Loss Of Epithelial Integrity Research Articles

The ciliary GTPase Arl3 maintains tissue architecture by directing planar spindle orientation during epidermal morphogenesis.

Arl/ARF GTPases regulate ciliary trafficking, but their tissue-specific functions are unclear. Here, we demonstrate that ciliary GTPase Arl3 is required for mitotic spindle orientation of mouse basal stem cells during skin development. Arl3 loss diminished cell divisions within the plane of the epithelium, leading to increased perpendicular divisions, expansion of progenitor cells and loss of epithelial integrity. These observations suggest that an Arl3-dependent mechanism maintains cell division polarity along the tissue axis, and disruption of planar spindle orientation has detrimental consequences for epidermal architecture. Defects in planar cell polarity (PCP) can disrupt spindle positioning during tissue morphogenesis. Upon Arl3 loss, the PCP signaling molecules Celsr1 and Vangl2 failed to maintain planar polarized distributions, resulting in defective hair follicle angling, a hallmark of disrupted PCP. In the absence of Celsr1 polarity, frizzled 6 lost its asymmetrical distribution and abnormally segregated to the apical cortex of basal cells. We propose that Arl3 regulates polarized endosomal trafficking of PCP components to compartmentalized membrane domains. Cell-cell communication via ciliary GTPase signaling directs mitotic spindle orientation and PCP signaling, processes that are crucial for the maintenance of epithelial architecture.

Probing Clostridium difficile Infection in Complex Human Gut Cellular Models.

Interactions of anaerobic gut bacteria, such as Clostridium difficile, with the intestinal mucosa have been poorly studied due to challenges in culturing anaerobes with the oxygen-requiring gut epithelium. Although gut colonization by C. difficile is a key determinant of disease outcome, precise mechanisms of mucosal attachment and spread remain unclear. Here, using human gut epithelial monolayers co-cultured within dual environment chambers, we demonstrate that C. difficile adhesion to gut epithelial cells is accompanied by a gradual increase in bacterial numbers. Prolonged infection causes redistribution of actin and loss of epithelial integrity, accompanied by production of C. difficile spores, toxins, and bacterial filaments. This system was used to examine C. difficile interactions with the commensal Bacteroides dorei, and interestingly, C. difficile growth is significantly reduced in the presence of B. dorei. Subsequently, we have developed novel models containing a myofibroblast layer, in addition to the epithelium, grown on polycarbonate or three-dimensional (3D) electrospun scaffolds. In these more complex models, C. difficile adheres more efficiently to epithelial cells, as compared to the single epithelial monolayers, leading to a quicker destruction of the epithelium. Our study describes new controlled environment human gut models that enable host–anaerobe and pathogen–commensal interaction studies in vitro.

Epithelial to Mesenchymal Transition and Reepithelialisation in Wound Healing: A review of Comparison

Skin wound healing is a complex physiological event, involving many cellular and molecular components. The event of wound healing is the coordinated overlap of a number of distinct phases, namely haemostasis, inflammatory, proliferative and remodelling. The molecular events surrounding wound healing, particularly the reepithelialisation, has been reported to be similar to the epithelial to mesenchymal transition (EMT). In this review, the mechanism between epithelialisation and EMT were compared. Both are characterised by the loss of epithelial integrity and increased motility. In terms of the signalling kinases, Smad and mitogen-activated protein kinase (MAPK) has been reported to be involved in both reepithelialisation and EMT. At the transcriptional level, SLUG transcription factor has been reported to be important for both reepithelialisation and EMT. Extracellular matrix proteins that have been associated with both events are collagen and laminin. Lastly, both events required the interplay between matrix metalloproteinases (MMPs) and its inhibitor. As a conclusion, both reepithelialisation and EMT shares similar signaling cascade and transcriptional regulation to exhibit decreased epithelial traits and increased motility in keratinocytes.

Aaquetzalli is required for epithelial cell polarity and neural tissue formation in Drosophila.

