Number Of PAS-positive Cells Research Articles

Clinical improvement of ocular surface parameters in dry eye patients following treatment with urea/crosslinked-hyaluronate eyedrops correlates with the secretion of MUC-4

ABSTRACT Objectives to assess the ocular surface status and patient’s satisfaction before and after treatment with urea/crosslinked hyaluronate (U-HACL) tear substitute in dry eye (DE) patients. Methods 17 DE patients were included in the study. They instilled U-HACL drops 3 times/day for two months. Symptoms (by OSDI questionnaire and VAS), osmolarity, tear film (through Schirmer test and tear film break-up time), corneal and conjunctival damage (with NEI and van Bijsterveld scores), impression cytology (number of PAS positive cells and MUC-4 immunostaining) and tear sampling were analyzed pre- and post-treatment. Results After the treatment, there was a significant improvement of VAS (7.16±2.59 vs. 3.94±2.23), OSDI (56.96±25.97 vs. 29.94±20.15), Schirmer (8.22±6.86 vs. 10.66±6.92), TFBUT (3.16±2.28 vs. 5.55±2.47), NEI (7.16±2.50 vs. 2.88±2.80) and van Bijsterveld (10.27±4.25 vs. 6.5±3.79) score, p<0.01. No significant changes in osmolarity were found. There was a significant increase of PAS positive cells and in the expression levels of MUC-4 (respectively, 44.21 ± 14.8 vs. 67.0 ± 10.1 and 49.79 ± 20.0 vs. 75.9 ± 20.7), p<0.01. Conclusion Patients showed significant clinical improvement following the administration of U-HACL eye drops. In particular, the rise of MUC-4 expression probably contributed to the increase in ocular wettability and amelioration of clinical parameters.

Histometric parameters of the large intestine of hybrid marmosets Callithrix sp. under the influence of seasonality.

Morphofunctional characteristics of the large intestine are rarely explored to understand the physiology, behavior and ecology of neotropical primates. In this study, we analyzed the histometric parameters of the large intestine of hybrid marmosets (Callithrix sp.) captured in forest fragments of Viçosa-Brazil, under seasonal interference. These animals were predominantly insectivorous in the rainy season and gummivores in the dry season. Large intestine fragments were collected and processed according to histological methods and stained with toluidine blue for general analysis, periodic acid of Schiff (PAS) and Alcian blue (AB) for goblet cells, Grimelius and Masson Fontana for argyrophil and argentaffin endocrine cells. Several histometric parameters were more expressive in the large intestine of the rainy season marmosets: greater thickness of the parietal layers, greater number of argyrophil and argentaffin endocrine cells, and AB-positive goblet cells, characteristics favor secretomotor functions and reduce the passage time of the fecal bolus, which is consistent with an insectivorous diet. In contrast, parameters such as crypt width, height of the absorptive cells and striated border, and the number of PAS-positive cells were more expressive in the dry season marmosets, reflecting the need for longer passage time for digestion and absorption of food items from tree gum, which are more complex and demand the action of microorganisms present in the large intestine, as well as greater protection against the abrasive action of dietary fibers and against microorganisms. Thus, it can be said that the marmoset's large intestine has morphological adaptations to maximize energy intake from the diet, which alternates under the influence of seasonality.

Abstract 928: Myofibroblasts induce poorly differentiated tumor-like spheroids in small intestinal organoids by Wnt-independent mechanism

