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1
Beagley, K. W., K. Fujihashi, A. S. Lagoo, S. Lagoo-Deenadaylan, C. A. Black, A. M. Murray, A. T. Sharmanov, M. Yamamoto, J. R. McGhee, and C. O. Elson. "Differences in intraepithelial lymphocyte T cell subsets isolated from murine small versus large intestine." Journal of Immunology 154, no. 11 (June 1, 1995): 5611–19. http://dx.doi.org/10.4049/jimmunol.154.11.5611.
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Abstract Intraepithelial lymphocytes (IELs) have been extensively studied in the murine small intestine. However, to date no studies have assessed IEL in the large intestine, despite the marked differences in function and lumenal environment. In the present study, we isolated IEL from both small and large intestine of three mouse strains (BALB/c, C3H/HeN, C57BL/6) and determined the frequency of CD2, CD4, and CD8 expression on CD3+ IEL, as well as the frequency of alpha beta and gamma delta TCR usage and V beta distribution. Higher numbers of IEL/unit length were always isolated from the small intestine (20-30 x 10(6)/5 mice) compared with large intestine (1.1-2.5 x 10(6)/5 mice). Interestingly, IEL from the large intestine of all strains were predominantly alpha beta TCR+ whereas gamma delta TCR+ IELs predominated in small intestine. Large intestinal IELs were mainly CD4+, in both BALB/c and C3H/HeN mouse strains. IELs from large intestine of C57BL/6 mice were mainly CD8+; however, the CD4+ subset was fourfold higher when compared with small intestine IEL. Potential functional differences between IEL subsets was assessed by determining the relative levels of mRNA for IL-1, 2, 4, 5, 10, IFN-gamma, TGF-beta, and TNF-gamma. Similar patterns of IL-1, IFN-gamma and TNF-alpha were seen while more IL-2, IL-4, IL-5, and IL-10 mRNA was noted in large intestinal IEL. Stimulation of C3H/HeJ IEL with anti-CD3 also resulted in higher levels of IL-3/GM-CSF, IL-4, and IL-6 by IEL from large intestine. These results show that marked differences occur among the T cell subsets present in IELs from mouse small and large intestine.
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Hummel, Jonas F., Patrice Zeis, Karolina Ebert, Jonas Fixemer, Philip Konrad, Christian Schachtrup, Sebastian J. Arnold, Dominic Grün, and Yakup Tanriver. "Single-cell RNA-sequencing identifies the developmental trajectory of C-Myc-dependent NK1.1− T-bet+ intraepithelial lymphocyte precursors." Mucosal Immunology 13, no. 2 (November 11, 2019): 257–70. http://dx.doi.org/10.1038/s41385-019-0220-y.
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Abstract Natural intraepithelial lymphocytes (IELs) are thymus-derived adaptive immune cells, which are important contributors to intestinal immune homeostasis. Similar to other innate-like T cells, they are induced in the thymus through high-avidity interaction that would otherwise lead to clonal deletion in conventional CD4 and CD8 T cells. By applying single-cell RNA-sequencing (scRNA-seq) on a heterogeneous population of thymic CD4−CD8αβ−TCRαβ+NK1.1− IEL precursors (NK1.1− IELPs), we define a developmental trajectory that can be tracked based on the sequential expression of CD122 and T-bet. Moreover, we identify the Id proteins Id2 and Id3 as a novel regulator of IELP development and show that all NK1.1− IELPs progress through a PD-1 stage that precedes the induction of T-bet. The transition from PD-1 to T-bet is regulated by the transcription factor C-Myc, which has far reaching effects on cell cycle, energy metabolism, and the translational machinery during IELP development. In summary, our results provide a high-resolution molecular framework for thymic IEL development of NK1.1− IELPs and deepen our understanding of this still elusive cell type.
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Laky, K., L. Lefrançois, and L. Puddington. "Age-dependent intestinal lymphoproliferative disorder due to stem cell factor receptor deficiency: parameters in small and large intestine." Journal of Immunology 158, no. 3 (February 1, 1997): 1417–27. http://dx.doi.org/10.4049/jimmunol.158.3.1417.
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Abstract Signaling through c-Kit/stem cell factor (SCF) is crucial for normal development of erythroid and myeloid hematopoietic precursors and of melanocytes and germ cells. While peripheral lymphoid populations of W/Wv and SI/SId mice appear normal, we demonstrated that the intraepithelial lymphocyte (IEL) populations of small (SI) and large (LI) intestine were significantly affected. IEL populations of young W/Wv animals were indistinguishable from those of their control littermates, but an age-dependent decrease in SI and LI TCRgamma delta IEL occurred in c-Kit mutant mice. In SI, but not in LI, this diminution was accompanied by gross expansion of TCRalpha beta IEL that resulted in significantly increased IEL:epithelial cell ratios in c-Kit mutant mice. Bromodeoxyuridine labeling studies revealed that the increase in cell numbers was due to lymphoproliferation that occurred in situ. Interestingly, TCRgamma delta IEL expressed cell surface c-Kit, while the expanding population of TCRalpha beta IEL did not. Analysis of radiation bone marrow chimeras demonstrated that the dysregulation required either disruption of stromal cell SCF or IEL c-Kit and showed that the effect on IEL or their precursors was not due to other changes in the intestinal microenvironment. Lamina propria T cell populations in these mice were unaffected, reinforcing the idea that the developmental requirements of these gut-resident lymphocyte populations are distinct. Overall, the results demonstrated that the development of intestinal TCRgamma delta IEL, regardless of location, shares common requirements for SCF, while SI and LI TCRalpha beta IEL may develop along distinct pathways. Possible mechanisms for the loss of proliferative regulation in gut T cells in c-Kit/SCF deficiency are discussed.
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Jiang, Wei, Isabel Ferrero, Elisa Laurenti, Andreas Trumpp, and H. Robson MacDonald. "c-Myc controls the development of CD8αα TCRαβ intestinal intraepithelial lymphocytes from thymic precursors by regulating IL-15–dependent survival." Blood 115, no. 22 (June 3, 2010): 4431–38. http://dx.doi.org/10.1182/blood-2009-11-254698.
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Abstract The murine gut epithelium contains a large population of thymus-derived intraepithelial lymphocytes (IELs), including both conventional CD4+ and CD8αβ+ T cells (expressing T-cell receptor αβ [TCRαβ]) and unconventional CD8αα+ T cells (expressing either TCRαβ or TCRγδ). Whereas conventional IELs are widely accepted to arise from recirculation of activated CD4+ and CD8αβ+ T cells from the secondary lymphoid organs to the gut, the origin and developmental pathway of unconventional CD8αα IELs remain controversial. We show here that CD4-Cre-mediated inactivation of c-Myc, a broadly expressed transcription factor with a wide range of biologic activities, selectively impairs the development of CD8αα TCRαβ IELs. In the absence of c-Myc, CD4− CD8− TCRαβ+ thymic precursors of CD8αα TCRαβ IELs are present but fail to develop on adoptive transfer in immunoincompetent hosts. Residual c-Myc–deficient CD8αα TCRαβ IEL display reduced proliferation and increased apoptosis, which correlate with significantly decreased expression of interleukin-15 receptor subunits and lower levels of the antiapoptotic protein Bcl-2. Transgenic overexpression of human BCL-2 resulted in a pronounced rescue of CD8αα TCRαβ IEL in c-Myc–deficient mice. Taken together, our data support a model in which c-Myc controls the development of CD8αα TCRαβ IELs from thymic precursors by regulating interleukin-15 receptor expression and consequently Bcl-2–dependent survival.
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Rostami, Kamran, Michael N. Marsh, Matt W. Johnson, Hamid Mohaghegh, Calvin Heal, Geoffrey Holmes, Arzu Ensari, et al. "ROC-king onwards: intraepithelial lymphocyte counts, distribution & role in coeliac disease mucosal interpretation." Gut 66, no. 12 (September 11, 2017): 2080–86. http://dx.doi.org/10.1136/gutjnl-2017-314297.
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ObjectivesCounting intraepithelial lymphocytes (IEL) is central to the histological diagnosis of coeliac disease (CD), but no definitive ‘normal’ IEL range has ever been published. In this multicentre study, receiver operating characteristic (ROC) curve analysis was used to determine the optimal cut-off between normal and CD (Marsh III lesion) duodenal mucosa, based on IEL counts on >400 mucosal biopsy specimens.DesignThe study was designed at the International Meeting on Digestive Pathology, Bucharest 2015. Investigators from 19 centres, eight countries of three continents, recruited 198 patients with Marsh III histology and 203 controls and used one agreed protocol to count IEL/100 enterocytes in well-oriented duodenal biopsies. Demographic and serological data were also collected.ResultsThe mean ages of CD and control groups were 45.5 (neonate to 82) and 38.3 (2–88) years. Mean IEL count was 54±18/100 enterocytes in CD and 13±8 in normal controls (p=0.0001). ROC analysis indicated an optimal cut-off point of 25 IEL/100 enterocytes, with 99% sensitivity, 92% specificity and 99.5% area under the curve. Other cut-offs between 20 and 40 IEL were less discriminatory. Additionally, there was a sufficiently high number of biopsies to explore IEL counts across the subclassification of the Marsh III lesion.ConclusionOur ROC curve analyses demonstrate that for Marsh III lesions, a cut-off of 25 IEL/100 enterocytes optimises discrimination between normal control and CD biopsies. No differences in IEL counts were found between Marsh III a, b and c lesions. There was an indication of a continuously graded dose–response by IEL to environmental (gluten) antigenic influence.
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Buzoni-Gatel, D., A. C. Lepage, I. H. Dimier-Poisson, D. T. Bout, and L. H. Kasper. "Adoptive transfer of gut intraepithelial lymphocytes protects against murine infection with Toxoplasma gondii." Journal of Immunology 158, no. 12 (June 15, 1997): 5883–89. http://dx.doi.org/10.4049/jimmunol.158.12.5883.
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Abstract Intraepithelial lymphocytes (IEL) of the gut represent a primary immune barrier against infection by orally acquired pathogens. Naturally acquired infection with Toxoplasma gondii induces the proliferation of CD8+ T cells in both the gut and spleen. Gut-derived CD8alpha/beta+ IEL exhibit MHC-restricted cytotoxicity against parasite-infected enterocytes and macrophages. In a murine model, we demonstrate that the adoptive transfer of IEL obtained from inbred mice at day 11 postinfection is able to protect against a virulent challenge in syngenic recipients. In CBA mice, the parasite cyst load within the brain of the recipients receiving primed IEL was reduced by 90%. In BALB/c and C57BL/6 mice, a 50% decrease in mortality was observed following adoptive transfer of primed IEL. To determine the T cell subset responsible for protective immunity, a purified CD8alpha/beta+ IEL population was isolated from infected mice at day 11 postinfection. These cells were able to protect naive mice by adoptive transfer against a lethal parasite challenge. RNA analysis by reverse-transcriptase PCR revealed that primed CD8alpha/beta+ IEL produce significant message for IFN-gamma, an essential cytokine for host protection against toxoplasmosis. Administration of anti-IFN-gamma at the time of adoptive transfer of primed IEL abrogated protection. The adoptive transfer of these protective IEL was not restricted to the Ld class I locus. These data demonstrate that IFN-gamma-producing IEL may be an important primary barrier against acute and perhaps recurrent infection with T. gondii.
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Osborne-Pellegrin, Mary, Carlos Labat, Nathalie Mercier, Pascal Challande, and Patrick Lacolley. "Changes in aortic stiffness related to elastic fiber network anomalies in the Brown Norway rat during maturation and aging." American Journal of Physiology-Heart and Circulatory Physiology 299, no. 1 (July 2010): H144—H152. http://dx.doi.org/10.1152/ajpheart.00040.2010.