Morphogenetic movements during embryogenesis require dynamic changes in epithelial cell polarity and cytoskeletal reorganization. Such changes involve, among others, rearrangements of cell-cell contacts and protein traffic. In Drosophila melanogaster, neuroblast delamination during early neurogenesis is a well-characterized process requiring a polarized neuroepithelium, regulated by the Notch signaling pathway. Maintenance of epithelial cell polarity ensues proper Notch pathway activation during neurogenesis. We characterize here aaquetzalli (aqz), a gene whose mutations affect cell polarity and nervous system specification. The aqz locus encodes a protein that harbors a domain with significant homology to a proline-rich conserved domain of nuclear receptor co-activators. aqz expression occurs at all stages of the fly life cycle, and is dynamic. aqz mutants are lethal, showing a disruption of cell polarity during embryonic ventral neuroepithelium differentiation resulting in loss of epithelial integrity and mislocalization of membrane proteins (shown by mislocalization of Crumbs, DE-Cadherin, and Delta). As a consequence, aqz mutant embryos with compromised apical-basal cell polarity develop spotty changes of neuronal and epithelial numbers of cells.

Candida albicans-Induced Epithelial Damage Mediates Translocation through Intestinal Barriers.

ABSTRACTLife-threatening systemic infections often occur due to the translocation of pathogens across the gut barrier and into the bloodstream. While the microbial and host mechanisms permitting bacterial gut translocation are well characterized, these mechanisms are still unclear for fungal pathogens such as Candida albicans, a leading cause of nosocomial fungal bloodstream infections. In this study, we dissected the cellular mechanisms of translocation of C. albicans across intestinal epithelia in vitro and identified fungal genes associated with this process. We show that fungal translocation is a dynamic process initiated by invasion and followed by cellular damage and loss of epithelial integrity. A screen of >2,000 C. albicans deletion mutants identified genes required for cellular damage of and translocation across enterocytes. Correlation analysis suggests that hypha formation, barrier damage above a minimum threshold level, and a decreased epithelial integrity are required for efficient fungal translocation. Translocation occurs predominantly via a transcellular route, which is associated with fungus-induced necrotic epithelial damage, but not apoptotic cell death. The cytolytic peptide toxin of C. albicans, candidalysin, was found to be essential for damage of enterocytes and was a key factor in subsequent fungal translocation, suggesting that transcellular translocation of C. albicans through intestinal layers is mediated by candidalysin. However, fungal invasion and low-level translocation can also occur via non-transcellular routes in a candidalysin-independent manner. This is the first study showing translocation of a human-pathogenic fungus across the intestinal barrier being mediated by a peptide toxin.

Potential role of periodontal pathogens in compromising epithelial barrier function by inducing epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells acquire a mesenchymal-like phenotype and this may be induced by exposure to gram-negative bacteria. It has been proposed that EMT is responsible for compromising epithelial barrier function in the pathogenesis of several diseases. However, the possible role of EMT in the pathogenesis of periodontitis has not previously been investigated. The aim of this study therefore was to investigate whether gram-negative, anaerobic periodontal pathogens could trigger EMT in primary oral keratinocytes in vitro. Primary oral keratinocytes were harvested from labial mandibular mucosa of Wistar Han rats. Cells were exposed to heat-killed Fusobacterium nucleatum and Porphyromonas gingivalis (100 bacteria/epithelial cell) and to 20μg/mL of Escherichia coli lipopolysaccharide over an 8-day period. Exposure to bacteria did not significantly change epithelial cell number or vitality in comparison with unstimulated controls at the majority of time-points examined. Expression of EMT marker genes was determined by semiquantitative RT-PCR at 1, 5, and 8days following stimulation. The expression of EMT markers was also assessed by immunofluorescence (E-cadherin and vimentin) and using immunocytochemistry to determine Snail activation. The loss of epithelial monolayer coherence, in response to bacterial challenge, was determined by measuring trans-epithelial electrical resistance. The induction of a migratory phenotype was investigated using scratch-wound and transwell migration assays. Exposure of primary epithelial cell cultures to periodontal pathogens was associated with a significant decrease in transcription (~3-fold) of E-cadherin and the upregulation of N-cadherin, vimentin, Snail, matrix metalloproteinase-2 (~3-5 fold) and toll-like receptor 4. Bacterial stimulation (for 8 days) also resulted in an increased percentage of vimentin-positive cells (an increase of 20% after stimulation with P.gingivalis and an increase of 30% after stimulation with F.nucleatum, compared with controls). Furthermore, periodontal pathogens significantly increased the activation of Snail (60%) and cultures exhibited a decrease in electrical impedance (P<.001) in comparison with unexposed controls. The migratory ability of the cells increased significantly in response to bacterial stimulation, as shown by both the number of migrated cells and scratch-wound closure rates. Prolonged exposure of primary rat oral keratinocyte cultures to periodontal pathogens generated EMT-like features, which introduces the possibility that this process may be involved in loss of epithelial integrity during periodontitis.

