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Simple Summary: Bacteria are responsible for a wide range of human diseases including cancer. In our study we identified changes in the microbial communities of the female upper genital tract correlated with genomic variation in genes seen in gynecological cancers, endometrial and ovarian. This supports our hypothesis that differences in bacterial communities in the upper genital tract epithelium may lead to selection of specific genomic variation at the cellular level of these tissues that may predispose to their malignant transformation. Pathway analyses including correlated genes with changing microbiome may help understand how changing bacterial landscapes could lead to these cancers. There are strong correlations between the microbiome and human disease, including cancer. However, very little is known about potential mechanisms associated with malignant transformation in microbiome-associated gynecological cancer, except for HPV-induced cervical cancer. Our hypothesis is that differences in bacterial communities in upper genital tract epithelium may lead to selection of specific genomic variation at the cellular level of these tissues that may predispose to their malignant transformation. We first assessed differences in the taxonomic composition of microbial communities and genomic variation between gynecologic cancers and normal samples. Then, we performed a correlation analysis to assess whether differences in microbial communities selected for specific single nucleotide variation (SNV) between normal and gynecological cancers. We validated these results in independent datasets. This is a retrospective nested case-control study that used clinical and genomic information to perform all analyses. Our present study confirms a changing landscape in microbial communities as we progress into the upper genital tract, with more diversity in lower levels of the tract. Some of the different genomic variations between cancer and controls strongly correlated with the changing microbial communities. Pathway analyses including these correlated genes may help understand the basis for how changing bacterial landscapes may lead to these cancers. However, one of the most important implications of our findings is the possibility of cancer prevention in women at risk by detecting altered bacterial communities in the upper genital tract epithelium. [ABSTRACT FROM AUTHOR]

Manipulation of the rumen microbial ecosystem in early life may affect ruminal fermentation and enhance the productive performance of dairy cows. The objective of this experiment was to evaluate the effects of dosing three different types of microbial inoculum on the rumen epithelium tissue (RE) transcriptome and the rumen epimural metatranscriptome (REM) in dairy calves. For this objective, 15 Holstein bull calves were enrolled in the study at birth and assigned to three different intraruminal inoculum treatments dosed orally once weekly from three to six weeks of age. The inoculum treatments were prepared from rumen contents collected from rumen fistulated lactating cows and were either autoclaved (control; ARF), processed by differential centrifugation to create the bacterial-enriched inoculum (BE), or through gravimetric separation to create the protozoal-enriched inoculum (PE). Calves were fed 2.5 L/d pasteurized waste milk 3x/d from 0 to 7 weeks of age and texturized starter until euthanasia at 9 weeks of age, when the RE tissues were collected for transcriptome and microbial metatranscriptome analyses, from four randomly selected calves from each treatment. The different types of inoculum altered the RE transcriptome and REM. Compared to ARF, 9 genes were upregulated in the RE of BE and 92 in PE, whereas between BE and PE there were 13 genes upregulated in BE and 114 in PE. Gene ontology analysis identified enriched GO terms in biological process category between PE and ARF, with no enrichment between BE and ARF. The RE functional signature showed different KEGG pathways related to BE and ARF, and no specific KEGG pathway for PE. We observed a lower alpha diversity index for RE microbiome in ARF (observed genera and Chao1 (p < 0.05)). Five microbial genera showed a significant correlation with the changes in host gene expression: Roseburia (25 genes), Entamoeba (two genes); Anaerosinus, Lachnospira, and Succiniclasticum were each related to one gene. sPLS-DA analysis showed that RE microbial communities differ among the treatments, although the taxonomic and functional microbial profiles show different distributions. Co-expression Differential Network Analysis indicated that both BE and PE had an impact on the abundance of KEGG modules related to acyl-CoA synthesis, type VI secretion, and methanogenesis, while PE had a significant impact on KEGGs related to ectoine biosynthesis and D-xylose transport. Our study indicated that artificial dosing with different microbial inocula in early life alters not only the RE transcriptome, but also affects the REM and its functions., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The human pathogen Neisseria gonorrhoeae ascends into the upper female reproductive tract to cause damaging inflammation within the Fallopian tubes and pelvic inflammatory disease (PID), increasing the risk of infertility and ectopic pregnancy. The loss of ciliated cells from the epithelium is thought to be both a consequence of inflammation and a cause of adverse sequelae. However, the links between infection, inflammation, and ciliated cell extrusion remain unresolved. With the use of ex vivo cultures of human Fallopian tube paired with RNA sequencing we defined the tissue response to gonococcal challenge, identifying cytokine, chemokine, cell adhesion, and apoptosis related transcripts not previously recognized as potentiators of gonococcal PID. Unexpectedly, IL-17C was one of the most highly induced genes. Yet, this cytokine has no previous association with gonococcal infection nor pelvic inflammatory disease and thus it was selected for further characterization. We show that human Fallopian tubes express the IL-17C receptor on the epithelial surface and that treatment with purified IL-17C induces pro-inflammatory cytokine secretion in addition to sloughing of the epithelium and generalized tissue damage. These results demonstrate a previously unrecognized but critical role of IL-17C in the damaging inflammation induced by gonococci in a human explant model of PID., (© 2024. The Author(s).)

Adhesion to mucosal surfaces is a critical step in many bacterial and fungal infections. Here, using a mouse model of oral infection by the human fungal pathobiont Candida albicans, we report the identification of a novel regulator of C. albicans adhesion to the oral mucosa. The regulator is a member of the regulatory factor X (RFX) family of transcription factors, which control cellular processes ranging from genome integrity in model yeasts to tissue differentiation in vertebrates. Mice infected with the C. albicans rfx1 deletion mutant displayed increased fungal burden in tongues compared to animals infected with the reference strain. High-resolution imaging revealed RFX1 transcripts being expressed by C. albicans cells during infection. Concomitant with the increase in fungal burden, the rfx1 mutant elicited an enhanced innate immune response. Transcriptome analyses uncovered HWP1, a gene encoding an adhesion protein that mediates covalent attachment to buccal cells, as a major RFX1-regulated locus. Consistent with this result, we establish that C. albicans adhesion to oral cells is modulated by RFX1 in an HWP1-dependent manner. Our findings expand the repertoire of biological processes controlled by the RFX family and illustrate a mechanism whereby C. albicans can adjust adhesion to the oral epithelium., (© 2024 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Direct laser metal forming (DLMF) is a procedure in which a high-power laser beam is directed on a metal powder bed and programmed to fuse particles according to a computer-aided design file, thus generating a thin metal layer. With DLMF, it is now possible to fabricate dental implants with a superficial porous surface. The aim of the present study was to evaluate the peri-implant soft tissues around human-retrieved DLMF dental implants. Collagen fibers, in the form of bundles, were oriented perpendicularly to a distance of 100 μm from the surface, where they became parallel, running in several directions. In some portions, only a few collagen fiber bundles appeared to be oriented perpendicularly or obliquely to the plane of the section. Collagen fibers appeared to form a dense chaotic three-dimensional network running in different, more or less parallel directions to the surface. Under scanning electron microscopy, an intimate contact of the fibrous matrix with the implant surface was evident, and some collagen bundles could be seen to bind directly to the metal surface. By changing the surface microtexture, it was possible to change the response of the peri-implant soft tissues. [ABSTRACT FROM AUTHOR]