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Adult Brown Norway (BN) rats exhibit numerous internal elastic lamina (IEL) ruptures in the abdominal aorta (AA) and a lower aortic elastin-to-collagen ratio (E/C) compared with other strains. We studied here AA mechanical properties in BN compared with control strains. AA stiffness (assessed by plotting elastic modulus/wall-stress curves obtained under anesthesia), thoracic aorta elastin and collagen contents, and IEL ruptures in AA were measured in male BN and LOU rats aged 6, 10, and 15 wk. The Long Evans (LE) control strain was compared with BN at more advanced ages (15, 28, and 64 wk). At all ages, aortic E/C was lower in BN than in control strains. At 6 wk, AA stiffness was greater in BN than in LOU. In both strains, AA stiffness decreased between 6 and 10 wk, more so in BN than in LOU, and then increased, reaching similar values at 15 wk. BN AA stiffness was not different from that of LE at 15 and 28 wk, but was significantly lower at 64 wk. The increased stiffness in young BN rat AA may be due to the decreased E/C. IEL rupture onset in the BN around 7–8 wk, which decreases stiffness, as suggested by its pharmacological modulation, abolished such differences by 15 wk. Thereafter, age-related AA stiffness increased less in BN than in LE, likely due to the numerous IEL ruptures. We conclude that, in the BN rat, the lower E/C and the presence of IEL ruptures have opposing effects on arterial stiffness.
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LefrançoisL and S. Olson. "Reconstitution of the extrathymic intestinal T cell compartment in the absence of irradiation." Journal of Immunology 159, no. 2 (July 15, 1997): 538–41. http://dx.doi.org/10.4049/jimmunol.159.2.538.
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Abstract Extrathymic development of intestinal intraepithelial lymphocytes was studied using a reconstitution model that does not require irradiation. WBB6F1/J-Kit(W)/Kit(W-v) mice were reconstituted with normal fetal liver cells. In this system, reduced c-Kit activity in host hemopoietic progenitors imparts normal precursors with a growth advantage and, thus, chimerism can be established without irradiation. In control mice, TCR gammadelta and TCR alphabeta intraepithelial lymphocytes (IEL) developed efficiently from fetal liver cells, with a predominance of TCR alphabeta over TCR gammadelta IEL. In contrast, development in reconstituted thymectomized mice was heavily skewed toward TCR gammadelta IEL generation. In thymectomized mice, development of CD4+ 8- and CD4+ 8+ TCR alphabeta IEL did not occur, while TCR alphabeta CD8 alphabeta development was nearly absent. The results indicated that without irradiation the majority of TCR alphabeta IEL were thymus dependent, whereas TCR gammadelta IEL developed extrathymically. Thus, the discrepancies observed between different models of athymic development may be explained by the induction of T cell development as a result of irradiation.
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Nijeboer, Petula, Georgia Malamut, Gerd Bouma, Nadine Cerf-Bensussan, Frits Koning, Jeroen van Bergen, Christophe Cellier, and Chris J. J. Mulder. "Therapy in RCDII: Rationale for Combination Strategies?" Digestive Diseases 33, no. 2 (2015): 227–30. http://dx.doi.org/10.1159/000381076.
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Refractory coeliac disease type II (RCDII) is characterized by a continuous gluten-independent duodenal immune activation with an extremely high risk of malignant transformation. It is therefore considered as an indolent lymphoma. RCDII is characterized by the presence of villous atrophy (Marsh III A-C) in combination with an aberrant intra- epithelial lymphocyte (IEL) population consisting of >20% sCD3-CD7+iCD3+ IELs. The sCD3-CD7+iCD3+ IELs are a heterogeneous lineage-negative cell population, consisting of cells that do or do not express CD127/IL7Rα. Experiments using IEL from non-RCDII patients have indicated that while the CD127- cells are IL-15 responsive, the CD127+ cells are not. Together with the observation that some patients express an aberrant (monoclonal) TCRγδ phenotype, this confirms the heterogeneity of the aberrant IEL population in RCDII and suggests that the aberrant cells are heterogeneous with respect to their response to common γ-chain cytokines. Although cladribine with or without autologous stem cell transplantation is effective in the treatment of signs and symptoms of RCDII and improves survival as compared to symptomatic topical steroid therapy, cladribine failures still bear a high risk of malignant transformation, and the rate of enteropathy-associated T-cell lymphoma (EATL) development in this subgroup is extremely high. It might be hypothesized that the heterogenous nature of aberrant IEL and the high risk of malignant transformation require a treatment strategy which is effective despite this heterogeneity. RCDII should be seen more in the light of a low-grade/no mass lymphoma or pre-EATL. We would suggest an upfront combination therapy approach integrating inhibition of downstream Jak-STAT signalling pathways with conventional therapy (2-CDA) to hopefully effectively treat signs and symptoms of RCDII and accomplish a more effective EATL prevention.
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Pai, Man-Hui, Jun-Jen Liu, Sung-Ling Yeh, Wei-Jao Chen, and Chiu-Li Yeh. "Glutamine modulates acute dextran sulphate sodium-induced changes in small-intestinal intraepithelial γδ-T-lymphocyte expression in mice." British Journal of Nutrition 111, no. 6 (November 14, 2013): 1032–39. http://dx.doi.org/10.1017/s0007114513003425.
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The present study investigated the effect of glutamine (Gln) on dextran sulphate sodium (DSS)-induced changes in the expression of small-intestinal intraepithelial lymphocyte (IEL) γδ-T cells in mice. Mice were randomly assigned to a normal control (NC) group and two DSS-treated groups. The NC group and one of the DSS-treated groups (DSS-C) were fed a common semi-purified diet, while the other DSS-treated group (DSS-G) was fed an identical diet, except that part of casein was replaced by Gln, which provided 25 % of total amino acid nitrogen. After being fed the diets for 10 d, mice in the NC group were given distilled water, while the DSS-treated groups were given distilled water containing 2·5 % DSS for 5 d. At the end of the experiment, the mice were killed. The small-intestinal IEL γδ-T-cell subset was isolated for further analysis. The results indicated that DSS treatment resulted in a lower percentage of small-intestinal IEL γδ-T cells and higher mRNA expressions of interferon-γ, TNF-α, IL-17, complement 5a receptor and keratinocyte growth factor in IEL γδ-T cells. Gln administration increased the proportion of small-intestinal IEL γδ-T cells, and the expression levels of immunomodulatory mediator genes in IEL γδ-T cells were lower in the DSS-treated mice. The histological findings indicated that the immunoreactive intensity of the tight junction protein ZO-1 in the small-intestinal mucosa was higher in the DSS-G group than in the DSS-C group. These results indicate that pretreatment with Gln increases the proportion of small-intestinal IEL γδ-T cells and down-regulates γδ-T-cell-expressed inflammatory mediators, which may consequently ameliorate the severity of DSS-induced small-intestinal epithelial injury.
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Abe, T., H. Sugaya, and K. Yoshimura. "Analysis of T cell populations and IL-3 mRNA expression in mesenteric lymph node cells and intestinal intraepithelial lymphocytes in Strongyloides ratti-infected mice." Journal of Helminthology 72, no. 1 (March 1998): 1–8. http://dx.doi.org/10.1017/s0022149x00000894.
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AbstractT cell populations and IL-3 mRNA expression were analysed in mesenteric lymph node cells and intestinal intraepithelial lymphocytes (IEL) in Strongyloides ratti- infected mice. On days 7 and 12 post-infection, 2.6 times as many mesenteric lymph node cells were present in S. ratti- infected mice compared with uninfected mice. Although the percentages of CD3+, CD4+ and CD8+ cells decreased during infection, the absolute numbers of these cell types increased on day 7 due to an overall increase in the mesenteric lymph node cell number. The CD4/CD8 ratio in IEL was increased on day 5, whereas no significant change in the CD4/CD8 ratio was observed in the mesenteric lymph node cells. Expression of IL-3 mRNA, which is an important cytokine for the induction of murine mucosal mastocytosis and S. ratti- expulsion, was examined in mesenteric lymph nodes and IEL of uninfected and infected mice. IL-3 mRNA was detected in mesenteric lymph nodes of S. ratti-infected mice but not detected in the lymph nodes of uninfected mice. IL-3 mRNA was detected in IEL from both infected and uninfected mice with an 20-fold increase in expression in IEL of infected mice. Overall, IL-3 mRNA levels were higher in IEL than in mesenteric lymph nodes following S. ratti- infection. Expression of IL-4, IL-10, stem cell factor (SCF or c-kit ligand) and IFN-γ mRNA was also examined in these two tissues. IL-10 mRNA was not detected in any tissue examined and IFN-γ mRNA levels were unaltered as a result of an S. ratti- infection. Elevated expression of mRNA for SCF (5-fold) and IL-4 (20-fold) was observed in the mesenteric lymph nodes of infected mice. In contrast, SCF mRNA levels were similar in IEL of uninfected and infected animals and only a modest increase in IL-4 mRNA was observed in IEL of infected mice.
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Van Kerckhove, C., G. J. Russell, K. Deusch, K. Reich, A. K. Bhan, H. DerSimonian, and M. B. Brenner. "Oligoclonality of human intestinal intraepithelial T cells." Journal of Experimental Medicine 175, no. 1 (January 1, 1992): 57–63. http://dx.doi.org/10.1084/jem.175.1.57.
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T cells bearing the T cell receptor alpha/beta (TCR-alpha/beta) are the predominant lymphocyte population in the human intestinal epithelium. To examine if normal intestinal intraepithelial lymphocytes (IEL) have a TCR repertoire distinct from the TCR-alpha/beta repertoire in peripheral blood lymphocytes (PBL), comparative analysis of relative V beta gene usage in IEL and PBL was performed by quantitative polymerase chain reaction. In each of the six individuals examined, one to three V beta families made up more than 40% of the total V beta transcripts detected in the IEL, whereas there was a more even distribution of V beta gene usage in the paired PBL. The predominant V beta families, especially V beta 1, V beta 2, V beta 3, and V beta 6, were frequently shared among IEL of different individuals. PCR cloning and sequence analysis of the predominant V beta 6 family in two individuals revealed an identical V-D-J-C sequence in 13 of 21 clones obtained from one donor, and a different repeated sequence in 18 of 27 clones examined in the second donor. These data suggest that the V beta skewing in IEL is due to an oligoclonal T cell expansion and may reflect the response of the intestinal mucosal immune system to a restricted set of as yet undefined antigens present in the gut.
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Dunmore, P. Joy, S. H. Song, and Margot R. Roach. "A comparison of the size of fenestrations in the internal elastic lamina of young and old porcine aortas as seen with the scanning electron microscope." Canadian Journal of Physiology and Pharmacology 68, no. 2 (February 1, 1990): 139–43. http://dx.doi.org/10.1139/y90-022.
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The size of the fenestrations (windows) in the internal elastic lamina (IEL) of arteries may be important in the functioning of the blood vessel wall. The fenestrations are filled with collagen, muscle, and (or) ground substance, which must be removed to make the fenestration visible with the scanning electron microscope. All of the nonelastic components are removed with a hot alkali solution. Our experiments were designed to compare the fenestration size in the IEL of the thoracic aorta of young (6–8 weeks) and old (6–9 months) pigs. A protocol for digestion of young pig tissue was developed and showed that fresh young aortas should be digested in 0.1 M NaOH at 75 °C for 2 h and fixed tissue should be digested for 5 h. The average area of the fenestrations for young pig thoracic aortas digested for 2 h was 1.8 ± 0.29 (SE) μm2 and for the old pig aortas digested for 2 h was 1.7 ± 0.11 (SE) μm2. These values were not significantly different (p > 0.05), but the IEL from young pigs appeared rougher than the previously reported smooth IEL of the adult pigs.Key words: elastin, fixation, digestion, fenestration, scanning electron microscope.