Immune response patterns in non-communicable inflammatory skin diseases.

Non‐communicable inflammatory skin diseases (ncISD) such as psoriasis or atopic eczema are a major cause of global disease burden. Due to their impact and complexity, ncISD represent a major challenge of modern medicine. Dermatology textbooks describe more than 100 different ncISD based on clinical phenotype and histological architecture. In the last decades, this historical description was complemented by increasing molecular knowledge – and this knowledge is now being translated into specific therapeutics. Combining the enormous advances made in lymphocyte immunology and molecular genetics with clinical and histological phenotyping reveals six immune response patterns of the skin – type I immune cells cause the lichenoid pattern characterized by immune‐mediated cell death of keratinocytes; type II immune cells underlie the eczematous pattern with impaired epidermal barrier, infection and eosinophils as well as the bullous pattern with loss of epithelial integrity; Th17 cells and ILC3 mediate the psoriatic pattern characterized by acanthosis, high metabolic activity and neutrophils; dysbalance of regulatory T cells causes either the fibrogenic pattern with rarefication of cells and dermal thickening or the granulomatous pattern defined by formation of granulomas. With more and more specific therapeutic agents approved, classifying ncISD also according to their immune response pattern will become highly relevant. This review defines the six immune response patterns of ncISD and highlights therapeutic strategies targeting key lymphocyte mediators.

Do advanced glycation end-products cause food allergy?

The aim of this study was to appraise the evidence relating to dietary advanced glycation end-products (AGEs), their influence on innate and adaptive immune responses and their possible role in the increasing rate of food allergy that is being observed globally. The western pattern of diet is high in both AGEs and their substrates and this has been increasing in the last 30 years. AGEs mimic alarmins such as S100 proteins and the high molecular group box 1 (HMBG1), binding to the Receptor for Advanced Glycation End Products (RAGE), which in turn influences innate and adaptive immune responses. AGEs can directly cause mast cell activation and degranulation. Population data from USA and Australia have correlates with the rise in food allergy and sources of AGEs in the diet and this is also reinforced by urban and regional data within countries. The function of the RAGE receptor is inter-twined with Toll-like receptor (TLR) signalling and vitamin D levels. Activation of the RAGE receptor results in loss of epithelial integrity. Animal models studies have shown that dietary compounds that reduce the formation of AGEs can protect from the development of food allergies; however, this has not been shown in humans. Dietary sources of AGEs and their substrates are a plausible contributor to the increasing prevalence of food allergy, which requires prospective evaluation in human populations.

Marked Enteropathy in an Accelerated Macaque Model of AIDS

Enteropathy in HIV infection is not eliminated with combination antiretroviral therapy and is possibly linked to microbial translocation. We used a rapidly progressing SIV/pigtailed macaque model of HIV to examine enteropathy and microbial translocation. Histologic evidence of intestinal disease was observed in only half of infected macaques during late-stage infection (LSI). Combination antiretroviral therapy initiated during acute infection prevented intestinal disease. In the ileum and colon, enteropathy was associated with increased caspase-3 staining, decreased CD3+ T cells, and increased SIV-infected cells. CD3+ T cells were preserved in LSI animals without intestinal disease, and levels of CD3 staining in all LSI animals strongly correlated with the number of infected cells in the intestine and plasma viral load. Unexpectedly, there was little evidence of microbial translocation as measured by soluble CD14, soluble CD163, lipopolysaccharide binding protein, and microbial 16s ribosomal DNA. Loss of epithelial integrity indicated by loss of the tight junction protein claudin-3 was not observed during acute infection despite significantly fewer T cells. Claudin-3 was reduced in LSI animals with severe intestinal disease but did not correlate with increased microbial translocation. LSI animals that did not develop intestinal disease had increased T-cell intracytoplasmic antigen 1-positive cytotoxic T lymphocytes, suggesting a robust adaptive cytotoxic T-lymphocyte response may, in part, confer resilience to SIV-induced intestinal damage.