Background: Clonorchis sinensis is a zoonotic liver fluke that inhabits the bile ducts of the human liver for prolonged periods, leading to cholangiocarcinoma. Recent research indicates associations between altered biliary microbiota and bile duct disorders. However, the impacts of C. sinensis infection on bile duct epithelium and subsequent effects on biliary microbiota remain unknown., Methods: Feline bile duct samples were collected from both uninfected and C. sinensis-infected cats. Histopathological examination was performed to assess epithelial changes, fibrosis, mucin and cell proliferation using hematoxylin-eosin staining and immunohistochemistry. Additionally, biliary microbiota composition was analyzed through 16S rRNA gene sequencing. Statistical analyses were conducted to compare the microbial diversity and relative abundance between infected and uninfected samples., Results: Histopathological analysis of infected feline bile ducts revealed prominent epithelial hyperplasia characterized by increased cell proliferation. Moreover, periductal fibrosis and collagen fibrosis were observed in infected samples compared to uninfected controls. Biliary microbial richness decreased with disease progression compared to uninfected controls. Streptococcus abundance positively correlated with disease severity, dominating communities in cancer samples. Predictive functional analysis suggested that C. sinensis may promote bile duct lesions by increasing microbial genes for carbohydrate metabolism, replication, and repair., Conclusions: This study provides comprehensive insights into the pathological effects of C. sinensis infection on feline bile duct epithelium and its influence on biliary microbiota composition. These novel findings provide insight into C. sinensis pathogenesis and could inform therapeutic development against human clonorchiasis. Further research is warranted to elucidate the underlying mechanisms driving these changes and their implications for host-parasite interactions., (© F. Li et al., published by EDP Sciences, 2024.)

Purpose: To determine the role of IQ-domain GTPase-activating protein1 (IQGAP-1) in tight junctions of human corneal epithelial cells (HCECs) and its effect against P. aeruginosa (PAK) invasion.Material and Methods: Primary human corneal epithelial cells (HCECs), immortalized HCECs, and IQGAP-1 RNA knockdown HCECs (siHCECs) were used. Confocal microscopy, transepithelial electrical resistance (TER), trypan blue exclusion assay and gentamicin invasion assay were done.Results: In primary and immortalized HCECs, IQGAP-1 co-localized with zonular occludin-1 (ZO-1) and actin. Enhanced actin and ZO-1 aggregation were seen in siHCECs. IQGAP-1 knockdown significantly increased TER of immortalized HCECs (P < .0001). Cell viability after PAK infection increased for siHCECs for up to 4 h after infection. PAK intracellular invasion was significantly lowered by 50% in siHCECs at 1 h post-infection.Conclusion: IQGAP-1 knockdown increased the strength and integrity of tight junctions and may provide an early protective effect against P. aeruginosa invasion. [ABSTRACT FROM AUTHOR]

Background: Sinonasal inverted papilloma (SIP) is a benign tumour originating from the sinonasal mucosa showing an extensive growth pattern, a high risk of recurrence and a 5–10% risk to malignify. Epstein-Barr virus (EBV) is an oncogenic herpesvirus which infects most individuals via the saliva eliciting a latent infection. Previous studies have been reporting variable data on EBV in SIP, and there is no present appreciation regarding the association between these. Aims/objectives: The aims were to investigate the presence and count of EBV in SIP and map the viral distribution in the epithelium versus the connective tissue. Material and method: Fifty-three SIP patients were identified in the Pathology Department register at the University Hospital of Umeå. The biopsies were analysed with Epstein-Barr Encoded Region (EBER) in situ hybridization. EBER-positive cells were counted in the epithelium and connective tissue. Results: We found EBER-stained cells in 30% of the cases, where 19% of these had an abundance of stained cells, and the rest showed a low count. Conclusions/significance: These findings demonstrate a low EBV count in SIP. EBV is less likely to be a causative agent in the formation of SIP, or its malignant transformation. [ABSTRACT FROM AUTHOR]

The article offers information on several methods in lung microbiome research sharon including the literature search is conducted with the goal of capturing broadly-defined lung microbiome studies with primary data; and anatomical structures are not strictly within the scope of pulmonary disorders and are being actively investigated as part of other organ system disorders.

Tissue-resident immune cells are important for organ homeostasis and defense. The epithelium may contribute to these functions directly or by cross-talk with immune cells. We used single-cell RNA sequencing to resolve the spatiotemporal immune topology of the human kidney. We reveal anatomically defined expression patterns of immune genes within the epithelial compartment, with antimicrobial peptide transcripts evident in pelvic epithelium in the mature, but not fetal, kidney. A network of tissue-resident myeloid and lymphoid immune cells was evident in both fetal and mature kidney, with postnatal acquisition of transcriptional programs that promote infection-defense capabilities. Epithelial-immune cross-talk orchestrated localization of antibacterial macrophages and neutrophils to the regions of the kidney most susceptible to infection. Overall, our study provides a global overview of how the immune landscape of the human kidney is zonated to counter the dominant immunological challenge. [ABSTRACT FROM AUTHOR]

The Wnt ligands display varied spatiotemporal expression in the epithelium and mesenchyme in the developing tooth. Thus far, the actions of these differentially expressed Wnt ligands on tooth development are not clear. Shh expression specifies the odontogenic epithelium during initiation and is consistently restricted to the dental epithelium during tooth development. In this study, we inactivate Wntless ( Wls), the key regulator for Wnt trafficking, by Shh-Cre to investigate how the Wnt ligands produced in the dental epithelium lineage act on tooth development. We find that conditional knockout of Wls by Shh-Cre leads to defective ameloblast and odontoblast differentiation. WlsShh-Cre teeth display reduced canonical Wnt signaling activity in the inner enamel epithelium and the underlying mesenchyme at the early bell stage, as exhibited by target gene expression and BAT-gal staining. The expression of Wnt5a and Wnt10b is not changed in WlsShh-Cre teeth. By contrast, Wnt10a expression is significantly increased in response to epithelial Wls deficiency. In addition, the expression of Hedgehog signaling pathway components Shh, Gli1, and Patched1 was greatly decreased in WlsShh-Cre teeth. Epithelial Wls loss of function in Shh lineage also leads to aberrant cell proliferation in dental epithelium and mesenchyme at embryonic day 16.5; however, the cell apoptosis is unaffected. Moreover, we find that Decorin and Col1a1, the key markers for odontoblast differentiation that are downregulated in WlsShh-Cre teeth, act as direct downstream targets of the canonical Wnt signaling pathway by chromatin immunoprecipitation analysis. Additionally, Decorin and Col1a1 expression can be increased by lithium chloride (LiCl) treatment in the in vitro tooth explants. Taken together, our results suggest that the spatial expression of Wnt ligands within the dental epithelial lineage regulates the differentiation of tooth structures in later stages. [ABSTRACT FROM AUTHOR]