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Nagler-Anderson, C., L. A. McNair, and A. Cradock. "Self-reactive, T cell receptor-gamma delta+, lymphocytes from the intestinal epithelium of weanling mice." Journal of Immunology 149, no. 7 (October 1, 1992): 2315–22. http://dx.doi.org/10.4049/jimmunol.149.7.2315.
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Abstract Intraepithelial T lymphocytes (IEL) are dispersed throughout the intestinal epithelial lining but their role in cellular immune defense is unknown. Their location suggests that their highly activated state may be due to constant exposure to bacterial Ag. To study IEL specificity and function we have prepared a panel of IEL-T cell hybridomas from both adult and weanling C57B1/6 mice. Many of these expressed TCR-gamma delta, a cell type rare in peripheral lymph nodes and spleen but predominant at epithelial surfaces. We have identified a subset of gamma delta T cells from weanling mice which is self reactive, i.e., these hybrids secrete IL-2 spontaneously, without antigenic stimulation or a requirement for APC. Self-reactive TCR-gamma delta+ hybrids and lines, all of which bear a particular TCR (V gamma 1.1C gamma 4V delta 6), have previously been derived from neonatal thymus and the skin. Northern blot and immunoprecipitation analyses suggest that the self-reactive IEL hybrids also bear a C gamma 4/V delta 6 TCR. Antibody inhibition experiments showed that the self-reactivity of the IEL hybrids is TCR mediated. Spontaneous IL-2 production was blocked by soluble anti-CD3 and anti-TCR-gamma delta antibodies but not by antibodies to the TCR-alpha beta. The self-reactive IEL hybrids lack class II MHC and the class I-like proteins CD1 and TLA but express class I MHC. IEL hybrids may also require the vitronectin receptor as an accessory molecule for their activation because spontaneous IL-2 production is blocked by antibody to the vitronectin receptor as well as by the extracellular matrix protein active site peptide RGDS, but not the control peptide RGES. V gamma 1.1C gamma 4V delta 6 T cells in the thymus, skin, and intestine may represent a small and unique subpopulation of lymphocytes with a potential for autoimmune reactivity at peripheral sites.
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Albors-Llorens, Albertina. "COURAGE V. CREHAN: JUDICIAL ACTIVISM OR CONSISTENT APPROACH?" Cambridge Law Journal 61, no. 1 (March 7, 2002): 1–52. http://dx.doi.org/10.1017/s0008197302341503.
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IN Case C-453/99 Courage Ltd. v. Crehan (judgment of 20 September 2001, not yet reported), the European Court of Justice has been confronted once more with the difficult task of reconciling the effectiveness of Community rights with national rules on remedies. By virtue of a series of agreements between Inntrepreneur Estates Ltd. (IEL), a company which owned public house estates, and Courage Ltd., a brewery with a 19% share of the United Kingdom market in sales of beer, all IEL tenants were required to purchase the whole of their beer requirements exclusively from Courage Ltd. In 1993, Courage Ltd. brought an action for the recovery from Mr. Crehan, a tenant of IEL, of a sum of more than £15,000 for unpaid deliveries of beer. Mr. Crehan contended that the exclusive purchasing obligation was anti-competitive because Courage Ltd. sold its beers to independent tenants of public houses at substantially lower prices than those imposed on IEL tenants. He claimed that the beer tie was therefore contrary to Article 81(1) EC and sought damages for loss caused to him by the imposition of the beer tie. Carnwath J. dismissed the counter-claim and found in favour of Courage Ltd. (Courage Ltd. v. Crehan [1998] E.G.C.S. 171). Mr. Crehan appealed.
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Kuhn, Kristi, Hanna M. Schulz, Alyssa K. Whitney, Eric L. Campbell, and Sean P. Colgan. "IL-6 producing intraepithelial lymphocytes in the colon modulate epithelial barrier integrity to protect against C. rodentium infection." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 200.11. http://dx.doi.org/10.4049/jimmunol.198.supp.200.11.
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Abstract Citrobacter rodentium infection of mice is frequently used as a model to study host responses during pathogenic gastrointestinal infection. IL-6 signaling in the colon is required for protection against C. rodentium infection; however the cellular source of IL-6 for this protection is unclear. Our previous work has identified IELs as one source of IL-6 in the colon. Therefore, we hypothesized that IL-6 produced by colonic IELs was sufficient to provide protection during C. rodentium infection. To test our hypothesis, we orally infected mice with C. rodentium following transfer of donor IL-6+/+ or IL-6−/− IELs into IL-6+/+ or IL-6−/− recipients. IL-6−/− mice that received IL-6−/− IELs had significantly more weight loss, increased intestinal histopathology, and increased bacterial translocation after 12 days of infection compared to transfer of IL-6+/+ IELs into recipient IL-6−/− mice and IL-6+/+ IELs into IL-6+/+ mice. IEL-derived IL-6 is functionally important in the maintenance of the epithelial barrier as IL-6−/− mice were noted to have increased paracellular permeability, decreased claudin-1 expression, and a thinner mucus-gel layer, all of which were reversed by transfer of IL-6+/+ IELs. In vitro studies utilizing model epithelia confirmed IL-6 was able to signal and increase claudin-1 and mucin-2 expression. Therefore, we conclude that IL-6 expression by IELs is sufficient to restore protection against C. rodentium infection through modulation of the epithelial barrier integrity.
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Pereira, P., D. Gerber, S. Y. Huang, and S. Tonegawa. "Ontogenic development and tissue distribution of V gamma 1-expressing gamma/delta T lymphocytes in normal mice." Journal of Experimental Medicine 182, no. 6 (December 1, 1995): 1921–30. http://dx.doi.org/10.1084/jem.182.6.1921.
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A hamster monoclonal antibody (mAb) recognizing an epitope in the V gamma 1-J gamma 4-C gamma 4 chain of the gamma/delta T cell receptor has been generated. Using this mAb, we have quantitated the occurrence of V gamma 1-bearing gamma/delta T cells in the developing thymus and in the lymphoid organs and several epithelia of adult mice. The V gamma 1-expressing cells constitute a minor gamma/delta T cell subpopulation during fetal and early postnatal life, but they constitute a major population of gamma/delta T cells in the thymus and in the peripheral lymphoid organs in adult mice. In addition, we found that V gamma 1-bearing cells comprise a large proportion (15-60%) of the gamma/delta T cells present in the intestinal epithelium (i-IEL) in all strains of mice tested. V gamma 1+ i-IEL are present in athymic (nude) mice and in antigen-free mice, demonstrating that they can develop extrathymically and that their presence in the intestinal epithelium is independent of the antigenic load of the gut. Our results show that V gamma 1-bearing lymphocytes account for the largest population of gamma/delta T cells in the mouse. This population includes a thymus-dependent component that homes to the secondary lymphoid organs and a thymus-independent component that constitutes a major fraction of the gamma/delta i-IELs.
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Guy-Grand, D., N. Cerf-Bensussan, B. Malissen, M. Malassis-Seris, C. Briottet, and P. Vassalli. "Two gut intraepithelial CD8+ lymphocyte populations with different T cell receptors: a role for the gut epithelium in T cell differentiation." Journal of Experimental Medicine 173, no. 2 (February 1, 1991): 471–81. http://dx.doi.org/10.1084/jem.173.2.471.
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Mouse gut intraepithelial lymphocytes (IEL) consist mainly (90%) of two populations of CD8+ T cells. One bears heterodimeric alpha/beta CD8 chains (Lyt-2+, Lyt-3+), a T cell receptor (TCR) made of alpha/beta chains, and is Thy-1+; it represents the progeny of T blasts elicited in Peyer's patches by antigenic stimulation. The other bears homodimeric alpha/alpha CD8+ chains, contains no beta chain mRNA, and is mostly Thy-1- and TCR-gamma/delta + or -alpha/beta +; it is thymo-independent and does not require antigenic stimulation, as shown by its presence: (a) in nude and scid mice; (b) in irradiated and thymectomized mice repopulated by T-depleted bone marrow cells bearing an identifiable marker; (c) in thymectomized mice treated by injections of monoclonal anti-CD8 antibody, which lead to total depletion of peripheral CD8+ T lymphocytes; and (d) in germ-free mice and in suckling mice. In young nude mice, alpha/alpha CD8 chains, CD3-TCR complexes, and TCR mRNAs (first gamma/delta) are found on IEL, while they are not detectable on or in peripheral or circulating lymphocytes or bone marrow cells. IEL, in contrast to mature T cells, contain mRNA for the RAG protein, which is required for the rearrangement of TCR and Ig genes. We propose that the gut epithelium (an endoderm derivative, as the thymic epithelium) has an inductive property, attracting progenitors of bone marrow origin, and triggering their TCR rearrangement and alpha/alpha CD8 chains expression, thus giving rise to a T cell population that appears to belong to the same lineage as gamma/delta thymocytes and to recognize an antigenic repertoire different from that of alpha/beta CD8+ IEL.
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Hosoe, Naoki, Soichiro Miura, Chikako Watanabe, Yoshikazu Tsuzuki, Ryota Hokari, Tokushige Oyama, Yoichi Fujiyama, Hiroshi Nagata, and Hiromasa Ishii. "Demonstration of functional role of TECK/CCL25 in T lymphocyte-endothelium interaction in inflamed and uninflamed intestinal mucosa." American Journal of Physiology-Gastrointestinal and Liver Physiology 286, no. 3 (March 2004): G458—G466. http://dx.doi.org/10.1152/ajpgi.00167.2003.
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It has recently been suggested that C-C chemokines may play a role in the organ-specific homing of lymphocytes, but there is not enough in vivo evidence in intestinal mucosa. The aim of this study was to examine whether thymus-expressed chemokine (TECK)/CCL25 and its ligand CCR9 are involved in T-lymphocyte interaction with microvessels of murine intestinal mucosa. T lymphocytes from the small intestine were fluorescence labeled, and their adhesion to mucosal microvessels was observed by intravital microscopy. Lamina proprial lymphocytes (LPL) and intraepithelial lymphocytes (IEL) adhered to both the small intestine and colon, and desensitization of CCR9 with TECK/CCL25 or anti-TECK/CCL25 antibody significantly inhibited these adhesions only in small intestine. At both sites, TNF-α significantly increased LPL adhesion but not IEL adhesion. Desensitization of CCR9 or anti-TECK/CCL25 antibody also attenuated the TNF-α-induced LPL adhesion in the small intestine. Increased expression of TECK/CCL25 by TNF-α was observed in the lamina propria of small intestine. TECK/CCL25 may thus play an important role in the adherence of mucosal lymphocytes to the microvessels of the small intestine but not the colon under uninflamed as well as inflamed conditions.
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Cebra, John J., Sangeeta Bhargava Periwal, Gwen Lee, Fan Lee, and Khushroo E. Shroff. "Development and Maintenance of the Gut-Associated Lymphoid Tissue (Galt): the Roles of Enteric Bacteria and Viruses." Developmental Immunology 6, no. 1-2 (1998): 13–18. http://dx.doi.org/10.1155/1998/68382.
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GALT can be subdivided into several compartments: (a) Peyer's patches (PP); (b) lamina propria (LP); and (c) intraepithelial leukocyte (IEL) spaces. The B-cell follicles of PP are quiescent in neonatal and germ-free (GF) adult mice. Germinal centers (GC), including sIgA+blasts, appear in the B follicles of formerly GF adult mice about 10-14 days after monoassociation with various gut commensal bacteria. The GC wax and wane over about a 3-week period, although the bacterial colonizers remain in the gut at high density. Neonatal mice, born of conventionally reared (CV), immunocompetent mothers, display GC reactions in PP postweaning, although pups of SCID mothers display precocious GC reactions at about 14 days of life. Normally, gut colonization of neonates with segmented filamentous bacteria (SFB) leads to explosive development of IgA plasmablasts in LP shortly after weaning. Commensal gut bacteria and the immunocompetency of mothers also appears to control the rate of accumulation of primary B cells from “virgin” B cells in neonates.Enteric reovirus infection by the oral route can cause the activation of CD8+T cells in the interfollicular regions of PP and the appearance of virus-specific precursor cytotoxic T lymphocytes (pCTL) in the IEL spaces. Such oral stimulation can also lead to “activation” of both CTL and natural killer (NK) cells in the IEL spaces. More normally, colonization of the gut with SFB also leads to similar activations of NK cells and “constitutively” cytotoxic T cells.
