This material was disrupted (Ultrasonic Processor XL, Misonix Incorporation, Farmingdale, NY) at 4C with six bursts of 10 s each, centrifuged at 10,000 Xgfor 25 min, and the supernatant was aspirated and used as the source of sonicate. == Cell Cultures == The intestinal epithelial cell line Caco-2, derived from a human colonic adenocarcinoma (HTB-37; ATCC, Manassas, VA), was grown in Dulbecco’s modified Eagle medium (DMEM) (Sigma Chemical Co., St. attached to the monolayers. Colonization increased with time, with the Caco-2 cell junctions being the initial targets of attachment. By electron microscopy, individual spirochete cells could be seen to have one cell end invaginated into the Caco-2 cell membranes, with the rest of the spirochete draped over the Caco-2 cell surface. After 6 h incubation, the monolayer was covered with a layer of spirochetes. Colonized monolayers demonstrated a time-dependent series of changes: staining with labelled phalloidin identified accumulation of actin at the cell junctions; ZO-1 staining revealed a loss of Caco-2 tight junction integrity; and Hoechst staining showed condensation and fragmentation of nuclear material consistent with apoptosis. Using quantitative reverse transcription PCR, the colonized monolayers demonstrated a significant up-regulation of interleukin-1 (IL-1) and IL-8 expression.B. pilosicolisonicates caused significant up-regulation of IL-1, TNF-, and IL-6, but culture supernatants and non-pathogenicBrachyspira innocensdid not alter cytokine expression. == Conclusions/Significance == The changes induced in the Caco-2 cells provide evidence thatB. pilosicolihas pathogenic potential, and give insights into the likelyin vivopathogenesis. == Introduction == The intestinal spirocheteBrachyspira pilosicolicolonizes the large intestine of a variety of species of animals and birds, as well as human beings[1]. Infection is common in intensively farmed chickens and pigs, in which the spirochete is considered to be an important enteric pathogen[2],[3]. In humans, infection is common in homosexual males and HIV patients in developed countries[4],[5], but also it occurs frequently in people in developing countries, especially those living in crowded and unhygienic conditions[6],[7]. Recently, large numbers of intestinal spirochetes have been found in stool samples from patients with cholera, and it has been suggested that they may exacerbate the disease[8]. A characteristic feature of colonization withB. pilosicoliis the intimate end-on or polar attachment of spirochete cells to the luminal surface of colonic and rectal epithelial cells, in a condition called intestinal spirochetosis or colonic spirochetosis[1]. This description was first made in colonic biopsy samples from humans where the associated dense layer of attached spirochetes was described as a false brush border[9]. Subsequently, a similar Norepinephrine condition was described in pigs[10], and eventually it was shown that strains of the same spirochete species (now calledB. pilosicoli) could cause the PGR condition in both humans and pigs[11],[12]. Humans also may be colonized with the distinct speciesBrachyspira aalborgi, which similarly attaches to colonic enterocytes by one cell end[13],[14]. Brachyspira pilosicoliis difficult to isolate as it is anaerobic and grows slowly, and, despite its potential importance as a pathogen, it has not been extensively studied. Very little is known about virulence determinants in this spirochete, apart from the fact that it appears to lack the attachment and invasion determinants encoded by theinv,ailandyadAgenes ofYersinia enterocolitica, theeaegene from enteropathogenicEscherichia coli, and a virulence plasmid ofShigella flexneri[15]. Progress has been hampered by a lack of genomic information for this spirochete, an absence of means for genetic manipulation, and a lack ofin vitromodels in which to study the pathogenesis of infection. Pigs, chickens and mice have been used experimentally as models ofB. pilosicoliinfection, using spirochete strains isolated from various species, including humans[16][21]. In these models, as in the natural infections, one cell end of the spirochetes can Norepinephrine be seen invaginating into the mature columnar cells without penetrating the host cell membrane. Often hundreds of individual spirochete cells can be seen attached to the surface of each enterocyte, forming a dense mat of spirochete cells overlaying the epithelium. In the only previous published study usingB. pilosicolito infect intestinal epithelial cell lines, a diffuse attachment of spirochetes was obtained, but the characteristic attachment by one cell end was not observed, and pathological changes similar Norepinephrine to those that occurin vivowere not induced[22]. The aim of the current study was to establish anin vitromodel to study the interactions ofB. pilosicoliwith enterocytes, and to gain insights into the pathogenesis of the infection that have not been documented previously. == Materials and Methods == == Spirochete Strains and Growth == Two Australian strains ofBrachyspira pilosicoliisolated from human beings (WesB and Karlton), and two from pigs (95/1000 and Cof-10), as well as theBrachyspira innocenstype strain B256T, were obtained as frozen stock from the culture collection held at the Australian Reference Centre for Intestinal Spirochetes, School of Veterinary and Biomedical Sciences, Murdoch University. The cells were thawed and grown in Kunkle’s pre-reduced anaerobic broth, containing 2% (v/v) fetal bovine serum and 1% (v/v) ethanolic cholesterol solution[23]. Broth cultures were incubated at 37C on a rocking platform for 35 days, and spirochete growth was monitored daily by examining aliquots under a phase contrast microscope. Cell.