Portion Of Epithelium Research Articles

Suture Materials: A Perio Perspective

Sutures are natural or synthetic biomaterial devices used to connect blood vessels and align tissues [1]. Its primary function is to reunite and secure tissues after separation due to surgery or trauma. The purposes of wound closure include erasing the dead space, regular distribution of tension along the deep suture lines, keeping the tractile strength across the wound until the tissue tractile strength is sufficient and aiming to approximate the epithelial portion of the closure. Sutures are the most commonly used material for mechanical wound closure, even though there are other methods like staples, tape, and adhesive [2].

Histologic and histomorphometric analysis of connective tissue grafts harvested by the parallel incision method: a pilot randomized controlled trial comparing macro- and microsurgical approaches.

The aim of this pilot randomized controlled trial was to assess the efficacy of macro- and microsurgical procedures in removing the epithelial tissue layer of subepithelial connective grafts (SCTGs) harvested by the parallel incision method. Sixteen patients were randomized to receive macro-SCTG harvesting (n = 10, control group) or micro-SCTG harvesting (n = 10, test group) by the parallel incision technique. Histologic and histomorphometric analysis of the SCTG evaluated the percentage remnant of epithelium and connective tissue. The presence of remnant portions of the epithelium was identified in eight samples (three in the macro- and five in the microsurgery groups). Sixteen participants with 20 sites were included and 20 SCTG were collected and analyzed. SCTG harvested by microsurgical approaches displayed more portions of remnant epithelium compared to the conventional removal (50% versus 30%). There were no significant differences in mean remnant epithelial thickness for test (147.3 ± 89.3 μm) and control (209.0 ± 127.5 μm) groups (P = .57). Likewise, nonsignificant differences were identified in terms of the connective tissue thickness (macrosurgery: 1,511.0 ± 1,160.0 μm; microsurgery: 1,472.0 ± 1,063.0 μm) between groups (P = .96). The samples harvested by microsurgery had greater remaining epithelial portions than those harvested by macrosurgery, and similar connective layer thickness.

Expression and localization of Sox10 during hair follicle morphogenesis and induced hair cycle.

Sox transcription factors play many diverse roles during development, including regulating stem cell states, directing differentiation, and influencing the local chromatin landscape. Sox10 has been implicated in the control of stem/progenitor activity and epithelial-mesenchymal transition, yet it has not been studied in relation to the hair follicle cycle or hair follicle stem cell (HFSC) control. To elucidate the role of Sox10 in hair follicle cycle control, we performed immunohistochemical and immunofluorescence analysis of its expression during hair morphogenesis, the postnatal hair cycle, and the depilation-induced murine hair follicle cycle. During hair follicle morphogenesis, Sox10 was expressed in the hair germ and peg. In telogen, we detected nuclear Sox10 in the hair bulge and germ cell cap, where HFSCs reside, while in anagen and catagen, Sox10 was detected in the epithelial portion, such as the strands of keratinocytes, the outer root sheath (ORS) in anagen, and the regressed epithelial strand of hair follicle in catagen. These results suggest that Sox10 may be involved in early hair follicle morphogenesis and postnatal follicular cycling.

Automated Cervical Digitized Histology Whole-Slide Image Analysis Toolbox

Background: Cervical intraepithelial neoplasia (CIN) is regarded as a potential precancerous state of the uterine cervix. Timely and appropriate early treatment of CIN can help reduce cervical cancer mortality. Accurate estimation of CIN grade correlated with human papillomavirus type, which is the primary cause of the disease, helps determine the patient's risk for developing the disease. Colposcopy is used to select women for biopsy. Expert pathologists examine the biopsied cervical epithelial tissue under a microscope. The examination can take a long time and is prone to error and often results in high inter-and intra-observer variability in outcomes. Methodology: We propose a novel image analysis toolbox that can automate CIN diagnosis using whole slide image (digitized biopsies) of cervical tissue samples. The toolbox is built as a four-step deep learning model that detects the epithelium regions, segments the detected epithelial portions, analyzes local vertical segment regions, and finally classifies each epithelium block with localized attention. We propose an epithelium detection network in this study and make use of our earlier research on epithelium segmentation and CIN classification to complete the design of the end-to-end CIN diagnosis toolbox. Results: The results show that automated epithelium detection and segmentation for CIN classification yields comparable results to manually segmented epithelium CIN classification. Conclusion: This highlights the potential as a tool for automated digitized histology slide image analysis to assist expert pathologists.

