Progress 10/01/04 to 09/30/09
Outputs
OUTPUTS: In this project (Oct 2005-Sept 2009), two porcine primary intestinal epithelial cell lines derived from jejunum (IPEC-J2) and small intestine (IPEC-1) of 1-day old pigs were extensively characterized for their biochemical and immunological properties. Varying number of cells in both cell lines stained positive for galectin-4, cytokeratin, vimentin, alpha-smooth muscle actin, and desmin. Next we investigated lectin binding profile of these cells using 21 biotinylated lectins. Most lectins except UEA-1 stained a large percentage of IPEC-J2 and IPEC-1 cells. A variable number of both porcine cell lines expressed MHC-I, MHC-II, CD44 and CD11b/c. Both type of cells expressed comparable levels of toll-like receptors' (TLR 2, -4, -5 and -9) transcripts. A variable number of both IPEC-J2 and IPEC-1 also expressed TLR-2, -4 and -9 proteins but did not express TLR-5 protein. We also investigated lectin binding profile and TLRs expression in ileal tissues and changes in intestinal microflora in pigs under normal, feed antibiotic chlortetracycline (CTC)-fed and gnotobiotic conditions. No significant differences in TLR-2, 4, 5 and 9 transcripts and proteins were observed among three groups of pigs. Gnotobiotic piglets expressed significantly weaker lectin binding for many lectins. CTC-fed pigs showed some significant differences in lectin binding profile compared to normal-fed pigs. Furthermore, significant changes were observed in microbial community between the ileum of normal-fed and CTC-fed pigs. We also investigated the adherence of various enterotoxigenic Escherichia coli (ETEC) strains to IPEC-J2 and IPEC-1 cell lines to develop an in vitro model for studying ETEC pathogenesis. ETEC expressing K88ab, K88ac or K88ad adhesins equally bound to both IPEC-1 and IPEC-J2 cells. ETEC expressing F18 strongly bound to IPEC-1 cells. We also found that both IPEC-J2 and IPEC-1 cells effectively bind heat-labile toxin (LT) of ETEC. In further studies we showed that LT promotes ETEC binding to IPEC-2 cells and various strains of ETEC induce apoptosis in these cells. We also evaluated the changes in transepithelial resistance (TER) and tight junction (TJ) protein localization in IPEC-J2 cells in response to various ETEC strains. Significant LT-independent reduction in TER was observed in the absence of TJ proteins mislocalization. In another study, both IPEC-J2 and IPEC-1 cells were successfully infected with a microsporidium Encephalitozoon intestinalis. TER in infected polarized cultures decreased after E. intestinalis infection and this pathogen also induced significant amount of apoptosis in these cells. We also successfully infected gnotobiotic piglets with E. intestinalis for studying microspordial pathogenesis. We performed another study to develop intestinal myoepithelial and myofibroblast-like cultures from young lambs and we successfully established these cultures. The findings of these studies were presented in various scientific conferences and published in various scientific journals. Two graduate students completed their master programs and five undergraduate students and four graduate students worked/trained on this project. PARTICIPANTS: Individual who worked on the project: Five under graduate students: YeJin Oh, Matthew J Anderson, Camille Moen, Jordan Hass and Brandon Wegehaupt. Four graduate students: Sajan George, Gopakumar Moorkanat, Amber Johnson and Seung Koh. Principal investigator: Radhey S Kaushik. Partner organizations: South Dakota State University. Collaborators and consultants: Drs. David Francis, Philip Hardwidge, Volker Brozel, Mike Hildreth, Alan Young and Artur Rosa; South Dakota State University, Brookings, SD 57007 USA. Training and professional development: Trained five undergraduate students and four graduate students. TARGET AUDIENCES: Target audiences: Students, producers, veterinarians and faculty involved in Animal Health and Microbiology research. Efforts: Some of the research findings of this project were submitted in the form of thesis: 1. George, S. 2006. Molecular and biochemical changes in the ileum of weanling pigs fed antibiotics as growth promotant and in germ-free conditions, Master of Science thesis for Major in Biological Sciences (Microbiology), South Dakota State University, USA and 2. Gopakumar Moorkanat. 