Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells

Gefitinib, an EGFR Tyrosine Kinase inhibitor, Prevents Smoke-Mediated Ciliated Airway Epithelial Cell Loss and Promotes Their Recovery.

Cigarette smoke exposure is a major health hazard. Ciliated cells in the epithelium of the airway play a critical role in protection against the noxious effects of inhaled cigarette smoke. Ciliated cell numbers are reduced in smokers which weakens host defense and leads to disease. The mechanisms for the loss of ciliated cells are not well understood. The effects of whole cigarette smoke exposure on human airway ciliated ciliated cells were examined using in vitro cultures of normal human bronchial epithelial cells and a Vitrocell® VC 10® Smoking Robot. These experiments showed that whole cigarette smoke causes the loss of differentiated ciliated cells and inhibits differentiation of ciliated cells from undifferentiated basal cells. Furthermore, treatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, Gefitinib, during smoke exposure prevents ciliated cell loss and promotes ciliated cell differentiation from basal cells. Finally, restoration of ciliated cells was inhibited after smoke exposure was ceased but was enhanced by Gefitinib treatment. These data suggest that inhibition of EGFR activity may provide therapeutic benefit for treating smoke related diseases

IL-13 Inhibits Multicilin Expression and Ciliogenesis via Janus Kinase/Signal Transducer and Activator of Transcription Independently of Notch Cleavage

Loss of ciliated cells and increases in goblet cells are seen in respiratory diseases such as asthma. These changes result in part from reduced differentiation of basal progenitor cells to ciliated cells during injury and repair. The T helper 2 cytokine, IL-13, has been shown to inhibit ciliated cell differentiation, but the mechanism is not clearly understood. We recently showed that Notch signaling inhibits ciliated cell differentiation in submerged culture by repressing multicilin and forkhead box J1 (FOXJ1) expression, genes required for ciliogenesis. Using a novel method to study ciliated cell differentiation, we investigated the relationship between IL-13 and Notch signaling pathways. We found that IL-13 inhibits ciliated cell differentiation by repressing multicilin and FOXJ1 expression but does so independent of Notch signaling. In addition, we show that pharmacological inhibition of Janus kinase/signal transducer and activator of transcription, but not mitogen activated protein kinase kinase, signaling rescues multicilin and FOXJ1 expression and ciliated cell differentiation in the presence of IL-13. These findings indicate that regulation of multicilin expression by two distinct signaling pathways affects ciliated cell differentiation. In addition, the requirement for Janus kinase activation in IL-13-induced inhibition of ciliogenesis provides a potential therapeutic target for the treatment of respiratory disease

Forskolin increases neuregulin receptors in human Schwann cells without increasing receptor mRNA

Forskolin and heregulin synergistically drive human Schwann cell (HSC) proliferation in vitro, but the role of forskolin is not completely understood. To learn how forskolin might affect receptor levels in HSC cultured from adult nerve roots, we first studied expression and localization of HER2 and HER3 in intact roots, using Western blotting and light and electron microscopic immunocytochemistry. We then determined the effect of forskolin and heregulin on receptor expression in HSC cultured from nerve roots using Western blotting and RNase protection assays. HER2 and HER3 were expressed in nonmyelinating Schwann cells in roots and in cultured HSCs before exposure to forskolin. HER2, but not HER3, was also expressed in endoneurial fibroblasts and in cultured nerve root-derived fibroblasts. Treatment with forskolin for 24 h consistently increased HER2 and HER3 protein levels in HSCs but did not alter HER2 and HER3 mRNA levels. In addition, 24-h treatment with heregulin alone decreased HER2 and HER3 protein levels, an effect not previously described. When both heregulin and forskolin were present, HER2 and HER3 protein levels were similar to initial control values. The effect of forskolin on receptor levels was mimicked by dibutyryl-cAMP and receptor levels in both untreated and forskolin treated HSCs were decreased by treatment with the protein kinase A inhibitor H-89. Following pretreatment of HSCs with forskolin, increased receptor levels were correlated with increased rates of thymidine incorporation into HSCs. These results suggest that forskolin/heregulin synergy might derive, at least in part, from post-transcriptional effects leading to increased steady-state receptor levels

Characterization of the expression and steroid hormone control of sialomucin complex in the rat uterus: Implications for uterine receptivity

