Activation of MTOR in pulmonary epithelium promotes LPS-induced acute lung injury

MTOR (mechanistic target of rapamycin [serine/threonine kinase]) plays a crucial role in many major cellular processes including metabolism, proliferation and macroautophagy/autophagy induction, and is also implicated in a growing number of proliferative and metabolic diseases. Both MTOR and autophagy have been suggested to be involved in lung disorders, however, little is known about the role of MTOR and autophagy in pulmonary epithelium in the context of acute lung injury (ALI). In the present study, we observed that lipopolysaccharide (LPS) stimulation induced MTOR phosphorylation and decreased the expression of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β)-II, a hallmark of autophagy, in mouse lung epithelium and in human bronchial epithelial (HBE) cells. The activation of MTOR in HBE cells was mediated by TLR4 (toll-like receptor 4) signaling. Genetic knockdown of MTOR or overexpression of autophagy-related proteins significantly attenuated, whereas inhibition of autophagy further augmented, LPS-induced expression of IL6 (interleukin 6) and IL8, through NFKB signaling in HBE cells. Mice with specific knockdown of Mtor in bronchial or alveolar epithelial cells exhibited significantly attenuated airway inflammation, barrier disruption, and lung edema, and displayed prolonged survival in response to LPS exposure. Taken together, our results demonstrate that activation of MTOR in the epithelium promotes LPS-induced ALI, likely through downregulation of autophagy and the subsequent activation of NFKB. Thus, inhibition of MTOR in pulmonary epithelial cells may represent a novel therapeutic strategy for preventing ALI induced by certain bacteria.     

A novel in vitro retinal differentiation model by co-culturing adult human bone marrow stem cells with retinal pigmented epithelium cells

Human retinal pigment epithelium (HRPE) cells are important in maintaining the normal physiology within the neurosensory retina and photoreceptors. Recently, transplantation of HRPE has become a possible therapeutic approach for retinal degeneration. By negative immunoselection (CD45 and glycophorin A), in this study, we have isolated and cultivated adult human bone marrow stem cells (BMSCs) with multilineage differentiation potential. After a 2- to 4-week culture under chondrogenic, osteogenic, adipogenic, and hepatogenic induction medium, these BMSCs were found to differentiate into cartilage, bone, adipocyte, and hepatocyte-like cells, respectively. We also showed that these BMSCs could differentiate into neural precursor cells (nestin-positive) and mature neurons (MAP-2 and Tuj1-positive) following treatment of neural selection and induction medium for 1 month. Furthermore, the plasticity of BMSCs was confirmed by initiating their differentiation into retinal cells and photoreceptor lineages by co-culturing with HRPE cells. The latter system provides an ex vivo expansion model of culturing photoreceptors for the treatment of retinal degeneration diseases.     

Proteomic analysis of the response of human lung cells to uranium

The industrial use of uranium and particularly of depleted uranium, has pinpointed the need to review its chemical impact on human health. A proteomic approach was used to evaluate the response of a human lung cell line (A549) to uranium. We established the first 2-D reference map of the A549 cell line, identifying 87 spots corresponding to 81 major proteins. Uranium treatment triggered differential expression of 18 spots, of which 14 corresponded to fragments of cytokeratin 8 (CK8) and cytokeratin (CK18) and 1 to peroxiredoxin 1. We probed several hypotheses regarding CK cleavage, and observed that it did not result from caspase or calpain activity. Furthermore, we showed that the fragments are recognised by an anti-ubiquitin antibody (KM691). These results suggest a regulatory pathway involving CK ubiquitinylation or dysfunction in the proteasome-ubiquitin system in response to uranium exposure in human lung cells.     

Morphogenesis and nature of the pigment granules in the adult human retinal pigment epithelium

Summary The pigment epithelial cells of the retina are a layer of highly specialized melanocytes. Beginning in the early embryonic period they produce melanin throughout the entire life. The Golgi apparatus plays a key role in the biosynthesis of melanin. The following steps can be distinguished morphologically: (a) Golgi-vesicles, (b) intermediate vesicles, (c) melanosomes, (d) melanin granules. Structures with a ringlike appearance that are described as lipofuscin granules in the literature prove to be altered intermediate vesicles and melanosomes.This investigation was carried out in part at the Francis I. Proctor Foundation for Research in Ophthalmology, San Francisco, California, U.S.A., and supported by United States Public Health Service Program Project Grant EY 00310, and Deutsche Forschungsgemeinschaft, Training Grant Nr. Sp 102/1.     