The adaptor protein Cindr regulates JNK activity to maintain epithelial sheet integrity

Epithelia are essential barrier tissues that must be appropriately maintained for their correct function. To achieve this a plethora of protein interactions regulate epithelial cell number, structure and adhesion, and differentiation. Here we show that Cindr (the Drosophila Cin85 and Cd2ap ortholog) is required to maintain epithelial integrity. Reducing Cindr triggered cell delamination and movement. Most delaminating cells died. These behaviors were consistent with JNK activation previously associated with loss of epithelial integrity in response to ectopic oncogene activity. We confirmed a novel interaction between Cindr and Drosophila JNK (dJNK), which when perturbed caused inappropriate JNK signaling. Genetically reducing JNK signaling activity suppressed the effects of reducing Cindr. Furthermore, ectopic JNK signaling phenocopied loss of Cindr and was partially rescued by concomitant cindr over-expression. Thus, correct Cindr-dJNK stoichiometry is essential to maintain epithelial integrity and disturbing this balance may contribute to the pathogenesis of disease states, including cancer.

Comprehensive evaluation of poly(I:C) induced inflammatory response in an airway epithelial model.

Respiratory viruses invade the upper airway of the lung, triggering a potent immune response that often exacerbates preexisting conditions such as asthma and COPD. Poly(I:C) is a synthetic analog of viral dsRNA that induces the characteristic inflammatory response associated with viral infection, such as loss of epithelial integrity, and increased production of mucus and inflammatory cytokines. Here, we explore the mechanistic responses to poly(I:C) in a well-defined primary normal human bronchial epithelial (NHBE) model that recapitulates in vivo functions and responses. We developed functional and quantifiable methods to evaluate the physiology of our model in both healthy and inflamed states. Through gene and protein expression, we validated the differentiation state and population of essential cell subtypes (i.e., ciliated, goblet, club, and basal cells) as compared to the human lung. Assays for total mucus production, cytokine secretion, and barrier function were used to evaluate in vitro physiology and response to viral insult. Cells were treated apically with poly(I:C) and evaluated 48 h after induction. Results revealed a dose-dependent increase in goblet cell differentiation, as well as, an increase in mucus production relative to controls. There was also a dose-dependent increase in secretion of IL-6, IL-8, TNF-α, and RANTES. Epithelial barrier function, as measured by TEER, was maintained at 1501 ± 355 Ω*cm² postdifferentiation, but dropped significantly when challenged with poly(I:C). This study provides first steps toward a well-characterized model with defined functional methods for understanding dsRNA stimulated inflammatory responses in a physiologically relevant manner.

Bazooka/PAR3 is dispensable for polarity in Drosophila follicular epithelial cells.

Apico-basal polarity is the defining characteristic of epithelial cells. In Drosophila, apical membrane identity is established and regulated through interactions between the highly conserved Par complex (Bazooka/Par3, atypical protein kinase C and Par6), and the Crumbs complex (Crumbs, Stardust and PATJ). It has been proposed that Bazooka operates at the top of a genetic hierarchy in the establishment and maintenance of apico-basal polarity. However, there is still ambiguity over the correct sequence of events and cross-talk with other pathways during this process. In this study, we reassess this issue by comparing the phenotypes of the commonly used baz4 and baz815-8 alleles with those of the so far uncharacterized bazXR11 and bazEH747 null alleles in different Drosophila epithelia. While all these baz alleles display identical phenotypes during embryonic epithelial development, we observe strong discrepancies in the severity and penetrance of polarity defects in the follicular epithelium: polarity is mostly normal in bazEH747 and bazXR11 while baz4 and baz815-8 show loss of polarity, severe multilayering and loss of epithelial integrity throughout the clones. Further analysis reveals that the chromosomes carrying the baz4 and baz815-8 alleles may contain additional mutations that enhance the true baz loss-of-function phenotype in the follicular epithelium. This study clearly shows that Baz is dispensable for the regulation of polarity in the follicular epithelium, and that the requirement for key regulators of cell polarity is highly dependent on developmental context and cell type.

Parameters affecting efficiency of in ovo electroporation of the avian neural tube and crest.

