PAS-positive Cells Research Articles

Pituitary blastoma

A 13-month-old Korean female presented with Cushing disease and diabetes insipidus. On MRI, a 3.5-cm, focally cystic, contrast-enhancing, sellar and suprasellar mass was noted. Aside from blood adrenocorticotropin (ACTH) and cortisol elevation, other pituitary hormone blood levels were normal or markedly reduced. The subtotally resected lesion consisted of synaptophysin-immunoreactive lobules of (a) large, polygonal, amphophilic, PAS-positive cells immunoreactive for ACTH, beta-endorphin, alpha melanocyte stimulating hormone (MSH), and keratin (CAM5.2) in some cells showing Crooke hyaline change, (b) less frequent acidophilic, growth hormone (GH) immunoreactive cells, and (c) rare luteinizing hormone (LH) and/or alpha subunit immunopositive cells. Also conspicuous were smaller cells resembling Rathke-type epithelium forming rosettes to sizable glands immunoreactive for EMA, keratin, S-100 protein, galectin-3 and rarely for synaptophysin and/or one of the above-noted adenohypophysial hormones. Transcription factors, including Neuro-D1 and Pit-1, were present in ACTH- and GH-producing cells, respectively, but only in occasional Rathke-type cells. The MIB-1 labeling index (LI) was 1.5% in secretory cells and 39% in Rathke-type epithelium. Ultrastructurally, the tissue resembled fetal pituitary of 10-12 weeks gestation and contained fully differentiated corticotrophs and somatotrophs, scant cells of glycoprotein-hormone producing type with small secretory granules, and glandular epithelial cells consistent with committed, but largely undifferentiated Rathke-type epithelium. We consider the tumor as a pituitary blastoma, a lesion composed of multiple cell types common to the development of the affected organ based upon (a) prominence of primitive Rathke-type epithelium, (b) disposition of secretory cells in lobules rather than acini, (c) the limited range of secretory cells represented, (d) the presence of their corresponding transcription factors, and (e) ultrastructural features indicating orderly development of the 10- to 12-week embryonic stage.

Intracellular calcium dynamics and acetylcholine-induced triggered activity in the pulmonary veins of dogs with pacing-induced heart failure

Heart failure increases autonomic nerve activities and changes intracellular calcium (Ca(i)) dynamics. The purpose of this study was to investigate the hypothesis that abnormal Ca(i) dynamics are responsible for triggered activity in the pulmonary veins (PVs) during acetylcholine infusion in a canine model of heart failure. Simultaneous optical mapping of Ca(i) and membrane potential was performed in isolated Langendorff-perfused PV-left atrial (LA) preparations from nine dogs with ventricular pacing-induced heart failure. Mapping was performed at baseline, during acetylcholine (1 micromol/L) infusion (N = 9), and during thapsigargin and ryanodine infusion (N = 6). Acetylcholine abbreviated the action potential. In four tissues, long pauses were followed by elevated diastolic Ca(i), late phase 3 early afterdepolarizations, and atrial fibrillation (AF). The incidence of PV focal discharges during AF was increased by acetylcholine from 2.4 +/- 0.6 beats/s (N = 4) to 6.5 +/- 2.2 beats/s (N = 8; P = .003). PV focal discharge and PV-LA microreentry coexisted in 6 of 9 preparations. The spatial distribution of dominant frequency demonstrated a focal source pattern, with the highest dominant frequency areas colocalized with PV focal discharge sites in 35 (95%) of 37 cholinergic AF episodes (N = 8). Thapsigargin and ryanodine infusion eliminated focal discharges in 6 of 6 preparations and suppressed the inducibility of AF in 4 of 6 preparations. PVs with focal discharge have higher densities of parasympathetic nerves than do PVs without focal discharges (P = .01), and periodic acid-Schiff (PAS)-positive cells were present at the focal discharge sites. Ca(i) dynamics are important in promoting triggered activity during acetylcholine infusion in PVs from pacing-induced heart failure. PV focal discharge sites have PAS-positive cells and high densities of parasympathetic nerves.