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Lambolez, Florence, Orly Azogui, Anne-Marie Joret, Corinne Garcia, Harald von Boehmer, James Di Santo, Sophie Ezine, and Benedita Rocha. "Characterization of T Cell Differentiation in the Murine Gut." Journal of Experimental Medicine 195, no. 4 (February 11, 2002): 437–49. http://dx.doi.org/10.1084/jem.20010798.
Full textAbstract:
Gut intraepithelial CD8 T lymphocytes (T-IEL) are distinct from thymus-derived cells and are thought to derive locally from cryptopatch (CP) precursors. The intermediate stages of differentiation between CP and mature T-IEL were not identified, and the local differentiation process was not characterized. We identified and characterized six phenotypically distinct lineage-negative populations in the CP and the gut epithelium: (a) we determined the kinetics of their generation from bone marrow precursors; (b) we quantified CD3-ϵ, recombination activating gene (Rag)-1, and pre-Tα mRNAs expression at single cell level; (c) we characterized TCR-β, -γ, and -α locus rearrangements; and (d) we studied the impact of different mutations on the local differentiation. These data allowed us to establish a sequence of T cell precursor differentiation in the gut. We also observed that the gut differentiation varied from that of the thymus by a very low frequency of pre-Tα chain mRNA expression, a different kinetics of Rag-1 mRNA expression, and a much higher impact of CD3 ϵ/δ and pre-Tα deficiencies. Finally, only 3% of CP cells were clearly involved in T cell differentiation, suggesting that these structures may have additional physiological roles in the gut.
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Godbout, J., M. Wulczynski, H. J. Galipeau, M. Constante, T. Ribeiro, D. Sloboda, and E. Verdu. "A10 EARLY LIFE SENSITIZATION TO GLUTEN INDUCES SUSTAINED IMMUNOPATHOLOGY IN DR3-DQ2 MICE." Journal of the Canadian Association of Gastroenterology 5, Supplement_1 (February 21, 2022): 11–13. http://dx.doi.org/10.1093/jcag/gwab049.009.
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Abstract Background Celiac disease (CeD) is an autoimmune T-cell mediated enteropathy, triggered by gluten, a group of proteins found in wheat, barley, and rye. The defined role of gluten as a dietary trigger, necessary genes (HLA-DQ2 and/or DQ8), and tissue transglutaminase (TG2) as the autoantigen together, are unique features of CeD. Although CeD onset can occur at any age, first dietary introduction of gluten during infancy is a critical window of exposure, especially in infants homozygous for the HLA DQ2.5 allele. While adult sensitization studies have been recently performed in DR3-DQ2 mice, the consequences of early life gluten sensitization timing remain unexplored. Aims Our aim was to characterize gluten-immunopathology and CeD-specific serology in specific pathogen free (SPF) DR3-DQ2 transgenic mice sensitized to gluten, 1 week before weaning. Methods Seven-week-old SPF DR3-DQ2 transgenic mice, kept on a gluten-free diet (GFD), were paired for breeding. At post-natal day 3, pups were standardized to 4 per litter (n=2 male, n=2 female) to ensure equal nutrition across litters. At 14 days of age, pups were sensitized with pepsin-trypsin digested gliadin and cholera toxin (CT) three times in one week (n=15). At 21 days of age, pups were weaned and placed either on a gluten-containing diet (n=7), (equivalent of 20g/d of gluten in a human diet -high dose-) or an isocaloric GFD (n=8) until 10 weeks of age. Non-sensitized controls (n=7) received only CT and were kept on the GFD. At sacrifice, serum was collected for anti-TG2 and anti-gliadin antibodies (AGA). Jejunal tissue was collected for histological analysis using villus-to-crypt (V/C) ratios and CD3+ intraepithelial lymphocytes (IEL) counts. Results Gluten-sensitized mice placed on a gluten-containing diet post-weaning had lower V/C ratios and higher CD3+ IEL counts compared with controls (p<0.01). Pre-weaning sensitized mice that were kept on a GFD post-weaning had sustained decreases in V/C ratios and higher CD3+ IEL counts (p<0.01). Out of 15 sensitized mice, 7 developed positive anti-gliadin IgA (p=0.02) and 4 had positive anti-TG2 IgA antibodies (p=0.01) in intestinal contents, irrespective of gluten in the diet. None of the controls had detectable AGA or anti-TG2 antibodies. Conclusions Pre-weaning gluten sensitization of DR3-DQ2 mice induced prolonged gluten immunopathology that did not reverse after 5 weeks on a GFD. Our results indicate that young DR3-DQ2 mice are susceptible to gluten sensitization, with sustained immunopathology, suggesting a critical window of vulnerability in familial carriers of DQ2.5. This novel model will be useful to investigate environmental cofactors at the first time of gluten introduction to the diet. Funding Agencies CIHR
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Clarizio, A. V., H. J. Galipeau, J. Jury, L. Rondeau, J. Godbout, L. Williams, B. Anderson, and E. Verdu. "A19 NOVEL HLA-DQ2 TRANSGENIC MICE DEVELOP GLUTEN-IMMUNOPATHOLOGY FOLLOWING GLUTEN SENSITIZATION." Journal of the Canadian Association of Gastroenterology 3, Supplement_1 (February 2020): 22–23. http://dx.doi.org/10.1093/jcag/gwz047.018.
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Abstract Background Celiac disease (CeD), an autoimmune and chronic inflammatory enteropathy triggered by the ingestion of gluten, is associated with HLA-DQ2 (~90%) and, to a lesser extent, HLA-DQ8. We previously characterized a humanized mouse model of gluten sensitivity that expresses the HLA-DQ8 allele, and that develops mild innate and adaptive immune pathways related to DQ8+ CeD patients. Gluten peptides recognized in the context of DQ2 differ from those bound to DQ8 molecules and homozygous carriage of HLA-DQ2 provides higher risk for CeD development. Thus, characterization of a transgenic HLA-DQ2 model would allow for the investigation of disease pathways that affect the majority of celiac patients. Aims Our aim was to determine gluten-immune responses and small intestinal pathology in C57BL/6 mice humanized with the HLA DR3-DQ2 gene. Methods To break oral tolerance to gluten, DR3-DQ2 mice were orally sensitized with gliadin (5mg/mL) and cholera toxin (CT; 2.5mg/mL) before being challenged with gluten (10mg/mL) for three weeks. Non-sensitized DR3-DQ2 controls mice received CT and a sham challenge. To determine the effects of long-term exposure to gluten, gliadin sensitized DR3-DQ2 mice were placed on a gluten-containing diet for twelve weeks. Controls were given CT and remained on a gluten-free diet. Pathology was evaluated by CD3+ intraepithelial lymphocytes (IELs) counts; villus-crypt (V/C) ratios. Gluten-induced immune responses were evaluated by anti-tissue transglutaminase-2 (tTG) and anti-gliadin antibodies, inflammatory gene expression by Nanostring Technology, and CD4+ T cell proliferation using flow cytometry. Intestinal permeability was measured in vitro by Ussing Chamber. Results DR3-DQ2 mice that were sensitized and challenged with gluten for three weeks had higher IEL counts, lower V/C ratios, higher anti-gliadin and tTG antibodies, which are used in the serological diagnosis of the disease. Gluten sensitized mice also has higher expression of pro-inflammatory genes, and an induction of gluten specific T cells, but no changes in permeability compared to control mice. DR3-DQ2 mice given a gluten-containing diet for 12 weeks also had higher IEL counts, lower V/C ratios, and higher anti-gliadin and tTG antibodies, but had no changes in permeability compared to controls. Conclusions These results indicate that mice humanized with the HLA DR3-DQ2 gene show innate and gluten-specific immune responses following sensitization. Thus, mice expressing HLA-DQ2 represents a novel model of gluten sensitivity that can be used to investigate celiac disease pathways related to gluten peptide repertoire that binds to DQ2 MHC class II, encoded by the HLA gene expressed by the majority of celiac patients. Supported by CIHR to EFV and OGS to AVC Funding Agencies CIHROGS
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Sacirbegovic, Faruk, Jieqing Zhu, Jinling Liu, Sarah Rosenberger, Mark J. Shlomchik, and Warren D. Shlomchik. "Identifying Tissue-Resident Memory T Cells in Graft-Versus-Host Disease." Blood 128, no. 22 (December 2, 2016): 4544. http://dx.doi.org/10.1182/blood.v128.22.4544.4544.
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Abstract Tissue-resident memory T cells (TRM) are a newly described subset of transcriptionally-distinct memory CD4 and CD8 cells that persist in barrier and non-barrier tissues. They are non-circulating, able to facilitate the recruitment of circulating effector cells and elicit rapid recall responses. The majority of TRM cells can be identified by the expression of CD69 and αE integrin, CD103. However, CD69+CD103- TRM cells have also been described. In graft-vs-host disease (GVHD), alloreactive effector T cells enter GVHD target tissues and mediate tissue damage through direct and indirect mechanisms.The recruitment of effector T cells into tissues is in general not dependent on the tissue expressing the target antigen; and even if a target antigen is available in a tissue, there could be niches free of presented alloantigen. We therefore hypothesized that even in GVHD where alloantigen is ubiquitous, TRM may develop. We first explored TRM formation in a CD4 T cell receptor (TCR) transgenic (Tg) GVHD system wherein donor BALB/c RAG2-/- TS1 TCR Tg T cells target the S1 peptide derived from HA, which is expressed ubiquitously in BALB/c RAG2-/- HA104 mice. In this model, GVHD is induced by <1000 TS1 cells and is manifest by weight loss, death, and TS1-infiltrative pathology of the skin, liver, small bowel and colon. We harvested tissues from GVHD mice at days 21 and 28 post-transplant and quantitated TS1 cells with a TRM phenotype. At day 21, a fraction of TS1 cells expressed CD69+CD103+ (as % of total TS1 cells) in the epidermis (2.8% ± 1.2), dermis (11.3% ±7.9), colon (11.5% ±4.5) and small intestine (SI) intraepithelial (IEL) (33.4% ±5.8) and lamina propria (LP) (6.8% ±0.8). At day 28, CD69+CD103+ TS1 cells (% of total TS1) were present in the epidermis (13.6% ± 1.9), dermis (15.9% ± 7.7), colon (30.8% ± 6.7) and the SI IEL (69.1% ± 9.2) and SI LP (18.7 ±3.2). While there were no CD69+CD103+ TS1 in the spleen, bone marrow (BM) or liver (days 21 and 28), a small number (1.5% ±0.7, day 21; 1.7% ± 0.3, day 28) were found in the mesenteric lymph node (mLN). CD103- TRM have been described in the liver and secondary lymphoid organs. Consistent with this, CD69+CD103- TS1 cells (% of total TS1) were found in the liver (24.6% ±6.3, day21; 31.5% ±3.1, day 28), BM (23.1% ±3.1, day 21; 35.4% ±3.2, day 28), spleen (1.8% ±0.6, day 21; 9.1% ±0.7, day 28) and mLN (9.9% ±4.5, day 21; 23.5% ± 3.7, day 28). We are currently confirming the TRM identity of TS1 cells based on their transcriptional and migratory profiles and these data will be presented. Alloreactive TRM were also identified in the B6 (H-2b) into 129 (H-2b) MHC-matched, multiple minor histocompatibility antigen (miHA)-mismatched model in which GVHD is induced by a mix of CD4 and CD8 cells. A fraction of CD8 cells target the Kb-restricted miHA LTFNYRNL derived from H60, which can be tracked with tetramers (TetH60). At day 22 post-transplant, CD69+CD103+ CD4 cells (% of total CD4 T cells) were found in the epidermis (22.9% ±1.2), dermis (19.7% ±2.8), colon (9.4% ±3.1), SI IEL (26.5% ±2.6), SI LP (16.1% ± 3.7) and mLN (6.9% ±3.0). CD8+TetH60+ T cells (% of total CD8T cells) were detected in the epidermis (7.5% ± 3.5), dermis (10.6% ± 6.7), colon (6.9% ± 2.7), SI IEL (8.9% ±6.2), SI LP (10.2% ±3.8), mLN (3.0 ± 0.7) and spleen (9.0% ±2.9). A fraction of CD8+TetH60+ cells in the dermis (7.3% ±1.9), colon (18.1% ±12.2), SI IEL (50.1% ±4.3), SI LP (52.6% ±13.6), and mLN (6.3% ± 0.8) were CD69+CD103+, suggesting that alloreactive H60-directed CD8 T cells acquired a TRM phenotype. Using two different murine models, we found GVHD-inducing T cells with TRM phenotypes. Future experiments will confirm the TRM identity of these cells and will determine their importance in the maintenance of GVHD, perhaps by serving as a reservoir of cells that maintain GVHD locally. Disclosures No relevant conflicts of interest to declare.
