bovine udders, quarters were intramammary inoculated with the LAB mixture at dry-off. After slaughtering, the teats were dissected and histopathologically analysed. No modifications were identified post-inoculation in the structure of the epithelial, subepithelial and connective tissues of the mammary gland. Probiotic strains L. lactis subsp lactis CRL1655 and S. perolens CRL1724 were both able to inhibit the adherence of a number of bovine mastitis pathogens in vitro, and that the intramammary inoculation of these strains at the established dose and concentration did not cause significant alterations in the mammary epithelium nor had undesirable effects on tissues, and may therefore be considered harmless. The promising findings demonstrated in this work support the potential of probiotic micro-organisms as a natural and effective alternative to prevent bovine mastitis during the dry-off period.

Surface pre-reacted glass-ionomer eluate protects gingival epithelium from penetration by lipopolysaccharides and peptidoglycans via transcription factor EB pathway.

Surface pre-reacted glass-ionomer (S-PRG) filler, produced by PRG technology for use with various dental materials, is bioactive and known to release ions from a glass-ionomer phase. We previously reported that coxsackievirus and adenovirus receptor (CXADR), a tight junction associated protein, was located in the epithelial barrier of gingival epithelium. In the present study, the tissue protective effects of an S-PRG eluate prepared with S-PRG filler were investigated using a three-dimensional human gingival epithelial tissue model. The results showed that the S-PRG eluate specifically induced CXADR expression at the transcriptional level of messenger RNA as well as the protein level, and also nuclear translocation of transcription factor EB (TFEB) in gingival epithelial cells. Furthermore, shigyakusan, a TFEB inhibitor, canceled induction of the CXADR protein by the S-PRG eluate. Additionally, gingival epithelial permeation by 40-kDa dextran, lipopolysaccharide, and peptidoglycan in the 3D-tissue models was prevented by the eluate, with those effects abrogated by knockdown of CXADR. These findings suggest that S-PRG eluate increases CXADR expression via the TFEB pathway, thus inhibiting penetration of bacterial virulence factors into subepithelial tissues.

Immunohistochemical expression of angiotensin-converting enzyme 2 in superficial and deep maxillofacial tissues: A cross-sectional study.

Background and AimsThe involvement of maxillofacial tissues in SARS‐CoV‐2 infections ranges from mild dysgeusia to life‐threatening tissue necrosis, as seen in SARS‐CoV‐2‐associated mucormycosis. Angiotensin‐converting enzyme 2 (ACE2) which functions as a receptor for SARS‐CoV‐2 was reported in the epithelial surfaces of the oral and nasal cavities; however, a complete understanding of the expression patterns in deep oral and maxillofacial tissues is still lacking.MethodsThe immunohistochemical expression of ACE2 was analyzed in 95 specimens from maxillofacial tissues and 10 specimens of pulmonary alveolar tissue using a semiquantitative immunohistochemical scoring procedure, taking into account all superficial and deep maxillofacial tissue cells. We also explored the associations of age, gender, and anatomical site with expression scores.ResultsACE2 was detected in keratinized epithelia (57.34%), non‐keratinized epithelia (46.51%), nasal respiratory epithelial cells (73.35%), pulmonary alveolar cells (82.54%), fibroblasts (63.69%), vascular endothelial cells (58.43%), mucous acinar cells (59.88%), serous acinar cells (79.49%), salivary duct cells (86.26%) skeletal muscle fibers (71.01%), neuron support cells (94.25%), and bone marrow cells (72.65%). Age and gender did not affect the expression levels significantly in epithelial cells (p = 0.76, and p = 0.7 respectively); however, identical cells expressed different protein levels depending on the site from which the specimens were obtained. For example, dorsal tongue epithelia expressed significantly lower ACE2 scores than alveolar epithelia (p < 0.001). A positive correlation was found between ACE2 expression in fibroblasts and epithelial cells (r = 0.378, p = 0.001), and between vascular endothelial and epithelial cells (r = 0.395, p = 0.001).ConclusionACE2 is expressed by epithelial cells and subepithelial tissues including fibroblasts, vascular endothelia, skeletal muscles, peripheral nerves, and bone marrow. No correlation was detected between ACE2 expression and patient age or sex while the epithelial expression scores were correlated with stromal scores.

