In this study, we utilized the reverse transcriptase component of telomerase, hTERT, and human papillomavirus type 16 (HPV-16) E6 and E7 genes to transform normal and cystic fibrosis (CF) human airway epithelial (HAE) cells. One cell line, designated NuLi-1 (normal lung, University of Iowa), was derived from HAE of normal genotype; three cell lines, designated CuFi (cystic fibrosis, University of Iowa)-1, CuFi-3, and CuFi-4, were derived from HAE of various CF genotypes. When grown at the air-liquid interface, the cell lines were capable of forming polarized differentiated epithelia that exhibited transepithelial resistance and maintained the ion channel physiology expected for the genotypes. The CF transmembrane conductance regulator defect in the CuFi cell lines could be corrected by infecting from the basolateral surface using adenoviral vectors. Using nuclear factor-kappaB promoter reporter constructs, we also demonstrated that the NuLi and CuFi cell lines retained nuclear factor-kappaB responses to lipopolysaccharide. These cell lines should therefore be useful as models for studying ion physiology, therapeutic intervention for CF, and innate immunity.
Cystic fibrosis (CF) airway epithelia exhibit defective transepithelial electrolyte transport: cAMP-stimulated Cl-secretion is abolished because of the loss of apical membrane cystic fibrosis transmembrane conductance regulator (CFTR) Clchannels, and amiloride-sensitive Na+ absorption is increased two-to threefold because of increased amiloride-sensitive apical Na+ permeability. These abnormalities are thought to alter respiratory tract fluid, thereby contributing to airway disease, the major source of mortality in this genetic disease. However, the underlying hypothesis, that fluid transport is abnormal in CF airway epithelia, has not been tested. Most conjecture about fluid transport is based on measurements of Na + and Cltransport performed under short circuit conditions in Ussing chambers. But such studies differ from in vivo conditions in that transepithelial voltage and mucosal fluid composition are held constant. Therefore, we measured fluid transport and mucosal electrolyte composition in primary cultures of CF airway epithelia without holding transepithelial voltage and ion concentration gradients at zero. In normal epithelia, cAMP agonists plus amiloride stimulated NaCl and fluid secretion. In CF epithelia, cAMP agonists failed to stimulate fluid or electrolyte secretion, changes consistent with the loss of CFIR Cl -channels. But in striking contrast to predictions based on Ussing chamber studies, CF epithelia absorbed fluid at a rate no greater than normal epithelia. Moreover, amiloride, which inhibits Na' channels, failed to inhibit fluid absorption by CF epithelia. These results have important implications for understanding the pathogenesis of CF airway disease and for the design and evaluation of therapy. (J. Clin. Invest. 1994.
Pharmacologic- and gene-based therapies have historically been developed as two independent therapeutic platforms for cystic fibrosis (CF) lung disease. Inhibition of the dysregulated epithelial Na channel (ENaC) is one pharmacologic approach to enhance airway clearance in CF. We investigated pharmacologic approaches to enhance CFTR gene delivery with recombinant adeno-associated virus (rAAV) and identified compounds that significantly improved viral transduction while simultaneously inhibiting ENaC activity through an unrelated mechanism. Treatment of human CF airway epithelia with proteasome modulating agents (LLnL and doxorubicin) at the time of rAAV2 or rAAV2/5 infection dramatically enhanced CFTR gene delivery and correction of CFTR-mediated short-circuit currents. Surprisingly, these agents also facilitated long-term (15-day) functional inhibition of ENaC currents independent of CFTR vector administration. Inhibition of ENaC activity was predominantly attributed to a doxorubicin-dependent decrease in gamma-ENaC subunit mRNA expression and an increase in gamma-ENaC promoter methylation. This is the first report to describe the identification of compounds with dual therapeutic action that are able to enhance the efficacy of CFTR gene therapy to the airway while simultaneously ameliorating primary aspects of CF disease pathophysiology. The identification of such compounds mark a new area for drug development, not only for CF, but also for other gene therapy disease targets.