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Khaldi, Samira, Gilles Gargala, Laetitia Le Goff, Simon Parey, Arnaud Francois, Jean Fioramonti, Jean-Jacques Ballet, Jean-Paul Dupont, Philippe Ducrotté, and Loïc Favennec. "Cryptosporidium parvum Isolate-Dependent Postinfectious Jejunal Hypersensitivity and Mast Cell Accumulation in an Immunocompetent Rat Model." Infection and Immunity 77, no. 11 (August 17, 2009): 5163–69. http://dx.doi.org/10.1128/iai.00220-09.
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ABSTRACT Cryptosporidium spp. are a cause of self-limited diarrhea in immunocompetent hosts. In immunocompetent rats, Cryptosporidium parvum infection induced digestive hypersensitivity, a key pathophysiological factor in functional digestive disorders such as irritable bowel syndrome (IBS). In such a rat model, we sought to document whether jejunal hypersensitivity depends on C. parvum isolate and is associated with a mast cell accumulation. Five-day-old rats were orally administered 105 oocysts of either Nouzilly (NoI) or Iowa (IoI) C. parvum isolate. NoI-infected rats exhibited the lowest food intake on days 7 and 14 postinfection (p.i.). On day 7 p.i., small intestine villus atrophy, crypt hyperplasia, and inflammatory cell infiltration were prominent in NoI-infected rats, with higher numbers of Cryptosporidium forms than in IoI-infected rats. Compared to uninfected control rats, jejunal intraepithelial lymphocytes (IELs) were increased only in NoI-infected rats on day 14 p.i. On day 50 p.i., jejunal hypersensitivity to distension was found only in NoI-infected rats; this hypersensitivity is associated with activated mast cell accumulation. The number of mast cells in the jejunal lamina propria was increased from day 36 p.i. in NoI-infected rats and only at day 120 p.i. in IoI-infected rats. Our data suggest that both the severity of infection (weight loss, reduced food intake, villus atrophy, and IEL accumulation) and the onset of a jejunal hypersensitivity after infection in association with an activated mast cell accumulation are isolate dependent and related to NoI infection. This cryptosporidiosis rat model is a relevant model for the study of underlying mechanisms of postinfectious IBS-like symptoms.
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Shammas, Nicolas W., W. John Shammas, Susan Jones-Miller, James T. Torey, Ehrin J. Armstrong, Qais Radaideh, and Gail A. Shammas. "Optimal Vessel Sizing and Understanding Dissections in Infrapopliteal Interventions: Data From the iDissection Below the Knee Study." Journal of Endovascular Therapy 27, no. 4 (May 18, 2020): 575–80. http://dx.doi.org/10.1177/1526602820924815.
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Purpose: To investigate if imaging with intravascular ultrasound (IVUS) yields a more accurate estimate of vessel diameter and the presence of dissections than angiography after intervention in the infrapopliteal arteries. Materials and Methods: A prospective, single-center study enrolled 20 consecutive patients (mean age 74.1±12.4 years; 12 women) with infrapopliteal disease who were treated with percutaneous transluminal angioplasty (PTA; n=10) or orbital atherectomy (OA) followed by PTA (n=10). The majority of patients were hypertensive and half were diabetic. The overall lesion length was 7.3±6.3 cm, and the diameter stenosis was 80.3%±22.1%. The baseline characteristics did not differ between the groups. Vessel diameters were measured using IVUS from the internal elastic lamina (IEL) to the IEL. IVUS was performed at baseline, post PTA or OA, and post OA+PTA. Quantitative vascular angiography (QVA) and IVUS were analyzed by a core laboratory. Dissections on cine images were categorized based on the National Heart Lung and Blood Institute (NHLBI) classification, while the arc and depth were used to characterize dissections on IVUS images. Results: Mean vessel diameter by QVA was 2.9±0.6 vs 4.0±1.0 mm by IVUS according to the core laboratory (mean difference 1.1±0.9, p<0.001). On angiography, there were 7 dissections after PTA (6 C, 1 D), 1 dissection after OA (1 B), and 2 dissections after OA+PTA (1 A, 1 B; p=0.028 vs post PTA). IVUS uncovered 3.8 times more dissections than seen on angiography. There were 23 dissections after PTA (18 intima, 3 media, 2 adventitia), 12 dissections after OA (8 intima, 1 media, 3 adventitia), and 11 dissections following OA+PTA (7 intima, 1 media, 3 adventitia; p=0.425 vs PTA). Bailout stenting (all due to angiographic dissections ≥C) was necessary in 6 of the PTA cohort and none of the OA+PTA group. Conclusion: In addition to underestimating the infrapopliteal vessel diameter by ~25%, angiography underappreciated the presence and severity of post-intervention dissections vs IVUS, particularly in the OA+PTA group.
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Sacirbegovic, Faruk, Sarah Rosenberger, Jieqing Zhu, Jinling Liu, Mark J. Shlomchik, and Warren D. Shlomchik. "Identifying the Clonal Origins of Gvhd-Causing T Cells." Blood 128, no. 22 (December 2, 2016): 497. http://dx.doi.org/10.1182/blood.v128.22.497.497.
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Abstract In graft-versus-host disease (GVHD) donor αβ T cells in the allograft recognize host tissues as non-self and cause multi-organ damage through direct and indirect mechanisms. This multi-organ disease is mediated by T cells acquiring diverse phenotypes, including expression of homing receptors, adhesion molecules, cytokines and other effector molecules. How this diversity is generated at a clonal level is not understood. In the one extreme, each type of effector could be derived from a single T cell expressing a unique antigen receptor that may even target a unique antigen. Alternatively, a single T cell targeting a single antigen may be able to differentiate into a variety of effectors (Fig. 1). A second related question is whether GVHD is maintained within affected organs. That is, what is the importance of the continued recruitment of new blood-derived alloreactive effectors in maintaining GVHD. To answer these questions, we needed 1) a system in which GVHD-inducing T cells could be unambiguously tracked, as in polyclonal systems it is difficult if not impossible to know which T cells are disease causing; and 2) a way to distinguish GVHD-inducing T cells at the clonal level. To address the first point, we used a CD4 T cell receptor (TCR) transgenic (Tg) model of GVHD wherein GVHD is induced by the transfer of <1000 BALB/c RAG2-/- TS1 TCR Tg T cells, which recognize the S1 peptide derived from HA, into BALB/c RAG2-/- HA104 Tg recipients that ubiquitously express HA. GVHD is manifest by weight loss, death, and TS1-infiltrative pathology of the skin, liver, small intestine and colon. In order to determine the clonal contributions, we crossed TS1 Tg mice to combinations of CD45.1, CD45.2, Thy1.1, Thy1.2 and GFP backgrounds to create a matrix of up to 9 distinguishable TS1 cells. Using this model, we first characterized the variety of TS1 effector phenotypes being generated across tissues at day 14 and 21 post-transplant and will be presenting data detailing these TS1 phenotypes, including the expression of CCR9, α4β7 integrin, and cutaneous lymphocyte antigen (CLA). To confirm that different matrix cells perform similarly, we assessed the development of total TS1 effector cells in mice that had received an equal number of 5 distinct TS1 cell combinations. We found that within individual mice (n=5), there is comparable distribution of GVHD-causing TS1 effector cells (% of total TS1 cells) from all 5 TS1cell matrices across tissues 14-21 days post-transplant (Table 1). We next tested our ability to detect total effector cells derived from transferring low numbers of TS1 cells (5-500 cells) of a given matrix combination. We found that within individual mice (n=9), the transfer of 5 and 10 TS1 clones of a given matrix results in disparate distribution of TS1 effectors (% of total TS1 cells) across tissues 14-21 days post-transplant. As an example for one mouse at day 14, the fraction of TS1effectors derived from 10 TS1clones of one matrix was 0.8% (spleen), 1.9% (mLN), 1.4% (BM), 0.6% (colon), 4.4% (SI IEL), 1.1% (SI LP), 1.2% (skin) and 1.5% (liver). In that same mouse, the fraction of TS1effectors derived from 5 TS1clones of a different matrix was 0.7% (spleen), 0.9% (mLN), 0.4% (BM), 1.1% (colon), 1.1% (SI IEL), 0.6% (SI LP), 0.6% (skin) and 2.7% (liver). We made similar observations in mice at day 21, where the percentage of TS1 effectors derived from 5 TS1 clones in one mouse was 2.5% (spleen), 2.0% (BM), 2.8% (mLN), 0.2% (SI IEL), 2.3% (SI LP), 1.1% (skin) and 1.6% (liver). When focusing on specific TS1 phenotypes derived from 5 and 10 TS1clones, we also found unequal distributions of these cells across different tissues. For example, the distribution of CD69+CD103+ TS1 cells (% of total TS1 cells) derived from 5 TS1clones was 2.9% (colon), 1.5% (SI IEL) and 1.9% (SI LP) in one mouse at day 14. Similarly, TS1 cells expressing α4β7 integrin also varied in distribution across tissues, with one mouse having 2.2% (spleen), 0.5% (mLN) and 0% (BM) α4β7+ TS1 cells. These data suggest that tissue GVHD effectors are not simply in equilibrium with blood, consistent with some local GVHD maintenance. Furthermore, the presence of 5 TS1 clones in more than one effector type suggests that single cells can differentiate into multiple effectors. We are now directly addressing these possibilities by analyzing progeny derived from many sorted single cells. Disclosures No relevant conflicts of interest to declare.
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Wulczynski, M., J. Blom, G. H. Rueda, M. Pinto-Sanchez, M. Constante, S. Rahmani, J. Murray, D. Armstrong, H. Galipeau, and E. Verdu. "A36 EFFECT OF FIBRE SUPPLEMENTATION IN CELIAC DISEASE: IMPLICATIONS FOR MUCOSAL HEALING." Journal of the Canadian Association of Gastroenterology 7, Supplement_1 (February 14, 2024): 20–21. http://dx.doi.org/10.1093/jcag/gwad061.036.