2008. Infectivity and pathogenesis of Encephalitozoon intestinalis in an in vivo and in vitro porcine model, Master of Science thesis for Major in Biological Sciences (Microbiology), South Dakota State University, USA. The research findings of this project were presented at Third international Rushmore conference on enteric diseases, South Dakota in 2005; Conference of Research Workers in Animal Diseases (CRWRD), Chicago in 2005, 2006, and 2007; ETEC Retreat, South Dakota in 2008, and the American Society for Microbiology North Central Branch Annual Meetings in 2006 and 2008. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Young food animals (pigs, lambs and calves) are susceptible to various enteric pathogens. Intestinal epithelial and fibroblast-like cells play a crucial role in mediating enteric mucosal immunity and in the pathogenesis of enteric pathogens but their roles in pigs and ruminants are not clearly defined. One approach to study the role of these cells is to establish and use intestinal epithelial/fibroblast-like cultures from various animal species for studying pathogenesis of enteric pathogens. In this study, we characterized two pig intestinal epithelial cell lines (IPEC-J2 and IPEC-1) for important biochemical and immunological epithelial cell markers, lectin binding profile, and toll-like receptors (TLRs). We also established polarized cultures of these cells to simulate in vivo conditions. We also studied the adherence of porcine enterotoxigenic Escherichia coli (ETEC) to these cells which cause severe watery diarrhea in newborn and weaned pigs. ETEC infections are of significant economic importance in the pig industry. Both these cell types adhered to various ETEC strains. We showed that heat-labile toxin (LT) effectively binds to these cells and promotes the adherence of ETEC. Further, various ETEC strains induced apoptosis in IPEC-J2 cells and LT-independent reduction in TER in polarized cultures. We also showed that both IPEC-J2 and IPEC-1 cells could be effectively infected with a microsporidium Encephalitozoon intestinalis (causes diarrhea in both humans and pigs) and this pathogen induced apoptosis in these cells. These findings suggested that both IPEC-J2 and IPEC-1 cells may act as antigen presenting cells and provide suitable and biologically relevant porcine cell culture models for studying the intestinal immune mechanisms. Further, these cells can be effectively used as a cell culture model for studying and developing preventive and therapeutic approaches for protecting pigs and humans against various enteric diseases. We also conducted some in vivo studies in pigs. Feed antibiotics are widely used as growth promotant in the pig industry but their use has been banned in Europe because of human health concerns. We studied the effect of chlortetracycline (CTC) and gut microflora on the physiology, immunity and biochemistry of intestine in antibiotic-fed and gnotobiotic pigs and observed some biologically important significant differences such changes in the lectin binding profile of CTC-fed and gnotobiotic pigs compared to normal-fed pigs. These findings will help in developing alternatives to feed antibiotics. In another study we successfully infected gnotobiotic pigs with E. intestinalis and established pigs as a model for studying the pathogenesis of microsporidial infections. In another study, we successfully established ileal myoepithelial and myofibroblast-like cultures from young lambs. These cultures may be used for studying the pathogenesis of various enteric diseases of ruminants such as scrapie and Johne's disease (JD). We established some local collaboration with SDSU faculty (Drs. David Francis, Philip Hardwidge, Volker Brozel, Mike Hildreth, Alan Young and Artur Rosa) for the successful execution of this project.
Publications
- Johnson, A.M., Kaushik, R.S. and Hardwidge, P.R. 2009. Disruption of transepithelial resistance by enterotoxigenic Escherichia coli. Vet. Microbiol. In press. doi:10.1016.j.vetmic.2009.08.020.