Abstract Recently, three dimensional (3D) organoid cultures were established from different organs; however those organoid cultures are exclusively composed of epithelial cells, thus missing the stromal niche. The later one is believed to play a crucial role during all stages of tumor development and resistance to therapy. One of the most abundant and the most important stromal cell in the tumor microenvironment is a myofibroblast. The contribution of myofibroblasts to tumor progression is well established, however little is known about their role during epithelial cell homeostasis and tumor initiation. The niche was reconstructed by combining small intestinal (SI) crypts together with different types of mesenchymal cells in 3D culture systems. As mesenchymal cells the following cells were used: murine small intestinal fibroblasts, murine gastric carcinoma associated fibroblasts (CAFs), human fetal esophageal fibroblasts and human cardia myofibroblasts. While SI organoid culture is almost exclusively composed of budding structures, in the co-culture with intestinal myofibroblasts ∼50% of crypts were growing as non-budding spheroids. Those spheroids were positive for Ki-67, and independent of external R-Spondin, EGF and Noggin. Surprisingly, no differences between CAFs and other mesenchymal cells could be observed. Gene expression analysis revealed that myofibroblasts significantly upregulated Sox-9 (∼3 fold) and CD44 (∼4-fold) mRNA levels, and promoted proliferation rather than differentiation in the SI organoids. Clonogenicity assay showed that in the co-culture crypt cells exhibited about 3 times greater self-renewal capacity. Interestingly, by morphology and decreased number of PAS positive cells, the spheroids resembled intestinal organoids from Apc +/1638N mouse tumors. However, in the spheroids induced by co-culture the expression of Axin-2 was not increased, and Wnt inhibitor studies (IWP-2, C59) provided additional evidence that spheroid induction can be mediated by other mechanism than canonical Wnt. Indirect co-culture and conditioned media experiments revealed that spheroid formation was mediated by soluble factors. Mass spectrometric analysis of the secretome from the co-culture suggested the involvement of extracellular matrix-receptor pathway and focal adhesion pathway. Taken together, our studies demonstrate that the microenvironment shapes intestinal crypts in a similar way as cell-autonomous genetic alterations. The data point out to the contribution of myofibroblasts to tumor initiation phenotype in the intestinal crypts by a mechanism independent of canonical Wnt. The precise signaling pathway remains to be elucidated in the future. Increased knowledge on the cellular communication between stroma and epithelium can contribute to the development of anti-cancer therapeutics that target not only cancer cells, but also the stromal niche. Citation Format: Agnieszka Pastula, Klaus-Peter Janssen, Stefanie Hauck, Roland M. Schmid, Michael Quante. Myofibroblasts induce poorly differentiated tumor-like spheroids in small intestinal organoids by Wnt-independent mechanism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 928.

Enhanced hepatogenic transdifferentiation of human adipose tissue mesenchymal stem cells by gene engineering with Oct4 and Sox2.

Adipose tissue mesenchymal stem cells (ATMSCs) represent an attractive tool for the establishment of a successful stem cell-based therapy in the field of liver regeneration medicine. ATMSCs overexpressing Oct4 and Sox2 (Oct4/Sox2-ATMSCs) showed enhanced proliferation and multipotency. Hence, we hypothesized that Oct4 and Sox2 can increase “transdifferentiation” of ATMSCs into cells of the hepatic lineage. In this study, we generated Oct4- and Sox2-overexpressing human ATMSCs by liposomal transfection. We confirmed the expression of mesenchymal stem cell surface markers without morphological alterations in both red-fluorescent protein (RFP) (control)- and Oct4/Sox2-ATMSCs by flow cytometry. After induction of differentiation into hepatocyte-like cells, the morphology of ATMSCs changed and they began to appear as round or polygonal epithelioid cells. Hepatic markers were evaluated by reverse transcription-polymerase chain reaction and confirmed by immunofluorescence. The results showed that albumin was strongly expressed in hepatogenic differentiated Oct4/Sox2-ATMSCs, whereas the expression level of α-fetoprotein was lower than that of RFP-ATMSCs. The functionality of hepatocytes was evaluated by periodic acid-Schiff (PAS) staining and urea assays. The number of PAS-positive cells was significantly higher and urea production was significantly higher in Oct4/Sox2-ATMSCs compared to that in RFP-ATMSCs. Taken together, the hepatocyte-like cells derived from Oct4/Sox2-ATMSCs were mature hepatocytes, possibly functional hepatocytes with enhanced capacity to store glycogen and produce urea. In this study, we demonstrated the enhanced transdifferentiation of Oct4- and Sox2-overexpressing ATMSCs into hepatocyte-like cells that have enhanced hepatocyte-specific functions. Therefore, we expect that Oct4/Sox2-ATMSCs may become a very useful source for hepatocyte regeneration or liver cell transplantation.

