Immortalized human middle ear epithelial cell lines

Details Immortalized human middle ear epithelial cell lines Immortalized human middle ear epithelial cell lines

2000-05-26

2002-03-19

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S467000, C435S371000

Reexamination Certificate

active

06358688

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The present invention relates to novel immortalized middle ear epithelial cell lines and their use in screening assays.
BACKGROUND OF THE INVENTION
Otitis media (OM), or inflammation of the middle ear, is the second most frequent illness resulting in visits to physicians following the common cold and the most common cause of hearing impairment in children. According to the Centers for Disease Control and Prevention/National Center for Health Statistics, OM accounts for an estimated 31 million annual visits to the doctor's office. Eighty percent of the children born each year experience at least one episode of OM by their third birthday, and one in three have repeated bouts of the disease. Although the fraction of health care expenditure taken up by OM is unknown, it is estimated to have a yearly cost exceeding $5 billion.
Acute OM (AOM) generally refers to the rapid onset of signs and symptoms of an acute infection in the middle ear. Chronic otitis media with effusion (OME), also known as persistent middle ear effusion, is the major sequela of acute OM. OME is characterized by the accumulation of serous, mucoid or purulent fluid in the middle ear space, without compromising the intactness of the tympanic membrane (Bluestone, C. and Klein, J O. 1995. Otitis media in infants and children: W.B. Saunders Company). In approximately 40% of the cases, middle ear effusion can still be seen one month after antibiotic treatment and in 20% of them, even after three months. In the US alone, about one million tympanostomy tubes are inserted per year, for the treatment of persistent or recurring OM. The course of OME is generally benign and self-limiting. The deafness caused by the effusions, however, if not treated in time, could adversely affect the child's development and educational progress.
OM is caused mainly by three pathogens:
Streptococcus pneumoniae, Moraxella catarrhalis
and non-typeable
Haemophilus influenzae
(NTHI) [Block, S. L. 1997. Causative pathogens, antibiotic resistance and therapeutic considerations in acute otitis media. Pediatr Infect Dis J. 16: 449-56; Brook, I. 1994. Otitis media: microbiology and management. J Otolaryngol. 23: 269-75; Maxson, S., T. Yamauchi. 1996. Acute otitis media. Pediatr Rev. 17: 191-5; and Strausbaugh, L. 1997. Haemophilus influenzae infections in adults: a pathogen in search of respect. Postgrad Med. 101:191-200]. The inflammatory reaction caused by the interaction of bacterial surface components with the epithelial cells of the middle ear is one of the most crucial steps in the development of otitis media. Many of the processes involved however, remain poorly understood [DeMaria, T. F., Yamaguchi, T., Bakaletz, L. O., and Lim, D. J. (1992). Serum and middle ear antibody response in the chinchilla during otitis media with effusion induced by nonviable nontypeable Haemophilus influenzae. J Infect Dis 165, S196-.; Ernst, (1999). Review article: the role of inflammation in the pathogenesis of gastric cancer. Aliment Pharmacol Ther 13, 13-18; Giebink, G. S. (1999). Otitis media: the chinchilla model. Microb Drug Resist 5, 57-72; Patel, J., Faden, H., Sharma, S., and Ogra, P. L. (1992). Effect of respiratory syncytial virus on adherence, colonization and immunity of non-typable Haemophilus influenzae: implications for otitis media. Int J Pediatr Otorhinolaryngol 23, 15-23; and Weinberg, A., Krisanaprakornkit, S., and Dale, B. A. (1998) Epithelial antimicrobial peptides: review and significance for oral applications. Crit Rev Oral Biol Med 9, 399-414]. In order elucidate the cellular and molecular mechanisms involved in the pathogenesis of otitis media, it is important to understand the biology of the middle ear epithelium and such studies have traditionally required large numbers of middle ear epithelial cells. Although there have been several reports on the establishment of primary cultures and cell lines of middle ear epithelial cells in rodents, to date there have been no reports of immortalized human middle ear epithelial cells. Thus, there is a need for cell lines derived from normal human middle ear that can be used, to study the molecular mechanisms involved in the pathogenesis of otitis media.