Background: Evaluate the effect of low-temperature plasma (LTP) on an anaerobic biofilm and on the biological response of an in vitro reconstituted gingival epithelium tissue.Methods: Porphyromonas gingivalis W83 biofilm was cultured on titanium discs and reconstituted gingival tissues were submitted to similar treatment conditions.Treatments: LTP1-plasma treatment for 1 minute, LTP3-plasma treatment for 3 minute, CHX-0.2% chlorhexidine for 1 minute, GAS-gas only (no plasma) for 3 minute, and NEGATIVE-no treatment. TRITON group was included as a positive control for tissue analysis. Counting of viable colony forming units (CFU/mL) and confocal laser scanning microscopy were performed to evaluate LTP's antimicrobial effect. EpiGingival tissue was evaluated through cytotoxocity, viability, histology, and imunnohistochemistry (Ki67, vascular endothelial growth factor-A vascular endothelial growth factor A [VEGF-A], and terminal deoxynucleotidyl transferase dUTP nick end labeling terminal deoxynucleotidyl transferase dutp nick end labeling [TUNEL] expression).Results: LTP1 and LTP3 presented significantly different reduced CFU/mL reduction in comparison to the negative control (Ρ < 0.001), but it was not as effective as the positive control (CHX). Low cytotoxicity and high viability were observed in gingival epithelium of NEGATIVE, GAS, CHX, and both LTP groups. The morphologic analysis of gingival epithelium revealed minor cell damage in the plasma groups (score 1). LTP1, LTP3, GAS, and NEGATIVE groups exhibited less than 5% of basal layer positive cells. LTP1, LTP3, GAS, and CHX groups were not positive for TUNEL assay. LTP1 and LTP3 showed the most positivity for VEGF.Conclusions: LTP treatment can be considered as an effective method for reducing P. gingivalis biofilm on implant surfaces, while being safe for the gingival epithelium. Furthermore, plasma treatment may be associated with cell repair. [ABSTRACT FROM AUTHOR]

Inflammatory bowel diseases (IBD) are chronic conditions of the gastrointestinal tract-the main site of host-microbial interaction in the body. Development of IBD is not due to a single event but rather is a multifactorial process where a patient's genetic background, behavioral habits, and environmental exposures contribute to disease pathogenesis. IBD patients exhibit alterations to gut bacterial populations "dysbiosis" due to the inflammatory microenvironment, however whether this alteration of the gut microbiota precedes inflammation has not been confirmed. Emerging evidence has highlighted the important role of gut microbes in developing measured immune responses and modulating other host responses such as metabolism. Much of the work on the gut microbiota has been correlative and there is an increasing need to understand the intimate relationship between host and microbe. In this review, we highlight how commensal and pathogenic bacteria interact with host intestinal epithelial cells and explore how altered microenvironments impact these connections. [ABSTRACT FROM AUTHOR]

Objective: Cystic fibrosis (CF) patients are exposed to respiratory system pathogens mostly forming biofilms, and therefore therapeutic interventions frequently fail. In this study, we aimed to investigate the resulting phenotypic changes in Pseudomonas aeruginosa due to interaction between bacteria and respiratory epithelium of CF patients. Methods: In vitro investigation of some phenotypic characteristics of P. aeruginosa such as biofilm formation, adhesion, and its growth rate was made in normal and CF bronchial (BEAS-2B and IB3-1) and tracheal (1HAEo- and CFTE29o-) epithelium. P. aeruginosa PAO1, a green fluorescent protein expressing strain, was used in the experiments. Results: Adhesion of P. aeruginosa to CF bronchial epithelium had decreased and biofilm formation had increased in CF bronchial epithelium compared to normal bronchial epithelium. Moreover, P. aeruginosa was found to be the cause of morphological changes in the CF bronchial epithelium. Conclusions: Our findings support that the changes in respiratory epithelium with CF directly affect bacterial phenotypic characteristics in infected CF patients, and these phenotypic changes may be the major determinant of unresponsiveness to antimicrobial agents during treatment of infections. [ABSTRACT FROM AUTHOR]

Background: The airway epithelium is the first barrier interacting with Aspergillus fumigatus conidia after their inhalation, suggesting that this structure functions as point of entry of this fungus to initiate pulmonary aspergillosis.Methods: To study epithelial entry by A fumigatus, primary human reconstituted pseudostratified epithelium cultured in air-liquid interface as well as bronchial epithelial cell monolayers were infected with conidia.Results: Under these experimental conditions, we found that A fumigatus hyphae traversed the bronchial epithelium through a mechanism involving the recruitment of actin, which formed a tunnel that allows hyphae to enter the cells without disturbing their integrity.Conclusions: These findings describe a new mechanism by which A fumigatus hyphae penetrate the airway epithelial barrier and can infect its human host. [ABSTRACT FROM AUTHOR]

Emerging data have underscored the significance of exogenous supplementation of butyrate in the regulation of rumen development and homeostasis. However, the effects of other short-chain fatty acids (SCFAs), such as acetate or propionate, has received comparatively less attention, and the consequences of extensive exogenous SCFA infusion remain largely unknown. In our study, we conducted a comprehensive investigation by infusion of three SCFAs to examine their respective roles in regulating the rumen microbiome, metabolism, and epithelium homeostasis. Data demonstrated that the infusion of sodium acetate (SA) increased rumen index while also promoting SCFA production and absorption through the upregulation of SCFA synthetic enzymes and the mRNA expression of SLC9A1 gene. Moreover, both SA and sodium propionate infusion resulted in an enhanced total antioxidant capacity, an increased concentration of occludin, and higher abundances of specific rumen bacteria, such as " Candidatus Saccharimonas," Christensenellaceae R-7, Butyrivibrio , Rikenellaceae RC9 gut, and Alloprevotella . In addition, sodium butyrate (SB) infusion exhibited positive effects by increasing the width of rumen papilla and the thickness of the stratum basale. SB infusion further enhanced antioxidant capacity and barrier function facilitated by cross talk with Monoglobus and Incertae Sedis . Furthermore, metabolome and transcriptome data revealed distinct metabolic patterns in rumen contents and epithelium, with a particular impact on amino acid and fatty acid metabolism processes. In conclusion, our data provided novel insights into the regulator effects of extensive infusion of the three major SCFAs on rumen fermentation patterns, antioxidant capacity, rumen barrier function, and rumen papilla development, all achieved without inducing rumen epithelial inflammation. IMPORTANCE The consequences of massive exogenous supplementation of SCFAs on rumen microbial fermentation and rumen epithelium health remain an area that requires further exploration. In our study, we sought to investigate the specific impact of administering high doses of exogenous acetate, propionate, and butyrate on rumen homeostasis, with a particular focus on understanding the interaction between the rumen microbiome and epithelium. Importantly, our findings indicated that the massive infusion of these SCFAs did not induce rumen inflammation. Instead, we observed enhancements in antioxidant capacity, strengthening of rumen barrier function, and promotion of rumen papilla development, which were facilitated through interactions with specific rumen bacteria. By addressing existing knowledge gaps and offering critical insights into the regulation of rumen health through SCFA supplementation, our study holds significant implications for enhancing the well-being and productivity of ruminant animals., Competing Interests: The authors declare no conflict of interest.

The author discusses the potential of microbiome-based therapeutics and diagnostics in generating excitement among scientists, clinicians, and entrepreneurs. He cites several studies that focused on microbiome therapeutics and their relationship with disease. The author also recognizes the advantage of meta-analysis in microbiome study and development as it prioritizes hypotheses without requiring significant additional investment.