Clinical and Pathological Implications of Increases in Tonsillar CD19+CD5+ B Cells, CD208+ Dendritic Cells, and IgA1-positive Cells of Immunoglobulin A Nephropathy

Several studies indicated that tonsillectomy can improve the prognosis of patients with immunoglobulin A nephropathy (IgAN). However, the relationship between tonsillar immunity and IgAN is still unclear. A total of 14 IgAN patients were recruited in the current study from May 2015 to April 2016 in Tongji Hospital. B cells, dendritic cells (DCs), and IgA1 positive cells in human tonsils were detected using immunofluorescence and immunohistochemistry. Correlations between these cells and clinicopathologic features were evaluated. CD19+CD5+ B cells were predominantly located in germinal centers and mantle zones of lymphoid follicles, the CD208+ DCs were distributed in the interfollicular and subepithelial area, and IgA1-positive cells were predominantly detected in mantle zones of lymphoid follicles and subepithelial tissues. The numbers of CD19+CD5+ B cells, CD208+ DCs, and IgA1-positive cells in tonsillar tissues from IgAN patients were significantly higher than those in the normal controls (P<0.01, respectively). CD19+CD5+ B cells, CD208+ DCs, and IgA1-positive cells in tonsillar tissues were significantly associated with 24-h proteinuria levels and tubular atrophy/interstitial fibrosis of IgAN. CD19+CD5+ B cells, CD208+ DCs, and IgA1-positive cells in tonsillar tissues might be involved in the pathogenesis of IgAN.

Neutrophil-Epithelial Crosstalk During Intestinal Inflammation.

Neutrophils are the most abundant leukocyte population in the human circulatory system and are rapidly recruited to sites of inflammation. Neutrophils play a multifaceted role in intestinal inflammation, as they contribute to the elimination of invading pathogens. Recently, their role in epithelial restitution has been widely recognized; however, they are also associated with bystander tissue damage. The intestinal epithelium provides a physical barrier to prevent direct contact of luminal contents with subepithelial tissues, which is extremely important for the maintenance of intestinal homeostasis. Numerous studies have demonstrated that transepithelial migration of neutrophils is closely related to disease symptoms and disruption of crypt architecture in inflammatory bowel disease and experimental colitis. There has been growing interest in how neutrophils interact with the epithelium under inflammatory conditions. Most studies focus on the effects of neutrophils on intestinal epithelial cells; however, the effects of intestinal epithelial cells on neutrophils during intestinal inflammation need to be well-established. Based on these data, we have summarized recent articles on the role of neutrophil–epithelial interactions in intestinal inflammation, particularly highlighting the epithelium-derived molecular regulators that mediate neutrophil recruitment, transepithelial migration, and detachment from the epithelium, as well as the functional consequences of their crosstalk. A better understanding of these molecular events may help develop novel therapeutic targets for mitigating the deleterious effects of neutrophils in inflammatory bowel disease.

Comparative Analysis of Preoperative and Postoperative Tympanometry Findings in Patients with Adenoid in Lagos State University Teaching Hospital, Ikeja

Background: Adenoid is the collection of sub-epithelial lymphoid tissues under the mucosa of the nasopharynx situated at the junction of the roof and posterior wall of the nasopharynx, forming part of the Waldeyer’s ring of lymphoid tissues of the nasopharynx. It is also called Nasopharyngeal Tonsils and is composed of vertical ridges of lymphoid tissue separated by deep clefts, covered by ciliated columnar epithelium. Aim: To identify the Tympanometric findings in patients with Adenoids pre and post Adenoidectomy in LASUTH, Ikeja. Methodology: This is a Prospective interventional hospital-based clinical study was carried out at the Department of Otorhinolaryngology (ORL) of the Lagos State University Teaching Hospital, Ikeja, Lagos State, Nigeria. A structured questionnaire was distributed to all consecutive eligible patients diagnosed with adenoid hypertrophy seen at the Otorhinolaryngology Clinic who were recruited for the study. Thorough examination of the Ear, Nose and Throat and tympanometry were done a week before the Adenoidectomy and six weeks post-surgery. The analysis of data collected was done using Statistical Package for Social Sciences (SPSS) version 20. Results: A total of 103 subjects participated with 59.2% (61) being males and 40.8% females. The male to female ratio was 1:0.7. The most frequently affected age range was ≤ 3 years accounting for 39.8% (41) of cases while the least frequently affected was 7-9 years i.e 21.4% (22) of cases. Tympanometric findings post operatively revealed type B tympanogram (OME) was found in 72.9% of the ears, with the right ear accounting for 35.0% while the left ear was at 37.9% (p = 0.314). Postoperatively, type B tympanogram had reduced to 34% with the right ear accounting for 11.7% while the left ear was 22.3% (p = 0.314). There were also differences between the preoperative and post-operative type A and C tympanometric patterns. These differences were statistically significant (p = 0.002, p = 0.004) on the right and left ear respectively. Conclusion: Adenoid hypertrophy was more prevalent in the ≤ 3 years age range and the reduction in the frequency of type B tympanometry findings in the right and left ears postoperative was significantly less from the preoperative tympanometry findings among the subjects.