Polyglactin 910 versus irradiated polyglactin 910 for subcuticular closure in elective surgeries: a randomized double-blind study

Background: All suture materials are treated as foreign bodies and bring out a reaction in the tissues. The severity and duration of response depends on the type of suture material used. The choice of suture material is based on the concept that it should provide adequate tensile strength across the wound until tissue tensile strength has adequately developed, approximation of the epithelial portion has occurred and that the suture material should be absorbed after serving its function. this study was done to compare irradiated polyglactin 910 (Vicryl Rapide) versus polyglactin 910 (Vicryl) for subcuticular skin closure in patients of elective surgeries and to note post-operative wound inflammation, pain, dehiscence and quality of scar.Methods: 60 consecutive patients undergoing elective surgeries in department of surgery SGRD hospital were taken up for the study from April 2020 to June 2020. After randomization the subjects were divided into two groups. Subcuticular skin closure at the end of surgery was done using a 3-0 polyglactin 910 in group A and 3-0 irradiated polyglactin 910 in group B with cutting needle in both groups. Postoperatively the wound was inspected on day 3, 5, 7 and again 3 weeks after surgery.Results: Postoperatively the wounds were observed for the signs of inflammation and infection. Each was given a wound score according to scoring system. Statistical analysis by SPSS 17.0 software and chi-square test. Irradiated polyglactin 910 showed less inflammation, pain and dehiscence along with good quality of scar as compared to polyglactin 910 on comparison.Conclusions: It was concluded that irradiated polyglactin 910 was a better choice for suture material than polyglactin 910 for subcuticular skin closure in terms of postoperative pain, wound dehiscence and quality of scar.

Immunolocalization of leptin and leptin receptor in colorectal mucosa of ulcerative colitis, Crohn\u2019s disease and control subjects with no inflammatory bowel disease

The expression of leptin and leptin receptor (Ob-R) has been partially elucidated in colon of patients with inflammatory bowel diseases (IBDs), even though leptin is involved in angiogenesis and inflammation. We previously reported overexpression of GLUT5 fructose transporter, in aberrant clusters of lymphatic vessels in lamina propria of IBD and controls. Here, we examine leptin and Ob-R expression in the same biopsies. Specimens were obtained from patients with ulcerative colitis (UC), Crohn’s disease (CD) and controls who underwent screening for colorectal cancer, follow-up after polypectomy or with a history of lower gastrointestinal symptoms. Immunohistochemistry revealed leptin in apical and basolateral membranes of short epithelial portions, Ob-R on the apical pole of epithelial cells. Leptin and Ob-R were also identified in structures and cells scattered in the lamina propria. In UC, a significant correlation between leptin and Ob-R in the lamina propria was found in all inflamed samples, beyond non-inflamed samples of the proximal tract, while in CD, it was found in inflamed distal samples. Most of the leptin and Ob-R positive areas in the lamina propria were also GLUT5 immunoreactive in inflamed and non-inflamed mucosa. A significant correlation of leptin or Ob-R expression with GLUT5 was observed in the inflamed distal samples from UC. Our findings suggest that there are different sites of leptin and Ob-R expression in large intestine and those in lamina propria do not reflect the status of mucosal inflammation. The co-localization of leptin and/or Ob-R with GLUT5 may indicate concomitance effects in colorectal lamina propria areas.

Challenge for macrophages and mast cells of Chelon ramada to counter an intestinal microparasite, Myxobolus mugchelo (Myxozoa).