608 The Effect of Natural Polymorphisms Upstream of the Helicobacter pylori Vacuolating Cytotoxin Gene, VacA, On VacA Transcription Levels

and corresponding littermate control (Pdx1) mice were examined by HEVilCre and Pdx1mice. Results: The Pdx1;VilCremice are fertile, have no gross or profound histological abnormalities. Pdx1 protein expression is eliminated in enterocytes, goblet, enteroendocrine and paneth cells in the duodenal epithelium of the Pdx1;VilCre mice. PAS-positive goblet cell number and the mRNA abundance for mucin 3 or mucin 13 gene are similar between the Pdx1;VilCre and Pdx1 mice. Although the number of enteroendocrine cells expressing chromogranin A and B appears unaffected in the Pdx1 ;VilCre mice, the mRNA expression of gastric inhibitory polypeptide, chromogranin A, chromogranin B and somatostatin is reduced 87%, 34%, 27% and 51%, respectively, as compared to the Pdx1 mice (p<0.05). Interestingly, lysozyme positive-paneth cell number is decreased 40% (p<0.05) in the Pdx1;VilCre mice and mRNA abundance for the paneth cell-specific defensin related cryptdin 4 gene is also reduced 44% (p<0.05). In enterocytes of the Pdx1;VilCremice, the expression of adenosine deaminase and alkaline phosphatase 3 mRNA is decreased 30% and 87%, respectively, while that of glutathione peroxidase 2mRNA is increased 2-fold (p<0.05). Conclusions: Pdx1 is necessary for patterning appropriate gene expression in duodenal epithelium, by regulating genes in enterocytes, paneth and enteroendocrine cells. Pdx1 may also play a role in paneth cell differentiation.

Modulation of mucus production by interleukin‐13 receptor α2 in the human airway epithelium

IL-13 induces goblet cell hyperplasia and mucus overproduction in airway epithelial cells. IL-13 receptor alpha2 (IL-13Ralpha(2)) has been suggested to act as a 'decoy receptor' in the airway epithelium by inhibiting the IL-13 signal. However, the regulatory mechanisms for mucus production by IL-13Ralpha(2) remain unclear. The aim of this study was to examine the role of IL-13Ralpha(2) in goblet cell hyperplasia and mucus overproduction by IL-13. Bronchi were obtained from patients who underwent a lung resection due to lung cancer or benign lung tumours. Normal human bronchial epithelial cells (NHBECs) were isolated and cultured using an air-liquid interface (ALI) method. The number of periodic acid-Schiff's (PAS)-positive cells, goblet cells and MUC5AC-positive cells increased after adding IL-13 into NHBECs. The concentrations of MUC5AC protein in the supernatant and the mRNA expression of MUC5AC significantly increased after adding IL-13, and returned to control levels at 21 days. The mRNA expression of IL-13Ralpha(2) significantly increased at 7 days and then continuously increased up to 21 days. The protein of a soluble form of IL-13Ralpha(2) in the supernatants significantly increased at 14 and 21 days. Anti-IL-13Ralpha(1) antibody and recombinant IL-13Ralpha(2) reduced the number of PAS-positive cells, goblet cells and MUC5AC-positive cells, and MUC5AC mRNA, while the anti-IL-13Ralpha(2) antibody increased the number of these cells and MUC5AC mRNA. The concentration of MUC5AC protein in the supernatant induced by IL-13 was reduced by anti- IL-13Ralpha(1) antibody and recombinant IL-13Ralpha(2). IL-13-induced signal transducer and activator of transcription (STAT) activation was inhibited by anti-IL-13Ralpha(1) antibody and recombinant IL-13Ralpha(2). In contrast, the IL-4-induced mucus production, mucus secretion and STAT activation were not inhibited by recombinant IL-13Ralpha(2). The soluble form of IL-13Ralpha(2) may therefore modulate mucus overproduction by IL-13 through the pathway including IL-13Ralpha(1) in NHBECs.