- Rettedal, E., Vilain, S., Lindblom, S., Lehnert, K., Scofield, C., George, S., Clay, S., Kaushik, R.S., Rosa, A. J. M. Francis, D. and Brozel, V.S. 2009. Alteration of the Ileal Microbiota of Weanling Piglets by the Growth-Promoting Antibiotic Chlortetracycline. Appl. Environ. Microbiol.75:5489-5495.
- Hemerka, J.N., Wang, D., Weng, Y., Lu, W., Kaushik, R.S., Jin, J., Harmon, A.F. and Li, F. 2009. Detection and characterization of influenza A virus PA-PB2 interaction through a bimolecular fluorescence complementation assay. J. Virol. 83(8):3944-55.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: In 2007 CRIS report we characterized two porcine, IPEC-J2 and IPEC-1 intestinal epithelial cells for the expression of Toll-like receptors and cell surface markers. We further extended these studies and investigated the susceptibility of these cultures to microsporidial infection. Both cell types were successfully infected with a microsporidium Encephalitozoon intestinalis. The effect of E. intestinalis infection on membrane integrity was evaluated by measuring transepithelial electrical resistance (TEER) in polarized IPEC-J2 cultures. TEER in infected cultures decreased after E. intestinalis infection; however, no pronounced changes in tight junction proteins (ZO-1 and occludin) were observed. E. intestinalis infection also induced the significant amount of apoptosis in these cells. We also infected gnotobiotic piglets with E. intestinalis in order to establish pigs as an animal model for studying microspordial pathogenesis. We were successful in infecting piglets with E. intestinalis based on the PCR analysis of fecal samples and certain tissues but infected piglets did not show any E. intestinalis specific clinical signs. These studies were conducted by a graduate student Gopakumar Moorkanat for his Master's thesis submitted in Sept 2008. One of the goals of another study was to develop intestinal epithelial and fibroblast-like cultures from young lambs. In this study, ileal tissues were collected from a 3 day old lamb. Cells obtained after enzymatic digestion were resuspended in EGF-10 media and incubated in cell culture flasks to remove adherent fibroblast-like cells. Adherent cells were further incubated with DMEM-10 media. Non-adherent cells were resuspended in EGF-10 media and transferred to Primaria flasks for growth of epithelial-like cells. The adherent cultures obtained from these non-adherent cells were further cultured in EGF-5 and EGF-2 media to inhibit the growth of fibroblast-like cells. Most cells in these cultures showed typical cuboidal epithelial cells morphology. However, cultures established with initially adherent cells in DMEM-10 displayed fibroblast-like morphology. Both types of cultures were further characterized by immunohistochemistry (IHC) using cytokeratin, vimentin, alpha-smooth muscle actin (alpha-SMA), and desmin specific monoclonal antibodies (MoAbs). The majority of cells in cultures established with initially non-adherent cells in EGF-2 media were positive for cytokeratin, vimentin and alpha SMA indicating their myoepithelial-like phenotype. The majority of cells cultured in DMEM-10 media did not stain for cytokeratin but were positive for vimentin and alpha-SMA indicating their myofibroblast-like phenotype. The primary ileal myoepithelial-like cultures were further immortalized using a vector expressing HPV16 E6 and E7. Immortalized cells expressed cytokeratin, vimentin and alpha-SMA similar to primary ileal epithelial-like cultures. The findings of this study were presented at the American Society for Microbiology North Central Branch 68th Annual Meeting, Oct 17-18, 2008, St Cloud, Minnesota and undergraduate student Camille Moen was awarded second prize in the students' poster competition. PARTICIPANTS: Individual who worked on the project: Camille Moen: Undergraduate student; Jordan Hass: Undergraduate student; Gopakumar Moorkanat: graduate student; Radhey S Kaushik: Principal investigator. Partner organization: South Dakota State University Collaborators and contracts: Drs. Mike Hildreth and David Francis, South Dakota State University. Training and professional development: Trained two undergraduate students and one graduate student. TARGET AUDIENCES: Target audiences: Students and faculty involved in Animal Health and Microbiology research. Efforts: Some of the reseach findings of this project were submitted as a thesis entitled ' Infectivity and pathogenesis of encephalitozoon intestinalis in an in vivo and in vitro porcine model' by the graduate student Gopakumar Moorkanat to South Dakota State University in Sept 2008. The findings of the sheep study were presented at the American Society for Microbiology North Central Branch 68th Annual Meeting, Oct 17-18, 2008, St Cloud, Minnesota by an undergraduate student Camille Moen. She was awarded second prize in the students' poster competition. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Most young food animals including pigs, lambs and calves are susceptible to various enteric pathogens. Both intestinal epithelial and fibroblast-like cells play a crucial role in the pathogenesis of enteric pathogens and mucosal immunity but their role in pigs and ruminants is not clearly defined. One approach to study the role of these cells is to establish primary and/or immortalized intestinal epithelial/fibroblast-like cultures from various animal species. These primary intestinal cultures may serve as suitable in vitro models for studying pathogenesis of enteric pathogens. Various microsporidial species have been shown to cause diarrheal diseases in both humans and pigs. We hypothesized that both porcine intestinal epithelial cells and pigs can be used as in vitro and in vivo animal model respectively for studying the pathogenesis of microsporidial infections. Therefore, in one the above mentioned project we used two porcine IPEC-J2 and IPEC-1 intestinal epithelial cells which were characterized in our lab in earlier studies, to study the pathogenesis of microsporidial infection. We found that both porcine intestinal epithelial cells and gnotobiotic pigs were successfully infected with microsporidium Encephalitozoon intestinalis. Thus, these studies established the use of porcine intestinal epithelial cells and gnotobiotic pigs as models for studying the pathogenesis of microsporidial infections in future studies. In another project, we successfully established the primary and/or immortalized cultures of ileal myoepithelial and myofibroblast-like cultures from young lambs. These cultures may be used to study the pathogenesis of various enteric diseases of ruminants in future projects. Some of the economically important and public health-related enteric diseases which effect sheep include scrapie and Johne's disease (JD) and these cell cultures may be successfully used to study the pathogenesis of these diseases.
Publications
- Amber M. Johnson, Radhey S. Kaushik, Nicholas J. Rotella, and Philip R. Hardwidge. 2009. Enterotoxigenic Escherichia coli modulates host intestinal cell asymmetry and metabolic activity. Infection and Immunity. 77(1):341-347.
- Amber M. Johnson, Radhey S. Kaushik, David H. Francis, James M. Fleckenstein, and Philip R. Hardwidge. 2009. Heat-labile enterotoxin promotes Escherichia coli adherence to intestinal epithelial cells. Journal of Bacteriology. 191(1):178-186.
- Seung Y Koh, Sajan George, Volker Brozel, Rodney Moxley, David Francis and Radhey S Kaushik. 2008. Porcine Intestinal epithelial cell lines as an in vitro model for studying pathogenesis of porcine Enterotoxogenic Escherichia coli. Veterinary Microbiology. 130:191-197.
- Kaushik, R.S., Begg, A.A., Wilson, H.L., Aich, P., Abrahamsen, M.S., Potter, A., Babiuk, L.A., and Griebel, P. 2008. Establishment of fetal bovine intestinal epithelial cell cultures susceptible to bovine rotavirus infection. Journal of Virological Methods. 148(1-2):182-196.