Thinlip mullet Chelon ramada is the most abundant mullet species found in the Comacchio lagoons (northern Adriatic Sea, Italy). Histological and ultrastructural sections of the intestine of C. ramada showed that over 83% of 48 mullets were infected with the intestinal parasite Myxobolus mugchelo (Myxozoa). In histological sections, plasmodia of M. mugchelo containing mature spores were situated closer to mucosal folds and were surrounded by numerous mast cells (MCs). Mature spores, generally oval in shape, were observed in the paracellular space among the enterocytes or within them. Near the infected epithelial cells, several MCs, rodlet cells and few neutrophils occurred. In intestinal epithelium, large cells resembling macrophages, some with spores of M. mugchelo inside, were observed. These macrophage-like cells were foamy and possessed elongate striated granules. The number of MCs and macrophages in the intestinal epithelium was significantly higher in parasitized fish. In some parasitized intestines, portions of epithelium were displaced by spores, or the spores were observed inside the damaged enterocytes. Immunohistochemical analysis of C. ramada infected or uninfected intestinal tissue revealed the presence of histamine, serotonin (5-HT), leu-enkephalin and inducible-nitric oxide synthase in epithelial macrophages. Several epithelial cells positive to proliferating cell-nuclear antigen were also observed in the proximity of the macrophages. The current study is the first to record the occurrence of intraepithelial macrophages which engulf myxozoan spores. A hypothesis on migration of spores from pancreas via intestinal wall to gut lumen is presented.

602 Sox13 is a novel marker for hair follicle development and differentiation

The Sry-related high-mobility-group box (Sox) gene encodes a transcription factor family that plays crucial roles in the determination of cell fate and organogenesis. Sox13, a group D member of the Sox family, is expressed in various tissues such as brain, cartilage, hair, kidney, pancreas, liver, kidney and blood vessels. However, knowledge of the biological roles of Sox13 is largely limited. To examine the precise expression domains of Sox13 protein and its role in mouse development, we generated Sox13-LacZ-knock-in mice (Sox13LacZ/+), in which the Sox13 coding sequence was disrupted by an in-frame insertion of the b-galactosidase (LacZ) gene. Using the knock-in mice, we examined spatiotemporal expression patterns of Sox13 in the hair follicle and its role in hair development. X-gal staining in Sox13LacZ/+ embryos revealed that Sox13 is initially expressed in the epithelial portion of the placode at E14.5, and subsequently in the hair germ and the hair peg during hair follicle morphogenesis. Immunohistochemistry with an anti-b-galactosidase antibody and anti-hair keratin antibodies that specifically mark the different layers of the hair follicle revealed that during the postnatal phases of hair development Sox13 was predominantly expressed in the outer root sheath. However, the integumentary structures of Sox13LacZ/LacZ mice were grossly and histologically normal. These results suggest that although Sox13 is dispensable for epidermal and adnexal development, Sox13 is a novel marker for early hair follicle development and differentiation.

Molecular profiling of microinvasive breast cancer microenvironment progression

BackgroundTumors develop by progression through a series of stages. Every cell of the tumor microenvironment is constantly changing in the flow of the cancer progression. It has become clear in recent years that stroma is essential for tumor maintenance and growth. Here, we aimed to give a chronological order of gene expression changes given in the dynamical framework of microinvasive breast cancer microenvironment.MethodsRNA-seq was performed on seven microinvasive breast cancers. For each of them we microdissected seven different portions of the tumor, four related to the breast epithelium and three to the stroma. Breast epithelium was chronologically subdivided in normal breast epithelium (NBE), carcinoma in situ (CIS), emerging invasive fingers (EIF) and invasive breast cancer (IBC). For each of the breast epithelium subdivisions we collected the adjacent stroma (S): S-NBE, S-EIF and S-IBC.ResultsThe overall differentially expressed genes (DEGs) in all the compartments were analysed and evaluated to understand the pathways involved in tumor progression. Then we analysed the DEGs of the epithelial and stromal portions in comparison with the normal portions. We observed that the stromal cells are necessary for the development and the maintenance of the tumor, especially in tumor progression. Moreover the most important genes involved in the main metabolic pathways were analysed and the communications within the different cell compartments were highlighted.ConclusionsAs a future perspective, a deeply study of the identified key genes, particularly in the stromal cells, will be crucial to develop an anticancer therapy that is undergoing a conversion from a cancer cell-centric strategy to a stroma-centric strategy, more genomically stable.