Central Nervous System-directed AAV2/5-Mediated Gene Therapy Synergizes with Bone Marrow Transplantation in the Murine Model of Globoid-cell Leukodystrophy

Globoid-cell leukodystrophy (GLD) is a rapidly progressing inherited neurodegenerative disorder caused by a deficiency in galactosylceramidase activity. Previous studies in the murine model of GLD (Twitcher mouse) have shown that both bone marrow transplantation (BMT) and central nervous system (CNS)-directed gene therapy can be moderately effective at ameliorating certain aspects of GLD. As BMT and CNS-directed gene therapy target fundamentally different tissues, we tested the hypothesis that combining these disparate therapies would be more efficacious than either therapy alone. Mice receiving myeloreductive conditioning at birth followed by syngeneic BMT had approximately 25-35% donor chimerism. Untreated Twitcher mice, Twitcher mice treated with BMT alone, AAV2/5 alone, or a combination of BMT and AAV2/5 had mean lifespans of 39, 44, 49, and 104 days, respectively. Twitcher mice treated with a combination of BMT and AAV2/5 also had significantly improved performance in several behavioral tests and greater reduction in demyelination, astrocytosis, and macrophage infiltration compared to untreated Twitcher mice or mice that received either therapy alone. These data suggest that CNS-directed gene therapy synergizes with BMT. The combination of these disparate therapeutic approaches may form the basis for more effective treatment of this inherited neurodegenerative disorder.

Anatomical descriptions and comparison of the reproductive tracts of Utterbackia imbecillis and Villosa iris (Bivalvia: Unionidae)

Summary The reproductive tracts of dioecious Villosa iris (Lea, 1829) and hermaphroditic Utterbackia imbecillis (Say, 1829) are described and compared in sections of viscera examined with light and transmission electron microscopy. In both species, gametes are transported via ciliated gonoducts to a collecting gonosinus, connected to a ciliated genital duct by a highly rugated and musculaturized gonopore. This gonopore may be sphinctural and important for retaining gametes in the gonosinus for timely release. The genital duct is separate from the kidney and leads to the suprabranchial chamber. Epithelia of the gonoducts and gonosinuses of U. imbecillis, but not V. iris, contain secretory cells whose granules stain positive with periodic acid/Schiff (PAS), indicating the presence of carbohydrates or glycoproteins. Examinations of reproductive tracts of other species revealed that those of Elliptio complanata (Lightfoot, 1786), Lexingtonia dolabelloides (Lea, 1840), Villosa vanuxemensis (Lea, 1838), and Lampsilis fasciola Rafinesque, 1820 are similar to those of V. iris and U. imbecillis, except that only the tracts of U. imbecillis contain PAS-positive secretory cells. The gonoducts and gonosinuses of all species examined had an abundance of cilia and microvilli. We hypothesize that the numerous microvilli in the reproductive tracts may indicate intra-luminal regulation of important ion and glucose concentrations, and that the PAS-positive secretions of U. imbecillis may temporarily inhibit selffertilization.

Immunohistochemical Analysis of Embryonal Sarcoma of the Liver

Embryonal sarcoma of the liver is a rare, aggressive malignant tumor that typically occurs in children and teenagers. Microscopic features include spindle, oval, or stellate cells with poorly defined cell borders, nuclear pleomorphism and multinucleation, and variable immunoreactivity to cytokeratin, vimentin, and alpha-1-antitrypsin. Intracellular and extracellular PAS-positive, diastase-resistant hyaline globules are commonly present. The authors evaluated a panel of IHC stains to better define the pattern of immunoreactivity in this tumor. Embryonal sarcomas of the liver were identified from archival files and were immunostained with antibodies: cytokeratin AE1/3, hepatocyte, SMMS, myogenin, calponin, h-caldesmon, desmin, S100, vimentin, CD34, C-kit (CD117), CD10, ALK-1, PE10, Bcl2, p53, and Ki-67. Six cases were identified. Patient age ranged from 6 to 24 years. Tumors ranged from 10 to 20 cm and contained spindled and epithelioid areas with PAS-positive, diastase-resistant globules and atypical cells with focal multinucleation. All cases showed immunoreactivity with vimentin and five showed immunoreactivity with Bcl2. Focal immunoreactivity was seen with cytokeratin AE1/3 in three cases, CD10 in four, calponin in two, desmin in one, and p53 in four. All tumors were negative with hepatocyte, myogenin, CD34, SMMS, h-caldesmon, PE10, ALK-1, and S100. No cytoplasmic staining was seen with C-kit. The proliferation index ranged from 30% to 95%. The diagnosis of embryonal sarcoma is based on typical morphologic features in a large liver tumor occurring in a young patient. The most useful IHC stains help to exclude tumors such as hepatoblastoma, hepatocellular carcinoma, embryonal rhabdomyosarcoma, and other sarcomas.

