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Yoda M, Takase S, Suzuki K, Murakami A, Namai F, Sato T, Fujii T, Tochio T, Shimosato T. Development of engineered IL-36γ-hypersecreting Lactococcus lactis to improve the intestinal environment. World J Microbiol Biotechnol 2024; 40:363. [PMID: 39446273 PMCID: PMC11502612 DOI: 10.1007/s11274-024-04157-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2024] [Accepted: 10/05/2024] [Indexed: 10/25/2024]
Abstract
Interleukin (IL) 36 is a member of the IL-1-like proinflammatory cytokine family that has a protective role in mucosal immunity. We hypothesized that mucosal delivery of IL-36γ to the intestine would be a very effective way to prevent intestinal diseases. Here, we genetically engineered a lactic acid bacterium, Lactococcus lactis, to produce recombinant mouse IL-36γ (rmIL-36γ). Western blotting and enzyme-linked immunosorbent assay results showed that the engineered strain (NZ-IL36γ) produced and hypersecreted the designed rmIL-36γ in the presence of nisin, which induces the expression of the recombinant gene. We administered NZ-IL36γ to mice via oral gavage, and collected the ruminal contents and rectal tissues. Colony PCR using primers specific for NZ-IL36γ, and enzyme-linked immunosorbent assay to measure the rmIL-36γ concentrations of the ruminal contents showed that NZ-IL36γ colonized the mouse intestines and secreted rmIL-36γ. A microbiota analysis revealed increased abundances of bacteria of the genera Acetatifactor, Eubacterium, Monoglobus, and Roseburia in the mouse intestines. Real-time quantitative PCR of the whole colon showed increased Muc2 expression. An in vitro assay using murine colorectal epithelial cells and human colonic cells showed that purified rmIL-36γ promoted Muc2 gene expression. Taken together, these data suggest that NZ-IL36γ may be an effective and attractive tool for delivering rmIL-36γ to improve the intestinal environment.
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Affiliation(s)
- Masahiro Yoda
- Department of Biomolecular Innovation, Institute for Biomedical Sciences, Shinshu University, Nagano, 399-4598, Japan
| | - Shogo Takase
- Department of Biomolecular Innovation, Institute for Biomedical Sciences, Shinshu University, Nagano, 399-4598, Japan
| | - Kaho Suzuki
- Department of Biomolecular Innovation, Institute for Biomedical Sciences, Shinshu University, Nagano, 399-4598, Japan
| | - Aito Murakami
- Department of Biomolecular Innovation, Institute for Biomedical Sciences, Shinshu University, Nagano, 399-4598, Japan
| | - Fu Namai
- Food and Feed Immunology Group, Laboratory of Animal Food Function, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, 980-8572, Japan
| | - Takashi Sato
- Department of Biomolecular Innovation, Institute for Biomedical Sciences, Shinshu University, Nagano, 399-4598, Japan
| | - Tadashi Fujii
- Department of Medical Research on Prebiotics and Probiotics, Fujita Health University, Toyoake, Aichi, 470-1101, Japan
| | - Takumi Tochio
- Department of Medical Research on Prebiotics and Probiotics, Fujita Health University, Toyoake, Aichi, 470-1101, Japan
| | - Takeshi Shimosato
- Department of Biomolecular Innovation, Institute for Biomedical Sciences, Shinshu University, Nagano, 399-4598, Japan.
- Institute for Aqua Regeneration, Shinshu University, Nagano, 399-4598, Japan.
- Department of Pharmacy, Medical Faculty, Universitas Brawijaya, Malang, 65145, Indonesia.
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Bandara M, MacNaughton WK. Protease-activated receptor-2 activation enhances epithelial wound healing via epidermal growth factor receptor. Tissue Barriers 2021; 10:1968763. [PMID: 34511032 DOI: 10.1080/21688370.2021.1968763] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
The intestinal barrier function relies on the presence of a single layer of epithelial cells. Barrier dysfunction is associated with the inflammatory bowel diseases (IBD). Understanding the mechanisms involved in intestinal wound healing in order to sustain the barrier function has a great therapeutic potential. Activation of protease-activated receptor-2 (PAR2) induces COX-2 expression in intestinal epithelial cells via EGFR transactivation. COX-2 is well known for its protective effects in the gastrointestinal tract. Therefore, we hypothesized that PAR-2 activation induces a wound healing response in intestinal epithelial cells through COX-2-derived lipid mediators and EGFR transactivation. Immunofluorescence and calcium assay were used to characterize CMT-93 mouse colonic epithelial cell line for PAR2 expression and its activity, respectively. Treatment with PAR2 activating peptide 2-furoyl-LIGRLO-NH2 (2fLI), but not by its inactive reverse-sequence peptide (2fO) enhanced wound closure in scratch wounded monolayers. The EGFR tyrosine kinase inhibitor (PD153035), broad-spectrum matrix metalloproteinase inhibitor (GM6001) and Src tyrosine kinase inhibitor (PP2) inhibited PAR2-induced wound healing. However, PAR2 activation did not induce COX-2 expression in CMT-93 cells and inhibition of COX-2 by COX-2 selective inhibitor (NS-398) did not alter PAR2-induced wound healing. In conclusion, PAR2 activation drives wound healing in CMT-93 cells via EGFR transactivation. Matrix metalloproteinases and Src tyrosine kinase activity may involve in EGFR transactivation and PAR2-induced wound healing is independent of COX-2 activity. These findings provide a mechanism whereby PAR2 can participate in the resolution of intestinal wounds in gastrointestinal inflammatory diseases.
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Affiliation(s)
- Mahesha Bandara
- Department of Physiology and Pharmacology, Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, Alberta Children's Hospital Research Institute for Child and Maternal Health, Cumming School of Medicine, University of Calgary, Calgary, Canada
| | - Wallace K MacNaughton
- Department of Physiology and Pharmacology, Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, Alberta Children's Hospital Research Institute for Child and Maternal Health, Cumming School of Medicine, University of Calgary, Calgary, Canada
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Nakayama H, Kitagawa N, Otani T, Iida H, Anan H, Inai T. Ochratoxin A, citrinin and deoxynivalenol decrease claudin-2 expression in mouse rectum CMT93-II cells. Microscopy (Oxf) 2018; 67:99-111. [PMID: 29474583 DOI: 10.1093/jmicro/dfy005] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2017] [Accepted: 01/16/2018] [Indexed: 01/01/2023] Open
Abstract
Intestinal epithelial cells are the first targets of ingested mycotoxins, such as ochratoxin A, citrinin and deoxynivalenol. It has been reported that paracellular permeability regulated by tight junctions is modulated by several mycotoxins by reducing the expression of specific claudins and integral membrane proteins in cell-cell contacts, accompanied by increase in phosphorylation of mitogen-activated protein kinases, including extracellular signal-related kinase (ERK) 1/2, p38 and c-Jun NH2-terminal protein kinase. Claudin-2 is expressed in the deep crypt cells, but not in the villus/surface cells in vivo. While Caco-2, T84 and IPEC-J2 cells, which are widely used intestinal epithelial cell lines to assess the influence of mycotoxins, do not express claudin-2, CMT93-II cells express claudin-2. We previously reported that inhibition of the ERK pathway reduced claudin-2 levels in cell-cell contacts in CMT93-II cells. In this study, we examined whether ochratoxin A, citrinin and deoxynivalenol affect claudin-2 expression and ERK1/2 phosphorylation in CMT93-II cells. We found that all mycotoxins reduced claudin-2 expression in cell-cell contacts, with reduction (by citrinin and deoxynivalenol) or no change (by ochratoxin A) in phosphorylated ERK1/2. All mycotoxins increased transepithelial electrical resistance, but did not affect flux of fluorescein. While ochratoxin A and citrinin are known to be nephrotoxic, only deoxynivalenol reduced claudin-2 expression in MDCK II cells derived from the renal tubule. These results suggest that claudin-2 expression is regulated not only by the ERK pathway, but also by other pathways in an organ-specific manner.
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Affiliation(s)
- Hideaki Nakayama
- Department of Odontology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan
| | - Norio Kitagawa
- Department of Morphological Biology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan
| | - Takahito Otani
- Department of Morphological Biology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan
| | - Hiroshi Iida
- Laboratory of Zoology, Graduate School of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
| | - Hisashi Anan
- Department of Odontology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan
| | - Tetsuichiro Inai
- Department of Morphological Biology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan
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Dicay MS, Hirota CL, Ronaghan NJ, Peplowski MA, Zaheer RS, Carati CA, MacNaughton WK. Interferon-γ suppresses intestinal epithelial aquaporin-1 expression via Janus kinase and STAT3 activation. PLoS One 2015; 10:e0118713. [PMID: 25793528 PMCID: PMC4405000 DOI: 10.1371/journal.pone.0118713] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2014] [Accepted: 01/09/2015] [Indexed: 12/29/2022] Open
Abstract
Inflammatory bowel diseases are associated with dysregulated electrolyte and water transport and resultant diarrhea. Aquaporins are transmembrane proteins that function as water channels in intestinal epithelial cells. We investigated the effect of the inflammatory cytokine, interferon-γ, which is a major player in inflammatory bowel diseases, on aquaporin-1 expression in a mouse colonic epithelial cell line, CMT93. CMT93 monolayers were exposed to 10 ng/mL interferon-γ and aquaporin-1 mRNA and protein expressions were measured by real-time PCR and western blot, respectively. In other experiments, CMT93 cells were pretreated with inhibitors or were transfected with siRNA to block the effects of Janus kinases, STATs 1 and 3, or interferon regulatory factor 2, prior to treatment with interferon-γ. Interferon-γ decreased aquaporin-1 expression in mouse intestinal epithelial cells in a manner that did not depend on the classical STAT1/JAK2/IRF-1 pathway, but rather, on an alternate Janus kinase (likely JAK1) as well as on STAT3. The pro-inflammatory cytokine, interferon-γ may contribute to diarrhea associated with intestinal inflammation in part through regulation of the epithelial aquaporin-1 water channel via a non-classical JAK/STAT receptor signalling pathway.
