The potential of microfluidic lung epithelial wounding: towards in vivo-like alveolar microinjuries

Idiopathic pulmonary fibrosis (IPF) remains a major clinical challenge to date. Repeated alveolar epithelial microinjuries are considered as the starting point and the key event in both the development and the progression of IPF. Various pro-fibrotic agents have been identified and shown to cause alveolar damage. In IPF, however, no leading cause of alveolar epithelial microinjuries can be identified and the exact etiology remains elusive. New results from epidemiologic studies suggest a causal relation between IPF and frequent episodes of gastric refluxes resulting in gastric microaspirations into the lung. The effect of gastric contents on the alveolar epithelium has not been investigated in detail. Here, we present a microfluidic lung epithelial wounding system that allows for the selective exposure of alveolar epithelial cells to gastric contents. The system is revealed to be robust and highly reproducible. The thereby created epithelial microwounds are of tiny dimensions and best possibly reproduce alveolar damage in the lung. We further demonstrate that exposure to gastric contents, namely hydrochloric acid (HCl) and pepsin, directly damages the alveolar epithelium. Together, this novel in vitro wounding system allows for the creation of in vivo-like alveolar microinjuries with the potential to study lung injury and alveolar wound repair in vitro.

Endothelial binding of beta toxin to small intestinal mucosal endothelial cells in early stages of experimentally induced Clostridium perfringens type C enteritis in pigs.

Beta toxin (CPB) is known to be an essential virulence factor in the development of lesions of Clostridium perfringens type C enteritis in different animal species. Its target cells and exact mechanism of toxicity have not yet been clearly defined. Here, we evaluate the suitability of a neonatal piglet jejunal loop model to investigate early lesions of C. perfringens type C enteritis. Immunohistochemically, CPB was detected at microvascular endothelial cells in intestinal villi during early and advanced stages of lesions induced by C. perfringens type C. This was first associated with capillary dilatation and subsequently with widespread hemorrhage in affected intestinal segments. CPB was, however, not demonstrated on intestinal epithelial cells. This indicates a tropism of CPB toward endothelial cells and suggests that CPB-induced endothelial damage plays an important role in the early stages of C. perfringens type C enteritis in pigs.

deltaNp63 has a role in maintaining epithelial integrity in airway epithelium.

The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its ΔN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the ΔN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that ΔNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium.

Retinal differentiation of human bone marrow-derived stem cells by co-culture with retinal pigment epithelium in vitro.

The goal of this study was to assess the in vitro differentiation capacity of human bone marrow-derived stem cells (hBMSCs) along retinal lineages. Mononuclear cells (MNC) were isolated from bone marrow (BM) and mobilized peripheral blood (mPB) using Ficoll-Paque density gradient centrifugation, and were sorted by magnetic-activated cell sorting (MACS) for specific stem cell subsets (CD34(+)CD38(+)/CD34(+)CD38(-)). These cells were then co-cultured on human retinal pigment epithelial cells (hRPE) for 7 days. The expression of stem cell, neural and retina-specific markers was examined by immunostaining, and the gene expression profiles were assessed after FACS separation of the co-cultured hBMSCs by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, in vitro functionality of the differentiated cells was analyzed by quantifying phagocytosis of CY5-labeled photoreceptor outer segments (POS). After 7 days of co-culture, hBMSCs adopted an elongated epithelial-like morphology and expressed RPE-specific markers, such as RPE65 and bestrophin. In addition, these differentiated cells were able to phagocytose OS, one of the main characteristics of native RPE cells. Our data demonstrated that human CD34(+)CD38(-) hBMSC may differentiate towards an RPE-like cell type in vitro and could become a new type of autologous donor cell for regenerative therapy in retinal degenerative diseases.

Mutation in the Monocarboxylate Transporter 12 Gene Affects Guanidinoacetate Excretion but Does Not Cause Glucosuria