- Pisal, D.S., Yellepeddi, V.K., Kumar, A., Kaushik, R.S., Hildreth, M.B., Guan, X., Palakurthi, S. 2008. Permeability of surface modified polyamidoamine (PAMAM) dendrimers across Caco-2 cell monolayers. International Journal of Pharmaceutics. 350(1-2):113-21.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: In earlier studies (2006 CRIS report) we investigated toll-like receptors' (TLRs) expression and lectin binding profile of enterocytes in response to use of chlortetracycline in pig feed and in the absence of normal intestinal microbiota in pigs. In the present report we further extended these studies to characterize the expression of various immunologically important cell surface markers and TLRs in/on the porcine intestinal epithelial cells for developing an in vitro cell culture model for future studies. Recently, intestinal epithelial cells have been shown to express MHC class I, class II and CD1 molecules consistent with their ability to present antigens to other immune cells. These cells also express other surface markers involved in various immunological processes including cell-cell communication and adhesion. Two porcine intestinal epithelial cell lines IPEC-J2 and IPEC-1 derived from jejunum and small intestine of one day old pigs respectively, have been used in
many studies but these cells have not been characterized for their surface markers. We assessed the expression of CD1, MHC-I, MHC-II, CD11b/c, CD14, CD18, wCD21, CD25, CD40, CD44, CD54, CD58, CD80, CD86, and CD172a on porcine IPEC-J2 and IPEC-1 cells by flow cytometry. As pigs are used as an animal model for many human enteric diseases, we checked the expression of these markers on human small intestinal cell line INT-407 for comparing and justifying the use of porcine cells for human enteric infections. A variable number of both human and porcine cells expressed MHC-I, MHC-II, and CD44. Both IPEC-J2 and IPEC-1 cells expressed CD11b/c but did not express CD54 and CD58; however, INT-407 cells expressed both CD54 and CD58 but no CD11b/c. None of the porcine and human cell lines expressed CD1, CD14, CD18, wCD21, CD25, CD40, CD80, CD86, and CD172a. Intestinal innate mechanisms are recognized as central protective mechanisms against enteric pathogens and include the barrier function of the
epithelium, the presence of TLRs, and secretion of mucus, antimicrobial peptides and cytokines. TLRs are a type of microbial pattern-recognition receptors that recognize certain microbes and microbial components and induce signals to activate genes responsible for host defense. Bacterial peptidoglycan and lipoteichoic acid are recognized by TLR2; lipopolysaccharide by TLR4; flagellin by TLR5, and the CpG motif of bacterial DNA by TLR9. In this study we also assessed the expression of TLR 2, -4, -5 and -9 in IPEC-J2 and IPEC-1 cells. The expression of TLR transcripts was quantified by real time reverse-transcriptase PCR. Both type of cells expressed comparable levels of TLR 2, -4, -5 and -9 transcripts. The presence of TLR proteins in both porcine cell lines was detected by immunohistochemistry using polyclonal antibodies against TLRs. Both IPEC-J2 and IPEC-1 expressed TLR-2, -4 and -9 proteins but did not express TLR-5 protein. The findings of this study were presented in the annual
meeting of South Dakota Academy of Science held at South Dakota State University on April 13-14, 07 and Conference of Research Workers in Animal Diseases (CRWAD) meeting held in Chicago, Illinois on Dec 2- 4, 2007.
PARTICIPANTS: Individuals who worked on the project: YeJin Oh: Undergraduate student Matthew J Anderson: Undergraduate student Gopakumar Moorkanat: Graduate student Radhey S Kaushik: Principal investigator Partner organizations: Nil Collaborators and contacts: Drs. David Francis and Philip Hardwidge, Dept. of Veterinary Science, South Dakota State University, Brookings, SD 57007 USA Training and profesional development: Trained two undergraduate students and one graduate student.
TARGET AUDIENCES: Target audiences: Students and faculty involved in animal health research. Efforts: The research findings of this project were presented in the annual meeting of South Dakota Academy of Science held at South Dakota State University on April 13-14, 07 and Conference of Research Workers in Animal Diseases (CRWAD) meeting held in Chicago, Illinois on Dec 2- 4, 2007.