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Affiliation(s)
- Michael S Dicay
- Inflammation Research Network and Department of Physiology and Pharmacology, University of Calgary, Calgary, Canada
| | - Christina L Hirota
- Inflammation Research Network and Department of Physiology and Pharmacology, University of Calgary, Calgary, Canada
| | - Natalie J Ronaghan
- Inflammation Research Network and Department of Physiology and Pharmacology, University of Calgary, Calgary, Canada
| | - Michael A Peplowski
- Inflammation Research Network and Department of Physiology and Pharmacology, University of Calgary, Calgary, Canada
| | - Raza S Zaheer
- Inflammation Research Network and Department of Physiology and Pharmacology, University of Calgary, Calgary, Canada
| | - Colin A Carati
- Department of Anatomy and Histology, Flinders University, Bedford Park, Australia
| | - Wallace K MacNaughton
- Inflammation Research Network and Department of Physiology and Pharmacology, University of Calgary, Calgary, Canada
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Hiemstra I, Klaver E, Vrijland K, Kringel H, Andreasen A, Bouma G, Kraal G, van Die I, den Haan J. Excreted/secreted Trichuris suis products reduce barrier function and suppress inflammatory cytokine production of intestinal epithelial cells. Mol Immunol 2014; 60:1-7. [DOI: 10.1016/j.molimm.2014.03.003] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2014] [Accepted: 03/10/2014] [Indexed: 12/26/2022]
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Kitahara S, Suzuki Y, Morishima M, Yoshii A, Kikuta S, Shimizu K, Morikawa S, Sato Y, Ezaki T. Vasohibin-2 modulates tumor onset in the gastrointestinal tract by normalizing tumor angiogenesis. Mol Cancer 2014; 13:99. [PMID: 24885408 PMCID: PMC4113181 DOI: 10.1186/1476-4598-13-99] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2014] [Accepted: 04/22/2014] [Indexed: 01/31/2023] Open
Abstract
Background Vasohibin-2 (VASH2) has been identified as an endogenous and vascular endothelial growth factor (VEGF)-independent angiogenic factor that is highly expressed in tumor cells. In the present study, we aimed to determine whether pre-existing vascular changes can be used to predict tumor transformation as benign or malignant. We sought to characterize microvascular changes and tumor development in the intestinal tract of ApcMin/+ mice and ApcMin/+/Vash2-/- mice. Methods ApcMin/+ mice provide a unique orthotopic model for the development of spontaneous adenomatous polyposis and subsequent carcinomas, a phenomenon termed the adenoma-carcinoma sequence. ApcMin/+ mice were mated with Vash2-/- mice with a mixed C57BL/6 background and the resulting pups were screened for the Min mutation and for the Vash2-/- gene by PCR. Intestinal tumors from ApcMin/+ mice and ApcMin/+/Vash2-/- mice were removed and either frozen or epon-embedded for subsequent analyses. For 3-dimensional imaging using confocal laser-scanning microscopy and transmission electron microscopy, cryosections were made, and immunofluorescent staining for various markers was performed. Results We found that structural abnormalities in tumor vessels from benign tumors resembled those in malignant tumors. In addition, a novel angiogenic factor, vasohibin-2 (VASH2) protein, was detected around tumor blood vessels in late-stage adenomas and adenocarcinomas, but was absent from early-stage adenomas in ApcMin/+ mice. Tumors used to examine endogenous VASH2 (derived from CMT93 colon carcinomas) were less vascularized in Vash2-/- mice and were more regular than those seen in wild-type (WT) mice. In addition, tumors in Vash2-/- mice were smaller than those in WT mice. Furthermore, cross-breeding of mice homozygous for a deletion of Vash2 with mice heterozygous for the APC mutation resulted in animals that showed a significant decrease in the number of polyps in the small intestine. Conclusion We propose that VASH2 may modulate the onset of tumors in the gastrointestinal tract by regulating tumor angiogenesis.
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Affiliation(s)
| | | | | | | | | | | | | | | | - Taichi Ezaki
- Department of Anatomy and Developmental Biology, School of Medicine, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.
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Gillen AE, Lucas CA, Haussecker PL, Kosak ST, Harris A. Characterization of a large human transgene following invasin-mediated delivery in a bacterial artificial chromosome. Chromosoma 2013; 122:351-61. [PMID: 23749207 DOI: 10.1007/s00412-013-0418-9] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2013] [Revised: 05/17/2013] [Accepted: 05/21/2013] [Indexed: 12/31/2022]
Abstract
Bacterial artificial chromosomes (BACs) are widely used in transgenesis, particularly for the humanization of animal models. Moreover, due to their extensive capacity, BACs provide attractive tools to study distal regulatory elements associated with large gene loci. However, despite their widespread use, little is known about the integration dynamics of these large transgenes in mammalian cells. Here, we investigate the post-integration structure of a ~260 kb BAC carrying the cystic fibrosis transmembrane conductance regulator (CFTR) locus following delivery by bacterial invasion and compare this to the outcome of a more routine lipid-based delivery method. We find substantial variability in integrated copy number and expression levels of the BAC CFTR transgene after bacterial invasion-mediated delivery. Furthermore, we frequently observed variation in the representation of different regions of the CFTR transgene within individual cell clones, indicative of BAC fragmentation. Finally, using fluorescence in situ hybridization, we observed that the integrated BAC forms extended megabase-scale structures in some clones that are apparently stably maintained at cell division. These data demonstrate that the utility of large BACs to investigate cis-regulatory elements in the genomic context may be limited by recombination events that complicate their use.
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Affiliation(s)
- Austin E Gillen
- Human Molecular Genetics Program, Lurie Children's Research Center, Chicago, IL, USA
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8
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Thanabalasuriar A, Kim J, Gruenheid S. The inhibition of COPII trafficking is important for intestinal epithelial tight junction disruption during enteropathogenic Escherichia coli and Citrobacter rodentium infection. Microbes Infect 2013; 15:738-44. [PMID: 23747681 DOI: 10.1016/j.micinf.2013.05.001] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2012] [Revised: 04/04/2013] [Accepted: 05/13/2013] [Indexed: 11/24/2022]
Abstract
Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are bacterial pathogens that cause severe illnesses in humans. Citrobacter rodentium is a related mouse pathogen that serves as a small animal model for EPEC and EHEC infections. EPEC, EHEC and C. rodentium translocate bacterial virulence proteins directly into host intestinal cells via a type III secretion system (T3SS). Non-LEE-encoded effector A (NleA) is a T3SS effector that is common to EPEC, EHEC and C. rodentium. NleA interacts with and inhibits the mammalian COPII complex, impairing cellular secretion; this interaction is required for bacterial virulence. Although diarrhea is a hallmark of EPEC, EHEC and C. rodentium infections, the underlying mechanisms are not well characterized. One of the essential functions of the intestine is to maintain a barrier between the lumen and submucosa. Tight junctions seal the space between adjacent epithelial cells creating this barrier. Consequently, it is thought that the disruption of intestinal epithelial tight junctions by EPEC, EHEC, and C. rodentium could result in a loss of barrier function. In this study, we demonstrate that NleA mediated COPII inhibition is required for EPEC- and C. rodentium-mediated disruption of tight junction proteins and increases in fecal water content.
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Affiliation(s)
- Ajitha Thanabalasuriar
- Department of Microbiology and Immunology, McGill University, 3649 Promenade Sir William Osler, Montreal, Quebec H3G 0B1, Canada
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Nod2 improves barrier function of intestinal epithelial cells via enhancement of TLR responses. Mol Immunol 2012; 52:264-72. [DOI: 10.1016/j.molimm.2012.06.007] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2012] [Revised: 06/04/2012] [Accepted: 06/05/2012] [Indexed: 12/12/2022]
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Young AM, Archibald KM, Tookman LA, Pool A, Dudek K, Jones C, Williams SL, Pirlo KJ, Willis AE, Lockley M, McNeish IA. Failure of translation of human adenovirus mRNA in murine cancer cells can be partially overcome by L4-100K expression in vitro and in vivo. Mol Ther 2012; 20:1676-88. [PMID: 22735379 DOI: 10.1038/mt.2012.116] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
Adaptive immune responses may be vital in the overall efficacy of oncolytic viruses in human malignancies. However, immune responses to oncolytic adenoviruses are poorly understood because these viruses lack activity in murine cells, which precludes evaluation in immunocompetent murine cancer models. We have evaluated human adenovirus activity in murine cells. We show that a panel of murine carcinoma cells, including CMT64, MOVCAR7, and MOSEC/ID8, can readily be infected with human adenovirus. These cells also support viral gene transcription, messenger RNA (mRNA) processing, and genome replication. However, there is a profound failure of adenovirus protein synthesis, especially late structural proteins, both in vitro and in vivo, with reduced loading of late mRNA onto ribosomes. Our data also show that in trans expression of the nonstructural late protein L4-100K increases both the amount of viral mRNA on ribosomes and the synthesis of late proteins, accompanied by reduced phosphorylation of eIF2α and improved anticancer efficacy. These results suggest that murine models that support human adenovirus replication could be generated, thus allowing evaluation of human adenoviruses in immunocompetent mice.