Sox13 is a novel early marker for hair follicle development

Sox13, a group D member of the Sry-related high-mobility group box (Sox) transcription factor family, is expressed in various tissues including the hair follicle. However, its spatiotemporal expression patterns in the hair follicle and its role in hair development remain to be elucidated. To address these questions, we generated Sox13-LacZ-knock-in mice (Sox13LacZ/+), in which the LacZ reporter gene was inserted in-frame into exon 2, which contains the translation initiation codon. X-gal staining in Sox13LacZ/+ embryos revealed that Sox13 is initially expressed in the epithelial portion of the placode, and subsequently in the hair germ and the hair peg during early hair follicle development. In postnatal catagen and anagen, Sox13 was detected in the epithelial sheath, whereas in telogen, Sox13 was localized in the bulge region, where hair follicle stem cells reside. Immunohistochemistry with an anti-β-galactosidase antibody and anti-hair keratin antibodies that specifically mark the different layers of the hair follicle revealed that Sox13 was predominantly expressed in the outer root sheath in anagen. However, the integumentary structures of Sox13LacZ/LacZ mice were grossly and histologically indistinguishable from those of wild type mice. These results suggest that although Sox13 is dispensable for epidermal and adnexal development, Sox13 is a useful marker for early hair follicle development.

Glucose transporter expression in the human colon.

AIMTo investigate by immunostaining glucose transporter expression in human colorectal mucosa in controls and patients with inflammatory bowel disease (IBD).METHODSColorectal samples were obtained from patients undergoing lower endoscopic colonoscopy or recto-sigmoidoscopy. Patients diagnosed with ulcerative colitis (n = 18) or Crohn’s disease (n = 10) and scheduled for diagnostic colonoscopy were enrolled. Patients who underwent colonoscopy for prevention screening of colorectal cancer or were followed-up after polypectomy or had a history of lower gastrointestinal symptoms were designated as the control group (CTRL, n = 16). Inflammatory status of the mucosa at the sampling site was evaluated histologically and/or endoscopically. A total of 147 biopsies of colorectal mucosa were collected and processed for immunohistochemistry analysis. The expression of GLUT2, SGLT1, and GLUT5 glucose transporters was investigated using immunoperoxidase labeling. To compare immunoreactivity of GLUT5 and LYVE-1, which is a marker for lymphatic vessel endothelium, double-labeled confocal microscopy was used.RESULTSImmunohistochemical analysis revealed that GLUT2, SGLT1, and GLUT5 were expressed only in short epithelial portions of the large intestinal mucosa. No important differences were observed in glucose transporter expression between the samples obtained from the different portions of the colorectal tract and between the different patient groups. Unexpectedly, GLUT5 expression was also identified in vessels, mainly concentrated in specific areas where the vessels were clustered. Immunostaining with LYVE-1 and GLUT5 antibodies revealed that GLUT5-immunoreactive (-IR) clusters of vessels were concentrated in areas internal to those that were LYVE-1 positive. GLUT5 and LYVE-1 did not appear to be colocalized but rather showed a close topographical relationship on the endothelium. Based on their LYVE-1 expression, GLUT5-IR vessels were identified as lymphatic. Both inflamed and non-inflamed mucosal colorectal tissue biopsies from the IBD and CTRL patients showed GLUT5-IR clusters of lymphatic vessels.CONCLUSIONGlucose transporter immunoreactivity is present in colorectal mucosa in controls and IBD patients. GLUT5 expression is also associated with lymphatic vessels. This novel finding aids in the characterization of lymphatic vasculature in IBD patients.

Spontaneous Peripheral Ameloblastic Odontoma in a Male Sprague-Dawley Rat

Peripheral ameloblastic odontoma is a rare variant of odontogenic tumor occurring in the extraosseous region. The present report describes a spontaneous tumor in male Sprague-Dawley (SD) rats. The clinically confirmed nodule in the right mandibular region was first observed when the rat was 42 weeks and remained until the terminal sacrifice date when the animal was 48 weeks of age. At necropsy, a well demarcated nodule, approximately 2.5 × 2.0 × 2.0 cm, protruded from the ventral area of the right mandible. The nodule was not attached to mandibular bone and was not continuous with the normal teeth. Histopathologically, the tumor was characterized by the simultaneous occurrence of an ameloblastomatous component and composite odontoma-like elements within the same tumor. The epithelial portion formed islands or cords resembling the follicle or plexiform pattern typical of ameloblastoma and was surrounded by mesenchymal tissue. Formation of eosinophilic and basophilic hard tissue matrix (dentin and enamel) resembling odontoma was observed in the center of the tumor. Mitotic figures were rare, and areas of cystic degeneration were present. Immunohistochemically, the epithelial component was positive for cytokeratin AE1/AE3 (CK AE1/AE3), and the mesenchymal component and odontoblast-like cells were positive for vimentin, in the same manner as in normal teeth. On the basis of these findings, the tumor was diagnosed as a peripheral ameloblastic odontoma in an extraosseous mandibular region in a SD rat. In the present study, we report the uncommon spontaneous peripheral ameloblastic odontoma in the SD rat. We also discuss here the morphological characteristics, origin, histochemical, and immunohistochemical features for the diagnosis of this tumor.