Endocrine cells in the human fetal small intestine

Summary In this report we describe the time of appearance and ultrastructural features of enteroendocrine cells (EECs) in the human fetal small intestine (SB) between 9 and 22 weeks gestation. Thirteen distinctive EECs were identified in fetal SB. Two of these, not found in normal adult SB, appeared within the stratified epithelium of the proximal SB at 9–10 weeks. They were arbitrarily termed primitive and precursor cells. As in all fetal EECs, the pale cytoplasm of the primitive cell contains a distinctive population of secretory granules (SGs). Primitive cell SGs average 200–330 nm; some have dense cores with lucent halos while others are filled with a homogeneous dense or flocculent material. The SGs of the precursor cells are larger, averaging up to 1 m in diameter and their contents vary in electron density. A third group of cells not described in normal adult SB was arbitrarily termed transitional cells. These have two populations of SGs; one resembles the SGs of the precursor cells, and the other resembles the SGs of some of the specific adult type EECs. Transitional EC, S, I and G cells are seen. In addition, mature appearing EC, S, G, I, L, D, and D₁ cells were identified by 12 weeks of gestation. The primitive, precursor, and transitional cells may represent sequential developmental precursors of adult type EECs.Supported by Research Grant AM-17537 from the National Institutes of Health, Besthesda, MarylandThe authors would like to thank Ms. Linda Barstein for her excellent technical assistance     

Ultrastructural similarity of endocrine-like cells of the human lung and some related cells of the gut

Summary The ultrastructure of endocrine-like cells of the human lung was compared to the ultrastructure of endocrine-like cells of the stomach and pancreas in both adult and foetal material.Three types of endocrine-like cells were found in the human foetal lung. Type 1 or P₁ cells contained very small granules (about 110 nm) of two varieties, cored and vesicular; type 2 or P₂ cells with cored granules measuring about 130 nm; and type 3 cells with cored granules of about 180–190 nm. In the adult lung only one type P_a cells with cored granules could be found.Cells resembling foetal P₁ cells were not found in foetal or adult gastric mucosa, or in the pancreas. In the gastric mucosa cells resembling pulmonary P_a or P₂ cells were moderately represented and often difficult to distinguish from each other. Thus, they were grouped together as gastric P cells. Cells with granules resembling those of pulmonary type 3 cells were found most numerous in the adult oxyntic mucosa. Cells resembling gastric P cells (and pulmonary P₂ cells) were rather numerous in foetal pancreas, but very rare in adult pancreas. Few cells containing granules somewhat resembling those of pulmonary type 3 cells were present in both foetal and adult pancreas.The results were discussed in respect to 1) the similarities between some gastric or pancreatic carcinoids and lung carcinoids, 2) the gastro-pancreatic P cells as a separate cell population, 3) the possible secretion by the lung endocrine-like cells of active substances, either amines or peptides, 4) the similarity between the secretory granules of P_a and P₁ cells and neurosecretory granules of the hypothalamus and between P₂ cells and some endocrine cells of the pituitary.Supported in part by the Italian National Research Council (Grants N. 75.00630.04 and N. 76.01558.04)     

Characteristics of a clone of endothelial cells derived from a line of normal adult rat lung cells

Summary A strain of endothelial cells derived from a single cell cloned from a line of normal adult rat lung parenchyma has been maintained in tissue culture for more than 3 years. These cells have been identified as endothelial cells based on the combination of their growth characteristics, cell morphology as observed with both light and electron microscopy, and their physiological properties. They have continued to produce granules, which stain specifically for glycosaminoglycans with Alcian blue, for over 2 1/2 years. During the same period of time, glycosaminoglycans were identified biochemically in both cells and medium. They have maintained the ability to degrade bradykinin over this period as well. This work was supported in part by NIH Program Project HL 15832.     