Relationship between Calcium-Activated Chloride Channel 1 and MUC5AC in Goblet Cell Hyperplasia Induced by Interleukin-13 in Human Bronchial Epithelial Cells

Background:Interleukin (IL)-13 has recently been reported as the major T-helper 2 cytokine involved in mucus overproduction and oversecretion in allergic airways. However, the relationship between human calcium-activated chloride channel-1 (hCLCA1) and MUC5AC induced by IL-13 in vitro has not been fully investigated. Objectives: The present study examines whether IL-13 induces the expression of hCLCA1 in normal human bronchial epithelial (NHBE) cells. We also investigated the relationship between hCLCA1 and MUC5AC expression and the development of goblet cell hyperplasia (GCH). Methods: NHBE cells were isolated from human bronchi, and cultured with an air-liquid interface. hCLCA1 and MUC5AC gene and protein expression, as well as GCH were examined in the cells after exposure to IL-13. Results: Incubation with IL-13 for 14 and 21 days increased the total number of epithelial cells, the number of periodic acid-Schiff (PAS)-stained epithelial cells, the number of goblet cells, as well as expression of mRNA and protein of hCLCA1 and MUC5AC. The number of goblet cells with secretory granules also increased after 21 days of incubation with IL-13. Niflumic acid, a chloride channel inhibitor, reduced mRNA expression of hCLCA1 and MUC5AC, and reduced the number of PAS-positive cells after incubation with IL-13. NHBE cells exposed or not to IL-13 expressed IL-13 receptor α₁ (IL-13Rα₁), and an antibody to IL-13 Rα₁ also reduced the number of PAS-positive cells after exposure to IL-13. Conclusions: IL-13 might induce the expression of MUC5AC and hCLCA1 gene and protein in well-differentiated NHBE cells. These cells might also differentiate into goblet cells and become hyperplastic.

Signet Ring Cells in Gastric Carcinomas Are Derived from Neuroendocrine Cells

Adenocarcinomas are malignant tumors with glandular growth and/or supposed intracellular mucin as identified by periodic acid-Schiff (PAS) positivity. Gastric signet ring cell carcinomas are classified as diffuse type. A proportion of diffuse-type adenocarcinomas have previously been suggested to be of neuroendocrine origin. In the present study we examined gastric signet ring cell carcinomas for neuroendocrine differentiation. Of 11 gastric signet ring cell carcinomas, 8 contained areas with PAS-positive signet ring cells that also were immunoreactive for one or several neuroendocrine markers: synaptophysin, chromogranin A, and histidine decarboxylase, the latter an enterochromaffin-like (ECL) cell marker. Whereas PAS positivity was located in the central cytoplasm, neuroendocrine immunoreactivity was often located as a rim surrounding an otherwise non-immunoreactive cytoplasm, presumed to represent the area with PAS-positive material. These findings indicate that signet ring cell carcinomas could be of neuroendocrine origin. We propose that signet ring cell carcinomas develop by gradual dedifferentiation from ECL cells via signet ring cells with neuroendocrine immunoreactivity toward signet ring cells where the cytoplasm mainly consists of PAS-positive material. This finding could have implications for the classification and understanding of gastric carcinogenesis.

The ontogeny of the alimentary tract during larval development in common pandora Pagellus erythrinus L.

