Distal Epithelial Cells Research Articles

Simvastatin ameliorates renal fibrosis via suppressing uterine sensitization‐associated gene‐1 (USAG‐1), a BMP‐7 antagonist, in mouse adenine‐induced tubulointerstitial injury model

Background & PurposeIt is known that BMP‐7 counteracts with TGF‐β, the dominant molecule promoting renal fibrosis, and that uterine sensitization‐associated gene‐1(USAG‐1), the dominant BMP‐7 antagonist expressing in distal tubular epithelial cells, promotes renal fibrosis. In this study, we investigated whether simvastatin (SIM), one of the HMG‐CoA reductase inhibitors, attenuates renal fibrosis by modulating BMP‐7 and USAG‐1 mediated pathway.Method & ResultsC57/BL6 mice fed by 0.2% adenine‐containing diet for 4 weeks showed significant increase of blood urea nitrogen (BUN) accompanied with severe tubulointerstitial fibrosis. SIM treatment for two weeks (50mg/kg/day) significantly reduced BUN compared with the control group (p<0.01). In pathological analysis, the area of fibrosis and the expression of alpha smooth muscle actin were significantly attenuated by SIM (p<0.05). In quantitative real‐time PCR, USGA‐1 expression was significantly lower in the SIM group (p<0.05). MDCK cells (distal epithelial tubular cells) incubated with TGF‐β (5ng/ml) for 72 hours showed increased expression of USAG‐1. SIM significantly reduced USAG‐1 expression (p<0.01). Several database analyses predicted the binding sites of HOXA‐13, a transcriptional factor, in the promoter region of USAG‐1 gene. The expression assay in vitro suggested that HOXA13 functions as a suppressor of the USAG‐1 gene and SIM decreased USAG‐1 expression by enhancing HOXA13.ConclusionOur data showed that SIM ameliorated renal fibrosis by suppressing USAG‐1. Decreased USAG‐1 levels may enhance the counteract effect of BMP‐7 against TGF‐β. We also demonstrated that HOXA13 plays a crucial role in the mechanism of this action by SIM.

The effects of Sutherlandia frutescens extracts in cultured renal proximal and distal tubule epithelial cells

Sutherlandia frutescens (SF), a medicinal plant indigenous to South Africa, is traditionally used to treat a diverse range of illnesses, including cancer and viral infections. The biologically active compounds of SF are polar, thus renal elimination increases susceptibility to toxicity in that organ. This study investigated the antioxidant potential, lipid peroxidation, mitochondrial membrane potential and apoptotic induction by SF extracts on proximal and distal tubule epithelial cells. Cell viability was determined using the MTT assay. Mitochondrial membrane potential was determined using a flow cytometric JC-1 Mitoscreen assay. Cellular glutathione and apoptosis were measured using the GSH-Glo™ Glutathione assay and Caspase-Glo® 3/7 assay, respectively. The IC50 values from the cell viability results for LLC-PK1 and MDBK were 15 mg/mL and 7 mg/mL, respectively. SF extracts significantly decreased intracellular glutathione in LLC-PK1 (p less than 0.0001) and MDBK (p less than 0.0001) cells, while lipid peroxidation increased in treated LLC-PK1 (p less than 0.0001) and MDBK (p less than 0.0001) cells. JC-1 analysis showed that SF extracts promoted mitochondrial membrane depolarization in both LLC-PK1 and MDBK cells by up to 80% (p less than 0.0001). The activity of caspase 3/7 increased in both LLC-PK1 (11.9-fold; p less than 0.0001) and MDBK (2.2-fold; p less than 0.0001) cells. SF extracts at high concentrations appear to increase oxidative stress, to alter mitochondrial membrane integrity, and to promote apoptosis in renal tubule epithelia.