Many variations in avian in ovo transfection of the neural tube/crest have been reported, but never compared quantitatively. Genome integrating pT2K-CAGGS-GFP and pCAGGS-T2TP transposase plasmids were co-electroporated into quail E2 embryo trunk neural tube and the proportion of GFP-expressing neural cells was counted 1 and 7 days later. Electroporation efficiency increased with plasmid concentration and pulse number but plateaued at, respectively, above 1.25 µg/µL and 3 pulses. Bilateral electroporation transfected more cells than unilateral but less than that anticipated by doubling the unilateral treatment. Holding the concentration of GFP plasmid constant and varying the transposase plasmid concentration revealed an optimum ratio of, in this case, 4:1 (1.2 µg/µL:0.3 µg/µL). Leaving transfected embryos to E9 confirmed that expression was maintained in vivo with the transposase system, but declined with non-integrated plasmid. Transfection of neural crest cells was low if electroporated less than 6-8 hr before emigration. We propose this indicates loss of epithelial integrity well prior to exit. We suggest this event be termed epithelio-mesenchymal transition sensu stricto, whereas the term delamination be reserved for the later emigration from the neural epithelium. Co-electroporation in ovo must take into account plasmid(s) concentration and ratio, pulse number, pulse directionality, and timing.

Amelioration of DSS-induced murine colitis by VSL#3 supplementation is primarily associated with changes in ileal microbiota composition.

Inflammatory bowel diseases encompass gastrointestinal illnesses typified by chronic inflammation, loss of epithelial integrity and gastrointestinal microbiota dysbiosis. In an effort to counteract these characteristic perturbations, we used stem cells and/or a probiotic therapy in a murine model of Dextran Sodium Sulfate induced colitis to examine both their efficacy in ameliorating disease and impact on niche-specific microbial communities of the lower GI tract. Colitis was induced in C57BL/6 mice by administering 3% DSS in drinking water for 10 days prior to administering one of three treatment plans: daily probiotic (VSL#3) supplementation for 3 days, a single tail vein injection of 1x106 murine mesenchymal stem cells, or both. Ileal, cecal and colonic sections were collected for microbiota and histological analyses. Microbiota profiling revealed distinct bacterial community compositions in the ileum, cecum and colon of control untreated animals, all of which were predicted in silico to be enriched for a number of discrete KEGG pathways, indicating compositional and functional niche specificity in healthy animals. DSS-treatment perturbed community composition in all three niches with ileal communities exhibiting the greatest change relative to control animals. Each treatment group exhibited treatment-specific alterations in microbiota composition in the lower GI tract, though disease scores were only improved in VSL#3-treated animals. The ileal microbiota were most profoundly altered in composition in this group of animals and characterized by significant Enterobacteriaceae enrichment compared with colitic mice (P < 0.05).

Revealing the Pathogenic and Aging-related Mechanisms of the Enigmatic Idiopathic Pulmonary Fibrosis. An Integral Model

A growing body of evidence indicates that aberrant activation of alveolar epithelial cells and fibroblasts in an aging lung plays a critical role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, the biopathological processes linking aging with IPF and the mechanisms responsible for the abnormal activation of epithelial cells and fibroblasts have not been elucidated. Many of the hallmarks of aging (e.g., genomic instability, telomere attrition, epigenetic alterations, mitochondrial dysfunction, and cellular senescence) have been proposed as essential mechanisms for the development of IPF; however, these disturbances are not restricted to IPF and also occur in other aging-related lung disorders, primarily chronic obstructive pulmonary disease (COPD). Therefore, an unanswered question is why a current/former smoker of about 60 years of age with shorter telomeres, alveolar epithelial senescence, excessive oxidative stress, and mitochondrial dysfunction develops IPF and not COPD; in other words, what makes old lungs specifically susceptible to develop IPF? In this Perspective, we propose an integral model in which the combination of some gene variants and/or gene expression in the aging lung results in the loss of epithelial integrity and consequently in the failure of the alveoli to correctly respond to injury and to face the stress associated with mechanical stretch. Afterward, a distinctive epigenetic "reprogramming" that affects both epithelial cells and fibroblasts provokes, among others, the recapitulation of developmental pathways and the aberrant activation and miscommunication between both cell types, resulting in the exaggerated production and accumulation of extracellular matrix and the subsequent destruction of the lung architecture.