PROJECT MODIFICATIONS: Major changes: Nil
Impacts
Intestinal epithelial cells play a significant role in mediating both innate and adaptive immune responses in the gut. Porcine intestinal cells have been shown to be involved in the pathogenesis and immunity to many porcine enteric diseases. These cells, in addition to their established roles in intestinal digestion and nutrients absorption, may act as antigen presenting cells by displaying immunologically relevant surface markers which play important roles in antigen presentation and cell-to-cell communication. Furthermore, intestinal epithelial cell lines that display various toll-like receptors (TLRs), the molecules which detect and sense the microbial products and pathogens may represent the valuable and biologically relevant cell culture models for studying the TLR-mediated mechanisms involved in defense against various enteric pathogens. The findings of this study suggest that both porcine intestinal epithelial IPEC-J2 and IPEC-1 cell lines may act as possible
antigen presenting cells and provide suitable and biologically relevant porcine cell culture models for studying the intestinal immune mechanisms and modulation of TLR-mediated intestinal innate immune defenses. Thus, these cells can be used effectively as a cell culture model for studying and developing preventive and therapeutic approaches for protecting pigs and humans against various enteric diseases including porcine enterotoxigenic Escherichia coli (ETEC) infections which are a major threat to pig industry. I have established some local collaboration with faculty (Drs. David Francis and Philip Hardwidge) in Veterinary Science Dept and Center for Infectious Disease Research and Vaccinology (CIDRV) for further investigating the role of porcine intestinal epithelial cells in ETEC pathogenesis. The findings of this study also support the use of porcine cell cultures as an alternative or replacement for many in vivo studies and use of animals in research.
Publications
- George, S., Oh, Y., Lindblom, S., Vilain, S., Rosa, A., Francis, D., Brozel, V., and Kaushik, R.S. 2007. Lectin binding profile on the small intestine of 5-week old pigs in response to the use of chlortetracycline as a growth promotant, and under gnotobiotic conditions. J. Anim Sci. 85: 1640-1650.
- Brown, E.A., Kaushik, R.S., and Philip R Hardwidge. 2007. Susceptibility of human enterotoxigenic Escherichia coli isolates to growth inhibition by porcine intestinal epithelial cells. FEMS Microbiol Lett. 274:95-101.
- Aich, P., Wilson, H.L., Kaushik, R.S., Potter, A., Babiuk, L.A., Griebel, P. 2007. Comparative analysis of innate immune responses following infection of newborn calves with bovine rotavirus and coronavirus. J Gen Virol. 88:2749-2761.
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Progress 01/01/06 to 12/31/06
Outputs
In earlier studies (2005 CRIS report), we characterized two porcine intestinal epithelial cell lines for the presence of sugar moieties and determined their ability to bind porcine enterotoxigenic E. coli. We further expanded these studies to investigate whether porcine enterocytes in vivo display similar sugar moieties as porcine epithelial cell lines. We also asked the question how various nutritional and biological factors such as use of feed antibiotics and gut microflora influence intestinal physiology and biochemistry including that of enterocytes. Feed antibiotics are widely used in the pig industry; however, recent human health concerns about the use of feed antibiotics have initiated considerable debate and a need for alternative strategies. The exact mechanism of action of feed antibiotics is not known and needs further investigation. We hypothesized that the use of chlortetracycline (CTC) as a growth promotant and intestinal microbiota mediate changes in
the expression of growth, metabolism and innate immunity related genes, proteins and carbohydrates in the pig intestine. Eighteen half-sib piglets obtained by cesarean section from three sows were randomly divided into three groups, namely antibiotic-fed, control and gnotobiotic groups (n=6). The antibiotic group pigs were fed with pig feed having 50 ppm CTC. Ileal tissues were collected at 5-weeks of age and were analyzed for differential expression of genes among the three groups of animals using porcine microarrays. The data showed that 135 genes were significantly different between the experimental groups. A variety of genes up-regulated in the antibiotic group as compared to the control and gnotobiotic groups were related to immune response, metabolism, DNA replication, cell cycle and cell signaling. Expression of innate immunity related toll-like receptor (TLR) genes (TLR-2, -4, -5 and -9) in the ileum was assessed by real time RT-PCR while the presence of TLR proteins was
detected by histochemistry (HC). No significant differences (p ≥0.5) in TLR transcripts and proteins were observed among three groups of pigs. Glycoconjugate composition on the ileal tissues was examined by HC using 23 biotinylated lectins. Ileal epithelium of control piglets showed similar lectin binding patterns as observed with porcine intestinal epithelial cell lines in earlier studies. LEL, Jacalin, PSA, LCA and SNA lectins showed significant differences among three groups in the binding intensities on the dome and villous epithelium. Gnotobiotic piglets expressed significantly weaker lectin binding for many lectins compared to control and antibiotic group piglets. This indicated that the presence of gut microbiota plays an important role in the expression of various sugar moieties on the intestinal epithelium. The findings of this study throw light on the molecular and biochemical mechanisms involved in growth regulation of weanling pigs in response to CTC and gnotobiotic
conditions. These findings will help in formulating alternatives for replacing the use of antibiotics as growth promoters in animal feed.