The use of in vitro barrier models is gaining relevance as an alternative to animal studies in risk assessment, pharmacokinetic, and toxicological studies in general. These models permit an easier evaluation of the underlying mechanisms taking place at the molecular and cellular levels on the barrier site. Here, we report several methodological modifications of the three-dimensional in vitro intestinal epithelial model Caco-2/HT29/Raji-B for its successful application in the Nanotoxicology field. In addition, new insights in the study of specific molecular markers and new confocal microscope approaches have also been incorporated. Due to the multiple variables and parameters playing a part when the model's complexity is increased, we have monitored the barrier's formation and cell differentiation over time. Finally, the practical usability of the proposed model was tested by evaluating the action of the food additives titanium dioxide and silica dioxide nanoparticles (TiO 2 NPs and SiO 2 NPs). The NPs-associated effects were evaluated by confocal microscopy. We have demonstrated the essential role of the mucus layer in the decrease of cellular uptake, avoiding potential NPs-cell nuclei interactions. [ABSTRACT FROM AUTHOR]

Livestock on the Qinghai-Tibetan Plateau are faced with extreme harsh winters and are often in negative energy balance during this period. Dietary supplementation can improve growth performance of Tibetan sheep and, consequently, we hypothesized that it would also increase microbial abundance and rumen epithelium development. To test this hypothesis, we examined the effect of feed supplementation during the cold season on rumen microbes, fermentation, epithelium development, and absorptive capability in Tibetan sheep. Eighteen 1-yr-old ewes (BW = 29.4 ± 1.79, kg) were offered oat hay ad libitum for 60 d and divided randomly into three groups: 1) no supplement; control group (CON); 2) urea-molasses lick block supplement (BS); and 3) concentrate feed supplement (CS). The ADG of CS ewes (143.3, g/d) was greater (P < 0.05) than BS ewes (87.9, g/d), which was greater (P < 0.05) than CON ewes (44.5, g/d). Serum concentrations of GH, IGF-1, and IGF-2 in the CS and BS groups were greater than in the CON group (P < 0.05). Greater relative abundance of protozoa, Ruminococcus albus, Fibrobacter succinogenes, Streptococcus bovis, and Ruminobacter amylophilus was observed in the CS and BS groups than in the CON group (P < 0.05), and relative abundances of rumen fungi, Butyrivibrio fibrisolvens, and Prevotella ruminicola in the CS group were greater than in the BS and CON groups (P < 0.05). Ruminal total VFA, ammonia, and microbial protein concentrations in the CS and BS groups were greater than in the CON group (P < 0.05), and in the CS group were greater than in the BS group (P < 0.05). Ruminal papillae width and surface area in the CS and BS groups were greater than in the CON group (P < 0.05), while in the CS group were greater than in the BS group (P < 0.05). The mRNA expressions of IGFBP5, NHE1 (sodium/hydrogen antiporter, isoform 1), DRA (downregulated in adenoma), and Na+/K+-ATPase (sodium/potassium ATPase pump) in ruminal epithelium were greater in the CS and BS groups than in the CON group (P < 0.05), and in the CS group was greater than in the BS group (P < 0.05), while NHE3 (sodium/hydrogen antiporter, isoform 3), MCT1 (monocarboxylate transporter 1), and MCT4 (monocarboxylate transporter 4) mRNA expressions in the CS group were greater than in the BS and CON groups (P < 0.05). It was concluded that supplementing Tibetan sheep during the cold season increases rumen microbial abundance and improves fermentation parameters, rumen epithelium development, and absorptive capability. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the type of the epithelium present on the epiglottis surface, through histological examination. The epithelium lining the pharyngeal side of epiglottis is keratinized stratified squamous. The thickness of the keratin layer is different from one subject to another. Usually the keratin layer extends to the apex of the epiglottis, but in some of the animals taken into study, the keratin layer extends to the apical third of the laryngeal side of epiglottis. The epithelium lining the laryngeal side is different. In most of the epiglottises examined, the surface is lined by non-keratinized stratified squamous epithelium in the apical third. The rest of the laryngeal side of the epiglottis is lined by respiratory epithelium type. In one subject, the entire laryngeal surface is lined by a non-keratinized stratified squamous epithelium. Our study highlights that the pharyngeal side of epiglottis is lined by a keratinized stratified squamous epithelium, whereas on the laryngeal side, the epithelium in the apical third is non-keratinized stratified squamous and at the rest of the surface the epithelium is pseudostratified. [ABSTRACT FROM AUTHOR]

An increasing number of reports suggests a role of hyaluronan (HA) in female reproduction and interest in its application in assisted reproduction is rising. However, there are contrasting data about the effectiveness of adding HA to the embryo-transfer medium on improving pregnancy rates. Using sheep as an experimental model, the studies reported here analysed the impact of HA infusion into the uterus on embryo attachment to uterine luminal epithelium (LE) and expression of selected markers of uterine receptivity. On Day 14 after natural mating (pre-attachment), uterine horns were infused with either (n = 4 each): PBS (control), HA (1mg mL-1), HA + hyaluronidase 2 (Hyal2; 300IU mL-1) or 4-methyl-umbelliferone (HA-synthesis inhibitor; 4MU, 1 mM). HA immunostaining on uterine sections collected on Day 17 was negative in the 4MU group and weak in the HA+Hyal2 group. In contrast to 4MU, which resulted in 100% attachment, HA infusion blocked embryo attachment in all treated animals. This was accompanied by the disappearance of mucin 1 and increased expression of osteopontin and CD44v6 in the LE of uteri with attached embryos. In conclusion, the presence of HA at the embryo-maternal interface during embryo implantation resulted in reduced endometrial receptivity and inhibited the interaction of trophoblasts with the LE, whereas clearance of HA favoured embryo attachment. [ABSTRACT FROM AUTHOR]

Citrobacter rodentium infection is a mouse model for the important human diarrheal infection caused by enteropathogenic E. coli (EPEC). The pathogenesis of both species is very similar and depends on their unique ability to form intimately epithelium-adherent microcolonies, also known as “attachment/effacement” (A/E) lesions. These microcolonies must be dynamic and able to self-renew by continuous re-infection of the rapidly regenerating epithelium. It is unknown whether sustained epithelial A/E lesion pathogenesis is achieved through re-infection by planktonic bacteria from the luminal compartment or local spread of sessile bacteria without a planktonic phase. Focusing on the earliest events as C. rodentium becomes established, we show here that all colonic epithelial A/E microcolonies are clonal bacterial populations, and thus depend on local clonal growth to persist. In wild-type mice, microcolonies are established exclusively within the first 18 hours of infection. These early events shape the ongoing intestinal geography and severity of infection despite the continuous presence of phenotypically virulent luminal bacteria. Mechanistically, induced resistance to A/E lesion de-novo formation is mediated by TLR-MyD88/Trif-dependent signaling and is induced specifically by virulent C. rodentium in a virulence gene-dependent manner. Our data demonstrate that the establishment phase of C. rodentium pathogenesis in vivo is restricted to a very short window of opportunity that determines both disease geography and severity. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae (Sp) is a commensal bacterium that normally resides on the upper airway epithelium without causing infection. However, factors such as co-infection with influenza virus can impair the complex Sp-host interactions and the subsequent development of many life-threatening infectious and inflammatory diseases, including pneumonia, meningitis or even sepsis. With the increased threat of Sp infection due to the emergence of new antibiotic resistant Sp strains, there is an urgent need for better treatment strategies that effectively prevent progression of disease triggered by Sp infection, minimizing the use of antibiotics. The complexity of the host-pathogen interactions has left the full understanding of underlying mechanisms of Sp-triggered pathogenesis as a challenge, despite its critical importance in the identification of effective treatments. To achieve a systems-level and quantitative understanding of the complex and dynamically-changing host-Sp interactions, here we developed a mechanistic mathematical model describing dynamic interplays between Sp, immune cells, and epithelial tissues, where the host-pathogen interactions initiate. The model serves as a mathematical framework that coherently explains various in vitro and in vitro studies, to which the model parameters were fitted. Our model simulations reproduced the robust homeostatic Sp-host interaction, as well as three qualitatively different pathogenic behaviors: immunological scarring, invasive infection and their combination. Parameter sensitivity and bifurcation analyses of the model identified the processes that are responsible for qualitative transitions from healthy to such pathological behaviors. Our model also predicted that the onset of invasive infection occurs within less than 2 days from transient Sp challenges. This prediction provides arguments in favor of the use of vaccinations, since adaptive immune responses cannot be developed de novo in such a short time. We further designed optimal treatment strategies, with minimal strengths and minimal durations of antibiotics, for each of the three pathogenic behaviors distinguished by our model. The proposed mathematical framework will help to design better disease management strategies and new diagnostic markers that can be used to inform the most appropriate patient-specific treatment options. [ABSTRACT FROM AUTHOR]