The ontogenesis of the alimentary tract and its associated structures (liver, pancreas, gall bladder) was studied histologically in common pandora from hatching (0 DAH, days after hatching) until day 50 (50 DAH). Larvae were obtained by natural spawning from a broodstock adapted to captivity. They were stocked in 1500 l tanks supplied with Isochrysis galbana and Tetraselmis suecica from hatching until the Artemia feeding stage, at a temperature of 18.5–20 °C. Larvae were fed Selco-enriched Brachionus plicatilis from day 3, Artemia nauplii from day 28 and formulated feed from day 35. At hatching, the digestive tract was a histologically undifferentiated straight tube lying dorsally to the yolk sac. At first feeding (3–4 DAH) both the mouth and anus had opened and the digestive tract was differentiated into four portions: buccopharynx, oesophagus, incipient stomach and intestine. The pancreas, liver and gall bladder were also differentiated at this stage. Within 2 days after the commencement of exogenous feeding, the anterior intestinal epithelium showed large vacuoles indicating the capacity for absorption of lipids, whereas supranuclear ninhydrin-Schiff (NS) positive inclusions indicating protein absorption were observed in the posterior intestinal epithelium. Both the bile and main pancreatic ducts had opened in the anterior intestine, just after the pyloric sphincter, at this stage. Intestinal coiling was apparent since 4 DAH, while mucosal folding began at 10 DAH. Scattered PAS-positive mucous cells occurred in the oral cavity and the intestine, while they were largely diffused in the oesophagus. Gastric glands and pyloric caeca appeared at 28 DAH, indicating the transition from larval to juvenile stage and the acquisition of an adult mode of digestion.

Intracellular Calcium Dynamics and Anisotropic Reentry in Isolated Canine Pulmonary Veins and Left Atrium

Rapid activations due to either focal discharge or reentry are often present during atrial fibrillation (AF) in the pulmonary veins (PVs). The mechanisms of these rapid activations are unclear. We studied 7 isolated, Langendorff-perfused canine left atrial (LA) and PV preparations and used 2 cameras to map membrane potential alone (Vm, n=3) or Vm and intracellular calcium simultaneously (Ca(i), n=4). Rapid atrial pacing induced 26 episodes of focal discharge from the proximal PVs in 5 dogs. The cycle lengths were 223+/-52 ms during ryanodine infusion (n=13) and 133+/-59 ms during ryanodine plus isoproterenol infusion (n=13). The rise of Ca(i) preceded Vm activation at the sites of focal discharge in 6 episodes of 2 preparations, compatible with voltage-independent spontaneous Ca(i) release. Phase singularities during pacing-induced reentry clustered specifically at the PV-LA junction. Periodic acid-Schiff (PAS) stain identified large cells with pale cytoplasm along the endocardium of PV muscle sleeves. There were abrupt changes in myocardial fiber orientation and increased interstitial fibrosis in the PV and at the PV-LA junction. PV muscle sleeves may develop voltage-independent Ca(i) release, resulting in focal discharge. Focal discharge may also be facilitated by the presence of PAS-positive cells that are compatible with node-like cells. During reentry, phase singularities clustered preferentially at sites of increased anisotropy such as the PV-LA junction. These findings suggest that focal discharge caused by spontaneous calcium release and anisotropic reentry both contribute to rapid activations in the PVs during AF.

Hepatocellular Carcinoma with Metastasis to the Spleen in a Holstein Cow

Hepatocellular carcinoma (HCC) with metastasis to the spleen in a Holstein cow was studied by histopathologic and immunohistochemical methods. The tumor was characterized by a pseudoglandular (acinar) pattern with an associated fibrous stroma. Individual cells often had a "hepatoid" appearance but were interspersed with scattered cells exhibiting a clear, periodic acid-Schiff (PAS)-positive cytoplasm and small eccentric nuclei. This pattern was present in nodules found in both liver and spleen. Moreover, hepatoid tumor cells were positive for alpha-fetoprotein. Immunohistochemical studies suggest that myofibroblasts were responsible for the production of fibrous septa surrounding the pseudoglandular structures of bovine HCC. In summary, our histologic and immunohistochemical findings support a diagnosis of primary HCC with splenic metastasis. Furthermore, the associated stromal response appears to be of a myofibroblast origin. The primary etiology of bovine HCC and the significance of the intralesional, PAS-positive clear cells remain undetermined.

Expression of HCLCA1 in cystic fibrosis lungs is associated with mucus overproduction.