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Affiliation(s)
- Anna-Mary Young
- Barts Cancer Institute, Queen Mary University of London, London, UK
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Kim JY, Park MS, Ji GE. Probiotic modulation of dendritic cells co-cultured with intestinal epithelial cells. World J Gastroenterol 2012; 18:1308-18. [PMID: 22493544 PMCID: PMC3319957 DOI: 10.3748/wjg.v18.i12.1308] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/09/2011] [Revised: 09/30/2011] [Accepted: 01/22/2012] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate cytokine production and cell surface phenotypes of dendritic cells (DC) in the presence of epithelial cells stimulated by probiotics.
METHODS: Mouse DC were cultured alone or together with mouse epithelial cell monolayers in normal or inverted systems and were stimulated with heat-killed probiotic bacteria, Bifidobacterium lactis AD011 (BL), Bifidobacterium bifidum BGN4 (BB), Lactobacillus casei IBS041 (LC), and Lactobacillus acidophilus AD031 (LA), for 12 h. Cytokine levels in the culture supernatants were determined by enzyme-linked immunosorbent assay and phenotypic analysis of DC was investigated by flow cytometry.
RESULTS: BB and LC in single-cultured DC increased the expression of I-Ad, CD86 and CD40 (I-Ad, 18.51 vs 30.88, 46.11; CD86, 62.74 vs 92.7, 104.12; CD40, 0.67 vs 6.39, 3.37, P < 0.05). All of the experimental probiotics increased the production of inflammatory cytokines, interleukin (IL)-6 and tumor necrosis factor (TNF)-α. However, in the normal co-culture systems, LC and LA decreased the expression of I-Ad (39.46 vs 30.32, 33.26, P < 0.05), and none of the experimental probiotics increased the levels of IL-6 or TNF-α. In the inverted co-culture systems, LC decreased the expression of CD40 (1.36 vs -2.27, P < 0.05), and all of the experimental probiotics decreased the levels of IL-6. In addition, BL increased the production of IL-10 (103.8 vs 166.0, P < 0.05) and LC and LA increased transforming growth factor-β secretion (235.9 vs 618.9, 607.6, P < 0.05).
CONCLUSION: These results suggest that specific probiotic strains exert differential immune modulation mediated by the interaction of dendritic cells and epithelial cells in the homeostasis of gastrointestinal tract.
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Zufferey C, Erhart D, Saurer L, Mueller C. Production of interferon-gamma by activated T-cell receptor-alphabeta CD8alphabeta intestinal intraepithelial lymphocytes is required and sufficient for disruption of the intestinal barrier integrity. Immunology 2010; 128:351-9. [PMID: 20067535 DOI: 10.1111/j.1365-2567.2009.03110.x] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Maintenance of intestinal epithelial barrier function is of vital importance in preventing uncontrolled influx of antigens and the potentially ensuing inflammatory disorders. Intestinal intraepithelial lymphocytes (IEL) are in intimate contact with epithelial cells and may critically regulate the epithelial barrier integrity. While a preserving impact has been ascribed to the T-cell receptor (TCR)-gammadelta subset of IEL, IEL have also been shown to attenuate the barrier function. The present study sought to clarify the effects of IEL by specifically investigating the influence of the TCR-alphabeta CD8alphabeta and TCR-alphabeta CD8alphaalpha subsets of IEL on the intestinal epithelial barrier integrity. To this end, an in vitro coculture system of the murine intestinal crypt-derived cell-line mIC(cl2) and syngeneic ex vivo isolated IEL was employed. Epithelial integrity was assessed by analysis of transepithelial resistance (TER) and paracellular flux of fluorescein isothiocyanate-conjugated (FITC-) dextran. The TCR-alphabeta CD8alphaalpha IEL and resting TCR-alphabeta CD8alphabeta IEL did not affect TER of mIC(cl2) or flux of FITC-dextran. In contrast, activated TCR-alphabeta CD8alphabeta IEL clearly disrupted the integrity of the mIC(cl2) monolayer. No disrupting effect was seen with activated TCR-alphabeta CD8alphabeta IEL from interferon-gamma knockout mice. These findings demonstrate that secretion of interferon-gamma by activated TCR-alphabeta CD8alphabeta IEL is strictly required and also sufficient for disrupting the intestinal epithelial barrier function.
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Affiliation(s)
- Christel Zufferey
- Institute of Pathology, Experimental Pathology, University of Bern, Bern, Switzerland
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Martinez C, Churchman M, Freeman T, Ilyas M. Osteopontin provides early proliferative drive and may be dependent upon aberrant c-myc signalling in murine intestinal tumours. Exp Mol Pathol 2010; 88:272-7. [PMID: 20053348 DOI: 10.1016/j.yexmp.2009.12.008] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2009] [Revised: 12/09/2009] [Accepted: 12/09/2009] [Indexed: 12/24/2022]
Abstract
Osteopontin is thought to play an important role in tumour metastasis. In a previous expression profiling study of murine intestinal adenomas, we found that Opn was up-regulated. We also found beta-catenin binding motifs in the Opn promoter implying that, contrary to current beliefs, induction of Opn may occur during early tumourigenesis. We studied 59 murine intestinal adenomas for Opn expression and every tumour showed up-regulation compared to normal mucosa confirming early deregulation in these tumours. To determine whether Opn makes a functional contribution to tumourigenesis, Opn was knocked down in the murine colorectal cancer cell line CMT93. Inhibition of Opn expression resulted in decreased cell numbers. To determine the mechanism of Opn induction in these tumours, the Opn promoter was cloned and each of the putative beta-catenin binding motifs was mutated. No major change in Opn promoter activity was observed thereby excluding Opn as a direct beta-catenin target gene. However, mutation of one of two putative c-myc binding sites in the Opn promoter led to near complete loss of promoter activity whilst mutation of one of four PEA3 binding sites led to a 50% reduction in promoter activity. We conclude that Opn deregulation is an early event in intestinal tumourigenesis which may promote tumour development by altering either proliferation or apoptosis to increase tumour cell numbers. Opn expression in the intestine is dependent on c-myc binding sites in the promoter. Since c-myc is a known beta-catenin target gene, deregulation of Opn may be a secondary effect of aberrant Wnt signalling.
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Affiliation(s)
- Cristina Martinez
- Digestive Diseases Research Unit, Institut Fundacio Recerca, Hospital General Vall D'hebron, 08035 Barcelona, Spain
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Ruiz P, Dunon D, Sonnenberg A, Imhof BA. Suppression of Mouse Melanoma Metastasis by EA-1, A Monoclonal Antibody Specific for α6 Integrins. ACTA ACUST UNITED AC 2009. [DOI: 10.3109/15419069309095682] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
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Abstract
Genetically engineered mice are essential tools in both mechanistic studies and drug development in colon cancer research. Mice with mutations in the Apc gene, as well as in genes that modify or interact with Apc, are important models of familial adenomatous polyposis. Mice with mutations in the beta-catenin signaling pathway have also revealed important information about colon cancer pathogenesis, along with models for hereditary nonpolyposis colon cancer and inflammatory bowel diseases associated with colon cancer. Finally, transplantation models (xenografts)have been useful in the study of metastasis and for testing potential therapeutics. This review discusses what models have been developed most recently and what they have taught us about colon cancer formation, progression, and possible treatment strategies.
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Affiliation(s)
- Makoto Mark Taketo
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, USA
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Flynn AN, Buret AG. Tight junctional disruption and apoptosis in an in vitro model of Citrobacter rodentium infection. Microb Pathog 2008; 45:98-104. [PMID: 18504087 DOI: 10.1016/j.micpath.2007.12.004] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2007] [Revised: 11/27/2007] [Accepted: 12/14/2007] [Indexed: 12/12/2022]
Abstract
The murine model of Citrobacter rodentium infection has been used to complement in vitro studies of enteropathogenic Escherichia coli (EPEC) infections of human intestinal epithelial cells (IECs). However, the differences in epithelial cell responses between these two models are not fully understood. We used an in vitro model of C. rodentium infection to examine important, yet incompletely understood, cellular responses of murine IECs to this pathogen. C. rodentium attached to CMT-93 cells and disrupted their tight junctional expression of claudins-4 and -5. This was associated with a loss of barrier function that required live bacteria and was partially prevented by the inhibition of Rho kinase. Furthermore, C. rodentium caused an upregulation of IEC apoptosis that was associated with the cytoplasmic accumulation of apoptosis-inducing factor, but not with the activation of caspase-3. These studies demonstrate for the first time that C. rodentium affects murine IECs in ways that may be similar, but distinct, to the effects of EPEC on human IECs.