Hypervolemia induces and potentiates lung damage after recruitment maneuver in a model of sepsis-induced acute lung injury

IntroductionRecruitment maneuvers (RMs) seem to be more effective in extrapulmonary acute lung injury (ALI), caused mainly by sepsis, than in pulmonary ALI. Nevertheless, the maintenance of adequate volemic status is particularly challenging in sepsis. Since the interaction between volemic status and RMs is not well established, we investigated the effects of RMs on lung and distal organs in the presence of hypovolemia, normovolemia, and hypervolemia in a model of extrapulmonary lung injury induced by sepsis.MethodsALI was induced by cecal ligation and puncture surgery in 66 Wistar rats. After 48 h, animals were anesthetized, mechanically ventilated and randomly assigned to 3 volemic status (n = 22/group): 1) hypovolemia induced by blood drainage at mean arterial pressure (MAP)≈70 mmHg; 2) normovolemia (MAP≈100 mmHg), and 3) hypervolemia with colloid administration to achieve a MAP≈130 mmHg. In each group, animals were further randomized to be recruited (CPAP = 40 cm H2O for 40 s) or not (NR) (n = 11/group), followed by 1 h of protective mechanical ventilation. Echocardiography, arterial blood gases, static lung elastance (Est,L), histology (light and electron microscopy), lung wet-to-dry (W/D) ratio, interleukin (IL)-6, IL-1β, caspase-3, type III procollagen (PCIII), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) mRNA expressions in lung tissue, as well as lung and distal organ epithelial cell apoptosis were analyzed.ResultsWe observed that: 1) hypervolemia increased lung W/D ratio with impairment of oxygenation and Est,L, and was associated with alveolar and endothelial cell damage and increased IL-6, VCAM-1, and ICAM-1 mRNA expressions; and 2) RM reduced alveolar collapse independent of volemic status. In hypervolemic animals, RM improved oxygenation above the levels observed with the use of positive-end expiratory pressure (PEEP), but increased lung injury and led to higher inflammatory and fibrogenetic responses.ConclusionsVolemic status should be taken into account during RMs, since in this sepsis-induced ALI model hypervolemia promoted and potentiated lung injury compared to hypo- and normovolemia.

Aedes aegypti cadherin serves as a putative receptor of the Cry11Aa toxin from Bacillus thuringiensis subsp. israelensis

Cry11Aa of Bacillus thuringiensis subsp. israelensis is the most active toxin to Aedes aegypti in this strain. We previously reported that, in addition to a 65 kDa GPI (glycosylphosphatidylinositol)-anchored ALP (alkaline phosphatase), the toxin also binds a 250 kDa membrane protein. Since this protein is the same size as cadherin, which in lepidopteran insects is an important Cry toxin receptor, we developed an anti-AaeCad antibody. This antibody detects a 250 kDa protein in immunoblots of larval BBMVs (brush border membrane vesicles). The antibody inhibits Cry11Aa toxin binding to BBMVs and immunolocalizes the cadherin protein to apical membranes of distal and proximal caecae and posterior midgut epithelial cells. This localization is consistent with areas to which Cry11Aa toxin binds and causes pathogenicity. Therefore, the full-length Aedes cadherin cDNA was isolated from Aedes larvae and partial overlapping fragments that covered the entire protein were expressed in Escherichia coli. Using toxin overlay assays, we showed that one cadherin fragment, which contains CR7-11 (cadherin repeats 7-11), bound Cry11Aa and this binding was primarily through toxin domain II loops alpha8 and 2. Cadherin repeats CR8-11 but not CR7 bound Cry11Aa under non-denaturing conditions. Cry11Aa bound the cadherin fragment with high affinity with an apparent Kd of 16.7 nM. Finally we showed that this Cry11Aa-binding site could also be competed by Cry11Ba and Cry4Aa but not Cry4Ba. These results indicate that Aedes cadherin is possibly a receptor for Cry11A and, together with its ability to bind an ALP, suggest a similar mechanism of toxin action as previously proposed for lepidopteran insects.

The Id2+ distal tip lung epithelium contains individual multipotent embryonic progenitor cells

The conducting airways (bronchi and bronchioles) and peripheral gas exchange (alveolar) regions of the mammalian lung are generated by a process of branching morphogenesis. Evidence suggests that during embryonic development, the undifferentiated epithelial progenitors are located at the distal tips of the branching epithelium. To test this hypothesis, we used an Id2-CreER(T2) knock-in mouse strain to lineage trace the distal epithelial tip cells during either the pseudoglandular or canalicular phases of development. During the pseudoglandular stage, the tip cells both self-renew and contribute descendents to all epithelial cell lineages, including neuroendocrine cells. In addition, individual Id2(+) tip cells can self-renew and contribute descendents to both the bronchiolar and alveolar compartments. By contrast, during the later canalicular stage, the distal epithelial tip cells only contribute descendents to the alveoli. Taken together, this evidence supports a model in which the distal tip of the developing lung contains a multipotent epithelial population, the fate of which changes during development.