Protocadherin-1: epithelial barrier dysfunction in asthma and eczema

Asthma and eczema result from the interaction between genetic susceptibility and environmental exposures. Over the last two decades, genetic studies have led to the identification of multiple susceptibility genes, increasing our understanding of the biological pathways leading to these diseases. Many asthma and eczema susceptibility genes are expressed in epithelial cells of airway mucosa and skin [1]. The epithelium constitutes the first line of defence against allergens and invading microbes. Recently, loss of epithelial integrity has been implicated as an important mechanism leading to both asthma [2] and eczema [3]. In this issue of the European Respiratory Journal , Mortensen et al. [4] report the association of genetic variants in the gene encoding protocadherin-1 ( PCDH1 ) with asthma, wheeze and eczema in (early) childhood in the Danish birth cohort study COPSAC (Copenhagen Studies on Asthma in Childhood). Their studies also suggest a gene–environment interaction of PCDH1 with passive smoke exposure in early childhood in asthma development. These studies expand on previous data reporting a role for PCDH1 polymorphisms in the susceptibility to both asthma [4–7] and eczema [4, 6, 8]. Given the replicated association of PCDH1 with both diseases, this gene might contribute to a biological pathway that constitutes a shared cause for both diseases. Here, we will summarise the cumulative data on PCDH1 polymorphisms in asthma and eczema, and make an attempt to interpret these genetic data in light of PCDH1 expression and function as a contributor to epithelial integrity. In 1993, PCDH1 was originally identified by Sano et al. [9] as protocadherin-42 and was reported to induce cell adhesion upon ectopic membrane expression in a mouse fibroblast L-cell assay. PCDH1 is a member of the δ1 subfamily of the …

Activation of the Epithelial-to-Mesenchymal Transition Factor Snail Mediates Acetaldehyde-Induced Intestinal Epithelial Barrier Disruption

Acetaldehyde (AcH) is mutagenic and can reach high concentrations in colonic lumen after ethanol consumption and is associated with intestinal barrier dysfunction and an increased risk of progressive cancers, including colorectal carcinoma. Snail, the transcription factor of epithelial-mesenchymal transition, is known to down-regulate expression of tight junction (TJ) and adherens junction (AJ) proteins, resulting in loss of epithelial integrity, cancer progression, and metastases. As AcH is mutagenic, the role of Snail in the AcH-induced disruption of intestinal epithelial TJs deserves further investigation. Our aim was to investigate the role of oxidative stress and Snail activation in AcH-induced barrier disruption in Caco-2 monolayers. The monolayers were exposed from the apical side to AcH±L-cysteine. Reactive oxygen species (ROS) generation and Snail activation were assessed by ELISA and immunofluorescence. Paracellular permeability, localization, and expression of ZO-1, occludin, E-cadherin, and β-catenin were examined using transepithelial electrical resistance (TEER), fluorescein isothiocyanate-labeled dextran 4 kDa (FITC-D4), immunofluorescence, and ELISA, respectively. Involvement of Snail was further addressed by inhibiting Snail using small interfering RNA (siRNA). Exposure to 25μM AcH increased ROS generation and ROS-dependently induced Snail phosphorylation. In addition, AcH increased paracellular permeability (decrease in TEER and increase in FITC-D4 permeation) in association with redistribution and decrease of TJ and AJ protein levels, which could be attenuated by L-cysteine. Knockdown of Snail by siRNA attenuated the AcH-induced redistribution and decrease in the TJ and AJ proteins, in association with improvement of the barrier function. Our data demonstrate that oxidative stress-mediated Snail phosphorylation is likely a novel mechanism contributing to the deleterious effects of AcH on the TJ and AJ, and intestinal barrier function.

OP092: Role of slug in head and neck squamous cell carcinoma (HNSCC)

Objective hypothesis Hand neck malignancies account for 6% of all cancers diagnosed in the United States and result in ∼14,000 deaths annually. Our previous oligonucleotide microarray study (Ginos et al., 2004 Cancer Res.) identified Slug (SNAI2) over-expression in a signature associated with recurrent head and neck squamous cell carcinoma (HNSCC) following definitive treatment. Slug, a zinc finger transcription factor family member, is critical in mediating epithelial to mesenchymal transitions (EMT) in developing organisms and in modulating sensitivity to apoptosis. Since acquisition of an invasive phenotype and resistance to apoptosis are primary characteristics of human cancer we hypothesized that Slug over-expression would induce EMT, increase invasion and evade apoptosis in HNSCC cell lines. Methods Baseline levels of Slug and E-cadherin were determined using Real time PCR and Western blotting in a variety of HNSCC cell lines. A cell line with high E-cadherin expression and low Slug expression was selected for transfection studies. We overexpressed Slug in a HNSCC cell line (SCC-15) and probed for epithelial and mesenchymal markers (E-cadherin, Vimentin and α -smooth muscle actin) using Immunofluorescence and Western blotting. Apoptosis evasion was tested using serum starvation and clonogenic assays. Results Epithelial marker, E-cadherin, expression was decreased significantly in the clones compared to mock transfectants. Increased mesenchymal markers Vimentin and α -smooth muscle actin expression was observed using both Immunofluorescence and western blotting. Slug-transfected cells showed reduced cell death in a serum starvation assay. Preliminary data also suggested that Slug-transfected clones show increased resistance to radiation-induced apoptosis using clonogenic assay. Conclusion Slug induces EMT when overexpressed in SCC-15 cell line. EMT induction includes loss of E-cadherin and therefore loss of epithelial integrity. Epithelial integrity loss leads to an altered phenotype and increased expression of mesenchymal markers Vimentin and α -smooth actin. Preliminary data from serum starvation and clonogenic assays suggest that Slug has a protective effect on the Slug-transfected clones when compared to the mock. The precise mechanism of this has yet to be explored in HNSCC cell lines. The above results suggest that Slug plays a strong role in recurrent disease through the malignant phenotype and evasion of apoptosis.