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Affiliation(s)
- Andrew N Flynn
- Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada
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17
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McNeal MM, Stone SC, Basu M, Clements JD, Choi AHC, Ward RL. IFN-gamma is the only anti-rotavirus cytokine found after in vitro stimulation of memory CD4+ T cells from mice immunized with a chimeric VP6 protein. Viral Immunol 2008; 20:571-84. [PMID: 18158731 DOI: 10.1089/vim.2007.0055] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
CD4+ T cells are the only lymphocytes required for protection of mice against rotavirus shedding after mucosal immunization with chimeric VP6 (MBP::VP6) and the adjuvant LT(R192G). One possible effector of protection is CD4+ T-cell cytokines. To determine if memory CD4+ T cells of immunized mice produce cytokines with direct anti-rotavirus activity, an in vitro infection model was developed using mouse CMT-93 cells and rhesus rotavirus (RRV). Spleen and lamina propria (LP) cells, as well as purified splenic CD4T cells obtained after intranasal immunization of BALB/c mice with MBP::VP6/LT(R192G) released large quantities of two cytokines (IL-17 and IFN-gamma) into cell supernatants when stimulated with MBP::VP6. Production of these same cytokines is rapidly upregulated in intestinal lymphocytes after rotavirus inoculation of immunized mice. IL-17 pretreatment of CMT-93 cells had no effect on subsequent RRV replication, but IFN-gamma was the most potent inhibitor within a panel of nine cytokines tested. Supernatants obtained after in vitro stimulation of splenic CD4+ T cells of immunized mice had high levels of anti-RRV activity and their pretreatment with mAb against IFN-gamma caused essentially complete loss of activity. Thus, IFN-gamma was the only cytokine identified in stimulated CD4+ T cells from immunized mice that directly inhibited rotavirus replication.
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Affiliation(s)
- Monica M McNeal
- Division of Infectious Diseases, Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA.
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18
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Comparative characterization of mouse rectum CMT93-I and -II cells by expression of claudin isoforms and tight junction morphology and function. Histochem Cell Biol 2007; 129:223-32. [PMID: 18034259 DOI: 10.1007/s00418-007-0360-0] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/07/2007] [Indexed: 01/28/2023]
Abstract
Recent studies suggest that the morphological and physiological properties of tight junctions (TJs) are determined by the combination and mixing ratios of claudin isoforms. In this study, we tried to characterize mouse cell lines by expression of claudin isoforms to use for studying epithelial TJs by overexpression or suppression of claudin(s) in the cells and found that claudin-2 was expressed in a few mouse rectum carcinoma cells, CMT93 cells. We have isolated CMT93-I and -II cells from CMT93 cells by immunohistochemical screening for the presence or absence of claudin-2 expression. Immunofluorescence and RT-PCR analyses showed that expression of claudin-4, -6, -7 and -12 was detected in both cell lines, but claudin-2 was only expressed in CMT93-II cells. There were no differences in paracellular permeability between CMT93-I and -II cells examined by 4 kDa FITC-dextran and fluorescein sodium, or in the number of TJ strands examined by freeze-fracture electron microscopy. However, the transepithelial electrical resistance (TER) of CMT93-I cells was approximately 6.5 times higher than that of CMT93-II cells, suggesting that expression of claudin-2 may be related to decreased TER. Comparative examinations of CMT93-I and -II cells provide a clue how the combination and mixing ratios of claudin isoforms regulate the paracellular permeability.
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19
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Cormier SA, Taranova AG, Bedient C, Nguyen T, Protheroe C, Pero R, Dimina D, Ochkur SI, O’Neill K, Colbert D, Lombari TR, Constant S, McGarry MP, Lee JJ, Lee NA. Pivotal Advance: eosinophil infiltration of solid tumors is an early and persistent inflammatory host response. J Leukoc Biol 2006; 79:1131-9. [PMID: 16617160 PMCID: PMC3496422 DOI: 10.1189/jlb.0106027] [Citation(s) in RCA: 131] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Tumor-associated eosinophilia has been observed in numerous human cancers and several tumor models in animals; however, the details surrounding this eosinophilia remain largely undefined and anecdotal. We used a B16-F10 melanoma cell injection model to demonstrate that eosinophil infiltration of tumors occurred from the earliest palpable stages with significant accumulations only in the necrotic and capsule regions. Furthermore, the presence of diffuse extracellular matrix staining for eosinophil major basic protein was restricted to the necrotic areas of tumors, indicating that eosinophil degranulation was limited to this region. Antibody-mediated depletion of CD4+ T cells and adoptive transfer of eosinophils suggested, respectively, that the accumulation of eosinophils is not associated with T helper cell type 2-dependent immune responses and that recruitment is a dynamic, ongoing process, occurring throughout tumor growth. Ex vivo migration studies have identified what appears to be a novel chemotactic factor(s) released by stressed/dying melanoma cells, suggesting that the accumulation of eosinophils in tumors occurs, in part, through a unique mechanism dependent on a signal(s) released from areas of necrosis. Collectively, these studies demonstrate that the infiltration of tumors by eosinophils is an early and persistent response that is spatial-restricted. It is more important that these data also show that the mechanism(s) that elicit this host response occur, independent of immune surveillance, suggesting that eosinophils are part of an early inflammatory reaction at the site of tumorigenesis.
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MESH Headings
- Animals
- CD4 Antigens/immunology
- CD4-Positive T-Lymphocytes/immunology
- Cells, Cultured
- Chemotactic Factors/metabolism
- Chemotaxis/drug effects
- Chemotaxis/physiology
- Culture Media, Conditioned/chemistry
- Culture Media, Conditioned/pharmacology
- Eosinophilia/etiology
- Eosinophilia/physiopathology
- Eosinophils/immunology
- Eosinophils/transplantation
- Immunologic Surveillance
- Immunotherapy, Adoptive
- Inflammation/immunology
- Inflammation/pathology
- Injections, Subcutaneous
- Interleukin-5/genetics
- Lymphocyte Depletion
- Melanoma, Experimental/complications
- Melanoma, Experimental/immunology
- Melanoma, Experimental/metabolism
- Melanoma, Experimental/pathology
- Melanoma, Experimental/therapy
- Mice
- Mice, Inbred C57BL
- Mice, Transgenic
- Microscopy, Confocal
- Necrosis
- Neoplasm Transplantation
- Th2 Cells/immunology
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Affiliation(s)
- Stephania A. Cormier
- Division of Hematology and Oncology, Mayo Clinic Arizona, 13400 East Shea Boulevard Scottsdale, AZ 85259
- Department of Biological Sciences, Louisiana State University, 202 Life Sciences Annex, Baton Rouge, LA 70803
| | - Anna G. Taranova
- Division of Pulmonary Medicine, Mayo Clinic Arizona, 13400 East Shea Boulevard Scottsdale, AZ 85259
| | - Carrie Bedient
- Division of Pulmonary Medicine, Mayo Clinic Arizona, 13400 East Shea Boulevard Scottsdale, AZ 85259
| | - Thanh Nguyen
- Division of Hematology and Oncology, Mayo Clinic Arizona, 13400 East Shea Boulevard Scottsdale, AZ 85259
| | - Cheryl Protheroe
- Division of Hematology and Oncology, Mayo Clinic Arizona, 13400 East Shea Boulevard Scottsdale, AZ 85259
| | - Ralph Pero
- Division of Hematology and Oncology, Mayo Clinic Arizona, 13400 East Shea Boulevard Scottsdale, AZ 85259
| | - Dawn Dimina
- Division of Pulmonary Medicine, Mayo Clinic Arizona, 13400 East Shea Boulevard Scottsdale, AZ 85259
| | - Sergei I. Ochkur
- Division of Pulmonary Medicine, Mayo Clinic Arizona, 13400 East Shea Boulevard Scottsdale, AZ 85259
| | - Katie O’Neill
- Division of Hematology and Oncology, Mayo Clinic Arizona, 13400 East Shea Boulevard Scottsdale, AZ 85259
| | - Dana Colbert
- Division of Hematology and Oncology, Mayo Clinic Arizona, 13400 East Shea Boulevard Scottsdale, AZ 85259
| | - Theresa R. Lombari
- Laboratory Animal Research Core (LARC) Facility, Mayo Clinic Arizona, 13400 East Shea Boulevard Scottsdale, AZ 85259
| | - Stephanie Constant
- Department of Microbiology and Tropical Medicine, George Washington University, 2300 Eye Street NW, Washington, DC 20037
| | - Michael P. McGarry
- Division of Pulmonary Medicine, Mayo Clinic Arizona, 13400 East Shea Boulevard Scottsdale, AZ 85259
| | - James J. Lee
- Division of Pulmonary Medicine, Mayo Clinic Arizona, 13400 East Shea Boulevard Scottsdale, AZ 85259
| | - Nancy A. Lee
- Division of Hematology and Oncology, Mayo Clinic Arizona, 13400 East Shea Boulevard Scottsdale, AZ 85259
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20
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van der Sluis M, Melis MHM, Jonckheere N, Ducourouble MP, Büller HA, Renes I, Einerhand AWC, Van Seuningen I. The murine Muc2 mucin gene is transcriptionally regulated by the zinc-finger GATA-4 transcription factor in intestinal cells. Biochem Biophys Res Commun 2004; 325:952-60. [PMID: 15541382 DOI: 10.1016/j.bbrc.2004.10.108] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2004] [Indexed: 11/29/2022]
Abstract
MUC2, the major mucin in the intestine, is expressed early during development and shows an altered expression pattern in intestinal bowel diseases. However, the mechanisms responsible for MUC2 expression in the intestine during these events are largely unknown. Having found putative GATA binding sites in the murine Muc2 promoter and that GATA-4 is expressed in Muc2-expressing goblet cells of the mouse small intestine, we undertook to study its regulation by this transcription factor. A panel of deletion mutants made in pGL3 vector and covering 2.2kb of the promoter were used to transfect the murine CMT-93 colorectal cancer cell line. The role of GATA-4 on Muc2 gene regulation was investigated by RT-PCR and co-transfections in the presence of expression vectors encoding either wild-type or mutated GATA-4 or by mutating the GATA-4 site identified within Muc2 promoter. Four GATA-4 cis-elements were identified in the promoter by EMSA and Muc2 promoter was efficiently activated when GATA-4 was overexpressed in the cells with a loss of transactivation when those sites were either mutated or a mutated form of GATA-4 was used. Altogether, these results identify Muc2, a goblet cell marker, as a new target gene of GATA-4 and point out an important role for this factor in Muc2 expression in the intestine.