Promotion of cardiovascular disease by exposure to the air pollutant ozone

in the decades following the discovery of the chemical nature of environmental air pollutants and the biochemical reactions they could undergo, it was viewed that the primary tissue target of air pollution would be our lungs. This made sense, as the principal constituents of photochemical air

A Protein Kinase Cδ-Dependent Protein Kinase D Pathway Modulates ERK1/2 and JNK1/2 Phosphorylation and Bim-Associated Apoptosis by Asbestos

Inhalation of asbestos and oxidant-generating pollutants causes injury and compensatory proliferation of lung epithelium, but the signaling mechanisms that lead to these responses are unclear. We hypothesized that a protein kinase (PK)Cdelta-dependent PKD pathway was able to regulate downstream mitogen-activated protein kinases, affecting pro- and anti-apoptotic responses to asbestos. Elevated levels of phosphorylated PKD (p-PKD) were observed in distal bronchiolar epithelial cells of mice inhaling asbestos. In contrast, PKCdelta-/- mice showed significantly lower levels of p-PKD in lung homogenates and in situ after asbestos inhalation. In a murine lung epithelial cell line, asbestos caused significant increases in the phosphorylation of PKCdelta-dependent PKD, ERK1/2, and JNK1/2/c-Jun that occurred with decreases in the BH3-only pro-apoptotic protein, Bim. Silencing of PKCdelta, PKD, and use of small molecule inhibitors linked the ERK1/2 pathway to the prevention of Bim-associated apoptosis as well as the JNK1/2/c-Jun pathway to the induction of apoptosis. Our studies are the first to show that asbestos induces PKD phosphorylation in lung epithelial cells both in vivo and in vitro. PKCdelta-dependent PKD phosphorylation by asbestos is causally linked to a cellular pathway that involves the phosphorylation of both ERK1/2 and JNK1/2, which play opposing roles in the apoptotic response induced by asbestos.

Surfactant-Associated Protein B Is Critical to Survival in Nickel-Induced Injury in Mice

The etiology of acute lung injury is complex and associated with numerous, chemically diverse precipitating factors. During acute lung injury in mice, one key event is epithelial cell injury that leads to reduced surfactant biosynthesis. We have previously reported that transgenic mice that express transforming growth factor alpha (TGFA) in the lung were protected during nickel-induced lung injury. Here, we find that the mechanism by which TGFA imparts protection includes maintenance of surfactant-associated protein B (SFTPB) transcript levels and epidermal growth factor receptor-dependent signaling in distal pulmonary epithelial cells. This protection is complex and not accompanied by a diminution in inflammatory mediator transcripts or additional stimulation of antioxidant transcripts. In mouse lung epithelial (MLE-15) cells, microarray analysis demonstrated that nickel increased transcripts of genes enriched in MTF1, E2F-1, and AP-2 transcription factor-binding sites and decreased transcripts of genes enriched in AP-1-binding sites. Nickel also increased Jun transcript and DNA-binding activity, but decreased SFTPB transcript. Expression of SFTPB under the control of a doxycycline-sensitive promoter increased survival during nickel-induced injury as compared with control mice. Together, these findings support the idea that maintenance of SFTPB expression is critical to survival during acute lung injury.

Intravenous glutamine decreases lung and distal organ injury in an experimental model of abdominal sepsis