Estimation of Salivary Glucose and Glycogen Content in Exfoliated Buccal Mucosal Cells of Patients with Type II Diabetes Mellitus.

Diabetes mellitus is a common metabolic disorder which shows an increasing incidence worldwide. Constant monitoring of blood glucose in diabetic patient is required which involves painful invasive techniques. Saliva is gaining acceptance as diagnostic tool for various systemic diseases which can be collected noninvasively and by individuals with limited training. The aim of the present study was to analyse the possibility of using salivary glucose and glycogen content of buccal mucosal cells as a diagnostic marker in Type II Diabetes mellitus patients which can be considered as adjuvant diagnostic tool to the gold standards. Sample consists of 30 study and 30 control groups. Saliva was collected by passive drool method.Intravenous blood samples were collected for glucose estimation. Exfoliated buccal mucosal cells were collected from apparently normal buccal mucosa, smeared on dry glass slide and stained with PAS. Blood and salivary glucose are estimated by Glucose Oxidase endpoint method. For Glycogen estimation, number of PAS positive cells in fifty unfolded cells was analysed. The results of the present study revealed a significant increase in the salivary glucose level and the number of PAS positive buccal mucosal cells in the diabetics than in the controls. The correlation between the fasting serum glucose and fasting salivary glucose and also that between fasting serum glucose and PAS positive cells was statistically significant. But the correlation between the staining intensity and fasting serum glucose was statistically insignificant. With the results of the present study it is revealed that salivary glucose and PAS positive cells are increased in diabetics which can be considered as adjuvant diagnostic tool for Diabetes mellitus.

Oestrus synchronization treatment induces histomorphological changes on the uterine tube epithelium of the gilt.

The aim of this study was to determine the histomorphological changes that occurred in response to two treatments for oestrus synchronization in three different regions of the gilt's uterine tubes epithelium: the ampulla (AMP), ampulla-isthmic junction (AIJ) and isthmus (IST). Nine prepuberal gilts were divided into three groups (n=3): (1) eCG 400IU and hCG 200IU (eCG/hCG), (2) progesterone agonist (P4) and (3) control group. The number of secretory cells (stained with periodic acid-Schiff reaction or PAS-positive cells) decreased in the AMP in the P4 treated group when compared to the control group, whereas, no difference was observed in the number of PAS-negative cells in the AMP of the three groups. A significant decrease in the number of PAS-positive cells was observed in the AIJ and IST of the P4 treated group when compared to the eCG/hCG and control groups. An increase in the number of PAS-negative cells was observed in the AIJ and IST in the P4 treated group. The epithelium height in the AMP and AIJ was increased in the eCG/hCG group when compared to the control and P4 groups. In this last group, we observed a reduced height compared with the other two groups for the AIJ. In the IST, there were no significant changes in the epithelium height of the control or the other two groups (eCG/hCG and P4). The epithelial cells of the P4 treated group had the least amount of cytoplasmic granules and the lowest intensity of PAS staining in the AMP, AIJ and IST. Animals treated with eCG/hCG showed an intermediate number of cytoplasmic granules and intensity in all regions evaluated. These data show that P4 treatment for synchronization induces a significant (P<0.001) decrease of PAS-positive cells and staining intensity of cytoplasmic granules in the different regions studied and an increased number of PAS-negative cells in the AIJ and IST epithelium. Moreover, eCG/hCG treatment increased the height of the epithelium in the AMP and AIJ, while in this last region, the P4 treatment decreased the epithelium height. These results show that synchronization treatments with P4 and in a smaller proportion with eCG/hCG can modify the amount of PAS-positive and PAS-negative cells, and the epithelium height. This has influence in the secretory activity of the epithelium and possibly alters the fluid microenvironment of the gilt's uterine tube. The biological impact of regional variations in the epithelial cells of the gilt's uterine tube needs further investigation to understand the implications that the reproductive processes can have in the uterine tube.