Porphyromonas gingivalis induces penetration of lipopolysaccharide and peptidoglycan through the gingival epithelium via degradation of coxsackievirus and adenovirus receptor.

Porphyromonas gingivalis is a major pathogen of human periodontitis and dysregulates innate immunity at the gingival epithelial surface. We previously reported that the bacterium specifically degrades junctional adhesion molecule 1 (JAM1), causing gingival epithelial barrier breakdown. However, the functions of other JAM family protein(s) in epithelial barrier dysregulation caused by P. gingivalis are not fully understood. The present results show that gingipains, Arg-specific or Lys-specific cysteine proteases produced by P. gingivalis, specifically degrade coxsackievirus and adenovirus receptor (CXADR), a JAM family protein, at R145 and K235 in gingival epithelial cells. In contrast, a gingipain-deficient P. gingivalis strain was found to be impaired in regard to degradation of CXADR. Furthermore, knockdown of CXADR in artificial gingival epithelium increased permeability to dextran 40 kDa, lipopolysaccharide and peptidoglycan, whereas overexpression of CXADR in a gingival epithelial tissue model prevented penetration by those agents following P. gingivalis infection. Together, these results suggest that P. gingivalis gingipains breach the stratified squamous epithelium barrier by degrading CXADR as well as JAM1, which allows for efficient transfer of bacterial virulence factors into subepithelial tissues. TAKEAWAYS: P. gingivalis, a periodontal pathogen, degraded coxsackievirus and adenovirus receptor (CXADR), a JAM family protein, in gingival epithelial tissues. P. gingivalis gingipains, cysteine proteases, degraded CXADR at R145 and K235. CXADR degradation by P. gingivalis caused increased permeability to lipopolysaccharide and peptidoglycan through gingival epithelial tissues.

Titanium dioxide particles modulate epithelial barrier protein, Claudin 7 in asthma

Epithelial barrier dysfunction is involved in allergic inflammation and asthma, due to increased exposure of sub-epithelial tissues to inhaled allergens and air pollutants. The tight junction proteins claudins (CLDNs) are important regulators of paracellular permeability. CLDN7 is expressed in the alveolar epithelium; however, its contribution to airway barrier function remains unclear. The aim of this study was to assess the effects of TiO2 on epithelial barrier function in asthma.Mice were sensitized and challenged with OVA or exposed to TiO2 on days 21–23. The effect of TiO2 on CLDN7 was assessed by ELISA, immunoblotting, and immunohistochemical analysis. The levels of CLDN7 in the plasma of patients with asthma and healthy individuals were also examined.CLDN7 levels were lower in plasma from patients with asthma compared with healthy individuals. CLDN7 levels were associated with FEV1/FVC and the blood eosinophils (%) in patients with asthma. Although CLDN7 expression was elevated in the lungs of mice with asthma and in NHBE cells treated with HDM extracts, its expression was suppressed by exposure to TiO2. p-AKT and p-ERK was increased in asthmatic mice and decreased in mice with TiO2 treatment. p-AKT and p-ERK was decreased in NHBE cells treated with TiO2 and HDM extracts. Trans-epithelial electrical resistance (TEER) was higher in NHBE cells treated with TiO2 or HDM extracts; however, this was decreased by concurrent TiO2 and HDM extracts treatment.Our data suggest that particulate matter contributes to airway epithelial barrier dysfunction and results in airway inflammation and responsiveness.