Genetically-engineered pigs are increasingly recognized as valuable models for the study of human disease. Immunohistochemical study of cellular markers of disease is an important tool for the investigation of these novel models so as to evaluate genotype and treatment differences. Even so, there remains a lack of validated markers for pig tissues that can serve as a translational link to human disease in organs such as the lung. Herein, we evaluate markers of cellular inflammation (CD3, CD79a, BCL6, IBA1, and myeloperoxidase) and those that may be involved with tissue remodeling (alpha-smooth muscle actin, beta-tubulin-III, lactoferrin, MUC5AC, MUC5B, and CFTR) for study of lung tissues. We compare the utility of these markers between pig and human lungs to validate translational relevance of each marker. Our results suggest these markers can be a useful addition in the pathological evaluation of porcine models of human disease.
I n r e c e n t y e a r s much discussion i n t h e f i e l d o f i n t e r n a t i o n a l development has centered on t h e i n a b i l i t y of t r a d it i o n a l development p o l i c i e s t o d e a l with t h e i n c r e a s i n g impoverishment of t h e r u r a l populations o f most Third World c o u n t r i e s . I t h a s become widely accept e d by i n t e r n a t i o n a l development i n s t it u i o n s , such as t h e World Bank, as w e l l as by n a t i o n a l development agencies, that i f the underdeveloped c o u n t r i e s are t o be able t o feed t h e i r evergrowing popu l a t i o n s , and t o c l o s e t h e widening gap between themselves and t h e i n d u s t r i a l i z e d world, a major upgrading of t h e standard of l i v i n g and p r o d u c t i v i t y of t h e i r r u r a l s e c t o r s i s an a b s o l u t e n e c e s s i t y ( 1 ) . Thus, t h e proposal t h a t an e s s e n t i a l a s p e c t of any development model must be an e f f e c t i v e s t r a t e g y t o promote broadbased r u r a l development h a s gained i n c r e a s i n g recognition. DESIGNING AN A P P R O P R I A T E STRATEGYI n designing an appropriate s t r a t e g y f o r r u r a l development it i s necessary simultaneously t o consider t h e g o a l s t o be f u r t h e r e d and t h e means The goals pursued through t h e strategy presented here s t e m from my percept i o n of t h e o v e r a l l development process. I n t h e most general way, I would d e f i n e development as t h e process by which an environment i s a t t a i n e d i n which a l l members of a s o c i e t y are given an opport u n i t y t o reach t h e i r f u l l p o t e n t i a l as human beings ( 3 ) . A n e s s e n t i a l condition f o r such f u l f i l l m e n t i s t h e provision of t h e following b a s i c human needs t o every member of t h e s o c i e t y : food, c l o t h i n g , s h e l t e r , a source o f meaningful prod u c t i v e a c t i v i t y , and a sense of belonging and self-esteem.I m p l i c i t w i t h i n t h e r u r a l development s t r a t e g y presented h e r e w i l l be f o u r c e n t r a l goals:(1) The e r a d i c a t i o n of poverty:( 2 ) The e l i m i n a t i o n of unemployment;( 3 ) The e l i m i n a t i o n of major ine q u a l i t i e s , both w i t h i n t h e r u r a l s e c t o r and between t h e r u r a l and urban s e c t o r s ; and ( 4 ) The p a r t i c i p a t i o n of t h e e n t i r e r u r a l population i n t h e development process.The f i r s t g o a l , e r a d i c a t i o n of r u r a l poverty, i s important simply because t h e incidence of poverty i n t h e r u r a l s e c t o r s of almost a l l Third World c o u n t r i e s i s alarmingly high. study notes t h a t 80 p e r c e n t of t h e poor i n t h e underdeveloped c o u n t r i e s of A s i a , Africa, and L a t i n America l i v e i n r u r a l areas(4). The r u r a l poor include s m a l l farmers, t e n a n t s , l a n d l e s s workers, s e l femployed craftsmen and a r t i s a n s , and s e r v i c ...
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