Primary cultures of untransformed human cells are generally difficult to propagate for extended periods. Their life span in culture is very limited and they usually become senescent after 4-5 passages. Although middle ear and Eustachian tube epithelial cell lines have been derived from laboratory animals [Portier F., et al. Oxygen modulates Na+ absorption in middle ear epithelium. Am J Physiol 1999 Feb;276(2 Pt 1):C312-7; van Blitterswijk C A, et al. Culture and characterization of rat middle-ear epithelium. Acta Otolaryngol (Stockh). 1986 May-Jun; 101 (5-6):453-66; Takeno S, et al. Tissue culture of middle ear epithelium using fibroblast-reorganized collagen gels. J Otolaryngol. 1993 Oct;22(5):3804; de Serres L M, et al. Bioelectric properties of gerbil middle ear epithelia. Arch Otolaryngol Head Neck Surg 1991 Apr;117(4):416-21; Herman P, et al. Ion transport by primary cultures of Mongolian gerbil middle ear epithelium. Am J Physiol 1992 Mar;262(3 Pt 2):F373-80; Ueyama S., et al. Immortalization of rat middle ear epithelial cells by Adenol2-SV40 hybrid. Ann Otol Rhinol Laryngol. (in print); Herman P., et al. Middle ear cell line that maintains vectorial electrolyte transport. J Cell Physiol. 1993 Mar;154(3):615-22.; and Nakamura A, et al. Serial culture and characterization of the chinchilla middle era epithelium. Ann Otol Rhinol Laryngol. 1991 Dec;100(12): 1024-31.], to date there have been no report of immortailized human middle ear epithelial cells.
Expression of viral genes is an effective way of immortalizing primary cells. Among the most commonly used and effective transforming viral sequences are the E6 and E7 genes of the human papilloma virus (HPV) type 16, which function to dysregulate cell growth. HPV is a site specific DNA virus that is known to infect the basal cell layers and replicate during epithelial cell differentiation [Woodworth CD, et al. Characterization of normal human exocervical epithelial cells immortalized in vitro by papillomavirus types 16 and 18 DNA. Cancer Res. 1988 Aug 15;48(16):4620-8; Pecoraro G, et al. Differential effects of human papilloma virus type 6, 16, and 18 DNAs on immortalization and transformation. Proc Natl Acad Sci USA. 1989 Jan; 86(2): 563-7; Band V, et al. Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements. Proc Natl Acad Sci U S A. 1990 Jan;87(1):463-7; Durst M, et al. Inverse relationship between human papillomavirus type 16 early gene expression and cell differentiation in nude mouse epithelial cysts and tumors induced by HPV-positive human cell lines. J Virol 1991 Feb;65(2):796-804; Merrick DT, et al. Altered expression of proliferation and differentiation markers in human papillomavirus 16 and 18 immortalized epithelial cells grown in organotypic culture. Am J Pathol. 1992 Jan;140(1):167-77; Pirisi L, et al. Continuous cell lines with altered growth and differentiation properties originate after transfection of human keratinocytes with human papillomavirus type 16 DNA. Carcinogenesis. 1988 Sep;9(9):1573-9; Willey J C, et al. Immortalization of normal human bronchial epithelial cells by human papillomaviruses 16 or 18. Cancer Res. 1991 Oct 1;51(19):5370-7; Woodworth C D, et al. Human cervical and foreskin epithelial cells immortalized by human papillomavirus DNAs exhibit dysplastic differentiation in vivo. Cancer Res 1990 Jun 15;50(12):3709-15; and Blanton R A, Perez-Reyes N, Merrick D T, McDougall J K. Epithelial cells immortalized by human papillomaviruses have premalignant characteristics in organotypic culture. Am J Pathol. 1991 Mar;138(3):673-85]. More than 80 different types of human papilloma viruses (HPVS) have now been isolated from a variety of squamous epithelial lesions, and approximately 18 of them have been associated

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