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Abstract Background Celiac disease (CeD) is a T-cell mediated enteropathy driven by gluten in genetically susceptible individuals. Mucosal inflammation persists for years even when treated with a gluten-free diet (GFD). Although patients may be advised to increase dietary fibre, some fibres are poorly tolerated leading, potentially, to suboptimal fibre intake and hampering dietary recommendations. Aims Our project aims to investigate whether and how the supplementation of two fibre types with distinct functional profiles affects the recovery of gluten-immunopathology in a mouse model of gluten-sensitivity. We hypothesize that: (1) HylonVII, but not inulin, accelerates mucosal recovery by increasing microbial carbolytic activity and production of short-chain fatty acids (SCFA); (2) fibre intake is decreased in CeD patients, leading to decreased SCFA production. Methods NOD-DQ8 mice were immunized to gliadin and then placed on a gluten-containing diet. Three weeks later, mice were returned to GFD supplemented with or without HylonVII or inulin to 15% total fibre, for 6, 10, or 12 weeks. Recovery from gluten-induced pathology was evaluated by counting CD3+ intraepithelial lymphocytes (IELs) in villi tips and measuring villus-to-crypt (V/C) ratios in the small intestine. Adult CeD patients (newly diagnosed n=6 and GFD-treated n=7) and healthy controls (n=5) completed a food frequency questionnaire (Victoria DQES v2) and provided fecal samples for evaluation of SCFA production. Fecal SCFA were measured with targeted single ion monitoring gas-chromatography/mass-spectrometry (Agilent) as markers of fibre metabolism. Results In gluten immunized mice, 3 weeks of wheat diet led to increased CD3+ IEL counts and decreased V/C ratios (pampersand:003C0.01), which remained abnormal after 6 weeks of GFD. Mice consuming GFD+inulin, but not HylonVII, normalized CD3+ IELs and V/C ratios, had higher fecal SCFAs (pampersand:003C0.05) (primarily acetate), and a higher carbolytic-proteolytic metabolite ratio (pampersand:003C0.05) compared with mice consuming a GFD alone for six weeks. Only 1/7 patients with CeD on GFD met the minimum recommendation for fibre intake (25 g/day, Health Canada), compared with 5/11 participants on gluten-containing diets. SCFA production was impaired in active but not in treated CeD patients (2563 vs 3744 μg/g, pampersand:003C0.01). Conclusions In gluten-immunized NOD-DQ8 mice, GFD+inulin accelerates mucosal recovery, which is associated with increased fecal SCFA. In our pilot clinical data, patients on a GFD did not meet the recommendation for fibre intake although they were capable of metabolizing fibre to produce SCFA that could facilitate mucosal recovery. Together these results suggest that CeD on GFD may benefit from increasing fibre, such as inulin, during the GFD. Funding Agencies CIHRCeliac Canada
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Stewart, Taryn M., Ilse Huijskens, Leslie G. Willis, and David A. Theilmann. "The Autographa californica Multiple Nucleopolyhedrovirus ie0-ie1 Gene Complex Is Essential for Wild-Type Virus Replication, but either IE0 or IE1 Can Support Virus Growth." Journal of Virology 79, no. 8 (April 15, 2005): 4619–29. http://dx.doi.org/10.1128/jvi.79.8.4619-4629.2005.
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ABSTRACT The immediate-early ie0-ie1 gene complex expresses the only baculovirus spliced gene that produces an alternate protein product. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) IE1 is a potent transcriptional transactivator that is essential for viral replication in transient assays. IE1 contains 582 amino acids that are arranged into different domains, including an acidic activation domain at the N terminus, a DNA binding domain, and an oligomerization domain at the C terminus. IE0 is a 52-amino-acid N-terminally elongated form of IE1. We investigated the functions of IE0 and IE1 in virus-infected cells by constructing the first ie1 open reading frame knockout virus. An infectious AcMNPV bacmid was used to generate the ie1 knockout, and the resulting virus, AcBacIE1KO, effectively deletes both ie0 and ie1. AcBacIE1KO does not infect Spodoptera frugiperda cells, showing that the ie0-ie1 gene complex is essential for viral infection. Rescue viruses of AcBacIE1KO were constructed that express only IE1, IE1 and IE0, or only IE0. Our results show that both IE0 and IE1 can function independently, but not equivalently, to support replication, producing infectious virus. Viruses expressing predominately, or only, IE0 produced significantly fewer cells with polyhedra than either the IE1 counterpart or wild-type virus. In addition, DNA replication was prolonged and budded virus and late gene expression were delayed. Viruses expressing only IE1 also produced fewer polyhedra, but replication was slightly faster and achieved higher levels than that of the wild-type virus. Both IE0 and IE1 are therefore required and must be expressed in the correct quantitative ratios to achieve a wild-type infection.
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Nakajima, P. B., C. J. Betz, and D. Hansburg. "Expression of identical V alpha V beta gene pairs by IE-alloreactive and IE-restricted, antigen-specific T cells from MHC disparate mice. Evidence for thymic selection of V(D)J combinations." Journal of Immunology 144, no. 1 (January 1, 1990): 348–57. http://dx.doi.org/10.4049/jimmunol.144.1.348.
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Abstract Alloreactive T cell hybridomas specific for IEk and/or IEb MHC Ag were obtained from IE-nonexpressor (IE alpha b) mice. The TCR V alpha and V beta gene segments used were identified by Northern blot and RNase protection. A large proportion (24 of 80 hybridomas tested) employed the same V alpha genes (V alpha 11.1 or V alpha 11.2) as are utilized in the IEk and IEb restricted response to the Ag cytochrome c. Of these 24 alloreactive hybridomas, 10 also expressed V beta genes utilized in the IE plus cytochrome c repertoire. Structural similarity between the two related sets of TCR indicates that V alpha segments can play a determining role in MHC specificity. These data also suggest that thymic selection based on TCR reactivity with self-MHC products acts on particular V(D)J combinations rather than on V alpha V beta pairings alone.
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Koh, Kyungmi, Karim Lee, Jin-Hyun Ahn, and Sunyoung Kim. "Human cytomegalovirus infection downregulates the expression of glial fibrillary acidic protein in human glioblastoma U373MG cells: identification of viral genes and protein domains involved." Journal of General Virology 90, no. 4 (April 1, 2009): 954–62. http://dx.doi.org/10.1099/vir.0.006486-0.
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Human cytomegalovirus (HCMV) has tropism for glial cells, among many other cell types. It was reported previously that the stable expression of HCMV immediate-early protein 1 (IE1) could dramatically reduce the RNA level of glial fibrillary acidic protein (GFAP), an astroglial cell-specific intermediate filament protein, which is progressively lost with an increase in glioma malignancy. To understand this phenomenon in the context of virus infection, a human glioblastoma cell line, U373MG, was infected with HCMV (strain AD169 or Towne). The RNA level of GFAP was reduced by more than 10-fold at an m.o.i. of 3 at 48 h post-infection, whilst virus treated with neutralizing antibody C23 or with UV light had a much-reduced effect. Treatment of infected cells with ganciclovir did not prevent HCMV-mediated downregulation of GFAP. Although the expression of GFAP RNA is downregulated in IE1-expressing cells, a mutant HCMV strain lacking IE1 still suppressed GFAP, indicating that other IE proteins may be involved. IE2 is also proposed to be involved in GFAP downregulation, as an adenoviral vector expressing IE2 could also reduce the RNA level of GFAP. Data from the mutational analysis indicated that HCMV infection might affect the expression of this structural protein significantly, primarily through the C-terminal acidic region of the IE1 protein.
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Matheny, Ronald W., Bradley C. Nindl, and Martin L. Adamo. "Minireview: Mechano-Growth Factor: A Putative Product of IGF-I Gene Expression Involved in Tissue Repair and Regeneration." Endocrinology 151, no. 3 (February 3, 2010): 865–75. http://dx.doi.org/10.1210/en.2009-1217.
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The discovery that IGF-I mRNAs encoding isoforms of the pro-IGF-I molecule are differentially regulated in response to mechanical stress in skeletal muscle has been the impetus for a number of studies designed to demonstrate that alternative splicing of IGF-I pre-mRNA involving exons 4, 5, and 6 gives rise to a unique peptide derived from pro-IGF-I that plays a novel role in myoblast proliferation. Research suggests that after injury to skeletal muscle, the IGF-IEb mRNA splice variant is up-regulated initially, followed by up-regulation of the IGF-IEa splice variant at later time points. Up-regulation of IGF-IEb mRNA correlates with markers of satellite cell and myoblast proliferation, whereas up-regulation of IGF-IEa mRNA is correlated with differentiation to mature myofibers. Due to the apparent role of IGF-IEb up-regulation in muscle remodeling, IGF-IEb mRNA was also named mechano-growth factor (MGF). A synthetically manufactured peptide (also termed MGF) corresponding to the 24 most C-terminal residues of IGF-IEb has been shown to promote cellular proliferation and survival. However, no analogous peptide product of the Igf1 gene has been identified in or isolated from cultured cells, their conditioned medium, or in vivo animal tissues or biological fluids. This review will discuss the relationship of the Igf1 gene to MGF and will differentiate actions of synthetic MGF from any known product of Igf1. Additionally, the role of MGF in satellite cell activation, aging, neuroprotection, and signaling will be discussed. A survey of outstanding questions relating to MGF will also be provided.
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Sanders, Rebecca L., Christia J. Del Rosario, Elizabeth A. White, and Deborah H. Spector. "Internal Deletions of IE2 86 and Loss of the Late IE2 60 and IE2 40 Proteins Encoded by Human Cytomegalovirus Affect the Levels of UL84 Protein but Not the Amount of UL84 mRNA or the Loading and Distribution of the mRNA on Polysomes." Journal of Virology 82, no. 22 (September 10, 2008): 11383–97. http://dx.doi.org/10.1128/jvi.01293-08.
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ABSTRACT The major immediate-early (IE) region of human cytomegalovirus encodes two IE proteins, IE1 72 and IE2 86, that are translated from alternatively spliced transcripts that differ in their 3′ ends. Two other proteins that correspond to the C-terminal region of IE2 86, IE2 60 and IE2 40, are expressed at late times. In this study, we used IE2 mutant viruses to examine the mechanism by which IE2 86, IE2 60, and IE2 40 affect the expression of a viral DNA replication factor, UL84. Deletion of amino acids (aa) 136 to 290 of IE2 86 results in a significant decrease in UL84 protein during the infection. This loss of UL84 is both proteasome and calpain independent, and the stability of the protein in the context of infection with the mutant remains unaffected. The RNA for UL84 is expressed to normal levels in the mutant virus-infected cells, as are the RNAs for two other proteins encoded by this region, UL85 and UL86. Moreover, nuclear-to-cytoplasmic transport and the distribution of the UL84 mRNA on polysomes are unaffected. A region between aa 290 and 369 of IE2 86 contributes to the UL84-IE2 86 interaction in vivo and in vitro. IE2 86, IE2 60, and IE2 40 are each able to interact with UL84 in the mutant-infected cells, suggesting that these interactions may be important for the roles of UL84 and the IE2 proteins. Thus, these data have defined the contribution of IE2 86, IE2 60, and IE2 40 to the efficient expression of UL84 throughout the infection.
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Ahn, Jin-Hyun, Edward J. Brignole, and Gary S. Hayward. "Disruption of PML Subnuclear Domains by the Acidic IE1 Protein of Human Cytomegalovirus Is Mediated through Interaction with PML and May Modulate a RING Finger-Dependent Cryptic Transactivator Function of PML." Molecular and Cellular Biology 18, no. 8 (August 1, 1998): 4899–913. http://dx.doi.org/10.1128/mcb.18.8.4899.