APUD-type recepto-secretory cells in the chicken lung

Summary The epithelium of the intrapulmonary airways of the chicken lung has been studied by fluorescence and electron microscopy. Numerous intensely yellow-fluorescent cells occur in the epithelium of the primary and secondary bronchi. The cell cytoplasm contains characteristic granular vesicles with an electron-dense central core. The vesicles react positively to chromaffm and argentaffin treatment, indicating that they are possible storage sites for amines. Synapse-like junctions occur between the granular cells and the intraepithelial nerve endings, filled with numerous mitochondria, suggesting that these granular cells may have a dual function as both receptor and endocrine cell.     

Isolation and long-term serial cultivation of endothelial cells from the microvessels of the adult human dermis

Sumamry A method to isolate and maintain microvascular endothelial cells from the cutaneous vessels of adult human skin in long-term culture has been developed. Endothelial cells lining the microvessels of the papillary dermis are released from surrounding tissue during a brief trypsin incubation (0.3% trypsin, 1% EDTA). Cells are plated onto a fibronectin substrate and maintained in Leibovitz (L15) culture medium containing pooled human serum (50%) and antibiotics. Proliferation is dependent upon the presence of several additional growth factors, cholera enterotoxin (1×10⁻⁹ M), isobutyl methylxanthine (3.3×10⁻⁵ M), and medium conditioned by explant culture of the mouse EHS sarcoma. Using this supplemented medium, cells proliferate readily and can be cultivated serially for more than 6 passages (3 months in vitro). These cells retain their characteristic endothelial cell morphology, stain positively for Factor VIII antigen, and contain Weibel-Palade bodies. This research was supported by grant AG 01312 from the U.S. Public Health Service, Washington, D.C.     

Outward-rectifying chloride channels in cultured adult and fetal human nasal epithelial cells

Summary The patch-clamp technique was used to characterize ion channels in the apical membranes of cultured human nasal epithelial cells, dissociated from fetal nasal mucosa and from adult nasal polyps. Outward-rectifying chloride channels were found in 4.3% of the cell-attached patches from fetal cells (n=258) and in 3.1% of the patches from adult cells (n=320). After exeision the number of patches containing active chloride channels increased threefold to 13% of the patches from the fetal cells and 10% from adult cells. The single-channel conductance at 0 mV in symmetrical 150mm NaCl solutions was 24.3 ±0.9 pS (n=28) and 26.0 ± 1.2 pS (n=30), respectively, in adult and fetal cells and showed outward rectification in the potential range from –80 to +80 mV. In fetal cells as well as in adult cells the channels were anion selective, and were almost impermeable for larger anions and monovalent cations. In cell-free patches the channels were Ca²⁺ independent. In most of the channels the open probability was voltage independent and high (±0.86); in 20% of the channels, however, the open probability increased with depolarization. In conclusion, fetal nasal epithelial cells contain chloride channels in their apical membranes with singlechannel properties and regulatory mechanisms similar to those found in cells from adults.     

Serotonin-immunoreactive cells of peculiar shape in the urethral epithelium of the human penis

Summary The present study deals with endocrine-like cells in the urethra of human penis. A large number of basal-granulated cells immunoreactive for serotonin were dispersed in the urethral epithelium. No cellular elements were stained positively with antisera against bioactive peptides. The serotonin-immunoreactive cells consisted of a small oval perikaryon and slender processes, and resembled neurons in shape. An apical process reached the urethral lumen. The basal processes frequently branched out in a dendritic fashion, some running laterally for a considerable distance. The number of cells immunoreactive for serotonin was remarkably reduced in subjects over 60 years of age.     