Estradiol increases mucus synthesis in bronchial epithelial cells.

Airway epithelial mucus hypersecretion and mucus plugging are prominent pathologic features of chronic inflammatory conditions of the airway (e.g. asthma and cystic fibrosis) and in most of these conditions, women have worse prognosis compared with male patients. We thus investigated the effects of estradiol on mucus expression in primary normal human bronchial epithelial cells from female donors grown at an air liquid interface (ALI). Treatment with estradiol in physiological ranges for 2 weeks caused a concentration-dependent increase in the number of PAS-positive cells (confirmed to be goblet cells by MUC5AC immunostaining) in ALI cultures, and this action was attenuated by estrogen receptor beta (ER-β) antagonist. Protein microarray data showed that nuclear factor of activated T-cell (NFAT) in the nuclear fraction of NHBE cells was increased with estradiol treatment. Estradiol increased NFATc1 mRNA and protein in ALI cultures. In a human airway epithelial (1HAE0) cell line, NFATc1 was required for the regulation of MUC5AC mRNA and protein. Estradiol also induced post-translational modification of mucins by increasing total fucose residues and fucosyltransferase (FUT-4, -5, -6) mRNA expression. Together, these data indicate a novel mechanism by which estradiol increases mucus synthesis in the human bronchial epithelium.

Promising Role of Exfoliative Cytology in the Evaluation of Glycaemic Status of Type II Diabetics: A Pilot Study.

The degree of metabolic control in diabetes mellitus influences the susceptibility of patients to oral diseases. It is mandatory to regularly monitor glycaemic status, however invasive methods may be contraindicated or intolerable to diabetic individuals. Thus, cytology, being a simple, non-invasive and rapid procedure, is a promising protocol for assessing diabetic status and assisting in management. To assess the number of PAS positive glycogen containing cells and associated cellular changes in buccal smears of type II diabetics and correlate the findings with their serum glucose levels. The study was conducted at the out patient Department of Oral and Maxillofacial Pathology, KLES Institute of Dental Sciences, Belgaum. Fifty known cases of type II diabetes mellitus and 50 healthy individuals were included in the study. Fasting blood glucose levels were estimated and buccal smears stained with Periodic Acid Schiff stain. The observed cellular changes were correlated with the glycaemic status of each patient. Statistical evaluations such as Student's t test (P<0.01-very significant; P<0.001-highly significant), correlation-coefficient and probability values were computed. Smears of diabetic patients depicted an increase in the number of PAS positive cells in significant correlation to their glycaemic status. Cellular and nuclear morphological alterations were also found in squames of diabetic individuals. Cytological findings and clinical observations, suggest a correlation between the extent of these changes and clinical parameters like glycaemic control. Further studies in this aspect can help in improving the reliability of oral cytology as a diagnostic tool in diabetes.

Asian Sand Dust Enhances Allergen-Induced Th2 Allergic Inflammatory Changes and Mucin Production in BALB/c Mouse Lungs