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ABSTRACT Both of the major immediate-early (IE) proteins IE1 and IE2 of human cytomegalovirus (HCMV) as well as input viral DNA and sites of viral IE transcription colocalize with or adjacent to punctate PML domains (PML oncogenic domains [PODs] or nuclear domain 10) in the nucleus within the first few hours after infection of permissive human fibroblasts. However, colocalization of IE1 and PML in PODs is only transient, with both proteins subsequently redistributing into a nuclear diffuse form. These processes are believed to promote efficient viral IE transcription and initiation of DNA synthesis especially at low multiplicities of infection. To examine the mechanism of PML displacement by IE1, we carried out indirect immunofluorescence assay experiments with plasmids expressing intact or deleted forms of PML and IE1 in DNA-transfected cells. The results demonstrated that deletion of the C-terminal acidic region of IE1 uncouples the requirements for displacement of both endogenous and coexpressed PML from those needed to target to the PODs. Mutant PML proteins containing either a Cys point mutation within the N-terminal RING finger domain or a small deletion (of positions 281 to 304) within the coiled-coil region did not localize to the PODs but instead gave a nuclear diffuse distribution, similar to that produced by intact PML in the presence of IE1. Endogenous PML also colocalized with IE1 in metaphase chromosomes in HCMV or recombinant adenovirus type 5-IE1-infected HF cells undergoing mitosis, implying that there may be a direct physical interaction between IE1 and PML. Indeed, a specific interaction between IE1 and PML was observed in a yeast two-hybrid assay, and the strength of this interaction was comparable to that of IE2 with the retinoblastoma protein. The RING finger mutant form of PML showed a threefold-lower interaction with IE1 in the yeast system, and deletion of the N-terminal RING finger domain of PML abolished the interaction. Consistent with the IFA results, a mutant IE1 protein that lacks the C-terminal acidic region was sufficient for interaction with PML in the yeast system. The two-hybrid interaction assay also showed that both the N-terminal RING finger domain and the intact coiled-coil region of PML are required cooperatively for efficient self-interactions involving dimerization or oligomerization. Furthermore, truncated or deleted GAL4/PML fusion proteins that retained the RING finger domain but lacked the intact coiled-coil region displayed an unmasked cryptic transactivator function in both yeast and mammalian cells, and the RING finger mutation abolished this transactivation property of PML. Therefore, we suggest that a direct interaction between IE1 and the N-terminal RING finger domain of PML may inhibit oligomerization and protein-protein complex formation by PML, leading to displacement of PML and IE1 from the PODs, and that this interaction may also modulate a putative conditional transactivator function of PML.
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Simon, Christian O., Rafaela Holtappels, Hanna-Mari Tervo, Verena Böhm, Torsten Däubner, Silke A. Oehrlein-Karpi, Birgit Kühnapfel, et al. "CD8 T Cells Control Cytomegalovirus Latency by Epitope-Specific Sensing of Transcriptional Reactivation." Journal of Virology 80, no. 21 (August 23, 2006): 10436–56. http://dx.doi.org/10.1128/jvi.01248-06.
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ABSTRACT During murine cytomegalovirus (mCMV) latency in the lungs, most of the viral genomes are transcriptionally silent at the major immediate-early locus, but rare and stochastic episodes of desilencing lead to the expression of IE1 transcripts. This low-frequency but perpetual expression is accompanied by an activation of lung-resident effector-memory CD8 T cells specific for the antigenic peptide 168-YPHFMPTNL-176, which is derivedfrom the IE1 protein. These molecular and immunological findings were combined in the “silencing/desilencing and immune sensing hypothesis” of cytomegalovirus latency and reactivation. This hypothesis proposes that IE1 gene expression proceeds to cell surface presentation of the IE1 peptide by the major histocompatibility complex (MHC) class I molecule Ld and that its recognition by CD8 T cells terminates virus reactivation. Here we provide experimental evidence in support of this hypothesis. We generated mutant virus mCMV-IE1-L176A, in which the antigenic IE1 peptide is functionally deleted by a point mutation of the C-terminal MHC class I anchor residue Leu into Ala. Two revertant viruses, mCMV-IE1-A176L and the wobble nucleotide-marked mCMV-IE1-A176L*, in which Leu is restored by back-mutation of Ala codon GCA into Leu codons CTA and CTT, respectively, were constructed. Pulmonary latency of the mutant virus was found to be associated with an increased prevalence of IE1 transcription and with events of IE3 transactivator splicing. In conclusion, IE1-specific CD8 T cells recognize and terminate virus reactivation in vivo at the first opportunity in the reactivated gene expression program. The perpetual gene expression and antigen presentation might represent the driving molecular force in CMV-associated immunosenescence.
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Akizuki, Kageo. "C Society in IEE Japan." IEEJ Transactions on Electronics, Information and Systems 121, no. 1 (2001): 5–6. http://dx.doi.org/10.1541/ieejeiss1987.121.1_5.
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Tamrakar, Sama, Anokhi J. Kapasi, and Deborah H. Spector. "Human Cytomegalovirus Infection Induces Specific Hyperphosphorylation of the Carboxyl-Terminal Domain of the Large Subunit of RNA Polymerase II That Is Associated with Changes in the Abundance, Activity, and Localization of cdk9 and cdk7." Journal of Virology 79, no. 24 (December 15, 2005): 15477–93. http://dx.doi.org/10.1128/jvi.79.24.15477-15493.2005.
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ABSTRACT Human cytomegalovirus infection in the presence of the cyclin-dependent kinase (cdk) inhibitor roscovitine leads to changes in differential splicing and the polyadenylation of immediate early IE1/IE2 and UL37 transcripts (V. Sanchez, A. K. McElroy, J. Yen, S. Tamrakar, C. L. Clark, R. A. Schwartz, and D. H. Spector, J. Virol. 78:11219-11232, 2004). To determine if this was associated with specific phosphorylation of the C-terminal domain (CTD) of the RNA polymerase II (RNAP II) large subunit by cdk7/cyclin H and cdk9/cyclin T1, we examined the expression and localization of these kinases and the various phosphorylated forms of RNAP II. Infection resulted in increased RNAP II CTD phosphorylated on serines 2 and 5 and increased levels of activity of cdk7 and cdk9. At early times, cdk9 localizes with input viral DNA, and aggregates of cdk9 and cdk7 and a subset of Ser2-phosphorylated RNAP II colocalize with IE1/IE2 proteins adjacent to promyelocytic leukemia protein oncogenic domains. Later, cdk9 and Ser2-phosphorylated RNAP II form a nuclear punctate pattern; cdk7 resides in replication centers, and Ser5-phosphorylated RNAP II clusters at the peripheries of replication centers. Roscovitine treatment leads to decreased levels of hyperphosphorylated RNAP II (RNAP IIo) in infected cells and of hypophosphorylated RNAP II in mock-infected and infected cells. The RNAP IIo decrease does not occur if roscovitine is added 8 h postinfection, as was previously observed for processing of IE transcripts. These results suggest that accurate IE gene expression requires specific phosphorylation of the RNAP II CTD early in infection.
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Zhang, Zhigang, David L. Evers, Joseph F. McCarville, Jean-Christophe Dantonel, Shu-Mei Huong, and Eng-Shang Huang. "Evidence that the Human Cytomegalovirus IE2-86 Protein Binds mdm2 and Facilitates mdm2 Degradation." Journal of Virology 80, no. 8 (April 15, 2006): 3833–43. http://dx.doi.org/10.1128/jvi.80.8.3833-3843.2006.
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ABSTRACT Levels of the p53 tumor suppressor protein are increased in human cytomegalovirus (HCMV)-infected cells and may be important for HCMV pathogenesis. In normal cells p53 levels are kept low due to an autoregulatory feedback loop where p53 activates the transcription of mdm2 and mdm2 binds and ubiquitinates p53, targeting p53 for proteasomal degradation. Here we report that, in contrast to uninfected cells, mdm2 was undetectable upon treatment of infected fibroblasts with the proteasome inhibitor MG132. Cellular depletion of mdm2 was reproducible in p53-null cells transfected with the HCMV IE2-86 protein, but not with IE172, independently of the endogenous mdm2 promoter. IE2-86 also prevented the emergence of presumably ubiquitinated species of p53. The regions of IE2-86 important for mdm2 depletion were those containing the sequences corresponding to the putative zinc finger and C-terminal acidic motifs. mdm2 and IE2-86 coimmunoprecipitated in transfected and infected cell lysates and in a cell-free system. IE2-86 blocked mdm2's p53-independent transactivation of the cyclin A promoter in transient-transfection experiments. Pulse-chase experiments revealed that IE2-86 but not IE1-72 or several loss-of-function IE2-86 mutants increased the half-life of p53 and reduced the half-life of mdm2. Short interfering RNA-mediated depletion of IE2-86 restored the ability of HCMV-infected cells to accumulate mdm2 in response to proteasome inhibition. Taken together, the data suggest that specific interactions between IE2-86 and mdm2 cause proteasome-independent degradation of mdm2 and that this may be important for the accumulation of p53 in HCMV-infected cells.
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Sacirbegovic, Faruk, Sarah T. Rosenberger, Daqiang Zhao, Matthias Günther, Martin H. Oberbarnscheidt, Fadi G. Lakkis, Thomas Höfer, and Warren D. Shlomchik. "GVHD Is Sustained By T Cell Maintenance within Target Tissues: Insights from Clonal Tracking and Parabiosis." Blood 132, Supplement 1 (November 29, 2018): 809. http://dx.doi.org/10.1182/blood-2018-99-119134.
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Abstract Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation (alloSCT). In GVHD, donor T cells recognize recipient tissues as non-self and mount a broad attack that results in multi-organ damage. While there has been some success in diminishing the incidence of GVHD, less progress has been made in treating established or steroid-refractory disease without severe global immunosuppression. This is in part due to a lack of understanding of the underlining mechanisms that sustain GVHD despite chronic T cell antigen exposure. To address this, we developed a mouse GVHD model that allows us to track the progeny of single alloreactive T cell clones. We used this model to test hypotheses on GVHD maintenance: 1) GVHD is driven by the continuous output and trafficking of alloreactive T cells from secondary lymphoid tissues (SLT) into GVHD target organs; and 2) once tissues are seeded with alloreactive T cells from SLT, GVHD is maintained locally within affected tissues. We reasoned that if GVHD is maintained by the continuous SLT output of T cells, the progeny of single alloreactive clones in a target tissue would come into equilibrium with those in SLT and other target tissues. Alternatively, if there is a degree of local tissue GVHD maintenance, our model predicts that clonal progeny should be unequally distributed and not in equilibrium with SLT. We first tested these possibilities using a GVHD model wherein BALB/c RAG2-/- TCR transgenic (Tg) CD4 T cells (TS1) that recognize the S1 peptide derived from influenza hemagglutinin (HA) induce GVHD in BALB/c RAG2-/- mice that ubiquitously express HA (HA104 mice). We generated TS1 TCR Tg mice on 9 congenic backgrounds based on the expression of CD45.1/2, Thy1.1/2 and GFP. We transferred 500 naïve TS1 cells of 1 clonotype (to induce GVHD) along with single naïve TS1 cells from the remaining 8 clonotypes and BALB/c RAG2-/- bone marrow (BM) into lethally irradiated HA104 mice. We recovered a total of 432 single-cell derived TS1 clones (72% of input clones) from tissues of 79 mice, analyzed 7-35 days after transfer. We enumerated the TS1 clonal composition of each tissue (expressed as the % of all TS1 of that tissue) and found disparate clonal distribution across tissues within individual mice. For example, in a representative mouse analyzed at day 33 (Figure 1), the fractions of a single TS1 clone were relatively high in the colon (1.4%) and small intestine intraepithelial lymphocyte (IEL) (4.6%) when compared to the spleen (0.07%), BM (0.02%) and liver (0.03%). These data support that TS1 clones are not equally distributed among tissues and are not in equilibrium with SLT, suggesting that GVHD is at least in part maintained locally. We also analyzed TS1 clonal frequency distribution in a second model. BALB/c RAG2-/- BM and polyclonal T cells were transplanted into F1 (BALB/c HA104xB10.D2) recipients along with 8 single distinct TS1 cells. In this system, GVHD is induced by polyclonal BALB/c cells and TS1 cells are trackers of reactivity to HA. Preliminary experiments also indicate unequal distributions of TS1 clones across tissues in individual mice. In a second approach to test whether GVHD is maintained locally, irradiated HA104 mice were reconstituted with either 500 Thy1.1 or 500 Thy1.2 TS1 cells and CD45.1 or CD45.2 BALB/c RAG2-/- BM. One partner also received congenic TS1 single cells. We performed parabiosis of mice from one group to the other 21-28 days later. We analyzed 4 pairs 4 weeks post-joining, looking first at the blood to establish a baseline for TS1 crossover from the parabiotic partner. In blood, 18.9% ±1.9 of all TS1 were derived from the partner. Importantly, relative to equilibration in blood, there were far fewer partner-derived TS1 cells in all other tissues (Figure 2). Only a few single cell-derived TS1 clones were detected in the partner mouse at very low frequencies, even when they were dominant in tissues of the corresponding partner. Together, these data indicate that once GVHD is established, local maintenance dominates over new TS1 entry. Consistent with this, TS1 cells incorporate BrdU in vivo even at late time points. We are combining proliferation and clonality data at multiple timepoints to develop a mathematical model of GVHD establishment and maintenance. We are also extending our observations in a polyclonal GVHD model wherein the progeny from single alloreactive CD8 cells can be enumerated. Disclosures Shlomchik: NapaJen: Consultancy.