IntroductionThe protective effect of glutamine, as a pharmacological agent against lung injury, has been reported in experimental sepsis; however, its efficacy at improving oxygenation and lung mechanics, attenuating diaphragm and distal organ injury has to be better elucidated. In the present study, we tested the hypothesis that a single early intravenous dose of glutamine was associated not only with the improvement of lung morpho-function, but also the reduction of the inflammatory process and epithelial cell apoptosis in kidney, liver, and intestine villi.MethodsSeventy-two Wistar rats were randomly assigned into four groups. Sepsis was induced by cecal ligation and puncture surgery (CLP), while a sham operated group was used as control (C). One hour after surgery, C and CLP groups were further randomized into subgroups receiving intravenous saline (1 ml, SAL) or glutamine (0.75 g/kg, Gln). At 48 hours, animals were anesthetized, and the following parameters were measured: arterial oxygenation, pulmonary mechanics, and diaphragm, lung, kidney, liver, and small intestine villi histology. At 18 and 48 hours, Cytokine-Induced Neutrophil Chemoattractant (CINC)-1, interleukin (IL)-6 and 10 were quantified in bronchoalveolar and peritoneal lavage fluids (BALF and PLF, respectively).ResultsCLP induced: a) deterioration of lung mechanics and gas exchange; b) ultrastructural changes of lung parenchyma and diaphragm; and c) lung and distal organ epithelial cell apoptosis. Glutamine improved survival rate, oxygenation and lung mechanics, minimized pulmonary and diaphragmatic changes, attenuating lung and distal organ epithelial cell apoptosis. Glutamine increased IL-10 in peritoneal lavage fluid at 18 hours and bronchoalveolar lavage fluid at 48 hours, but decreased CINC-1 and IL-6 in BALF and PLF only at 18 hours.ConclusionsIn an experimental model of abdominal sepsis, a single intravenous dose of glutamine administered after sepsis induction may modulate the inflammatory process reducing not only the risk of lung injury, but also distal organ impairment. These results suggest that intravenous glutamine may be a potentially beneficial therapy for abdominal sepsis.

Specific regulation of CYP27B1 and VDR in proximal versus distal renal cells

In this study, we utilized murine renal proximal (MPCT-G) and distal (DKC-8) tubular epithelial cell lines to compare the gene expressions and promoter activities of 1,25(OH) 2D 3 receptor (VDR) and 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) in response to 50 nM of parathyroid hormone (PTH) and changes in extracellular calcium (Ca 2+) concentration. In MPCT-G cells, VDR gene expression was suppressed by PTH, whereas CYP27B1 gene expression was elevated in response to PTH. In DKC-8 cells, treatment of PTH significantly increased the relative gene expression of VDR by 6.5-fold while CYP27B1 gene expression was unchanged. High Ca 2+ exposure stimulated VDR gene expression and repressed CYP27B1 gene expression in both dose and time-dependent fashion in MPCT-G but not DKC-8 cells. The analysis of promoter activities and VDR protein levels corresponded with the gene expression data. We conclude that PTH-mediated decrease in VDR and increase in renal CYP27B1 is proximal cell-specific.

S2003 Synergistic Effects of Progastrin (PG) and Citrobacter Rodentium (CR) On the Growth of Proximal and Distal Colonic Epithelial Cells in FVB/N Mice: Associated Changes in the Levels of β-Catenin, NF-κB and ERK1/2

S2003 Synergistic Effects of Progastrin (PG) and Citrobacter Rodentium (CR) On the Growth of Proximal and Distal Colonic Epithelial Cells in FVB/N Mice: Associated Changes in the Levels of β-Catenin, NF-κB and ERK1/2

Plasticity of epithelial cells derived from human normal and ADPKD kidneys in primary cultures

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation initiated by dedifferentiation and proliferation of renal tubular epithelial cells. Renal tubular epithelial cells (RTC, derived from normal kidney tissue) in primary cultures exhibit both homogeneous expression of gamma-glutamyl transferase and low molecular weight cytokeratin, two different markers for proximal and distal renal epithelial cells, respectively. RTC in cultures also abnormally express the dedifferentiation markers vimentin and PAX-2, which are proteins normally expressed in epithelial cells lining cysts in ADPKD kidneys but not tubular cells in normal kidneys. In contrast, different cultures of cystic epithelial cells (CEC, derived from the cysts walls of polycystic kidneys) display variable expression of cytokeratin, gamma-glutamyl transferase, and PAX-2, but a constant level of vimentin. Importantly, RTC and CEC exhibit the capacity to convert to their respective original structures by forming tubules and cysts, respectively, when cultured in a three-dimensional gel matrix, whereas HK-2, LLC-PK1, and MDCK renal epithelial cell lines form cell aggregates or cysts. Our study demonstrates that the marker expression of the various epithelial cell types is not highly stable in primary cultures. Their modulation is different in cells originating from normal and ADPKD kidneys and in cells cultured in monolayer and three-dimensions. These results indicate the plasticity of epithelial cells that display a mixed epithelial/dedifferentiated/mesenchymal phenotype during their expansion in culture. However, RTC and CEC morphogenic epithelial properties in three-dimensional cultures are similar to those in vivo. Thus, this model is useful for studying the mechanisms leading to tubulogenesis and cystogenesis.