α-Defensin 5 is important to both maintenance of a gut microbiota and host immunity. While previous reports have shown that gut bacteria are able to upregulate α-defensin 5 through Toll-like receptor signaling, we demonstrate here that α-defensin 5 expression can also be regulated by microbial metabolites. Among these, lactate appeared to significantly suppress α-defensin 5 gene transcription. Actually, fractions of <3 kD compounds obtained from the ceca of SPF mice were suppressed α-defensin 5 gene transcription at specific concentrations. Our results also suggest that cecal content may include as yet unidentified factors that can enhance α-defensin 5 expression. Our data point to a novel function for the gut microbial metabolites in controlling the expression of antimicrobial peptides in the intestine. [ABSTRACT FROM PUBLISHER]

The intestinal epithelium is an active barrier separating the host from its microbiota. It senses microbial compounds through expression of a wide range of receptors including the Toll-like receptors (TLRs). TLRs have been shown to regulate epithelium permeability or secretion of defensin by Paneth cells. However, the expression and function of TLRs in enteroendocrine L-cells, a specific subtype of intestinal cells secreting PYY and GLP-1, have not yet been assessed. PYY and GLP-1 are implicated in regulation of gut motility, food intake and insulin secretion, and are of great interest regarding obesity and type 2 diabetes. Using a cellular model of human L-cells and a reporter system for NF-κB activation pathway, we reported functional expression of TLRs in these cells. Stimulation with specific TLR-agonists increased expression of Pyy but not Proglucagon in an NF-κB-dependent manner. Moreover, the effect of TLR stimulation was additive to butyrate, a product of bacterial fermentation, on Pyy expression. Additionally, butyrate also increased Tlr expression, including Tlr4, and the NF-κB response to TLR stimulation. Altogether, our results demonstrated a role of TLRs in the modulation of Pyy expression and the importance of butyrate, a product of bacterial fermentation in regulation of microbial TLR-dependent sensing. [ABSTRACT FROM AUTHOR]

Background: Streptococcus agalactiae can cause urinary tract infection (UTI). The role of the S. agalactiae global virulence regulator, CovR, in UTI pathogenesis is unknown.Methods: We used murine and human bladder uroepithelial cell models of UTI and S. agalactiae mutants in covR and related factors, including β-hemolysin/cytolysin (β-h/c), surface-anchored adhesin HvgA, and capsule to study the role of CovR in UTI.Results: We found that covR-deficient serotype III S. agalactiae 874391 was significantly attenuated for colonization in mice and adhesion to uroepithelial cells. Mice infected with covR-deficient S. agalactiae produced less proinflammatory cytokines than those infected with wild-type 874391. Acute cytotoxicity in uroepithelial cells triggered by covR-deficient but not wild-type 874391 was associated with significant caspase 3 activation. Mechanistically, covR mutation significantly altered the expression of several genes in S. agalactiae 874391 that encode key virulence factors, including β-h/c and HvgA, but not capsule. Subsequent mutational analyses revealed that HvgA and capsule, but not the β-h/c, exerted significant effects on colonization of the murine urinary tract in vivo.Conclusions: S. agalactiae CovR promotes bladder infection and inflammation, as well as adhesion to and viability of uroepithelial cells. The pathogenesis of S. agalactiae UTI is complex, multifactorial, and influenced by virulence effects of CovR, HvgA, and capsule. [ABSTRACT FROM AUTHOR]

Background: The wide use of topical and systemic antifungal agents as the conventional treatment for oral candidiasis has caused Candida albicans to develop resistance to these agents. The aims of this study were to evaluate the effects of photodynamic therapy (PDT) and chitosan on buccal candidiasis and study the possible enhancive effect of chitosan on the photosensitizer methylene blue.Methods: Thirty-five DBA/2 immunosuppressed mice were orally inoculated with a suspension of Candida albicans. The animals were randomized into seven groups (n = 5 per group): Group 1 (control); Group 2 (nystatin); Group 3 (PDT); Group 4 (chitosan 1.5 mg/ml); Group 5 (chitosan 3 mg/ml); Group 6 (PDT + chitosan 1.5 mg/ml); and Group 7 (PDT + chitosan 3 mg/ml). The Candida albicans count was evaluated on days 3, 5, 7, and 11 after inoculation. At last, macroscopic and microscopic analyses of the tongue dorsa were performed.Results: On day 7 after inoculation, the control group showed a greater number of Candida albicans (5.25 ± 0.41 log10 CFU/ml), with significant differences compared to all other groups (P ≤ 0.05). On day 11 after inoculation, animals treated with PDT showed lower CFU/ml count. Groups 2, 3, 4, 5, and 6 showed fewer microscopic candidiasis lesions than Groups 1 and 7.Conclusions: PDT has an antifungal effect, even greater than nystatin. Chitosan has a powerful fungicide effect but did not possess any enhancive effect on methylene blue. [ABSTRACT FROM AUTHOR]

Human papillomavirus (HPV) is a double-helix DNA virus in the papillomaviridae family. HPV has an affinity for epithelial tissue and causes benign and malignant changes to the stratified epithelium of the epidermis and mucous membranes. HPV is an etiological factor in many benign and malignant lesions of the head, neck, urogenital organs, skin, and mucous membranes. The role of HPV in the pathogenicity of squamous cell carcinoma in the head and neck area was first recognised in 1983 after findings showed histopathologic similarities between oral cancer and HPV infection. Because HPV has been implicated as the etiological factor for oral squamous cell carcinoma, HPV infection can be regarded as a subgroup of cancers affecting the oropharyngeal region. Due to these characteristics, HPV infection has been of particular interest in the field of maxillofacial surgery and dentistry. Screening, oral findings, early diagnosis, and proper treatment are of paramount importance in many HPVrelated infections. [ABSTRACT FROM AUTHOR]

Microbiome-based stratification of healthy individuals into compositional categories, referred to as “enterotypes” or “community types”, holds promise for drastically improving personalized medicine. Despite this potential, the existence of community types and the degree of their distinctness have been highly debated. Here we adopted a dynamic systems approach and found that heterogeneity in the interspecific interactions or the presence of strongly interacting species is sufficient to explain community types, independent of the topology of the underlying ecological network. By controlling the presence or absence of these strongly interacting species we can steer the microbial ecosystem to any desired community type. This open-loop control strategy still holds even when the community types are not distinct but appear as dense regions within a continuous gradient. This finding can be used to develop viable therapeutic strategies for shifting the microbial composition to a healthy configuration. [ABSTRACT FROM AUTHOR]