The sialomucin complex (SMC), originally isolated as a heterodimeric glycoprotein complex from membranes of ascites sublines of a highly metastatic mammary adenocarcinoma, consists of a high Mr mucin subunit (ASGP‐1, ascites sialoglycoprotein) and a transmembrane subunit (ASGP‐2) with two epidermal growth factor‐like domains. SMC has been characterized in the mammary gland, where it is present in both membrane and nonmembrane (soluble) forms, the latter lacking its transmembrane domain. SMC in the mammary gland is observed during pregnancy and lactation, but not in the virgin gland, and is regulated by a posttranscriptional mechanism. Both membrane and nonmembrane forms of SMC are found in rat uterus, also as a complex of ASGP‐1 and ASGP‐2. Immunocytochemical analyses indicate that the primary site of expression is at the luminal surface of the endometrium. Approximately 40% of the SMC, corresponding to the nonmembrane fraction, is removed by rinsing uterine preparations with saline, indicating that the soluble form is adsorbed loosely to the cell surface. In contrast to mammary gland, SMC is most highly expressed in the virgin animal, and its expression varies during the estrous cycle with the steady state level of transcript. The complex is present in a location consistent with steric inhibition of blastocyst implantation and is abruptly lost at the beginning of the period of receptivity for implantation. Expression of SMC in the uterus is regulated by estrogen and progesterone and is inversely correlated with receptivity. Both implantation and loss of SMC expression can be blocked with RU486. We propose that the downregulation of SMC and its loss from the apical surface of the rat uterine lining contribute to the generation of the receptive state for uterine implantation. Furthermore, the presence of both membrane and soluble SMC at the luminal surface of the endometrium may provide a model for its proposed protective function in other accessible and vulnerable epithelia. J. Cell. Physiol. 176:110–119, 1998. © 1998 Wiley‐Liss, Inc

Inhibition of EGFR signaling prevents WCS induced loss of ciliated cells.

<p><b>(A–F)</b>, Representative confocal micrographs of differentiated human airway epithelial cells treated with Air <b>(A, D)</b>, Smoke <b>(B, E)</b> or Smoke + 500 nM Gefitinib <b>(C, F)</b> stained for FoxJ1 (green), acetylated-tubulin (white) or nuclei (blue) <b>(A, B, C)</b>. <b>D</b>, <b>E</b> and <b>F</b> represent the same microscopic fields as <b>A</b>, <b>B</b> and <b>C</b>, respectively showing only the FoxJ1 and nuclear staining. Scale bar = 25 μm <b>G</b>, Quantification of the percent FoxJ1 positive cells relative to the Air treated samples. N = 7 air and smoke, N = 5 smoke + Gefitinib; * P<0.05, one way ANOVA.</p

Inhibition of EGFR signaling restores ciliated cell differentiation during whole cigarette smoke exposure.

<p><b>(A–F)</b> Representative confocal micrographs of human airway epithelial cells after treatment with Air <b>(A, D)</b>, Smoke <b>(B, E)</b> or Smoke + Geftinib <b>(C, F)</b> during 21 d of differentiation using ALI conditions, stained for FoxJ1 (green), acetylated-tubulin (white) or nuclei (blue) <b>(A, B, C)</b>. Panels <b>D</b>, <b>E</b> and <b>F</b> represent the same microscopic fields as <b>A</b>, <b>B</b> and <b>C</b>, respectively showing only the FoxJ1 and nuclear staining. Scale bar = 30 μm <b>(G)</b> Quantification of the percent FoxJ1 positive cells after 21 d of differentiation using ALI conditions treated with Air, Smoke or Smoke + Gefitinib. N = 7 different lung donors, * P<0.05, one-way ANOVA.</p

Whole cigarette smoke exposure inhibits ciliated cell differentiation.

<p><b>(A–F)</b> Representative confocal micrographs of human airway epithelial cells treated with Air <b>(A, B, C)</b> or Smoke <b>(D, E, F)</b> for 14 d <b>(A, D)</b>, 21 d <b>(B, E)</b> or 27 d <b>(C, F)</b> during differentiation using ALI conditions and stained for FoxJ1 (green), acetylated-tubulin (white) or nuclei (blue). Scale bar = 30 μm. <b>(G)</b> Quantification of FoxJ1 positive cells treated with Air (white bars) or Smoke (grey bars) for 27 d during differentiation in ALI conditions. N = 3 different lung donors, * P = 0.06, two-tailed Student’s t test. <b>(H)</b> Quantitation of the average number of nuclei / microscopic field in cells treated with Air (white bar) or WCS (grey bar) for 27 days during differentiation. N = 3 different lung donors, N.S., not significant, two-tailed Student’s t-test. <b>(I)</b> Duplex qRT-PCR of FoxJ1 mRNA relative to GAPDH mRNA in cells treated with Air or Smoke for 27 d during differentiation using ALI conditions. N = 8 different lung donors, * P<0.05, two-tailed Student’s t-test. <b>(J)</b> Duplex qRT-PCR of MCIDAS mRNA relative to B2M in human airway epithelial cells treated with Air (white bars) or Smoke (grey bars) for 27 d of differentiation in ALI conditions. N = 8 different lung donors, N.S., not significant, two-tailed Student’s t-test.</p