Reorganization of centrosomal marker proteins coincides with epithelial cell differentiation in the vertebrate lens

The differentiation of epithelial cells in the vertebrate lens involves a series of changes that includes the degradation of all intracellular organelles and a dramatic elongation of the cells. The latter is accompanied by a substantial remodelling of the cytoskeleton and changes in the distribution of the actin, microtubule and intermediate filament cytoskeletons during lens cell differentiation have been well documented. There have, however, been no studies of microtubule organizing centres (MTOCs) and specifically centrosomes during lens cell differentiation. We have investigated the fate of the centrosomal MTOCs during cellular differentiation in the bovine lens using γ-tubulin, ninein, centrin 2 and centrin 3 as markers. Our studies show that these markers oscillate between a clear centrosome-based association in epithelial cells and a defocused cluster in lens fibre cells. Our data further reveal a transient loss of signal for the typical centrosomal marker γ-tubulin as the lens epithelial cells begin to differentiate into lens fibre cells. This marker apparently disappears in the most distal epithelial cells at the lens equator, only to reappear in early lens fibre cells. The changes in γ-tubulin distribution are mirrored by the other centrosomal markers, centrins 2 and 3 and ninein that also show a similar transient loss of their signals and subsequent clustering at the apical ends of differentiating fibre cells. The transient loss of staining for these centrosomal markers in the most posterior epithelial cells is a distinctive feature that precedes lens cell elongation. The dramatic reorganization of MTOC markers coincides with gap junction reorganization as seen by the loss of connexin 43 (α1-connexin) in these lens epithelial cells suggesting that these events mark a significant change preceding subsequent cell elongation and differentiation into fibre cells.

Effects of Cadmium on Differentiation and Cell Cycle Progression in Cultured Xenopus Kidney Distal Epithelial (A6) Cells

Cadmium (Cd) is an important industrial and environmental pollutant, and the kidney is the primary organ to be affected. To elucidate the effects of Cd on cell proliferation, an epithelial cell line (A6) originally derived from the distal part of the Xenopus laevis kidney was cultured in media containing 10% fetal bovine serum. The effects of Cd (added as CdCl(2)) on cellular growth and differentiation from single cells to confluent epithelia were investigated by visual inspection and by measurement of the degree to which living cells covered a unit area. Over a concentration range from 5 to 50 microM, Cd did not affect the settling and adherence of single cells to the bottom of the culture well. The addition of 5 microM Cd for 4 days did not affect the ability of the A6 cells to develop confluent epithelia, measured as the area covered by adherent living epithelial cells (99 +/- 4% of the control value). However, 10 microM Cd did effectively inhibit development of confluent epithelia to 13 +/- 5% compared to control. Visual inspection of adherent cells exposed to 50 microM Cd for 7 days revealed no increase in cell number or in cell death, which indicated the induction of cell cycle arrest. Flow cytometric analysis showed that treatment of cells with Cd (0.4mM) for 24 hours induced a significant increase in the proportion of G1 phase cells from 58.6 +/- 3.9 to 80.6 +/- 3.7%, and a corresponding reduction in the proportion of cells in both the S and G2 phases from 24.0 +/- 3.6 to 13.4 +/- 3.3% and 17.2 +/- 1.7 to 5.8 +/- 2.1%, respectively. This study showed that Cd stopped cell proliferation in a very narrow concentration range, between 5 and 10 microM, and cell cycle analysis indicated that Cd arrested the cells in the G1 phase of the cell cycle.

Effects of dietary soyabean meal, inulin and oxytetracycline on intestinal microbiota and epithelial cell stress, apoptosis and proliferation in the teleost Atlantic salmon (Salmo salar L.)