Mucus overproduction is typical in cystic fibrosis (CF) airway disease. The human calcium-activated chloride channel, hCLCA1, has been reported to be upregulated by interleukin (IL)-9 and to regulate the expression of mucins. Therefore, the expression of IL-9, IL-9 receptor (IL-9R) and hCLCA1 between the lungs of CF patients and healthy control subjects was compared. Endoscopic biopsy samples of bronchial mucosa from 10 CF patients and six control subjects were stained with periodic acid-Schiff. IL-9, IL-9R and hCLCA1 expression was determined by immunocytochemistry. Expression of hCLCA1 mRNA was also determined by in situ hybridisation. The present study found significant increases in IL-9, IL-9R and hCLCA1 immunoreactivity, hCLCA1 mRNA expression, and numbers of mucus-producing cells in the mucosa of CF patients compared to control subjects. Positive correlations were found between IL-9R-positive-cells with IL-9-positive cells and hCLCA1-positive cells, and between PAS-positive cells with hCLCA1-positive cells and IL-9R-positive cells. Expression of hCLCA1 mRNA was colocalised with IL-9R expression and PAS-positive staining in epithelial cells. Increased expression of interleukin-9 and interleukin-9 receptor, as well as an upregulation of the human calcium-activated chloride channel, hCLCA1, in mucus-producing epithelium of cystic fibrosis patients, support the hypothesis that interleukin-9 contributes to mucus overproduction in cystic fibrosis airway disease.

Cell proliferation and tumour suppressor gene expression in iodine unstained area surrounding oral squamous cell carcinoma

The purpose of this study was to investigate the relationship between epithelial dysplasia unstained with iodine and the expression of proliferating cell nuclear antigen (PCNA) and/or tumour suppressor gene (p53) and the existence of glycogen. Thirty cases of squamous cell carcinomas arising from the buccal mucosa and floor of the mouth were examined. Iodine unstained areas were diagnosed histopathologically as mild, moderate or severe epithelial dysplasia. Normal oral mucosa stained with iodine was used as a control group. There was no histochemical difference in the distribution or ratio of PAS-positive cells between the control and the mild epithelial dysplasia groups, however PAS stained areas of the moderate and the severe dysplasia groups were significantly decreased. Ultrastructurally, glycogen granules were not recognized in the moderate or severe dysplastic epithelia. Immunoreactive ratios of PCNA and p53 in the moderate and severe dysplastic groups were significantly higher than those of the control and the mild dysplasia groups. The positive ratio of PCNA was higher than that of p53, although the immunostaining patterns of PCNA- and p53-positive cells were quite similar. These results suggest that mild dysplastic epithelia that are stained with iodine may be in the category of normal epithelia, whereas both moderate and severe dysplasia that are un-stained with iodine may be suspected of malignant lesions.

Increased expression of interleukin-9, interleukin-9 receptor, and the calcium-activated chloride channel hCLCA1 in the upper airways of patients with cystic fibrosis.

Mucus overproduction is commonly found in airway disease in patients with cystic fibrosis. Interleukin-9 (IL-9) has been shown to mediate airway hyper-responsiveness and mucus overproduction. Recently, the calcium-activated chloride channel hCLCA1 has been described to be upregulated by IL-9 and has been thought to regulate the expression of soluble gel-forming mucins. We sought to examine the expression of IL-9, interleukin-9 receptor (IL-9R), and hCLCA1 in the upper airway of patients with cystic fibrosis in comparison to healthy control subjects and to demonstrate the relationship of IL-9, IL-9R, and hCLCA1 expression with mucus production. Prospective design. Biopsy samples from nasal polyps of four patients with cystic fibrosis, nasal mucosa of six patients with cystic fibrosis, sinus mucosa of eight patients with cystic fibrosis, and nasal mucosa of six healthy control subjects were stained with periodic acid-Schiff (PAS) to identify mucus glycoconjugates. IL-9, IL-9R, and hCLCA1 expression was determined by immunocytochemical study. We demonstrated significant increases in IL-9, IL-9R, and hCLCA1 immunoreactivity in the mucosa of patients with cystic fibrosis compared with that found in control subjects (P <.05). There were no significant differences between the different locations (nasal polyps, nasal mucosa, and sinus mucosa) in the patient group (P >.05). We also observed a significant increase in the number of mucus-producing cells in biopsy specimens from patients with cystic fibrosis in comparison to control subjects. A positive correlation was found between hCLCA1-positive cells and IL-9-positive cells (correlation coefficient [r] = 0.79, P <.05) or IL-9R-positive cells (r = 0.92, P <.05). Moreover, a positive correlation was also present between PAS-positive (mucus-producing) cells and hCLCA1-positive cells (r = 0.64, P <.05) or IL-9R-positive cells (r = 0.64, P <.05). Increased expression of IL-9 and IL-9R, as well as upregulation of hCLCA1, in mucus-overproducing epithelium of patients with cystic fibrosis supports the hypothesis that IL-9 contributes to mucus overproduction in cystic fibrosis. Expression of hCLCA1 may also be responsible, in part, for the overproduction of mucus. These preliminary findings suggest that hCLCA1 might be an interesting new therapeutic target to control mucus overproduction in airway disease in patients with cystic fibrosis.