Basaloid follicular hamartoma on the upper eyelid.

Basaloid follicular hamartoma on the upper eyelid.

Avaliação da imunomarcação de células-tronco tumorais em carcinossarcomas mamários e carcinomas em tumores mistos em cadelas

As células-tronco tumorais (CTTs) pertencem a uma pequena população de células dentro do tumor com propriedades de autorrenovação e diferenciação em outros tipos celulares. Neste estudo avaliou-se o comportamento tanto das porções mesenquimais quanto das epiteliais de seis carcinossarcomas (CSs), 11 carcinomas em tumores mistos (CTMs) grau I, 11 grau II e 10 grau III. Nas porções epiteliais dos CS e CTM foram observadas imunomarcações para os anticorpos CD44, CD24, Oct-4 e ALDH-1. Nas porções mesenquimais dos CS, nas porções epiteliais dos CTMs graus II e III não houve imunomarcação para o ALDH-1. Concluiu-se que as CTTs são expressas em proporções iguais tanto nas porções mesenquimais quanto nas epiteliais dos CSs e ausentes nas porções mesenquimais bem diferenciadas de CTMs.

DOP001 Epithelial polarity is disrupted by interleukin-22

Background: Interleukin-22 (IL-22), a cytokine secreted by TH17 cells, acts specifically on epithelia via the heterodimeric IL22R1/IL10 receptor. Depending on the context it is examined, IL-22 was shown to have either protective (wound healing) or deleterious effects (induction of ileitis) on intestinal epithelia. We have previously shown that it strongly disturbs the epithelial barrier by attacking the protein complex at tight junctions (TJ). Herein epithelial polarity is analyzed using the 3D cyst model as well as IL-22 knock-out mice. Methods: Caco-2 cells, seeded in Matrigel, were grown to develop 3-dimensional cysts. These cysts were treated with IL-22 (10 ng/ml) and analyzed for the number of lumina, proliferation and apoptosis (active caspase-3). Moreover, fully developed cysts, as well as cysts in early phases of cyst formation were immunostained for apical junctional as well as polarity complex proteins and examined with confocal LSM. To examine TJ assembly, Caco2 cells on filter supports were mounted to Ussing chambers, to perform a calcium switch assay. Ilea of wild-type and IL-22 knock-out mice, previously been treated with T. gondii to induce ileitis, were mounted to Ussing chambers to measure the epithelial portion (Repi) of the transmural electrical resistance by impedance spectroscopy. Results: IL-22-treated cysts revealed a multiple lumen phenotype as seen in stages of dyspolarity. Furthermore complex cysts with branched lumina were found. IL-22 treated cysts revealed a basolateral expression of the apical membrane marker Ezrin. The expression of polarity proteins Par-3 and Dlg-1 normally expressed at the TJ and the basolateral membrane, respectively were reduced and shifted to an intracellular vesicle pool.

DOP002 Conditional deletion of Prep1 in the intestinal epithelium alters epithelial homeostasis, intestinal development, and controls colitis susceptibility