Soyabean meal (SBM)-induced enteritis in the distal intestine of the teleost Atlantic salmon (Salmo salar L.) and other salmonids may be considered a model for diet-related mucosal disorders in other animals and man. The role of the intestinal microbiota in its pathogenesis was explored. Compared to diets containing fishmeal (FM) as the sole protein source, responses to extracted SBM or the prebiotic inulin, with or without oxytetracycline (OTC) inclusion, were studied following a 3-week feeding trial. Intestinal microbiota, organosomatic indices and histology, as well as immunohistochemical detection of proliferating cell nuclear antigen (PCNA), heat shock protein 70 (HSP70) and caspase-3-positive cells in the distal intestine, were studied. Distal intestine somatic indices (DISI) were higher in inulin and lower in SBM compared to FM-fed fish. The low DISI caused by SBM corresponded with histological changes, neither of which was affected by OTC, despite a significant decrease in adherent bacteria count. Image analysis of PCNA-stained sections showed a significant increase in the proliferative compartment length in SBM-fed fish, accompanied by apparent increases in reactivity to HSP70 and caspase-3 along the mucosal folds, indicating induction of cellular repair and apoptosis, respectively. Fish fed the SBM diet had higher total number as well as a more diverse population composition of adherent bacteria in the distal intestine. Thus SBM-induced enteritis is accompanied by induction of distal intestinal epithelial cell protective responses and changes in microbiota. Putative involvement of bacteria in the inflammatory response merits further investigation.

Airway Epithelial NF-κB Activation Modulates Asbestos-Induced Inflammation and Mucin Production In Vivo

To investigate the role of bronchiolar epithelial NF-kappaB activity in the development of inflammation and fibrogenesis in a murine model of asbestos inhalation, we used transgenic (Tg) mice expressing an IkappaBalpha mutant (IkappaBalphasr) resistant to phosphorylation-induced degradation and targeted to bronchial epithelium using the CC10 promoter. Sham and chrysotile asbestos-exposed CC10-IkappaBalphasr Tg(+) and Tg(-) mice were examined for altered epithelial cell proliferation and differentiation, cytokine profiles, lung inflammation, and fibrogenesis at 3, 9, and 40 days. KC, IL-6 and IL-1beta were increased (p < or = 0.05) in bronchoalveolar lavage fluid (BALF) from asbestos-exposed mice, but to a lesser extent (p < or = 0.05) in Tg(+) vs Tg(-) mice. Asbestos also caused increases in IL-4, MIP-1beta, and MCP-1 in BALF that were more elevated (p < or = 0.05) in Tg(+) mice at 9 days. Differential cell counts revealed eosinophils in BALF that increased (p < or = 0.05) in Tg(+) mice at 9 days, a time point corresponding with significantly increased numbers of bronchiolar epithelial cells staining positively for mucus production. At all time points, asbestos caused increased numbers of distal bronchiolar epithelial cells and peribronchiolar cells incorporating the proliferation marker, Ki-67. However, bronchiolar epithelial cell and interstitial cell labeling was diminished at 40 days (p < or = 0.05) in Tg(+) vs Tg(-) mice. Our findings demonstrate that airway epithelial NF-kappaB activity plays a role in orchestrating the inflammatory response as well as cell proliferation in response to asbestos.

Mesenchymal maintenance of distal epithelial cell phenotype during late fetal lung development

Classical tissue recombination experiments have reported that at early gestation both tracheal and distal lung epithelium have the plasticity to respond to mesenchymal signals. Herein we examined the role of epithelial-mesenchymal interactions in maintaining epithelial differentiation at late (E19-E21, term = 22 days) fetal gestation in the rat. Isolated distal lung epithelial cells were recombined with mesenchymal cells from lung, skin, and intestine, and the homotypic or heterotypic recombinant cell aggregates were cultured for up to 5 days. Recombining lung epithelial cells with mesenchyme from various sources induced a morphological pattern that was specific to the type of inducing mesenchyme. In situ analysis of surfactant protein (SP)-C, SP-B, and Clara cell secretory protein (CCSP) expression, as well as SP-C and CCSP promoter transactivation experiments, revealed that distal lung epithelium requires lung mesenchyme to maintain the alveolar, but not bronchiolar, phenotype. Incubation of lung recombinants with an anti-FGF7 antibody resulted in a partial inhibition of mesenchyme-induced SP-C promoter transactivation. Immunoreactivity for Delta and Lunatic fringe, components of the Notch pathway that regulates cell differentiation, was downregulated in the heterotypic recombinants. In contrast, Hes1 mRNA expression was increased in these recombinants. Cumulatively, these results suggest that at late fetal gestation, distal lung epithelial cells are not fully committed to a specific phenotype and still have the plasticity to respond to various signals. Their alveolar phenotype is likely maintained by Notch/Notch ligand interactions and mesenchymal factors, including FGF7.