Multilayer Primary Epithelial Cell Culture from Bovine Conjunctiva as a Model for in vitro Toxicity Tests

Objective: The purpose of this study was to obtain a primary cell culture of bovine origin similar to the conjunctiva in terms of morphology and cell types, which could be of use for in vitro toxicity studies. Methods: After separation from the stroma by enzymatic treatment, conjunctival epithelial cells were dissociated and plated onto collagen-coated Transwell^® filters (1.13 cm² area). One group of plates was maintained in immersion and another was cultured under air-lifted conditions. Anti-epithelial keratin antibodies (AE1/AE3, K4) and antidesmoplakin 1 and 2 were used to characterize the cells by indirect immunofluorescence. The cell layer was examined after histological processing of the Transwell filter. Ultrastructural analysis was carried out by scanning electron microscopy (SEM). The bioelectric parameters transepithelial electrical resistance (TEER), potential difference (PD), short circuit current and paracellular permeability profile of carboxyfluorescein were monitored as indices of the functional characteristics of these cultures. Cytotoxicity was evaluated on morphological and functional (TEER) grounds after treating the cultures with several test substances. Results: Morphological studies showed pure and homogeneous cell cultures. In the SEM analysis, we observed contiguous polygonal cells with numerous short microvilli, a characteristic proportion of light, medium and dark cells and a sparse population of rounded PAS-positive cells, i.e. resembling goblet cells. Air-lifted cultures also showed a tissue-like cellular organization (8–9 layers). Immersion cultures reached a maximum TEER value of around 2.95 kΩ·cm² 7 days after plating while in air-lifted cultures TEER peaked up to 5.59 kΩ·cm² 11 days after plating. With regard to the use of bovine conjunctival epithelial cells (BCECs) for cytotoxicity screening, the system responded finely to the insults and yielded morphological and functional results in accordance with data obtained in vivo. Conclusions: BCECs reproduce cell morphology and differentiation of the original tissue and should prove a useful tool for initial studies of drug toxicity.

Histomorphological changes in the oviduct epithelium of the rabbit during early pregnancy.

In this study, we determined the histomorphological changes in different regions of the oviduct (fimbriae, infundibulum, ampullae and isthmus) during early pregnancy (days 1-4) of the rabbit. It was observed that the number of secretory cells (PAS-positive cells) diminished in the fimbriae during the first 4 days of pregnancy with the corresponding increase in the number of non-secretory cells (PAS-negative cells). In contrast, there was an increase in the number of PAS-positive cells in the isthmus during early pregnancy. In both the infundibulum and the ampullae the number of PAS-positive cells was increased on day 3. A significant decrease in the height of the epithelium was observed in the fimbriae during early pregnancy. On the contrary, a significant increase in this parameter was observed in the isthmus during the first 3 days of pregnancy. A slight decrease in the height of the epithelium was observed in the infundibulum on day 4 of pregnancy, while in the ampullae it was significantly increased on days 1 and 3. The overall results indicate that during early pregnancy there is a selective modification in the histomorphological characteristics of the different regions of the rabbit oviduct.