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Affiliation(s)
- Maria van der Sluis
- Laboratory of Paediatrics, Department of Gastroenterology and Nutrition, Erasmus MC and Sophia Children Hospital, Rotterdam, The Netherlands
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21
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Hu JY, Li GC, Wang WM, Zhu JG, Li YF, Zhou GH, Sun QB. Transfection of colorectal cancer cells with chemokine MCP-3 (monocyte chemotactic protein-3) gene retards tumor growth and inhibits tumor metastasis. World J Gastroenterol 2002; 8:1067-72. [PMID: 12439927 PMCID: PMC4656382 DOI: 10.3748/wjg.v8.i6.1067] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To evaluate the possibility of the induction of anti-tumor immune response by transfecting the colorectal cancer cells with chemokine MCP-3 gene.
METHODS: Mouse MCP-3 gene was transduced into mouse colorectal cancer cells CMT93 by using of Liposome. G418-resistant clones were selected and the MCP-3 mRNA expression was detected by RT-PCR. The chemotactic activity of MCP-3 in the cell culture supernatant was detected by Chemotaxis assay. The tumorigenicity of wild type CMT93 and CMT93 gene transfectants were detected by in vivo experiments. The immune cell infiltrations in tumor tissue and tumor metastasis were detected histopathologically.
RESULTS: MCP-3 mRNA expression was detected by RT-PCR in gene-transfected cells (CMT93/MCP-3), but not in control groups. And MCP-3 secreted in the cell culture supernatant possessed chemotatic activity. The results from in vivo experiments showed that the tumorigenicity of CMT93/MCP-3 had not decreased, but the tumors derived from CMT93/MCP-3 cells grew more slowly than those from CMT93 cells (1.021 ± 0.253) cm2vs (1.769 ± 0.371) cm2, P < 0.05) or CMT93/mock cells (1.021 ± 0.253) cm2vs (1.680 ± 0.643) cm2, P < 0.05). Histophathological results showed few immune cells infiltrating in the tumor tissue derived from the controls. In the tumor tissue derived from CMT93/MCP-3, infiltrating immune cells increased. In addition, no tumor metastasis was found in all mice inoculated with CMT93/ MCP-3 tumor cells. But all mice had tumor metastasis in CMT93 controls and 4 in 5 mice had tumor metastasis in CMT93/mock controls.
CONCLUSION: The results suggested that the transfection of chemokine MCP-3 gene could promote the induction of anti-colorectal cancer immunity, but the tumor growth could not be inhibited completely by merely MCP-3 gene transfection.
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Affiliation(s)
- Jin-Yue Hu
- Lab of Tumor Immunobiology, Cancer Research Institute, Xiangya Medical School, Central South University, Hunan Province, China
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22
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Dredge K, Marriott JB, Macdonald CD, Man HW, Chen R, Muller GW, Stirling D, Dalgleish AG. Novel thalidomide analogues display anti-angiogenic activity independently of immunomodulatory effects. Br J Cancer 2002; 87:1166-72. [PMID: 12402158 PMCID: PMC2376196 DOI: 10.1038/sj.bjc.6600607] [Citation(s) in RCA: 235] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2002] [Revised: 08/21/2002] [Accepted: 08/28/2002] [Indexed: 01/18/2023] Open
Abstract
The anti-tumour effects of thalidomide have been associated with its anti-angiogenic properties. Second generation thalidomide analogues are distinct compounds with enhanced therapeutic potential. Although these compounds are beginning to enter trials for the treatment of cancer there is very little information regarding the anti-angiogenic activity of these clinically relevant compounds. Furthermore, it is not known how the various immunomodulatory activities of these compounds relate to anti-angiogenic activity. In this study we assessed the anti-angiogenic activity of compounds from both IMiD and SelCID classes of analogues using a novel in vitro multicellular human assay system and the established rat aorta assay. Our results show that both the IMiDs and SelCIDs tested are significantly more potent than thalidomide. The anti-angiogenic potency of the analogues was not related to inhibition of endothelial cell proliferation, nor their TNF-alpha/PDE type 4 inhibitory properties. However, anti-migratory effects in vitro and inhibition of tumour growth in vivo was observed with the analogue IMiD-1 (clinically known as REVIMID). Our results show that anti-angiogenic activity spans both currently defined classes of thalidomide analogue and is not related to their previously described immunomodulatory properties. Identification of the differential effects of these compounds will enable targeting of such compounds into the appropriate clinical setting.
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Affiliation(s)
- K Dredge
- Division of Oncology, St. George's Hospital Medical School, Tooting, London SW17 0RE, UK.
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23
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Medema JP, de Jong J, Peltenburg LT, Verdegaal EM, Gorter A, Bres SA, Franken KL, Hahne M, Albar JP, Melief CJ, Offringa R. Blockade of the granzyme B/perforin pathway through overexpression of the serine protease inhibitor PI-9/SPI-6 constitutes a mechanism for immune escape by tumors. Proc Natl Acad Sci U S A 2001; 98:11515-20. [PMID: 11562487 PMCID: PMC58761 DOI: 10.1073/pnas.201398198] [Citation(s) in RCA: 241] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2001] [Accepted: 07/30/2001] [Indexed: 11/18/2022] Open
Abstract
The concept for cellular immunotherapy of solid tumors relies heavily on the capacity of class I MHC-restricted cytotoxic T lymphocytes (CTLs) to eliminate tumor cells. However, tumors often have managed to escape from the cytolytic machinery of these effector cells. Therefore, it is very important to chart the mechanisms through which this escape can occur. Target-cell killing by CTLs involves the induction of apoptosis by two major mechanisms: through death receptors and the perforin/granzyme B (GrB) pathway. Whereas tumors previously were shown to exhibit mechanisms for blocking the death receptor pathway, we now demonstrate that they also can resist CTL-mediated killing through interference with the perforin/GrB pathway. This escape mechanism involves expression of the serine protease inhibitor PI-9/SPI-6, which inactivates the apoptotic effector molecule GrB. Expression of PI-9 was observed in a variety of human and murine tumors. Moreover, we show that, indeed, expression results in the resistance of tumor cells to CTL-mediated killing both in vitro and in vivo. Our data reveal that PI-9/SPI-6 is an important parameter determining the success of T cell-based immunotherapeutic modalities against cancer.
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Affiliation(s)
- J P Medema
- Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, Albinusdreef 2, 2333ZA Leiden, The Netherlands.
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24
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Kyuwa S, Ohsawa K, Sato H, Urano T. Replication of enterotropic and polytropic murine coronaviruses in cultured cell lines of mouse origin. Exp Anim 2000; 49:251-7. [PMID: 11109550 DOI: 10.1538/expanim.49.251] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/31/2022] Open
Abstract
To understand the virus-cell interactions that occur during murine coronavirus infection, six murine cell lines (A3-1M, B16, CMT-93, DBT, IC-21 and J774A.1) were inoculated with eight murine coronaviruses, including prototype strains of both polytropic and enterotropic biotypes, and new isolates. All virus strains produced a cytopathic effect (CPE) with cell-to-cell fusion in B16, DBT, IC-21 and J774A.1 cells. The CPE was induced most rapidly in IC-21 cells and was visible microscopically in all cell lines tested. In contrast, the coronaviruses produced little CPE in A3-1M and CMT-93 cells. Although most virus-infected cells, except KQ3E-infected A3-1M, CMT-93 and J774A.1 cells, produced progeny viruses in the supernatants when assayed by plaque formation on DBT cells, the kinetics of viral replication were dependent on both the cell line and virus strain; replication of prototype strains was higher than that of new isolates. There was no significant difference in replication of enterotropic and polytropic strains. B16 cells supported the highest level of viral replication. To determine the sensitivity of the cell lines to murine coronaviruses, the 50% tissue culture infectious dose of the coronaviruses was determined with B16, DBT, IC-21 and J774A.1 cells, and compared to that with DBT cells. The results indicate that IC-21 cells were the most sensitive to murine coronaviruses. These data suggest that B16 and IC-21 cells are suitable for large-scale preparation and isolation of murine coronaviruses, respectively.