A Cluster of Nosocomial Herpes Simplex Virus Type 1 Pneumonia in a Medical Intensive Care Unit

We report a cluster of 3 cases of nosocomial herpes simplex virus type 1 (HSV-1) pneumonia occurring in close temporal and physical proximity during a 1-week period, which suggested a common source. HSV-1 nosocomial pneumonia occurs in immunocompetent intubated patients and presents as otherwise unexplained profound and/or prolonged hypoxemia (decreased F(IO2), increased P(O2), and decreased A-a gradient) and "failure to wean." The diagnosis of HSV-1 pneumonia is determined by demonstration of characteristic cytopathologic findings (Cowdry type A inclusion bodies) in distal respiratory epithelial cells from bronchoscopic specimens. Acyclovir therapy results in rapid improvement and ability to wean.

Localization and diurnal variations of carbonic anhydrase mRNA expression in the inner ear of the rainbow trout Oncorhynchus mykiss

Physiological studies have suggested that carbonic anhydrase (CA) plays a central role in otolith biomineralization via ion transport. However, the presence and exact function of CA in the inner ear have not been determined. In the present study, to investigate the localization of CA and its involvement in otolith calcification, we cloned two cDNAs encoding CAs from the rainbow trout sacculus. These two cDNAs, designated rainbow trout CAa (rtCAa) and rtCAb, both had an open reading frame encoding 260 amino acids with a sequence identity of 78%. Remarkably, rtCAb has a high degree of homology (82%) with “high activity CA” in the zebrafish, and its mRNA expression showed variation in the range 1.9–11.4 × 10 4 copies/ng total RNA in the sacculus. In contrast, rtCAa mRNA was constantly expressed at approximately 3 × 10 4 copies/ng total RNA. In situ hybridization revealed that rtCAb mRNA was strongly expressed in the distal squamous epithelial cells and transitional epithelial cells, except the mitochondria-rich cells, whereas, rtCAa was localized in extrasaccular tissue. These results suggest that the rtCAb isozyme is involved in the daily increment formation and calcification of otoliths via phase and spatial differences of the bicarbonate supply to the endolymph.

Cells differentiated from mouse embryonic stem cells via embryoid bodies express renal marker molecules

Differentiation of mouse embryonic stem (ES) cells via embryoid bodies (EB) is established as a suitable model to study cellular processes of development in vitro. ES cells are known to be pluripotent because of their capability to differentiate into cell types of all three germ layers including germ cells. Here, we show that ES cells differentiate into renal cell types in vitro. We found that genes were expressed during EB cultivation, which have been previously described to be involved in renal development. Marker molecules characteristic for terminally differentiated renal cell types were found to be expressed predominantly during late stages of EB cultivation, while marker molecules involved in the initiation of nephrogenesis were already expressed during early steps of EB development. On the cellular level - using immunostaining - we detected cells expressing podocin, nephrin and wt-1, characteristic for differentiated podocytes and other cells, which expressed Tamm–Horsfall protein, a marker for distal tubule epithelial cells of kidney tissue. Furthermore, the proximal tubule marker molecules renal-specific oxido reductase, kidney androgen-related protein and 25-hydroxyvitamin D 3 α-hydroxylase were found to be expressed in EBs. In particular, we could demonstrate that cells expressing podocyte marker molecules assemble to distinct ring-like structures within the EBs. Because the differentiation efficiency into these cell types is still relatively low, application of fibroblast growth factor (FGF)-2 in combination with leukaemia inhibitory factor was tested for induction, but did not enhance ES cell-derived renal differentiation in vitro.