Expansion of Conjunctival Epithelial Progenitor Cells on Amniotic Membrane

Amniotic membrane (AM) reconstructed human conjunctival surfaces recover a goblet cell density higher than normal. Cultured rabbit conjunctival epithelial cells (RCE) on AM preferentially exhibit non-goblet epithelial differentiation. It was thus wondered if conjunctival progenitor cells that might have been preserved during ex vivo expansion on AM can still differentiate into conjunctival non-goblet epithelial and goblet cells under the influence of mesenchymal cells.Fourteen day old AM cultures of RCE were subcutaneously implanted in Balb/c athymic mice for 11 days and processed for PAS staining and immunostaining with monoclonal antibodies to conjunctival goblet cell mucin (MUC5AC, AM3), glycocalyx (AMEM2), cornea specific cytokeratins K3 (AE5) and K12 (AK2) and basal cell specific cytokeratin K14. Cell cycle kinetics were measured by BrdU labelling for 1 or 7 days. The 7 day labelled RCE were chased for 14 days in the same primary culture.After subcutaneous implantation, conjunctival non-goblet epithelial cells increased stratification and formed occasional cysts. The resultant epithelial phenotype was conjunctival with many PAS-positive, MUC5AC-positive, and AM3-positive goblet cells, AMEM2-positive suprabasal and superficial cells, and K14-positive basal cells, but was not corneal (negative to AE5 and AK2 staining). Twenty four hr BrdU labelling showed a labelling index of 42.5%. A higher labelling index or 69% was noted after continuous BrdU labelling for 7 days. A large number of label retaining basal cells with a labelling index of 84% were noted following 14 days of chase.Conjunctival epithelial progenitor cells for goblet and non-goblet cell differentiation are preserved by AM in vitro as evidenced by being able to differentiate into goblet cells in a permissive stromal environment, and being slow-cycling, and label retaining. This information is useful for future ex vivo expansion of conjunctival epithelial stem cells for conjunctival surface reconstruction.

Intraventricular administration of recombinant adenovirus to neonatal twitcher mouse leads to clinicopathological improvements.

Twitcher mouse is a murine model of human globoid cell leukodystrophy (Krabbe disease), which is characterized by a genetic deficiency in galactocerebrosidase (GALC) activity. The nervous system is affected early and severely by demyelination in the white matter. So far, there is no effective treatment for Krabbe disease except bone marrow transplantation (BMT). However, BMT has inherent limitations such as unavailability of donors and graft-versus-host disease. In this study, we injected recombinant adenovirus encoding GALC into the lateral ventricle of twitcher mice at postnatal day 0 (PND 0) and the therapeutic effects were evaluated. Our results showed slight, but significant improvements in motor functions, body weight and twitching and a prolonged life span. In brain, GALC activity was increased to 15% that of normal littermates and psychosine concentration was decreased to 55% that of untreated twitcher mice at PND 15. The number of PAS-positive globoid cells in brain stem was also reduced significantly at PND 35. In contrast, when adenoviruses were injected to the twitcher mice at PND 15, almost no improvements were observed. These results demonstrate that the timing of treatment may be of great importance in Krabbe disease.

Airway Goblet Cells and Respiratory Epithelial Injury in an Animal Model of Obliterative Airways Disease (OAD)

Goblet cells are important in the maintenance of the epithelial cell population in the airway, defense against injury and storage and release of mucins, which can protect the surface epithelial layer. In our rat tracheal model of acute rejection, there is injury and loss of respiratory epithelium in allografts. This loss of epithelium is associated with obliteration of the airway lumen. In small bowel allografts, studies have shown that the loss of goblet cells is an important histologic feature of rejection. The aims of this study were: (i) to examine for the first time the close time-course of goblet cell proliferation in acute rejection; and (ii) to compare the isograft vs. allograft morphometric changes associated with epithelial damage. Heterotopically transplanted rat tracheas (n = 45) were harvested at days 3,5,7, 10 and 12. Hematoxylin & eosin (H & E), Alcian blue and PAS staining was completed. Computerized image analysis was used to assess epithelial coverage. The mean number of PAS-positive goblet cells counted at 40x/field was determined, and 10 fields were counted per tracheal section. RESULTS There was a significant decrease in the number of goblet cells in the allografts between days 5 and 12 (p < 0.006). In the isografts, there was a gradual increase from day 3 to 10 (p < 0.05), then a sharp fall from day 10 to 12 (p < 0.03). In isografts from day 7 to 10, the goblet cell number increased, while the percentage respiratory epithelium remained the same. The percentage respiratory epithelial coverage and the number of goblet cells showed a direct correlation in the allografts (r2 = 0.57). Our study shows, for the first time, that goblet cell proliferation occurs in the epithelial repair phase in isografts, whereas in allografts the goblet cells are lost and do not recover.