Impacts
Feed antibiotics are widely used as growth promotant in the pig industry but their use has been banned in Europe because of emergence of drug resistant microbes and human health concerns. It is important to understand the mechanism of action of feed antibiotics for developing alternative strategies. We studied the effect of chlortetracycline and gut microflora on the physiology, immunity and biochemistry of intestine in antibiotic-fed and gnotobiotic pigs and observed some biologically important significant differences. These findings will help in developing alternatives to feed antibiotics.
Publications
- George, S. 2006. Molecular and biochemical changes in the ileum of weanling pigs fed antibiotics as growth promotant and in germ-free conditions. Master of Science thesis for Major in Biological Sciences (Microbiology), South Dakota State University, USA. Major advisor: Radhey S. Kaushik.
- Nichani, A.K., Dar, A., Krieg, A., Mirakhur, K.K., Kaushik, R.S., Griebel, P.J., Manuja, A., Townsend, H.G.G., Babiuk, L.A. and Mutwiri, G. 2007. Systemic innate immune responses following intrapulmonary delivery of CpG oligodeoxynucleotides in sheep. Vet. Immunol. Immunopathol. 115(3-4): 357-368.
- Nichani, A.K., Mena, A., Kaushik, R.S., Mutwiri, G.K., Townsend, H.G.G., Hecker, R., Krieg, A.M., Babiuk, L.A. and Griebel, P.J. 2006. Stimulation of innate immune responses by CpG oligodeoxynucleotide in newborn lambs can reduce bovine herpes virus-1 shedding. Oligonucleotides. 16:58-67.
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Progress 01/01/05 to 12/31/05
Outputs
Two porcine intestine epithelial cell lines derived from the jejunum (IPEC-J2) and small intestine (IPEC-1) of one day old pigs had been described earlier but had not been characterized for their use in studying host-pathogen interactions. Therefore as a first step these cells were characterized for the presence of various epithelial cell specific biochemical markers. Galectin-4, one of the major lactose-binding proteins, is primarily expressed in the porcine enterocytes in vivo and in vitro intestinal epithelial cell models are required to elucidate the role of galectin-4. As intestinal epithelial cell cultures in addition to cytokeratin may express other cytoskeletal proteins and galectin-4, we investigated the presence of galectin-4, cytokeratin, vimentin, alpha-smooth muscle actin (ASMA), and desmin proteins in these cells by immunohistochemistry using specific monoclonal antibodies. Both IPEC-J2 and IPEC-1 cells expressed galectin-4 mRNA and protein, and varying
number of cells in both cell lines stained positive for cytokeratin, vimentin, ASMA, and desmin. Many enteric bacterial pathogens display surface lectins (pili or fimbriae), which mediate bacterial adherence to intestinal epithelial cells. Thus lectin binding profile of intestinal epithelial cells has direct implication for understanding pathogenesis and prevention of enteric diseases. We investigated lectin binding profile of IPEC-J2 and IPEC-1cells by flow cytometry using 21 biotinylated lectins representing different carbohydrate specificities. Most lectins except Ulex europaeus I (UEA-1) stained a large percentage of IPEC-J2 and IPEC-1 cells. However, Dolichos biforus agglutinin (DBA), Griffonia simplicifolia lectin II (GSL-II), Erythrina cristagalli lectin (ECL) and Vicia villosa agglutinin (VVA) stained higher number of cells in the IPEC-J2 cells as compared to IPEC-1 cells. The fucose specific lectin UEA-1 showed negligible affinity for both cell types. Enterotoxigenic