Background: The pathogenesis of pulmonary oedema (PE) in patients with severe malaria is still unclear. It has been hypothesized that lung injury depends, in addition to microvascular obstruction, on an increased pulmonary capillary pressure and altered alveolar-capillary membrane permeability, causing pulmonary fluid accumulation. Methods: This study compared the histopathological features of lung injury in Southeast Asian patients (n = 43) who died from severe Plasmodium falciparum malaria, and correlated these with clinical history in groups with or without PE. To investigate the expression of mediators that may influence fluid accumulation in PE, immunohistochemistry and image analysis were performed on controls and sub-sets of patient with or without PE. Results: The expression of leukocyte sub-set antigens, bronchial interleukin (IL)-33, γ-epithelium sodium channel (ENaC), aquaporin (AQP)-1 and -5, and control cytokeratin staining was quantified in the lung tissue of severe malaria patients. Bronchial IL-33 expression was significantly increased in severe malaria patients with PE. Malaria patients with shock showed significantly increased bronchial IL-33 compare to other clinical manifestations. Bronchial IL-33 levels were positively correlated with CD68+ monocyte and elastase + neutrophil, septal congestion and hyaline membrane formation. Moreover, the expression of both vascular smooth muscle cell (VSMC) and bronchial γ-ENaC significantly decreased in severe malaria patients with PE. Both VSMC and bronchial γ-ENaC were negatively correlated with the degree of parasitized erythrocyte sequestration, alveolar thickness, alveolar expansion score, septal congestion score, and malarial pigment score. In contrast AQP-1 and -5 and pan cytokeratin levels were similar between groups. Conclusions: The results suggest that IL-33 may play a role in lung injury during severe malaria and lead to PE. Both VSMC and bronchial γ-ENaC downregulation may explain pulmonary fluid disturbances and participate in PE pathogenesis in severe malaria patients. [ABSTRACT FROM AUTHOR]

Anthrax, caused by Bacillus anthracis, a Gram-positive spore-forming bacterium, is initiated by the entry of spores into the host body. There are three types of human infection: cutaneous, inhalational, and gastrointestinal. For each form, B. anthracis spores need to cross the cutaneous, respiratory or digestive epithelial barriers, respectively, as a first obligate step to establish infection. Anthrax is a toxi-infection: an association of toxemia and rapidly spreading infection progressing to septicemia. The pathogenicity of Bacillus anthracis mainly depends on two toxins and a capsule. The capsule protects bacilli from the immune system, thus promoting systemic dissemination. The toxins alter host cell signaling, thereby paralyzing the immune response of the host and perturbing the endocrine and endothelial systems. In this review, we will mainly focus on the events and mechanisms leading to crossing of the respiratory epithelial barrier, as the majority of studies have addressed inhalational infection. We will discuss the critical gaps of knowledge that need to be addressed to gain a comprehensive view of the initial steps of inhalational anthrax. We will then discuss the few data available on B. anthracis crossing the cutaneous and digestive epithelia. [ABSTRACT FROM AUTHOR]

Summary The intestinal epithelial barrier plays a critical role in the mucosal immunity. However, it remains largely unknown how the epithelial barrier is maintained after damage. Here we show that growth factor FGF2 synergized with interleukin-17 (IL-17) to induce genes for repairing of damaged epithelium. FGF2 or IL-17 deficiency resulted in impaired epithelial proliferation, increased pro-inflammatory microbiota outgrowth, and consequently worse pathology in a DSS-induced colitis model. The dysregulated microbiota in the model induced transforming growth factor beta 1 (TGFβ1) expression, which in turn induced FGF2 expression mainly in regulatory T cells. Act1, an essential adaptor in IL-17 signaling, suppressed FGF2-induced ERK activation through binding to adaptor molecule GRB2 to interfere with its association with guanine nucleotide exchange factor SOS1. Act1 preferentially bound to IL-17 receptor complex, releasing its suppressive effect on FGF2 signaling. Thus, microbiota-driven FGF2 and IL-17 cooperate to repair the damaged intestinal epithelium through Act1-mediated direct signaling cross-talk. [ABSTRACT FROM AUTHOR]

The epithelium is the main entry point for many viruses, but the processes that protect barrier surfaces against viral infections are incompletely understood. Here we identified interleukin 22 (IL-22) produced by innate lymphoid cell group 3 (ILC3) as an amplifier of signaling via interferon-λ (IFN-λ), a synergism needed to curtail the replication of rotavirus, the leading cause of childhood gastroenteritis. Cooperation between the receptor for IL-22 and the receptor for IFN-λ, both of which were 'preferentially' expressed by intestinal epithelial cells (IECs), was required for optimal activation of the transcription factor STAT1 and expression of interferon-stimulated genes (ISGs). These data suggested that epithelial cells are protected against viral replication by co-option of two evolutionarily related cytokine networks. These data may inform the design of novel immunotherapy for viral infections that are sensitive to interferons. [ABSTRACT FROM AUTHOR]

The tumor suppressor p53 is a major regulator of genes important for cell cycle arrest, senescence, apoptosis, and innate immunity, and has recently been implicated in retinal aging. In this study we sought to identify the genetic networks that regulate p53 function in the retina using quantitative trait locus (QTL) analysis. First we examined age-associated changes in the activation and expression levels of p53; known p53 target proteins and markers of innate immune system activation in primary retinal pigment epithelial (RPE) cells that were harvested from young and aged human donors. We observed increased expression of p53, activated caspase-1, CDKN1A, CDKN2A (p16INK4a), TLR4, and IFNα in aged primary RPE cell lines. We used the Hamilton Eye Institute (HEI) retinal dataset () to identify genomic loci that modulate expression of genes in the p53 pathway in recombinant inbred BXD mouse strains using a QTL systems biology-based approach. We identified a significant trans-QTL on chromosome 1 (region 172-177 Mb) that regulates the expression of Cdkn1a. Many of the genes in this QTL locus are involved in innate immune responses, including Fc receptors, interferon-inducible family genes, and formin 2. Importantly, we found an age-related increase in FCGR3A and FMN2 and a decrease in IFI16 levels in RPE cultures. There is a complex multigenic innate immunity locus that controls expression of genes in the p53 pathway in the RPE, which may play an important role in modulating age-related changes in the retina. [ABSTRACT FROM AUTHOR]

The use of dental implants in replacing missing teeth is proven to be a valid treatment with a high success rate. To achieve the best treatment outcome in all implant systems, the implant has to be able to integrate with the surrounding tissue. However, dental implants are affected by peri-implant diseases and may fail as a result. As the number of implants placed continues to increase, the prevalence of peri-implant disease will also increase. This requires preventive measures to inhibit the development of the disease and stop its progression. Clinical Relevance: Understanding how to maintain healthy peri-implant tissue as well as diagnosis and treatment of disease are vital for every dentist and dental student. [ABSTRACT FROM AUTHOR]

Exfoliating infected bladder epithelium is a powerful host defense mechanism that reduces bacterial burden. In this issue of Immunity , Choi et al. (2016) present a function of mast cells that orchestrates the infected epithelial cell exfoliation via cytolytic granules. [ABSTRACT FROM AUTHOR]

Abstract: Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode that has been used as experimental model to investigate the factors determining the expulsion of intestinal helminths. We analyze the changes in the protein expression and glycosylation induced by E. caproni in Wistar rat, a host of low compatibility in which the parasites are rapidly rejected. To determine the changes in protein expression, two-dimensional difference gel electrophoresis was employed using protein extracts from the intestine of naïve and infected rats. The patterns of glycosylation were analyzed by lectin blotting. Those spots showing differential expression or glycosylation were analyzed by mass spectrometry. A total of 33 protein spots differentially expressed were identified (26 were found to be over-expressed and 7 down-regulated). Moreover, E. caproni induced changes in the glycosylation status of 8 proteins that were successfully identified. Most of these proteins were related to the cytoskeleton and the maintenance of the functional integrity of the ileal epithelium. This suggests that the regeneration of the intestinal tissue is a major effector mechanism responsible for the early expulsion of this helminth. Furthermore, several proteins involved in the energy metabolism were also altered in the ileum of rats as a consequence of the E. caproni infection. Biological significance: Our analysis provides essential new insights in the factors determining the natural expulsion of intestinal parasitic helminths from their hosts. The results obtained contribute to a better understanding of the effective mechanisms involved in the defense against the intestinal helminths. The identification of proteins in the intestine that become modified in their expression or glycosylation in hosts in which the parasite is rapidly rejected may serve for the development of tools for the control of these infections. [Copyright &y& Elsevier]