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Affiliation(s)
- S Kyuwa
- Division of Microbiology and Genetics, Kumamoto University, Japan
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25
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Todryk S, Melcher AA, Hardwick N, Linardakis E, Bateman A, Colombo MP, Stoppacciaro A, Vile RG. Heat Shock Protein 70 Induced During Tumor Cell Killing Induces Th1 Cytokines and Targets Immature Dendritic Cell Precursors to Enhance Antigen Uptake. THE JOURNAL OF IMMUNOLOGY 1999. [DOI: 10.4049/jimmunol.163.3.1398] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Abstract
Previously, we reported that killing tumor cells in vivo with the HSV thymidine kinase/ganciclovir system generates potent antitumor immunity, determined in part by the mechanism by which the cells die and by the levels of inducible heat shock protein (hsp) expression induced during the process of cell death. Here, we show that induction of hsp70 expression induces an infiltrate of T cells, macrophages, and predominantly dendritic cells (DCs) into the tumors as well as an intratumoral profile of Th1 cytokine expression (IFN-γ, TNF-α, and IL-12) and enhances immunogenicity via a T cell-mediated mechanism. In addition, the protection conferred by hsp70 is both tumor and cell specific. We also demonstrate that hsp70 targets immature APC to make them significantly more able to capture Ags. This is likely to optimize cross-priming of the infiltrating APC with tumor Ags, which are simultaneously being released by the dying cells. In addition, using an Myc epitope-tagged hsp70 expression vector, we present evidence that hsp70 released from dying tumor cells is taken up directly into DCs and may, therefore, be involved in direct chaperoning of Ags into DCs. Taken together, our data suggest that hsp70 induction serves to signal the immune system of the presence of an immunologically relevant (dangerous) situation against which an immune reaction should be raised.
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Affiliation(s)
- Stephen Todryk
- *Imperial Cancer Research Fund Laboratory of Molecular Therapy, Imperial Cancer Research Fund Oncology Unit, Imperial College of Science and Medicine, Hammersmith Hospital, London, United Kingdom
| | - Alan A. Melcher
- *Imperial Cancer Research Fund Laboratory of Molecular Therapy, Imperial Cancer Research Fund Oncology Unit, Imperial College of Science and Medicine, Hammersmith Hospital, London, United Kingdom
- †Molecular Medicine Program, Mayo Clinic, Rochester, MN 55905
| | - Nicola Hardwick
- *Imperial Cancer Research Fund Laboratory of Molecular Therapy, Imperial Cancer Research Fund Oncology Unit, Imperial College of Science and Medicine, Hammersmith Hospital, London, United Kingdom
| | - Emmanouela Linardakis
- *Imperial Cancer Research Fund Laboratory of Molecular Therapy, Imperial Cancer Research Fund Oncology Unit, Imperial College of Science and Medicine, Hammersmith Hospital, London, United Kingdom
| | - Andrew Bateman
- *Imperial Cancer Research Fund Laboratory of Molecular Therapy, Imperial Cancer Research Fund Oncology Unit, Imperial College of Science and Medicine, Hammersmith Hospital, London, United Kingdom
- †Molecular Medicine Program, Mayo Clinic, Rochester, MN 55905
| | - Mario P. Colombo
- ‡Experimental Oncology D, Istituto Nazionale Tumori, Milan, Italy; and
| | - Antonella Stoppacciaro
- *Imperial Cancer Research Fund Laboratory of Molecular Therapy, Imperial Cancer Research Fund Oncology Unit, Imperial College of Science and Medicine, Hammersmith Hospital, London, United Kingdom
- §Department of Experimental Medicine and Pathology, Second Chair of Pathology, University of Rome La Sapienza, Rome, Italy
| | - Richard G. Vile
- *Imperial Cancer Research Fund Laboratory of Molecular Therapy, Imperial Cancer Research Fund Oncology Unit, Imperial College of Science and Medicine, Hammersmith Hospital, London, United Kingdom
- †Molecular Medicine Program, Mayo Clinic, Rochester, MN 55905
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26
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Chong H, Hutchinson G, Hart IR, Vile RG. Expression of B7 co-stimulatory molecules by B16 melanoma results in a natural killer cell-dependent local anti-tumour response, but induces T-cell-dependent systemic immunity only against B7-expressing tumours. Br J Cancer 1998; 78:1043-50. [PMID: 9792148 PMCID: PMC2063155 DOI: 10.1038/bjc.1998.625] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Abstract
In an attempt to enhance the anti-tumour immune response, the co-stimulatory molecules B7-1 or B7-2 were expressed on the surface of B16 melanoma cells. B7-expressing tumours grew more slowly in both syngeneic immunocompetent mice and athymic T cell-immunodeficient nude mice. The delay in growth of B7-expressing tumours was dependent on natural killer (NK) cells, as reductions in tumour growth rates were minimized in mice depleted of NK cells. Systemic immunity to B16 melanoma was examined by vaccination with irradiated tumour cells. Inoculation with irradiated B16 B7-1 cells failed to protect against a subsequent challenge with live parental B16 cells, but conferred partial protection against challenge with live B16 B7-1 cells. In contrast to the local anti-tumour reaction, this protective response was dependent on T cells. The results presented here reveal some of the mechanisms involved in the in vivo response to a poorly immunogenic tumour modified to express co-stimulatory molecules.
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Affiliation(s)
- H Chong
- Department of Histopathology, United Medical and Dental Schools of Guy's and St. Thomas's Hospitals, London, UK
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27
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Chong H, Hutchinson G, Hart IR, Vile RG. Expression of co-stimulatory molecules by tumor cells decreases tumorigenicity but may also reduce systemic antitumor immunity. Hum Gene Ther 1996; 7:1771-9. [PMID: 8886848 DOI: 10.1089/hum.1996.7.14-1771] [Citation(s) in RCA: 26] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
Abstract
Many tumor cells do not express co-stimulatory molecules, and this may account, in part, for their poor ability to stimulate T cells directly. One strategy to enhance immune recognition would be to express such molecules on the tumor cell. Here, we show that expression of a member of the B7 family of co-stimulatory molecules by CMT93 murine colorectal tumor or 1735 murine melanoma cells resulted in a local antitumor response in immunocompetent mice. The antitumor effect was diminished in athymic nude mice, indicating that T cells played an important part in this response. The ability of the B7-expressing tumor cells to generate systemic protective immunity was investigated by excision of tumors that developed from the initial inoculation followed by rechallenge with parental tumor cells. CMT93 is a poorly immunogenic tumor and no significant systemic immunity was elicited by the expression of B7-1 in these cells. 1735 melanoma is a mildly immunogenic tumor. Unexpectedly, the systemic immunity obtained with 1735 tumors expressing B7-1 or B7-2 was weaker than that generated by parental 1735 cells (p < 0.001, stratified logrank test), even when coexpression of interferon-gamma in the B7-1 cells produced high levels of surface MHC class I expression. These results suggest that some caution is appropriate when considering the use of these molecules in the gene therapy of cancer.
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MESH Headings
- Animals
- Antigens, CD/analysis
- Antigens, CD/genetics
- Antigens, CD/immunology
- B7-1 Antigen/analysis
- B7-1 Antigen/genetics
- B7-1 Antigen/immunology
- B7-2 Antigen
- Cell Division
- Colorectal Neoplasms
- Genetic Vectors/genetics
- Histocompatibility Antigens Class I/analysis
- Immunity, Cellular
- Interferon-gamma/genetics
- Melanoma
- Membrane Glycoproteins/analysis
- Membrane Glycoproteins/genetics
- Membrane Glycoproteins/immunology
- Mice
- Mice, Inbred C3H
- Mice, Inbred C57BL
- Mice, Nude
- Neoplasm Transplantation
- Neoplasms, Experimental/genetics
- Neoplasms, Experimental/immunology
- Neoplasms, Experimental/pathology
- Retroviridae/genetics
- Tumor Cells, Cultured
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Affiliation(s)
- H Chong
- Imperial Cancer Research Fund Laboratory of Cancer Gene Therapy, Rayne Institute, St. Thomas' Hospital, London, UK
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28
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Darro F, Camby I, Kruczynski A, Pasteels JL, Martinez J, Kiss R. Characterisation of the influence of anti-gastrin, anti-epidermal growth factor, anti-oestradiol, and anti-luteinising hormone releasing hormone antibodies on the proliferation of 27 cell lines from the gastrointestinal tract. Gut 1995; 36:220-30. [PMID: 7883221 PMCID: PMC1382408 DOI: 10.1136/gut.36.2.220] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
Numerous data from published reports prove that the proliferation of gastrointestinal tumour cell lines are under the control of many hormones or growth factors, or both. Most of these publications report the influence on a very small number of cell lines of one or two such factors only. This work deals with the in vitro characterisation of the influence of the anti-gastrin, the anti-epidermal growth factor (EGF), the anti-oestradiol (E2), and the anti-luteinising hormone releasing hormone (LHRH) antibodies on the proliferation of a large series of gastrointestinal cell lines. Cell proliferation was assessed by means of the colorimetric MTT assay on a series of 27 gastrointestinal cell lines obtained from the American Type Culture Collection (ATCC). Of the 27 cell lines, the anti-gastrin, the anti-EGF, the anti-E2, and the anti-LHRH neutralising antibodies considerably influenced the proliferation of 13, 25, 12, and 16. No gastrointestinal cell line was unresponsive to the four antibodies simultaneously. The anti-gastrin and anti-EGF antibody induced effects on the 27 gastrointestinal cell line proliferation were significantly correlated, as was also the case for the anti-E2 and anti-LHRH antibody induced effects. Of the anti-gastrin, the anti-EGF, the anti-E2, and the anti-LHRH antibodies, it was the anti-EGF one that had the greatest influence, both quantitatively and qualitatively, on gastrointestinal cell proliferation. The correlation of the effects of definite anti-hormone antibodies is suggestive of a common mechanism of action for the corresponding hormones and casts some doubt on the efficiency of anti-hormone monotherapy.