A heterozygous mutation (c.643C>A; p.Q215X) in the monocarboxylate transporter 12-encoding gene MCT12 (also known as SLC16A12) that mediates creatine transport was recently identified as the cause of a syndrome with juvenile cataracts, microcornea, and glucosuria in a single family. Whereas the MCT12 mutation cosegregated with the eye phenotype, poor correlation with the glucosuria phenotype did not support a pathogenic role of the mutation in the kidney. Here, we examined MCT12 in the kidney and found that it resides on basolateral membranes of proximal tubules. Patients with MCT12 mutation exhibited reduced plasma levels and increased fractional excretion of guanidinoacetate, but normal creatine levels, suggesting that MCT12 may function as a guanidinoacetate transporter in vivo. However, functional studies in Xenopus oocytes revealed that MCT12 transports creatine but not its precursor, guanidinoacetate. Genetic analysis revealed a separate, undescribed heterozygous mutation (c.265G>A; p.A89T) in the sodium/glucose cotransporter 2-encoding gene SGLT2 (also known as SLC5A2) in the family that segregated with the renal glucosuria phenotype. When overexpressed in HEK293 cells, the mutant SGLT2 transporter did not efficiently translocate to the plasma membrane, and displayed greatly reduced transport activity. In summary, our data indicate that MCT12 functions as a basolateral exit pathway for creatine in the proximal tubule. Heterozygous mutation of MCT12 affects systemic levels and renal handling of guanidinoacetate, possibly through an indirect mechanism. Furthermore, our data reveal a digenic syndrome in the index family, with simultaneous MCT12 and SGLT2 mutation. Thus, glucosuria is not part of the MCT12 mutation syndrome.

Insulin activates Na(+)/H(+) exchanger 3: biphasic response and glucocorticoid dependence.

Insulin is an important regulator of renal salt and water excretion, and hyperinsulinemia has been implicated to play a role in hypertension. One of the target proteins of insulin action in the kidney is Na(+)/H(+) exchanger 3 (NHE3), a principal Na(+) transporter responsible for salt absorption in the mammalian proximal tubule. The molecular mechanisms involved in activation of NHE3 by insulin have not been studied so far. In opossum kidney (OK) cells, insulin increased Na(+)/H(+) exchange activity in a time- and concentration-dependent manner. This effect is due to activation of NHE3 as it persisted after pharmacological inhibition of NHE1 and NHE2. In the early phase of stimulation (2-12 h), NHE3 activity was increased without changes in NHE3 protein and mRNA. At 24 h, enhanced NHE3 activity was accompanied by an increase in total and cell surface NHE3 protein and NHE3 mRNA abundance. All the effects of insulin on NHE3 activity, protein, and mRNA were amplified in the presence of hydrocortisone. These results suggest that insulin stimulates renal tubular NHE3 activity via a biphasic mechanism involving posttranslational factors and an increase in NHE3 gene expression and the effects are dependent on the permissive action of hydrocortisone.

Transcriptome analysis reveals a classical interferon signature induced by IFNλ4 in human primary cells.

The IFNL4 gene is negatively associated with spontaneous and treatment-induced clearance of hepatitis C virus infection. The activity of IFNλ4 has an important causal role in the pathogenesis, but the molecular details are not fully understood. One possible reason for the detrimental effect of IFNλ4 could be a tissue-specific regulation of an unknown subset of genes. To address both tissue and subtype specificity in the interferon response, we treated primary human hepatocytes and airway epithelial cells with IFNα, IFNλ3 or IFNλ4 and assessed interferon mediated gene regulation using transcriptome sequencing. Our data show a surprisingly similar response to all three subtypes of interferon. We also addressed the tissue specificity of the response, and identified a subset of tissue-specific genes. However, the interferon response is robust in both tissues with the majority of the identified genes being regulated in hepatocytes as well as airway epithelial cells. Thus we provide an in-depth analysis of the liver interferon response seen over an array of interferon subtypes and compare it to the response in the lung epithelium.

Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

BACKGROUND Mammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMECUS) or Swiss Holstein-Friesian (bMECCH) cows, and of primary bovine mammary alveolar epithelial cells stably transfected with simian virus-40 (SV-40) large T-antigen (MAC-T) for in vitro analyses. This was evaluated by testing their expression pattern of cytokeratin (CK) 7, 18, 19, vimentin, and α-smooth muscle actin (α-SMA). RESULTS The expression of the listed markers was assessed using real-time quantitative PCR, flow cytometry and immunofluorescence microscopy. Characteristic markers of the mesenchymal (vimentin), myoepithelial (α-SMA) and glandular secretory cells (CKs) showed differential expression among the studied cell cultures, partly depending on the analytical method used. The relative mRNA expression of vimentin, CK7 and CK19, respectively, was lower (P < 0.05) in immortalized than in primary mammary cell cultures. The stain index (based on flow cytometry) of CK7 and CK19 protein was lower (P < 0.05) in MAC-T than in bMECs, while the expression of α-SMA and CK18 showed an inverse pattern. Immunofluorescence microscopy analysis mostly confirmed the mRNA data, while partly disagreed with flow cytometry data (e.g., vimentin level in MAC-T). The differential expression of CK7 and CK19 allowed discriminating between immortal and primary mammary cultures. CONCLUSIONS The expression of the selected widely used cell type markers in primary and immortalized MEC cells did not allow a clear preference between these two cell models for in vitro analyses studying aspects of milk composition. All tested cell models exhibited to a variable degree epithelial and mesenchymal features. Thus, based on their characterization with widely used cell markers, none of these cultures represent an unequivocal alveolar mammary epithelial cell model. For choosing the appropriate in vitro model additional properties such as the expression profile of specific proteins of interest (e.g., transporter proteins) should equally be taken into account.