The article deals with the direct molecular effects of parasitic helminths on host tissue. Topics include disruption of epithelial barrriers, reinforcement of epithelial barriers, alteration of lymphoid tisue, modulation of tissue vascularity and tuning of granulomatous responses. The helminths studied include Schistosoma haematobium, Trichuris trichiura, Strongyloided stercoralis, Schistosoma japonicum, Nippostrongylus brasiliensis, Heligmosomoides polygyrus and Dirofilaria immitis.

The innate immune system is the first line in the defense system and prevents the body from further bacteria, virus, or fungal infections.Most of the innateimmune systemis relevant tomucosaimmunity. Lactotransferrin is secreted fromthe humanmammal breast duct epithelial tissue and strengthens infant immunity to defensewith regard to outward pathogens. Splunc-1 is also an innate material secreted from the soft palate, lung, nasal cavity epithelium, and mucosa. It helps with mucosa defense against bacterial, virus, and even fungus. LPS is the main etiology of Gram-negative bacilla infection source. And studies of lactoferricin and slpunc- 1 both can combine with LPS and subsequently cause insults to the mucosa. Although, we know that both of them partake in an important role in innate immunity, we do not know the effects when they work together. In this study, we just overview silicon stimulation to examine the combination of Lactoferricin and Splunc-1 and the effect with regard to LPS. [ABSTRACT FROM AUTHOR]

We evaluated a hypothesis that horses are susceptible to avian influenza viruses by in vitro testing, using explanted equine tracheal epithelial cultures, and in vivo testing by aerosol inoculation of ponies. Results showed that several subtypes of avian influenza viruses detectably replicated in vitro. Three viruses with high in vitro replication competence were administered to ponies. None of the three demonstrably replicated or caused disease signs in ponies. While these results do not exhaustively test our hypothesis, they do highlight that the tracheal explant culture system is a poor predictor of in vivo infectivity. [ABSTRACT FROM AUTHOR]

Abstract: Colonization of the gastric epithelium by Helicobacter pylori induces the transcription factor nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) and the innate immune response. Virulent strains of H. pylori carry a cag pathogenicity island (cagPAI), which encodes a type IV secretion system (T4SS). Recent publications have shown controversial data regarding the role of the T4SS and the effector protein cytotoxin associated gene A (CagA), which becomes translocated by the T4SS into the eukaryotic epithelial cell, in H. pylori-induced NF-κB activation. Thus, this study analyses by using three different H. pylori strains (P1, B128 and G27) whether CagA is required to initiate activation of different molecules of inhibitors of kappa B (IκB) and the NF-κB transcription factor RelA. We provide experimental evidence that H. pylori induces phosphorylation of NF-κB inhibitors IκBα, IκBβ and IκBɛ, and degradation of IκBα. Further, H. pylori stimulates phosphorylation of RelA at amino acids S536, S468 and S276, promotes DNA binding of RelA, and interleukin 8 (IL-8) gene expression in a T4SS-, but CagA-independent manner at early time points. [Copyright &y& Elsevier]

Conjugation of therapeutic proteins with high molecular weight polyethylene glycols (HMW PEGs) is used to extend the half-life of biologics. To evaluate the effects of HMW PEGs in animals, we used an immunohistochemical procedure to study the tissue distribution and toxicity of unconjugated HMW PEGs in rats given 100 mg/kg 10KPEG, 20KPEG, or 40KPEG intravenously. Both the PEG cellular distribution and the histology were different between groups. In 10KPEG and 20KPEG groups, PEG immunoreactivity was most prominent in the renal tubule epithelium and in alveolar macrophages and hepatic Kupffer cells and cellular vacuolation was absent. In contrast, rats given 40KPEG had strong PEG immunoreactivity in splenic subcapsular red pulp macrophages, renal interstitial macrophages, and choroid plexus epithelial cells that was frequently associated with cytoplasmic vacuolation. While the vacuolation appeared to be an adaptive response, there was focal renal tubular epithelial degeneration associated with strong PEG immunoreactivity in one rat given 40KPEG. These data indicate that both the tissue distribution and the vacuolation observed with unconjugated HMW PEGs are markedly influenced by the molecular weight of the PEG and that when vacuolation is observed it is likely an adaptive change that is associated with PEG cytoplasmic immunoreactivity. [ABSTRACT FROM AUTHOR]