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Affiliation(s)
- F Darro
- Division de Cancérologie Expérimentale 1, Centre de Recherche Pierre Fabre, Castres, France
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29
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Durbin H, Young S, Stewart LM, Wrba F, Rowan AJ, Snary D, Bodmer WF. An epitope on carcinoembryonic antigen defined by the clinically relevant antibody PR1A3. Proc Natl Acad Sci U S A 1994; 91:4313-7. [PMID: 7514303 PMCID: PMC43775 DOI: 10.1073/pnas.91.10.4313] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
The monoclonal antibody PR1A3 has been used successfully for in vivo imaging of colorectal cancers, and several properties associated with this antibody, including minimal reactions of the antibody with circulating antigen in patients' sera, differentiate it from anti-carcinoembryonic antigen (CEA) antibodies used in similar studies. However, the antigen bound by PR1A3 was identified as CEA by analysis of somatic cell hybrids and by antigen expression from yeast artificial chromosomes, cosmids, and cDNA clones. The molecular weight, presence of a glycosyl-phosphatidylinositol anchor, elevation of surface expression by gamma-interferon, and N-terminal amino acid sequence all confirmed the antigen identification as CEA. A series of biliary glycoprotein-CEA hybrid proteins was produced which demonstrated that the epitope bound by the antibody was at the site of membrane attachment and involved parts of the glycosyl-phosphatidylinositol anchor and the B3 domain of CEA to form a conformational epitope. Access to this epitope, although possible when the antigen was on the cell surface, appeared to be blocked when CEA was released from the cell. The nature and location of the epitope on CEA are proposed to be responsible for the unique properties of the antibody.
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Affiliation(s)
- H Durbin
- Cancer Genetics Laboratory, Imperial Cancer Research Fund, London, United Kingdom
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30
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Brown I, Mitchell JS. The development of a [211At]-astatinated endoradiotherapeutic drug: Part II. Therapeutic results for transplanted adenocarcinoma of the rectum in mice and associated studies. Int J Radiat Oncol Biol Phys 1994; 29:115-24. [PMID: 8175418 DOI: 10.1016/0360-3016(94)90233-x] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
PURPOSE 6-[211At]-astato-MNDP is of a class of a high linear energy transfer endoradiotherapeutic drug, which selectively targets to an onco-APase isoenzyme expressed by certain epithelial and germ cell tumors. The therapeutic efficacy and acute toxicity of its endogenous alpha-particle emissions have been studied in a murine tumor model. METHODS AND MATERIALS 211At was produced by the 207Bi(alpha,2n)211 At cyclotron-based nuclear reaction. High specific activity 6-[211At]-astato-MNDP was rapidly synthesized by in vacuo thermal heterogeneous isotopic exchange. The therapeutic potential of 6-[211At]-astato-MNDP and 211At- was determined in mice bearing a transplanted CMT-93 rectal carcinoma which exhibited onco-APase activity. RESULTS Significant therapeutic effects due to targeted alpha-particle emissions have been confirmed for the activity dose range, 10-750 kBq 6-[211At]-astato-MNDP. A therapeutic window has been identified, whereby cure rates of approximately 45-65% were achieved following administration of 55-300 kBq 6-[211At]-astato-MNDP. Estimated tumor absorbed radiation doses were not inconsistent with clinical response. Irreversible hematoxicity or stigmata of acute radiation damage in other critical normal tissues were not encountered. Nonspecifically internalized 211At- exerted no therapeutic effect. CONCLUSION Therapeutic results for 6-[211At]-astato-MNDP have confirmed the profound in vivo cytotoxicity of its targeted alpha-radiations in the CMT-93 tumor. Acute normal tissue toxicity was acceptable. A rationale for optimal fractionation of targeted 6-[211At]-astato-MNDP endoradiotherapy is discussed, and its putative role in the possible individualized management of certain human tumors has been proposed.
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Affiliation(s)
- I Brown
- Research Laboratories, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, UK
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31
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Roberts K, Kilshaw PJ. The mucosal T cell integrin alpha M290 beta 7 recognizes a ligand on mucosal epithelial cell lines. Eur J Immunol 1993; 23:1630-5. [PMID: 8100775 DOI: 10.1002/eji.1830230735] [Citation(s) in RCA: 78] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
The integrin alpha M290 beta 7 is expressed at high levels on mucosal T cells, particularly on those within the epithelium of the gut. We now report that a mouse T cell hybridoma, MTC-1, with similar surface expression of this molecule, adhered strongly to cells of the mouse rectal carcinoma line CMT93 and that adhesion was blocked completely by the monoclonal antibody (mAb) M290. Other mAb to the alpha M290 or beta 7 subunits had little or no inhibitory effect. M290 also inhibited adhesion of the hybridoma to cells of the mouse lung carcinomas CTM64/61 and KLN205 but had little or no effect on adhesion to seven other mouse epithelial cell lines or to the human colon carcinoma line, HT29. Intraepithelial lymphocytes (IEL) isolated from the small intestine of BALB/c mice displayed potent T cell receptor-dependent cytotoxic effector function against CMT93 in the presence of low concentrations of Phytolacca americana lectin. This cytotoxic activity also was inhibited by the M290 mAb. Treatment of CMT93 cells with tumor necrosis factor-alpha and interferon-gamma induced expression de novo of ICAM-1 and reduced the inhibitory effect of M290 in tests both for adhesion and cytotoxicity. In further experiments cytotoxic activity of IEL against the mastocytoma P815 was investigated. This target cell was considered not to possess a ligand for the integrin. In this case cytotoxic effector function was triggered by anti-CD3 mAb and, in contrast to results with CMT93, target cell lysis was increased in the presence of M290 and other antibodies to the integrin, suggesting a co-stimulatory effect. These results show that alpha M290 beta 7 recognizes a ligand on the surface of certain epithelial cell lines. Further, they provide the first clear indication that this integrin may play an important role in functional interactions between T cells and the mucosal epithelium.
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Affiliation(s)
- K Roberts
- Department of Cell Biology, AFRC Institute of Animal Physiology and Genetics Research, Cambridge
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32
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Brown I, Carpenter RN, Mitchell JS. The development of A [211At]-astatinated endoradiotherapeutic drug: Part I. Localization by alpha-particle autoradiography in a murine tumor model. Int J Radiat Oncol Biol Phys 1992; 23:563-72. [PMID: 1612957 DOI: 10.1016/0360-3016(92)90012-7] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Alpha-particle track autoradiography has been used to define the in vivo cellular and intracellular distribution of radioactivity from the potential high linear energy transfer endoradiotherapeutic drug, 6-[211At]-astato-2-methyl-1,4-naphthoquinol bis(diphosphate) in tumor and relevant critical normal tissues of mice bearing a transplanted murine rectal carcinoma. A strikingly selective uptake of this compound into tumor cells, particularly into specific tumor cell nuclei, has been demonstrated. Its localization in certain tumor cells appears to depend on the presence of an onco-product, in this case an alkaline phosphatase isoenzyme, which is synthesized in some tumor cells and to which the compound targets. In curable tumors, it selectively concentrates in cells which may be regarded as tumor stem cells. There is low uptake into normal cells, particularly those in bone marrow, colon, and lung, where its sequestration is mainly extranuclear.
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Affiliation(s)
- I Brown
- Research Laboratories, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, UK
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33
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Ringwald M, Baribault H, Schmidt C, Kemler R. The structure of the gene coding for the mouse cell adhesion molecule uvomorulin. Nucleic Acids Res 1991; 19:6533-9. [PMID: 1754391 PMCID: PMC329213 DOI: 10.1093/nar/19.23.6533] [Citation(s) in RCA: 63] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
We have recently shown that the Ca2+ dependent cell adhesion molecule uvomorulin is encoded by a single gene, localized on mouse chromosome 8. Here we describe the organization of the uvomorulin gene and give an initial characterization of the uvomorulin promoter. Uvomorulin is encoded by 16 exons, which are distributed over a region of more than 40 kb genomic DNA. The exon structure of the genes for uvomorulin and its chicken homologue L-CAM are nearly identical and thus highly conserved. The relationship between the exon structure and the structure of the uvomorulin protein is analysed. The initiation site of transcription of the uvomorulin gene is located 127 bp upstream of the translation start site in a GC-rich region with no TATA-box, but with a GC-box in position -48 and a CCAAT-box starting at position -65 with respect to the transcription start site. 1.6 kb of the uvomorulin promoter (-1492 to + 92) confer cell type specific promoter activity to the CAT reporter gene. Homologies to known cis acting elements of other promoters are discussed.