Evaluation and Assessment of Efficient Psidium Guajava and Mentha Piperita Fresh Leaves Juice for Perio Pathogens

The research was assessed to evaluate the efficacy of fresh juice of Psidium guajava and Mentha piperita against selected aerobic and anaerobic bacterial strain. The fresh juice was extracted and evaluated for its antimicrobial activity on anaerobic pathogens like Fusobacterium nucleatum (Fn), Porphyromonas gingivalis (Pg) and Prevotella intermedia (Pi). Antimicrobial activity was evaluated by the disc diffusion method and minimum inhibitory concentration. Long before since from the ancient time, humankind depended on the trees and herbs for medicines to alleviate ailments, search for better health, fragrance, flavours and food. In ancient time human beings depended on animals and plants for his food, shelter demand etc. Over 5000 years, peoples of India and China kept on the use of plants for food as well as to protect from disease. Most of the dental problems are due to microorganism (Oral Pathogens) like Fusobacterium, Actinomyces, Porphyromonas, Prevotella, Eubacterium, Bacteroids, Staphylococcus, Streptococcus, Enterococcus etc. Microbial debridement might uproot pathogenic living being due to their area. Previously, subepithelial gingival tissues, crevicular epithelial cells, other anatolian dialect features entangling sufficient defiant. The results indicate that Psidium guajava and Mentha piperita leaves shows significant Antimicrobial activity due to the presence of flavonoids. This work is more useful in health Mentha Piperita Fresh Leaves Juice for Perio Pathogens.

Abstract 3987: Direct cancer cell/cancer-associated fibroblast interaction elicits resistance to oncolytic virus therapy in squamous cell carcinoma

Abstract The interaction between different cells in the tumor microenvironment leads to changes in a plethora of cytokines secreted both by malignant and non-malignant cells. Invading epithelial tumors are able to break the basal membrane, which allows for direct cell-to-cell contact between cancer cells and fibroblasts present in the sub-epithelial tissues. To imitate this event, we performed co-culture experiments using human squamous cancer cells (SCC) and patient-derived cancer-associated fibroblasts (CAFs) of gynecological origin. This direct co-culture led to production of IFNβ and interferon-stimulated genes (ISGs), whereas co-culture using a transwell did not. Using siRNA, confocal microscopy and DNA damage compounds, we showed that CAFs can sense genotoxic stress present in the cancer cells (CC).Transfer of cytoplasmic material containing cGAMP from CC to CAFs triggers the STING-TBK-IRF3 pathway that induces an IFN type I response. Oncolytic viruses are approved in melanoma and are being tested in multiple clinical trials to treat human malignancies. There is very scarce information on how the interaction among cells in the tumor microenvironment affects the efficacy of viral therapy. We hypothesized that ISGs produced in the direct co-culture would impair the efficacy of oncolytic virus infection. Indeed, using a modified herpes virus (HSV1716), we showed that direct co-culture of CC and CAFs elicits resistance to viral infection. We were able to replicate this phenomenon using cancer cells and CAFs from gynecological and head and neck origin. Moreover, direct co-culture also decreased infectivity of other oncolytic viruses, such as vaccinia and reovirus. This resistance was dependent on IRF3 expression in CAFs, as silencing IRF3 in these cells abrogated this protection. We were able to rescue this effect by transfecting a siRNA-resistant form of IRF3 into the CAFs. Overexpression of IRF3 in CAFs further increased resistance to HSV infection in the cancer cells. Using a xenograft in vivo model of human gynecological SCC, we observed that co-injection of cancer cells and CAFs promoted resistance to HSV treatment. Transfection of siRNA targeting IRF3 into CAFs dramatically decreased tumor growth after HSV infection. This decrease was not seen when using CAFs expressing the siRNA-resistant form of IRF3. An additional model of human Head and Neck SCC demonstrated a gain-of-function effect of IRF3 overexpression on tumor growth after oncolytic virus treatment. Finally, through analysis of a single-cell RNA sequencing dataset and histological staining we were able to show IRF3 expression in CAFs present in the tumor microenvironment. Our data highlight the relevance of cancer cell/cancer-associated fibroblast crosstalk in the TME. Changes in cytokine and chemokines present in the tumor may result in changes of susceptibility to treatments like oncolytic viruses. Additionally, this could have a broader impact in the interaction between the tumor and the immune infiltrate. Citation Format: Antonio Rullan, Emma Milford, Esther Arwert, Alan Melcher, Kevin Harrington, Erik Sahai. Direct cancer cell/cancer-associated fibroblast interaction elicits resistance to oncolytic virus therapy in squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3987.