Objective: Implication of human caspase-4 in periodontitis and in sensing periodontal pathogens by gingival epithelial cells (GECs) is unclear. This study aimed to determine caspase-4 and interleukin (IL)-18 expressions in gingival tissues affected with periodontitis and to investigate caspase-4 involvement in mediating innate immune responses in GECs., Design: Ex vivo, caspase-4 and IL-18 expressions in gingival biopsies, obtained from healthy participants with periodontitis or clinically healthy gingiva (N = 20 each), were determined by immunohistochemistry. In vitro, caspase-4 activation in cultured GECs stimulated with Porphyromonas gingivalis or Fusobacterium nucleatum was analyzed by immunoblotting. mRNA expressions of human β-defensin-2 (hBD-2), IL-8, and IL-18 in stimulated GECs in the presence or absence of a caspase-4 inhibitor were assayed by RT-qPCR., Results: Ex vivo, compared with healthy gingival epithelium, the epithelium affected with periodontitis displayed a significant decrease in caspase-4 expression (P = 0.015), whereas IL-18 expression was significantly increased (P = 0.012). Moreover, the expression of caspase-4, but not IL-18, was found to be a predictor of periodontitis (P = 0.007). In vitro, caspase-4 was activated in cultured GECs challenged with P. gingivalis, but not F. nucleatum. mRNA upregulations of hBD-2, IL-8, and IL-18 upon P. gingivalis stimulation were significantly reduced when caspase-4 was inhibited (P < 0.05), whereas the inhibitor failed to suppress those inductions by F. nucleatum., Conclusions: Caspase-4 expression is diminished in the epithelium affected with periodontitis while that of IL-18 is enhanced. Caspase-4 activation in P. gingivalis-infected GECs upregulates the three innate immune effector molecules, suggesting a possible sensing mechanism of caspase-4 in GECs in periodontal disease pathogenesis., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Lactobacillus-deficient cervicovaginal microbiota, including Gardnerella vaginalis, are implicated in cervical remodeling and preterm birth. Mechanisms by which microbes drives outcomes are not fully elucidated. We hypothesize that Gardnerella vaginalis induces matrix metalloproteinases through TLR-2, leading to epithelial barrier dysfunction and premature cervical remodeling. Cervicovaginal cells were treated with live Gardnerella vaginalis or Lactobacillus crispatus or their bacteria-free supernatants for 24 h. For TLR-2 experiments, cells were pretreated with TLR-2 blocking antibody. A Luminex panel was run on cell media. For human data, we conducted a case-control study from a prospective pregnancy cohort of Black individuals with spontaneous preterm (sPTB) (n = 40) or term (n = 40) births whose vaginal microbiota had already been characterized. Cervicovaginal fluid was obtained between 20 and 24 weeks' gestation. Short cervix was defined as < 25 mm by second trimester transvaginal ultrasound. MMP-9 was quantified by ELISA. Standard analytical approaches were used to determine differences across in vitro conditions, as well as MMP-9 and associations with clinical outcomes. Gardnerella vaginalis induced MMP-1 in cervical cells (p = 0.01) and MMP-9 in cervical and vaginal (VK2) cells (p ≤ 0.001 for all). TLR-2 blockade mitigated MMP-9 induction by Gardnerella vaginalis. MMP-9 in cervicovaginal fluid is higher among pregnant individuals with preterm birth, short cervix, and Lactobacillus-deficient microbiota (p < 0.05 for all). MMP-9 is increased in the cervicovaginal fluid of pregnant individuals with subsequent sPTB. Our in vitro work ascribes a potential mechanism by which a cervicovaginal microbe, commonly associated with adverse pregnancy outcomes, may disrupt the cervicovaginal epithelial barrier and promote premature cervical remodeling in spontaneous preterm birth., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Damage to the lung epithelium is a unifying feature of disease caused by the saprophytic fungus Aspergillus fumigatus . However, the mechanistic basis and the regulatory control of such damage is poorly characterized. Previous studies have identified A. fumigatus mediated pathogenesis as occurring at early (≤ 16 hours) or late (>16 hours) phases of the fungal interaction with epithelial cells, and respectively involve direct contact with the host cell or the action of soluble factors produced by mature fungal hyphae. Both early and late phases of epithelial damage have been shown to be subject to genetic regulation by the pH-responsive transcription factor PacC. This study sought to determine whether other transcriptional regulators play a role in modulating epithelial damage. In particular, whether the early and late phases of epithelial damage are governed by same or distinct regulators. Furthermore, whether processes such as spore uptake and hyphal adhesion, that have previously been documented to promote epithelial damage, are governed by the same cohorts of epithelial regulators. Using 479 strains from the recently constructed library of A. fumigatus transcription factor null mutants, two high-throughput screens assessing epithelial cell detachment and epithelial cell lysis were conducted. A total of 17 transcription factor mutants were found to exhibit reproducible deficits in epithelial damage causation. Of these, 10 mutants were defective in causing early phase damage via epithelial detachment and 8 mutants were defective in causing late phase damage via epithelial lysis. Remarkably only one transcription factor, PacC, was required for causation of both phases of epithelial damage. The 17 mutants exhibited varied and often unique phenotypic profiles with respect to fitness, epithelial adhesion, cell wall defects, and rates of spore uptake by epithelial cells. Strikingly, 9 out of 10 mutants deficient in causing early phase damage also exhibited reduced rates of hyphal extension, and culture supernatants of 7 out of 8 mutants deficient in late phase damage were significantly less cytotoxic. Our study delivers the first high-level overview of A. fumigatus regulatory genes governing lung epithelial damage, suggesting highly coordinated genetic orchestration of host-damaging activities that govern epithelial damage in both space and time., Competing Interests: MB is the director and shareholder of PIQ Laboratories Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rahman, van Rhijn, Papastamoulis, Thomson, Carter, Fortune-Grant, Rattray, Bromley and Bignell.)

Objective:To introduce the application of confocal endomicroscopy during microlaryngoscopy, to enable intra-operative evaluation of human laryngeal epithelium.Methods:A rigid endoscope was connected to the scanner head of a Heidelberg Retina Tomograph II confocal laser scanning system via an adapter. The endoscope was gently placed on the surface of a vocal fold through a laryngoscope during microlaryngoscopy.Results:The application of confocal endomicroscopy using a rigid endoscope enabled technical improvements (i.e. improved image quality, automatic volume scan, and reduced tissue pressure due to the presence of a perforation plate with central hole at the end of the endoscope) which permitted greater sensitivity and improved handling. Confocal endomicroscopy provided good quality, in vivo, en-face images and enabled an assessment of laryngeal epithelium volume.Conclusion:This method enables the surgeon to monitor epithelial changes in pre-malignant lesions. The combination of confocal endomicroscopy together with optical coherence tomography (as a complementary technique that provides optical cross-sections) should be further explored in a formal clinicopathological study. [ABSTRACT FROM PUBLISHER]

Background: The toxicity of many xenobiotic compounds is believed to involve oxidative injury to cells. Direct assessment of mechanistic events involved in xenobiotic-induced oxidative stress is not easily achievable. Development of genetically encoded probes designed for monitoring intracellular redox changes represents a methodological advance with potential applications in toxicological studies. Objective: We tested the utility of redox-sensitive green fluorescent protein (roGFP)-based redox sensors for monitoring real-time intracellular redox changes induced by xenobiotics in toxicological studies. Methods: roGFP2, a reporter of the glutathione redox potential (EGSH), was used to monitor EGSH in cultured human airway epithelial cells (BEAS-2B cells) undergoing exposure to 0.15-1.0 ppm ozone (O3). Cells were imaged in real time using a custom-built O3 exposure system coupled to a confocal microscope. Results: O3 exposure induced a dose- and time-dependent increase of the cytosolic EGSH. Additional experiments confirmed that roGFP2 is not directly oxidized, but properly equilibrates with the glutathione redox couple: Inhibition of endogenous glutaredoxin 1 (Grx1) disrupted roGFP2 responses to O3, and a Grx1-roGFP2 fusion protein responded more rapidly to O3 exposure. Selenite-induced up-regulation of GPx (glutathione peroxidase) expression-enhanced roGFP2 responsiveness to O3, suggesting that (hydro)peroxides are intermediates linking O3 exposure to glutathione oxidation. Conclusion: Exposure to O3 induces a profound increase in the cytosolic EGSH of airway epithelial cells that is indicative of an oxidant-dependent impairment of glutathione redox homeostasis. These studies demonstrate the utility of using genetically encoded redox reporters in making reliable assessments of cells undergoing exposure to xenobiotics with strong oxidizing properties. [ABSTRACT FROM AUTHOR]

Abstract: Bacillus thuringiensis (Bt) Cry1A toxin-binding sites in the Douglas fir tussock moth (DFTM) larval gut were localized using immunofluorescence microscopy. Cry1Aa, Cry1Ab and Cry1Ac all bound strongly to the DFTM peritrophic membrane (PM); weaker binding of the Cry1A toxins was observed along the apical brush border of the midgut epithelium. Comparative analysis of the Cry1A toxin-binding molecules in the PM and brush border membrane vesicles (BBMVs) showed that a similar toxin-binding complex was present in both. The Cry1A toxin-binding substance, a broad band with an apparent size of 180kDa, consisted of a closely spaced doublet. The doublet was present in peritrophins, proteins tightly bound to the PM. Lectin binding studies of the PM and BBMV toxin-binding components revealed that they are glyconjugates with terminal α-GalNAc residues comprised exclusively of O-linked oligosaccharides in their glycan structures. Mild periodate oxidation, release of O-linked glycans by β-elimination, and enzymatic removal of terminal α-linked GalNAc residues with N-acetyl-α-d-galactosaminidase digestion abolished Cry1A toxin-binding to the PM and BBMV components. These data provide strong evidence that O-linked glycans are the target structures on the toxin-binding glycoconjugates for the Cry1A class of insecticidal proteins in DFTM larvae. [Copyright &y& Elsevier]