Does dienogest influence the inflammatory response of endometriotic cells? A systematic review.

OBJECTIVE AND DESIGN A systematic review of all literature was done to assess the ability of the progestin dienogest (DNG) to influence the inflammatory response of endometriotic cells. MAIN OUTCOME MEASURES In vitro and in vivo studies report an influence of DNG on the inflammatory response in eutopic or ectopic endometrial tissue (animal or human). RESULTS After strict inclusion criteria were satisfied, 15 studies were identified that reported a DNG influence on the inflammatory response in endometrial tissue. These studies identified a modulation of prostaglandin (PG) production and metabolism (PGE2, PGE2 synthase, cyclo-oxygenase-2 and microsomal PGE synthase-1), pro-inflammatory cytokine and chemokine production [interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-α, monocyte chemoattractant protein-1 and stromal cell-derived factor-1], growth factor biosynthesis (vascular endothelial growth factor and nerve growth factor) and signaling kinases, responsible for the control of inflammation. Evidence supports a progesterone receptor-mediated inhibition of the inflammatory response in PR-expressing epithelial cells. It also indicated that DNG inhibited the inflammatory response in stromal cells, however, whether this was via a PR-mediated mechanism is not clear. CONCLUSIONS DNG has a significant effect on the inflammatory microenvironment of endometriotic lesions that may contribute to its clinical efficacy. A better understanding of the specific anti-inflammatory activity of DNG and whether this contributes to its clinical efficacy can help develop treatments that focus on the inhibition of inflammation while minimizing hormonal modulation.

Vitamin D represses rhinovirus replication in cystic fibrosis cells by inducing LL-37.

Vitamin D has immunomodulatory properties in the defence against pathogens. Its insufficiency is a widespread feature of cystic fibrosis (CF) patients, which are repeatedly suffering from rhinovirus (RV)-induced pulmonary exacerbations.To investigate whether vitamin D has antiviral activity, primary bronchial epithelial cells from CF children were pre-treated with vitamin D and infected with RV16. Antiviral and anti-inflammatory activity of vitamin D was assessed. RV and LL-37 levels were measured in bronchoalveolar lavage (BAL) of CF children infected with RV.Vitamin D reduced RV16 load in a dose-dependent manner in CF cells (10(-7 )M, p<0.01). The antiviral response mediated by interferons remained unchanged by vitamin D in CF cells. Vitamin D did not exert anti-inflammatory properties in RV-infected CF cells. Vitamin D increased the expression of the antimicrobial peptide LL-37 up to 17.4-fold (p<0.05). Addition of exogenous LL-37 decreased viral replication by 4.4-fold in CF cells (p<0.05). An inverse correlation between viral load and LL-37 levels in CF BAL (r=-0.48, p<0.05) was observed.RV replication in primary CF bronchial cells was reduced by vitamin D through the induction of LL-37. Clinical studies are needed to determine the importance of an adequate control of vitamin D for prevention of virus-induced pulmonary CF exacerbations.

Lung structure at preterm and term birth

When lung development is not interrupted by premature birth and unaffected by genetic or environmental disturbances, all components develop with complex control to form a functional organ with a predictable timeline during fetal development. In this chapter we describe the relationship between morphological development and function in both physiological and pathological conditions in human lung development. Tree-like growth of the lung begins during the first few weeks postconception, with the embryonic stage characterized by branching morphogenesis in both the airways and blood vessels, separately in the left and right lung buds, which appear near day 26 postcoitus (p.c.). Branching continues through the embryonic stage, with proliferation of mesenchymal and epithelial cells and apoptosis near branch points and in the areas of new formation. The pseudoglandular stage (weeks 5–17 p.c.) is characterized by accelerated cellular proliferation and airway and vascular branching, with epithelial differentiation in proximal and distal airways. Further epithelial differentiation, angiogenesis of the parenchymal capillary network, and the first formation of the air–blood barrier characterize the canalicular stage (16–26 weeks p.c.), just before the completion of branching morphogenesis (saccular stage, weeks 24–38 p.c.) and the start of alveolarization (week 36 through adolescence).