Incorporation of biodegradable nanoparticles into human airway epithelium cells-in vitro study of the suitability as a vehicle for drug or gene delivery in pulmonary diseases

PURPOSE: Nanoparticles are able to enhance drug or DNA stability for purposes of optimised deposition to targeted tissues. Surface modifications can mediate drug targeting. The suitability of nanoparticles synthesised out of porcine gelatin, human serum albumin, and polyalkylcyanoacrylate as drug and gene carriers for pulmonary application was investigated in vitro on primary airway epithelium cells and the cell line 16HBE14o-. METHODS: The uptake of nanoparticles into these cells was examined by confocal laser scan microscopy (CLSM) and flow cytometry (FACS). Further the cytotoxicity of nanoparticles was evaluated by an LDH-release-test and the inflammatory potential of the nanoparticles was assessed by measuring IL-8 release. RESULTS: CLSM and FACS experiments showed that the nanoparticles were incorporated into bronchial epithelial cells provoking little or no cytotoxicity and no inflammation as measured by IL-8 release. CONCLUSIONS: Based on their low cytotoxicity and the missing inflammatory potential in combination with an efficient uptake in human bronchial epithelial cells, protein-based nanoparticles are suitable drug and gene carriers for pulmonary application.     

Ontogeny and distribution of certain endocrine cells in the human fetal large intestine

Summary In 9 fetuses, 9 to 24 weeks-old, the occurrence and relative distribution of argentaffin cells, as well as of cells immunoreactive to somatostatin (SRIF), glucagon-like polypeptide (GLI), pancreatic polypeptide (PP) and substance P (SP) were studied in five segments of the colon (appendix, cecum, ascending colon, descending colon, and rectosigmoid). For each colonic segment, data concerned with the occurrence of endocrine cells were expressed either as mean absolute numbers of specific cells per entire mucosal section, or as cell densities per mm³ of mucosa after calculation of the mucosal volume of the sections. Argentaffin, GLI, SRIF and PP immunoreactive cells are all present in relatively large numbers, scattered along the entire length of the colonic mucosa as early as the 9th–10th week of gestation, whereas substance P-containing cells occur sporadically and first appear during the 14th–17th week. Until the 20th week, with progressing embryonic development, an increase was determined in absolute numbers per section of all types of endocrine cells in all segments of the colon. This observation is clearly related to the general growth of the colonic mucosa, since cell densities per mm³ of mucosa do not greatly change or even decrease during gestation. However, it is possible that densities of argentaffin, GLI and BPP cells increase in the appendix around the 14th–17th week of gestation. Between the 20th and 24th week, absolute numbers of cells per section remain stable or slightly increase, while cell densities tend rather to decrease in all segments. These data demonstrate that some endocrine cells are present very early in the human fetal colon, but their functional significance remains to be elucidated.This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM)     

Quantitative characteristics of the Feyrter cells and neuroepithelial bodies of the fetal rabbit lung in normoxia and short term chronic hypoxia

We report here quantitative data on the Feyrter (single) cells (APUD cells) and neuroepithelial bodies (grouped Feyrter cells), in the lungs of rabbit fetuses at 26, 27.5 and 29 days gestational age, during normoxia and short term chronic hypoxia. The apparent number of these cells declines during this period; we suggest that this might be due to increased hypoxemia. Moreover, the number of cells in the lungs of fetuses from short term chronically hypoxic mothers is lower than in the normoxic animals. These findings are in agreement with our previous studies in short term chronically hypoxic neonatal rabbits, and suggest that the increased hypoxemia in the fetus, caused by the induction of hypoxia in the mother, constitutes a stimulus for secretory activity of the Feyrter cells and neuroepithelial bodies (NEBs). This in turn could be part of the mechanism responsible for maintaining the pulmonary vasoconstriction due to hypoxemia. Our results from fetuses of normoxic does provide base line data on the chronological importance of the Feyrter cells and NEBs.     