Background: Interleukin-22 (IL-22), a cytokine secreted by TH17 cells, acts specifically on epithelia via the heterodimeric IL22R1/IL10 receptor. Depending on the context it is examined, IL-22 was shown to have either protective (wound healing) or deleterious effects (induction of ileitis) on intestinal epithelia. We have previously shown that it strongly disturbs the epithelial barrier by attacking the protein complex at tight junctions (TJ). Herein epithelial polarity is analyzed using the 3D cyst model as well as IL-22 knock-out mice. Methods: Caco-2 cells, seeded in Matrigel, were grown to develop 3-dimensional cysts. These cysts were treated with IL-22 (10 ng/ml) and analyzed for the number of lumina, proliferation and apoptosis (active caspase-3). Moreover, fully developed cysts, as well as cysts in early phases of cyst formation were immunostained for apical junctional as well as polarity complex proteins and examined with confocal LSM. To examine TJ assembly, Caco2 cells on filter supports were mounted to Ussing chambers, to perform a calcium switch assay. Ilea of wild-type and IL-22 knock-out mice, previously been treated with T. gondii to induce ileitis, were mounted to Ussing chambers to measure the epithelial portion (Repi) of the transmural electrical resistance by impedance spectroscopy. Results: IL-22-treated cysts revealed a multiple lumen phenotype as seen in stages of dyspolarity. Furthermore complex cysts with branched lumina were found. IL-22 treated cysts revealed a basolateral expression of the apical membrane marker Ezrin. The expression of polarity proteins Par-3 and Dlg-1 normally expressed at the TJ and the basolateral membrane, respectively were reduced and shifted to an intracellular vesicle pool.

Molecular characterization and in vitro differentiation of feline progenitor-like amniotic epithelial cells

IntroductionWhile amniotic mesenchymal cells have been isolated and characterized in different species, amniotic epithelial cells (AECs) have been found only in humans and horses and are recently considered valid candidates in regenerative medicine. The aim of this work is to obtain and characterize, for the first time in the feline species, presumptive stem cells from the epithelial portion of the amnion (AECs) to be used for clinical applications.MethodsIn our study, we molecularly characterized and induced in vitro differentiation of feline AECs, obtained after enzymatic digestion of amnion.ResultsAECs displayed a polygonal morphology and the mean doubling time value was 1.94 ± 0.04 days demonstrating the high proliferating capacity of these cells. By RT-PCR, AECs expressed pluripotent (Oct4, Nanog) and some mesenchymal markers (CD166, CD44) suggesting that an epithelial-mesenchymal transition may occur in these cells that lack the hematopoietic marker CD34. Cells also showed the expression of embryonic marker SSEA-4, but not SSEA-3, as demonstrated by immunocytochemistry and flow cytometry. Moreover, the possibility to use feline AECs in cell therapies resides in their low immunogenicity, due to the absence of MHC-II antigen expression. After induction, AECs differentiated into the mesodermic and ectodermic lineages, demonstrating high plasticity.ConclusionsIn conclusion, feline AECs appear to be a readily obtainable, highly proliferative, multipotent and non-immunogenic cell line from a source that may represent a good model system for stem cell biology and be useful in allogenic cell-based therapies in order to treat tissue lesions, especially with loss of substance.

Role of p53-mediated apoptotic pathway in oral lichen planus: Relationship among pro-apoptotic, anti-apoptotic, and keratinocytic markers

ObjectivesThe aim of the study is to investigate the relationship between oral lichen planus (OLP) pathogenesis, apoptosis and keratinocyte markers. Study designThe study employed 98 subjects. In the epithelial portion of OLP biopsies expression of pro-apoptotic molecules, i.e. caspase-3, Bax, p53 and p63, or anti-apoptotic factor such as Bcl-2, involucrin as a keratinocyte-maturation marker and p63 as a keratinocyte stem cell marker were determined immunohistochemically. ResultsAnti-apoptotic Bcl-2 expression showed no change. p53 expression rate among examined cells was significantly higher in the OLP group than in control group. p53 expression was significantly correlated with expression of p63 in total OLP group: the both were increased considerably after onset of more than a year. Bax expression was significantly higher in total OLP group. On the other hand, caspoase-3 expression was significantly lower in the total OLP group than in the control group, especially in subjects who were evaluated less than a year after onset. ConclusionAlthough pro-apoptotic factors are active, and anti-apoptotic factor is also active, the above results indicate that OLP might be resistant to p53-mediated caspase-3 dependent apoptosis, specially at less than a year following onset.

Abstract 3502: Proximity ligation assays reveal protein-protein interactions associated with oncogenic signaling and drug sensitivity.