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Affiliation(s)
- M Ringwald
- Max-Planck-Institut für Immunbiologie, Freiburg, FRG
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34
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Pignatelli M, Bodmer WF. Genetics and biochemistry of collagen binding-triggered glandular differentiation in a human colon carcinoma cell line. Proc Natl Acad Sci U S A 1988; 85:5561-5. [PMID: 2840666 PMCID: PMC281798 DOI: 10.1073/pnas.85.15.5561] [Citation(s) in RCA: 74] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
We have examined the interaction between collagen binding and epithelial differentiation by using a human colon carcinoma cell line (SW1222) that can differentiate structurally when grown in a three-dimensional collagen gel to form glandular structures. As much as 66% inhibition of glandular differentiation can be achieved by addition to the culture of a synthetic peptide (2 mg/ml) containing the Arg-Gly-Asp-Thr (RGDT) sequence, which is a cell recognition site found in collagen. Arg-Gly-Asp-Thr also inhibited the cell attachment to collagen-coated plates. A control peptide containing the Arg-Gly-Glu-Thr (RGET) sequence had no effect on cell adhesion or cell differentiation. Chromosome 15 was found in all human-mouse hybrid clones [from a cross between SW1222 and a mouse rectal carcinoma cell line (CMT-93)] that could differentiate in the collagen gel and bind collagen. Both binding to collagen and glandular differentiation of the hybrid cells were also inhibited by Arg-Gly-Asp-Thr as for the parent cell line SW1222. The ability of SW1222 cells to express the differentiated phenotype appears, therefore, to be determined by an Arg-Gly-Asp-directed collagen receptor on the cell surface that is controlled by a gene on chromosome 15.
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Affiliation(s)
- M Pignatelli
- Director's Laboratory, Imperial Cancer Research Fund, London, United Kingdom
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35
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Smith AL, Winograd DF, Burrage TG. Comparative biological characterization of mouse adenovirus strains FL and K 87 and seroprevalence in laboratory rodents. Arch Virol 1986; 91:233-46. [PMID: 3022678 PMCID: PMC7086991 DOI: 10.1007/bf01314283] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/1986] [Accepted: 03/23/1986] [Indexed: 01/03/2023]
Abstract
The growth, stability and seroprevalence in laboratory rodents of the two known strains of mouse adenovirus were compared. The FL strain of mouse adenovirus grew in both L 929 murine fibroblasts and in CMT-93 murine rectal carcinoma cells, whereas the K 87 strain grew only in CMT-93 cells. The bulk of the FL progeny virus was released from the host cells. K 87 virus was largely cell-associated. Both virus strains were stable at 37 degrees C in liquid medium. The K 87 strain was completely inactivated after 5-15 minutes at 56 degrees C, whereas FL infectivity was still detected after two hours at this temperature. Both virus strains were stable in the dessicated state for 14 days, although FL viability was more dependent on the presence of protein in the virus diluent. Seroepidemiologic data suggest that viruses antigenically related to mouse adenovirus are more prevalent among laboratory rats than among laboratory mice and that the virus(es) infecting rats differ from those infecting mice. Results of retrospective serologic testing suggest an association between mouse adenovirus and an outbreak of disease in a mouse breeding colony.
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36
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Brown I. Astatine-211: its possible applications in cancer therapy. INTERNATIONAL JOURNAL OF RADIATION APPLICATIONS AND INSTRUMENTATION. PART A, APPLIED RADIATION AND ISOTOPES 1986; 37:789-98. [PMID: 3021680 DOI: 10.1016/0883-2889(86)90273-x] [Citation(s) in RCA: 55] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
The cyclotron-produced radiohalogen, 211At, is eminently suitable as a possible therapeutic radionuclide. It decays by the emission of 6.8 MeV mean energy alpha-particles, which from a radiobiological viewpoint are of near optimal therapeutic LET. This paper reviews developments in the possible application of [211At]astato-labelled molecules as potential anti-tumour agents. Additionally, radio-dosimetric evidence is presented, and its implications for human cancer therapy are discussed.
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37
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Mitchell JS, Brown I, Carpenter RN. Alpha-particle track autoradiographic study of the distribution of a [211At]-astatinated drug in normal tissues of the mouse. EXPERIENTIA 1985; 41:925-8. [PMID: 4007129 DOI: 10.1007/bf01970016] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
The microscopic distribution of the potential endoradiotherapeutic drug, 6-[211At]-astato-2-methyl-1,4- naphthoquinol bis (diphosphate salt) in normal tissues of the mouse has been studied by alpha-particle track autoradiography. The uptake into critical radiosensitive tissues, especially bone marrow, colon and lung, was low.
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38
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Brown I, Carpenter RN, Mitchell JS. Biodistribution of 6-[211At]astato-2-methyl-1,4-naphthoquinol bis(diphosphate salt) and 211At- in mice with a transplanted rectal adenocarcinoma. THE INTERNATIONAL JOURNAL OF APPLIED RADIATION AND ISOTOPES 1984; 35:843-7. [PMID: 6480147 DOI: 10.1016/0020-708x(84)90019-x] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
6-[211At]astato-MNDP is currently being investigated as a potential high LET endoradiotherapeutic drug. Biodistribution and whole-body radiation retention studies have been carried out with 6-[211At]astato-MNDP and 211At- in a murine rectal tumour model; results indicate that the 211At-C bond in the compound is metabolically stable for at least 6 h. The Mean Biological Concentration of 6-[211At]astato-MNDP in tumour tissue ranged from 170-253% over an initial 12 h period; this was higher than that observed for the [211At]astatide anion. Conversely, the uptake of compound into radiobiologically critical organs was significantly lower.
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Mitchell JS, Brown I, Carpenter RN. alpha-Particle track autoradiography for localization of a 211At-astatinated drug. EXPERIENTIA 1983; 39:337-9. [PMID: 6825808 DOI: 10.1007/bf01955337] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
A potential endoradiotherapeutic drug, 6-211At-astato-2-methyl-1,4-naphthoquinol bis (diphosphate salt), incorporating the alpha-emitting radio-halogen astatine-211 of half-life 7.2 h, is shown to be valuable for localization studies by means of alpha-particle track autoradiography in malignant and normal cells and tissues in the mouse with transplanted adenocarcinoma of the rectum.
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40
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Brown I, Carpenter RN, Mitchell JS. 6-125I-iodo-2-methyl-1,4-naphthoquinol bis (diammonium phosphate) as a potential radio-halogenated anti-cancer agent: in vitro investigations and possible clinical implications. EUROPEAN JOURNAL OF NUCLEAR MEDICINE 1982; 7:115-20. [PMID: 7067713 DOI: 10.1007/bf00256398] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
6-125-I-iodo-2-methyl-1,4-naphthoquinol bis (diammonium phosphate) (6-125I-iodo-MNDP) has been synthesised and studied as the prototype of a class of potential radio-halogenated anti-cancer agents. The incorporated 125I provides Auger electron radiations which behave like high LET radiations in the treatment of tumours, though the accompanying X- and gamma-radiations make an undesirable contribution to the total body dose. The in vitro experiments reported show that 6-125I-iodo-MNDP is selectively concentrated in the cells of some human malignant tumours by factor of about 15 to 20 or more in relation to the cells of normal origin studied. On the basis of dosimetric considerations and comparison with clinical treatment with tritiated methylnaphthoquinol diphosphate, practical dosage of 6-125I-iodo-MNDP is suggested and clinical indications and safety of use are discussed. The types of tumour of particular interest are inoperable cases of carcinoma of the colon, carcinoma of the pancreas, malignant melanoma and osteosarcoma. Further investigations are in progress.
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41
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Childs RA, Kapadia A, Feizi T. Expression of blood group I and i active carbohydrate sequences on cultured human and animal cell lines assessed by radioimmunoassays with monoclonal cold agglutinins. Eur J Immunol 1980; 10:379-84. [PMID: 6157539 DOI: 10.1002/eji.1830100512] [Citation(s) in RCA: 36] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
Human monoclonal anti-I and anti-i antibodies, reactive with known carbohydrate sequences, have been used as reagents to quantitate (by radioimmunoassay) and visualize (by immunofluorescence) the expression of the various blood group I and i antigenic determinants in a variety of cultured cell lines commonly used in laboratory investigations. It has been shown that the antigens they recognize are widely distributed on the surface of human and animal cell lines, expressed in varying amounts in different cell lines and on individual cells within a given cell line. In two cell lines, a transformation-associated increase in the expression of I antigen was observed. Because of their precise specificity for defined carbohydrate chain domains, these autoantibodies have become valuable reagents in biological chemistry.
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Franks LM, Hamilton E, Hemmings VJ. "Epithelial" foci of presumptive neural origin in cultures of normal mouse colon. J Pathol 1978; 124:19-22. [PMID: 722369 DOI: 10.1002/path.1711240105] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Patches of cells, which at the light-microscope level appear to be epithelial, persist for several months in cultures of normal mouse colon. Ultrastructural studies showed that these cells were nerve-associated and were probably derived from the Schwann cell-satellite cell group. True epithelial cells all died out within the first 2 wk of culture, and epithelial cell lines could not be established.
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