A prospective study to assess in vivo optical coherence tomography imaging for early detection of chemotherapy‐induced oral mucositis

Oral mucositis (OM) is a common and severe complication of many cancer therapies. Currently, prediction and early detection are not possible and objective monitoring remains problematic. Goal of this prospective study is to assess non-invasive imaging using optical coherence tomography (OCT) for early detection and evaluation of chemotherapy-induced OM in 48 patients, 12 of whom developed clinical mucositis. In 48 patients receiving neoadjuvant chemotherapy for primary breast cancer, oral mucosal health was assessed clinically, and imaged using non-invasive OCT. Images were evaluated for mucositis using an imaging-based scoring system ranging from 0 to 6. Conventional clinical assessment using the OM assessment scale (OMAS) was used as the gold standard. Patients were evaluated on Days 0-11 after commencement of chemotherapy. OCT images were visually scored by three blinded investigators. The following events were identified from OCT images (1) change in epithelial thickness and subepithelial tissue integrity (beginning on Day 2), (2) loss of surface keratinized layer continuity (beginning on Day 4), (3) loss of epithelial integrity (beginning on Day 4). Imaging data gave higher scores compared to clinical scores early in treatment, suggesting that the imaging-based diagnostic scoring was more sensitive to early mucositic change than the clinical scoring system. Once mucositis was established, imaging and clinical scores converged. Using OCT imaging and a novel scoring system, earlier, more sensitive detection of mucositis was possible than using OMAS. Specific imaging-based changes were a consistent predictor of clinical mucositis.

Role of Recipient Derived Myd88 in Small Bowel Allograft Rejection

Background: Accumulating evidence suggests that Myd88, a common adaptor protein for signal transduction of most TLRs, plays an important role in ischemia/reperfusion injury and skin and heart allograft rejection. However, little is known concerning its role in small bowel (SB) transplantation. The purpose for this study was to determine whether MyD88 deficiency in recipients alleviates SB allograft rejection. Methods: Small bowels from B6 were transplanted into either wild type (WT) or Myd88−/− BALB/c mice (allografts) heterotopically. The recipients were followed up on POD7 and POD11. Frequencies of CD4+, CD8+, CD11b+, Gr-1+, IFN-g producing T cells, IL-17 producing cells, as well as CD4+Foxp3+ cells in spleen and SB allografts were measured by flow cytometry analysis on POD7 and POD11. Morphological changes of small bowel grafts were examined by histology and phenotypes of cellular infiltrates including CD4, CD8 were identified by immunohistochemical staining on POD7 and POD11. Results: B6 to BALB/c allografts developed severe rejection manifested by loss of epithelial integrity along with diffuse infiltration by CD11b+, CD8+ and Gr-1+ cells. In contrast, Myd88−/− allograft has minimal morphological changes with a marked decrease in the number of CD11b+, CD8+ and Gr1+ cell infiltrates on both POD7 and POD11. Moreover, data from phonotypical analysis of SB infiltrating cells reveal that compared to WT allografts on both POD7 and POD11, Myd88−/− SB allografts had decreased frequencies of CD8+ cells, IFN-g producing cells and IL-17 producing cells, whereas numbers of CD4+Foxp3+ T cells were markedly increased in both spleens and SB infiltrating cells of Myd88−/− allografts. Conclusion: Our results suggest that MyD88 dependent TLR signaling plays an important role in T cell differentiation by shifting the balance between effector T cells and Tregs, thereby influencing cellular infiltration and small bowel allograft rejection.