Abstract Predictive biomarkers are critical to optimize the use of receptor tyrosine kinase (RTK) inhibitors for cancer therapy. Because multi-protein complexes are critical to maintain oncogenic signaling, we hypothesized that assays that measure protein-protein interactions could identify patients with RTK activity and predict RTK inhibitor response. We developed in situ proximity ligation assays (PLA) to assess the degree of active EGFR signaling in both non-small cell lung cancer (NSCLC) cell lines and formalin-fixed paraffin-embedded (FFPE) patient specimens with the goal of establishing PLA assays as biomarkers of erlotinib sensitivity. Using in situ PLA, we evaluated EGFR association with its signaling adaptors, GRB2 and SHC1. By confocal microscopy we observed increased PLA signals in EGFR mutant NSCLC cell lines (PC9, H1650, HCC4006, HCC827) relative to EGFR wildtype (H23, H1299, H322, A549), indicative of constitutively active signaling complexes in mutant cell lines. PLA signal intensity was independent of EGFR, GRB2 and SHC1 protein expression levels, but did correlate with co-IP and pEGFR immunoblot results. Erlotinib dose-dependently reduced PLA signals in PC9 cells. H1650 xenografts exhibited high PLA signals that were reduced by erlotinib, corresponding with tumor shrinkage. Conversely, low PLA signals were observed in xenografts from EGFR wildtype cell lines (H322, H157). In human xenografts, PLA signals were significantly higher in EGFR mutant xenografts and reduced by erlotinib. In human FFPE specimens, PLA signals were absent in normal non-epithelial tissues and low in epithelial portions. FFPE lung tumor specimens (N = 221) revealed a wide range of PLA signal intensities (scored as 0+ to 3+), which localized to cytokeratin-positive regions. High PLA signals were found in approximately 25% of specimens across multiple histological classifications, including adenocarcinoma, squamous and large cell carcinoma. Furthermore, specimens from metastatic brain lesions were highly enriched for high PLA signal (p<.0001). Automated Quantitative Analysis (AQUA) of EGFR expression revealed multiple examples of EGFR expression-PLA signal discordance - we observed both high PLA signal (>2+) in specimens with relatively low EGFR protein expression and low PLA signal (<1+) in specimens with relatively high EGFR protein expression. Additionally, PLA signal was observed in both EGFR mutant and wildtype specimens, indicating that PLA provides additional information beyond immunohistochemistry and mutational analysis. Our results establish PLA as a powerful technique to detect signaling activity by monitoring protein complexes in clinical specimens. PLA-based approaches will likely have utility in biomarker studies for other receptor tyrosine kinase systems and their respective inhibitors. Citation Format: Matthew A. Smith, Richard Hall, Scott Haake, Jiannong Li, Eric B. Haura. Proximity ligation assays reveal protein-protein interactions associated with oncogenic signaling and drug sensitivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3502. doi:10.1158/1538-7445.AM2013-3502

Mesenchymal stem cells from amnion and amniotic fluid in the bovine

Amnion and amniotic fluid (AF) are noncontroversial and inexhaustible sources of mesenchymal stem cells (MSCs) that can be harvested noninvasively at low cost. As in humans, also in veterinary field, presumptive stem cells derived from these tissues reveal as promising candidates for disease treatment, specifically for their plasticity, their reduced immunogenicity, and high anti-inflammatory potential. The aim of this work is to obtain and characterize, for the first time in bovine species, presumptive MSCs from the epithelial portion of the amnion (AECs) and from the AF (AF-MSCs) to be used for clinical applications. AECs display a polygonal morphology, whereas AF-MSCs exhibit a fibroblastic-like morphology only starting from the second passage, being heterogeneous during the primary culture. For both lines, the proliferative ability has been found constant over the ten passages studied and AECs show a statistically lower (P<0.05) doubling time with respect to AF-MSCs. AECs express MSC-specific markers (ITGB1 (CD29), CD44, ALCAM (CD166), ENG (CD105), and NT5E (CD73)) from P1 to P3; in AF-MSCs, only ITGB1, CD44, and ALCAM mRNAs are detected; NT5E is expressed from P2 and ENG has not been found at any passage. AF-MSCs and AECs are positive for the pluripotent markers (POU5F1 (OCT4) and MYC (c-Myc)) and lack of the hematopoietic markers. When appropriately induced, both cell lines are capable of differentiating into ectodermal and mesodermal lineages. This study contributes to reinforce the emerging importance of these cells as ideal tools in veterinary medicine. A deeper evaluation of the immunological properties needs to be performed in order to better understand their role in cellular therapy.