Escherichia coli (ETEC) cause severe watery diarrhea in neonatal and weaned pigs and fimbriae-mediated bacterial adherence to intestinal epithelial cells is the first important step in ETEC pathogenesis. In vitro models of adherence of ETEC to intestinal epithelial cells provide excellent experimental platforms for studying various aspects of ETEC pathogenesis; however, no homologous porcine intestinal epithelial cell lines have been described which bind to porcine ETEC in vitro. In this study, adherence of various ETEC strains to IPEC-J2 and IPEC-1 cell lines was investigated. ETEC expressing K88ac or K88ad adhesins equally bound to both IPEC-1 and IPEC-J2, but ETEC expressing K88ab adhesin more heavily adhered to IPEC-1. E. coli expressing K99 fimbriae heavily bound to both epithelial cell lines. E. coli expressing F18 strongly bound to IPEC-1 but did not adhere to IPEC-J2 cells. E. coli that expressed 987P failed to bind either cell line. These findings show that porcine IPEC-J2
and IPEC-1 cell lines express important biochemical markers of intestinal epithelial cells, show similar lectin binding profile as observed in vivo and may be useful model for studying porcine ETEC pathogenesis in vitro.
Impacts
One approach for studying the role of intestinal epithelial cells in enteric diseases is to use intestinal epithelial cell cultures which display most properties of epithelial cells observed in the animal. In this study, we characterized two pig intestinal epithelial cell lines for important biochemical epithelial cell markers, lectin binding profile and adherence of enterotoxigenic Escherichia coli (ETEC) bacteria to these cells which cause severe watery diarrhea in newborn and weaned pigs. The findings of this study show that both pig intestinal cell lines display similar biochemical properties and lectin binding profile as described for enterocytes present in the pigs. Furthermore, these cells adhered to various ETEC strains. These findings suggest that these porcine intestinal epithelial cell lines may provide a valuable cell culture model for further studying the biology of intestinal epithelial cells and their role in enteric diseases of pigs.
Publications
- Kaushik, R.S., George, S., Lin, J. and Erickson A. 2005. Biochemical characterization of porcine intestinal epithelial cell lines for the expression of galectin-4, cytokeratin, vimentin, alpha-smooth muscle actin and desmin. 86th Conference of Research Workers in Animal Diseases (CRWAD). St. Louis, Missouri, USA, Dec 4-6, 2005, P51.
- Koh, S.Y., George, S., Brozel, V., Francis, D. and Kaushik, R.S. 2005. Porcine intestinal epithelial cell lines as an in vitro model for studying pathogenesis of enterotoxigenic Escherichia coli. Third International Rushmore Conference on Enteric Diseases, Rapid City, South Dakota, USA, Sept 29-31, 2005, P40. 86th Conference of Research Workers in Animal Diseases (CRWAD). St. Louis, Missouri, USA, Dec 4-6, 2005, #90.
- Kaushik, R.S., George, S., Young, A. and Francis, D. 2005. Lectin binding profile of porcine intestinal epithelial cell lines. Third International Rushmore Conference on Enteric Diseases, Rapid City, South Dakota, USA, Sept 29-31, 2005, P37. 86th Conference of Research Workers in Animal Diseases (CRWAD). St. Louis, Missouri, USA, Dec 4-6, 2005, #100.
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