PurposeRecent studies have reported that Asian sand dust (ASD) has a potential risk of aggravating airway inflammation. The purpose of this study was to investigate the effect of ASD on inflammation and mucin production in the airways of allergic mice.MethodsForty BALB/c female mice were divided into four groups: saline (group 1); ASD (group 2); ovalbumin (OVA) alone (group 3); and OVA+ASD (group 4). OVA-specific immunoglobulin E (IgE) in serum and interleukin (IL)-4, IL-5, IL-13, and interferon-gamma (IFN-γ) in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay (ELISA). Hematoxylin & eosin (H&E) and Periodic acid-Schiff (PAS) staining was performed on lung tissues. In addition, immunohistochemical staining for IL-4, IL-5, MUC5AC, and transforming growth factor alpha (TGF-α) was conducted.ResultsSerum IgE levels were significantly higher in group 4 than in group 3 (P<0.05). IL-4 and IL-5 in BALF were significantly higher in group 4 than in group 3 (P<0.05, respectively). Based on H&E staining, inflammatory cell numbers were significantly greater in group 4 than in the other groups (P<0.05). The number of PAS-positive cells was also significantly greater in groups 3 and 4 than in groups 1 and 2 (P<0.05). The numbers of IL-4 and IL-5-positive cells were higher in group 4 than in group 3 (P<0.05). The number of MUC5AC and TGF-α-positive cells were also higher in group 4 than in group 3 (P<0.05).ConclusionsOur data suggest that ASD increases cytokine expression and mucin production in an allergic murine model. The increased inflammatory reactions were related to cytokine production.

The effect of diethylstilboestrol on the in vitro growth of human ectocervical cells.

The effect of chronic exposure to diethylstilboestrol (DES) over the concentration range 10(-4)-10(-6) M on the in vitro growth of cells derived from the squamo -columnar junction of the human cervix has been examined. DES over the dose range 10(-4)-2 X 10(-5) M inhibits cervical epithelial cell (HCE) growth in terms of colony formation and colony expansion. This effect involves both an effect on cell proliferation, as measured by cell number and mitotic index, and an induction of differentiation as measured by an increase in the number of PAS positive cells (glycogen accumulating cells) and in the number of cells with cornified envelopes. At lower doses of DES, 10(-5)-5 X 10(-6) M, the efficiency of colony formation and colony size are not inhibited but mitotic index and cell number are decreased and the fraction of differentiating cells is increased. The effects of DES on HCE cell growth can be modified to some extent by epidermal growth factor (EGF). EGF at 10 ng/ml, increases the fraction of proliferating cells, induces cellular hypertrophy, reduces stratification and the fraction of differentiating cells in HCE colonies. DES, at all concentrations other than 10(-6) M, in the presence of EGF inhibits cell proliferation but does not prevent the EGF induced cellular hypertrophy, reduction in stratification or reduction in the fraction of differentiating cells.

Air Pollution and Respiratory Infection

Increased incidence of respiratory infections has been mentioned in air polluted area. The incidence rates of pharygolaryngitis and common cold + bronchitis have been increased and are much higher in the air polluted area in Yokkaichi than control nonpolluted area. However, the level of SO2 in this area has decreased and averaged 0.03 ppm in 1972 compared with 0.08 ppm in 1964. The prevalence rates of rhinitis and tonsillitis of school children was significantly higher in this area in 1972.A distinct increase in DNA labeling index of the epithelial cells of the trachea was observed at the first week of exposure period to SO2 in mice. The labeling index returned to preexposure level after the second week of the exposure. In a period from the third week to 15th, the tracheal epithelium was replaced by tall ciliated columnar one which contained a large number of PAS positive cells. The mice exposed to SO2 for 4 weeks were inoculated influenza virus PR8 intranasally. The inflammatory changes in the nasal cavity in the SO2 + virus group were severer and spread much rapidly than virus control. In addition, HI titer developed much rapidly and reached higher level than virus control.The mean concentration of parotid salivary IgA of the normal school children in the polluted area was not significantly different than that of nonpolluted area. But the variance was significantly different.Individual value of serum IgG of the mice exposed to NO2 from intrauterine to the 4th week pcstpartum varied considerably compared with consistent value of control.The incresed severity of virus infection in SO2 mice appeared to result from interaction between effects of SO2 and infection. This interaction could increase the severity of infection. Although the manner of this interaction still needs to be answered, it should be considered that air pollutants may affect the immune response.