A Comparative Study of the Effect of Melatonin Versus Vitamin C on Aged Labial Tissues in Albino Rats

Background: Structural changes occur with aging, associated with fibrosis and inflammation in body tissues. Aging of oral mucosa causes alterations in oral epithelium and sub-epithelial tissues, due to reduction in certain growth factors, endogenous anti-oxidant enzymes, or both. Aim: to assess anti-aging potential of melatonin & vitamin C on albino rats’ lips.Materials and Methods: 28 male albino rats were used, divided into: Group I: 7 rats aged 6 months (18 years in humans), Group II: 21 rats aged 24 months (72 years in humans), were equally subdivided into 3 subgroups 7 rats each as follows: Subgroup IIA: positive control for other subgroups; Subgroup IIB: received melatonin via gastric gavage (10mg/kg/day) for six consecutive weeks; Subgroup IIC: received vitamin C via gastric gavage (2mg/ml/day) for six consecutive weeks. At the end of the experiment, rats were sacrificed by a high dose of anesthesia. The lips were excised. Specimens were processed for histological examination, stained by H&E, Masson’s Trichrome& Anti-VEGF, examined by light microscope.Results: Subgroup IIA showed statistically significant increase in number of vacuolated epithelial cells, statistically significant reduction in VEGF positive cells, apparent decrease in epithelial thickness, statistically significant reduction in immature collagen fibers’ percentage within connective tissue (CT). Subgroups IIB & IIC, showed significant reduction in number of vacuolated epithelial cells and significant increase in VEGF positive cells and area percentage of immature collagen fibers in CT. However, subgroup IIB’s epithelial structure showed better image and subgroup IIC’s CT structure presented better image.Conclusion: Aging showed structural changes in skin & mucous membrane sides of rat lips, observed within both epithelium and CT. Melatonin & vit. C exhibited efficiency in counteracting these effects. Melatonin presented superior effect on keratinocytes, vit. C showed superiority in repairing senile CT components.

PLGA nanocapsules improve the delivery of clarithromycin to kill intracellular Staphylococcus aureus and Mycobacterium abscessus

Drug delivery systems are promising for targeting antibiotics directly to infected tissues. To reach intracellular Staphylococcus aureus and Mycobacterium abscessus, we encapsulated clarithromycin in PLGA nanocapsules, suitable for aerosol delivery by nebulization of an aqueous dispersion. Compared to the same dose of free clarithromycin, nanoencapsulation reduced 1000 times the number of intracellular S. aureus in vitro. In RAW cells, while untreated S. aureus was located in acidic compartments, the treated ones were mostly situated in non-acidic compartments. Clarithromycin-nanocapsules were also effective against M. abscessus (70–80% killing efficacy). The activity of clarithromycin-nanocapsules against S. aureus was also confirmed in vivo, using a murine wound model as well as in zebrafish. The permeability of clarithromycin-nanocapsules across Calu-3 monolayers increased in comparison to the free drug, suggesting an improved delivery to sub-epithelial tissues. Thus, clarithromycin-nanocapsules are a promising strategy to target intracellular S. aureus and M. abscessus.

Porphyromonas gingivalis induces penetration of lipopolysaccharide and peptidoglycan through the gingival epithelium via degradation of junctional adhesion molecule 1

Porphyromonas gingivalis is a major pathogen in severe and chronic manifestations of periodontal disease, which is one of the most common infections of humans. A central feature of P. gingivalis pathogenicity is dysregulation of innate immunity at the gingival epithelial interface; however, the molecular basis underlying P. gingivalis–dependent abrogation of epithelial barrier function remains unknown. Gingival epithelial cells express junctional adhesion molecule (JAM1), a tight junction–associated protein, and JAM1 homodimers regulate epithelial barrier function. Here we show that Arg-specific or Lys-specific cysteine proteases (gingipains) secreted by P. gingivalis can specifically degrade JAM1 at K134 and R234 in gingival epithelial cells, resulting in permeability of the gingival epithelium to 40 kDa dextran, lipopolysaccharide (LPS), and proteoglycan (PGN). A P. gingivalis strain lacking gingipains was impaired in degradation of JAM1. Knockdown of JAM1 in monolayer cells and a three-dimensional multilayered tissue model also increased permeability to LPS, PGN, and gingipains. Inversely, overexpression of JAM1 in epithelial cells prevented penetration by these agents following P. gingivalis infection. Our findings strongly suggest that P. gingivalis gingipains disrupt barrier function of stratified squamous epithelium via degradation of JAM1, allowing bacterial virulence factors to penetrate into subepithelial tissues.