Stem cells of the beetle midgut epithelium

At the completion of metamorphosis, adult insect cells have traditionally been assumed to halt cell divisions and terminally differentiate. While this model of differentiation holds for adult ectodermal epithelia that secrete cuticular specializations of exoskeletons, adult endodermal epithelia are populated by discrete three-dimensional aggregates of stem cells that continue to divide and differentiate after adult emergence. Aggregates of these presumptive adult stem cells are scattered throughout larval and pupal midgut monolayers. At the beginning of adult development (pupal-adult apolysis), the number of cells within each aggregate begins to increase rapidly. Dividing cells form three-dimensional, coherent populations that project as regenerative pouches of stem cells into the hemocoel surrounding the midgut. Stem cell pouches are regularly spaced throughout endodermal monolayers, having adopted a spacing pattern suggesting that each incipient pouch inhibits the formation of a similar pouch within a certain radius of itself—a process referred to as lateral inhibition. At completion of adult development (pupal-adult ecdysis), a distinct basal-luminal polarity has been established within each regenerative pouch. Dividing stem cells occupying the basal region are arranged in three-dimensional aggregates. As these are displaced toward the lumen, they transform into two-dimensional monolayers of differentiated epithelial cells whose apical surfaces are covered by microvilli. This organization of stem cell pouches in insect midguts closely parallels that of regenerative crypts in mammalian intestines.     

Mast cells in the human lung at high altitude

Mast cell densities in the lung were measured in five native highlanders of La Paz (3600 m) and in one lowlander dying from high-altitude pulmonary oedema (HAPO) at 3440 m. Two of the highlanders were mestizos with normal pulmonary arteries and the others were Aymara Indians with muscular remodelling of their pulmonary vasculature. The aim of the investigation was to determine if accumulation of mast cells in the lung at high altitude (HA) is related to alveolar hypoxia alone, to a combination of hypoxia and muscularization of the pulmonary arterial tree, or to oedema of the lung. The lungs of four lowlanders were used as normoxic controls. The results showed that the mast cell density of the two Mestizos was in the normal range of lowlanders (0.6–8.8 cells/mm²). In the Aymara Indians the mast cell counts were raised (25.6–26.0 cells/mm²). In the lowlander dying from HAPO the mast cell count was greatly raised to 70.1 cells/mm² lung tissue. The results show that in native highlanders an accumulation of mast cells in the lung is not related to hypoxia alone but to a combination of hypoxia and muscular remodelling of the pulmonary arteries. However, the most potent cause of increased mast cell density in the lung at high altitude appears to be high-altitude pulmonary oedema.     

On argyrophil reactions of endocrine cells in the human fetal pancreas

Summary The endocrine cells in the pancreas of five human fetuses with gestational ages of 18–20 weeks were examined by light and electron microscopy with special regard to argyrophil reactions. B-cells and typical A and D-cells were easily identified electron microscopically on the basis of their typical secretory granules. In the Grimelius argyrophil silver stain, a concentration of silver grains over the less electron dense peripheral mantle of the A-cell secretory granules was observed by electron microscopy. In the Hellerström and Hellman modification of the argyrophil Davenport alcoholic silver stain, silver grains were concentrated over the internal structures of the D-cell secretory granules. With this stain an accumulation of silver grains was also seen at the surface of the A-cell secretory granules. The argyrophil reaction of the A-granules was less pronounced than in the D-cells. In addition to B-cells and A- and D-cells, two other types of endocrine cell were observed by electron microscopy. These cells were argyrophil with the silver impregnation method of Grimelius. The electron microscopic findings at least partly explain the frequent overlapping between the two staining methods observed at the light microscope level.This study was supported by the Swedish Medical Research Council (Project No. 102)     

Neural stem cells in the adult ciliary epithelium express GFAP and are regulated by Wnt signaling

The identification of neural stem cells with retinal potential in the ciliary epithelium (CE) of the adult mammals is of considerable interest because of their potential for replacing or rescuing degenerating retinal neurons in disease or injury. The evaluation of such a potential requires characterization of these cells with regard to their phenotypic properties, potential, and regulatory mechanisms. Here, we demonstrate that rat CE stem cells/progenitors in neurosphere culture display astrocytic nature in terms of expressing glial intermediate neurofilament protein, GFAP. The GFAP-expressing CE stem cells/progenitors form neurospheres in proliferating conditions and generate neurons when shifted to differentiating conditions. These cells express components of the canonical Wnt pathway and its activation promotes their proliferation. Furthermore, we demonstrate that the activation of the canonical Wnt pathway influences neuronal differentiation of CE stem cells/progenitors in a context dependent manner. Our observations suggest that CE stem cells/progenitors share phenotypic properties and regulatory mechanism(s) with neural stem cells elsewhere in the adult CNS.