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Awasthi, Sita, Jennifer A. Isler, and James C. Alwine. "Analysis of Splice Variants of the Immediate-Early 1 Region of Human Cytomegalovirus." Journal of Virology 78, no. 15 (August 1, 2004): 8191–200. http://dx.doi.org/10.1128/jvi.78.15.8191-8200.2004.
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ABSTRACT The major immediate-early (MIE) gene of human cytomegalovirus (HCMV) produces multiple mRNAs through differential splicing and polyadenylation. Reverse transcriptase PCR was used to characterize transcripts from exons 1, 2, 3, and 4 (immediate-early 1 [IE1]). The expected IE72 and IE19 mRNAs were detected, as well as two heretofore-uncharacterized transcripts designated IE17.5 and IE9. The IE72, IE19, and IE17.5 transcripts utilized the same 5′-splice site in exon 3. IE9 utilized a cryptic 5′-splice site within exon 3. The IE19, IE17.5, and IE9 transcripts all used different 3′-splice sites within exon 4. These spliced species occur in infected human foreskin fibroblast (HFF) cells, with accumulation kinetics similar to those of IE72 mRNA. IE19 and IE9 RNAs were much more abundant than IE17.5 RNA. Transfection of CV-1 cells with cDNAs resulted in IE19 and IE17.5 proteins detectable by antibodies to either N-terminal or C-terminal epitopes. No IE9 protein product has been detected. We have not been able to detect IE19, IE17.5, or IE9 proteins during infection of HFF, HEL, or U373MG cells. Failure to detect IE19 protein contrasts with a previous report (M. Shirakata, M. Terauchi, M. Ablikin, K. Imadome, K. Hirai, T. Aso, and Y. Yamanashi, J. Virol. 76:3158-3167, 2002) of IE19 protein expression in HCMV-infected HEL cells. Our analysis suggests that an N-terminal breakdown product of IE72 may be mistaken for IE19. Expression of IE19 or IE17.5 from its respective cDNA results in repression of viral gene expression in infected cells. We speculate that expression of these proteins during infection may be restricted to specific conditions or cell types.
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Ahn, Jin-Hyun, Yixun Xu, Won-Jong Jang, Michael J. Matunis, and Gary S. Hayward. "Evaluation of Interactions of Human Cytomegalovirus Immediate-Early IE2 Regulatory Protein with Small Ubiquitin-Like Modifiers and Their Conjugation Enzyme Ubc9." Journal of Virology 75, no. 8 (April 15, 2001): 3859–72. http://dx.doi.org/10.1128/jvi.75.8.3859-3872.2001.
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ABSTRACT The human cytomegalovirus (HCMV) major immediate-early protein IE2 is a nuclear phosphoprotein that is believed to be a key regulator in both lytic and latent infections. Using yeast two-hybrid screening, small ubiquitin-like modifiers (SUMO-1, SUMO-2, and SUMO-3) and a SUMO-conjugating enzyme (Ubc9) were isolated as IE2-interacting proteins. In vitro binding assays with glutathioneS-transferase (GST) fusion proteins provided evidence for direct protein-protein interaction. Mapping data showed that the C-terminal end of SUMO-1 is critical for interaction with IE2 in both yeast and in vitro binding assays. IE2 was efficiently modified by SUMO-1 or SUMO-2 in cotransfected cells and in cells infected with a recombinant adenovirus expressing HCMV IE2, although the level of modification was much lower in HCMV-infected cells. Two lysine residues at positions 175 and 180 were mapped as major alternative SUMO-1 conjugation sites in both cotransfected cells and an in vitro sumoylation assay and could be conjugated by SUMO-1 simultaneously. Although mutations of these lysine residues did not interfere with the POD (or ND10) targeting of IE2, overexpression of SUMO-1 enhanced IE2-mediated transactivation in a promoter-dependent manner in reporter assays. Interestingly, many other cellular proteins identified as IE2 interaction partners in yeast two-hybrid assays also interact with SUMO-1, suggesting that either directly bound or covalently conjugated SUMO moieties may act as a bridge for interactions between IE2 and other SUMO-1-modified or SUMO-1-interacting proteins. When we investigated the intracellular localization of SUMO-1 in HCMV-infected cells, the pattern changed from nuclear punctate to predominantly nuclear diffuse in an IE1-dependent manner at very early times after infection, but with some SUMO-1 protein now associated with IE2 punctate domains. However, at late times after infection, SUMO-1 was predominantly detected within viral DNA replication compartments containing IE2. Taken together, these results show that HCMV infection causes the redistribution of SUMO-1 and that IE2 both physically binds to and is covalently modified by SUMO moieties, suggesting possible modulation of both the function of SUMO-1 and protein-protein interactions of IE2 during HCMV infection.
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Hagemeier, C., S. M. Walker, P. J. G. Sissons, and J. H. Sinclair. "The 72K IE1 and 80K IE2 proteins of human cytomegalovirus independently trans-activate the c-fos, c-myc and hsp70 promoters via basal promoter elements." Journal of General Virology 73, no. 9 (September 1, 1992): 2385–93. http://dx.doi.org/10.1099/0022-1317-73-9-2385.
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Ahn, Jin-Hyun, Won-Jong Jang, and Gary S. Hayward. "The Human Cytomegalovirus IE2 and UL112-113 Proteins Accumulate in Viral DNA Replication Compartments That Initiate from the Periphery of Promyelocytic Leukemia Protein-Associated Nuclear Bodies (PODs or ND10)." Journal of Virology 73, no. 12 (December 1, 1999): 10458–71. http://dx.doi.org/10.1128/jvi.73.12.10458-10471.1999.
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ABSTRACT During human cytomegalovirus (HCMV) infection, the periphery of promyelocytic leukemia protein (PML)-associated nuclear bodies (also known as PML oncogenic domains [PODs] or ND10) are sites for both input viral genome deposition and immediate-early (IE) gene transcription. At very early times after infection, the IE1 protein localizes to and subsequently disrupts PODs, whereas the IE2 protein localizes within or adjacent to PODs. This process appears to be required for efficient viral gene expression and DNA replication. We have investigated the initiation of viral DNA replication compartment formation by studying the localization of viral IE proteins, DNA replication proteins, and the PML protein during productive infection. Localization of IE2 adjacent to PODs between 2 and 6 h after infection was confirmed by confocal microscopy of human fibroblasts (HF cells) infected with both wild-type HCMV(Towne) and with an IE1-deletion mutant HCMV(CR208) that fails to disrupt PODs. In HCMV(Towne)-infected HF cells at 24 to 48 h, IE2 also accumulated in newly formed viral DNA replication compartments containing the polymerase processivity factor (UL44), the single-stranded DNA binding protein (SSB; UL57), the UL112-113 accessory protein, and newly incorporated bromodeoxyuridine (BrdU). Double labeling of the HCMV(CR208)-infected HF cells demonstrated that formation of viral DNA replication compartments initiates within granular structures that bud from the periphery of some of the PODs and subsequently coalesce into larger structures that are flanked by PODs. In transient DNA transfection assays, both the N terminus (codons 136 to 290) and the C terminus (codons 379 to 579) of IE2 exon 5, but not the central region between them, were found to be necessary for both the punctate distribution of IE2 and its association with PODs. Like IE2, the UL112-113 accessory replication protein was also distributed in a POD-associated pattern in both DNA-transfected and virus-infected cells beginning at 6 h. Furthermore, when all six replication core machinery proteins (polymerase complex, SSB, and helicase-primase complex) were expressed together in the presence of UL112-113, they also accumulated at POD-associated sites, suggesting that the UL112-113 protein (but not IE2) may play a role in recruitment of viral replication fork proteins into the periphery of PODs. These results show that (i) subsequent to accumulating at the periphery of PODs, IE2 is incorporated together with the core proteins into viral DNA replication compartments that initiate from the periphery of PODs and then grow to fill the space between groups of PODs, and (ii) the UL112-113 protein appears to have a key role in assembling and recruiting the core replication machinery proteins in the initial stages of viral replication compartment formation.
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Horváth, Tibor, and Levente Tábi. "Counter-Improvised Explosive Devices (C-IED) mission support capabilities." Hadtudomány 33, no. 3 (December 22, 2023): 15–31. http://dx.doi.org/10.17047/hadtud.2023.33.3.15.
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Actions and activities taken against the IED threat comprise complex tasks. In thebeginning, the focus was on the deactivation and destruction of IEDs, as the only wayto fight against them. As time has passed, more existing capabilities were included inthe field of C-IED, which were able to contribute to the mitigation of the effectivenessof an IED attack.Due to the fact that the first type of response to IED-threat was tasked to combatengineers, particularly to EOD personnel, that was rather obvious that C-IED missionsand responsibilities fell to combat engineers. Nowadays, however, capabilities andcapacities in the C-IED can be identified that exceed the competence of engineer unitsand commanders. The C-IED requires a comprehensive approach. In a staff andheadquarters lots of capabilities and capacities should be involved in C-IED activities,which requires coordination and cooperation. The involvement of those capabilitiesdepends on the type of mission, its phases, and the nature of IED threat. Thus amission commander needs to understand which are the C-IED enablers that cancontribute to C-IED tasks, when and how they influence or mitigate IED threats.
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Horváth, Tibor, and Levente Tábi. "Support Capabilities to Supplement C-IED Mission. Part 1." Land Forces Academy Review 27, no. 2 (June 1, 2022): 154–61. http://dx.doi.org/10.2478/raft-2022-0020.
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Abstract Actions and activities taken against the IED threat comprise complex tasks. In the beginning, the focus was on the deactivation and destruction of IEDs, as the only way to fight against them. As time has passed more existing capabilities were included in the field of C-IED, which were able to contribute to the mitigation of the effectiveness of an IED attack. Due to the fact that the first type of response to IED-threat was tasked to combat engineers, particularly to EOD personnel, that was rather obvious that C-IED missions and responsibilities fell to combat engineers. Nowadays, however, capabilities and capacities in the C-IED can be identified that exceed the competence of engineer units and commanders. C-IED requires a comprehensive approach. In a staff and headquarters lots of capabilities and capacities should be involved in C-IED activities, which requires coordination and cooperation. The involvement of those capabilities depends on the type of mission, its phases, and the nature of IED threat. Thus a mission commander needs to understand which are the C-IED enablers that can contribute to C-IED tasks, when and how they influence or mitigate IED threats.
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Tábi, Levente. "Evolution of the C-IED Staff Officers Course." Hadmérnök 14, no. 1 (April 8, 2019): 61–71. http://dx.doi.org/10.32567/hm.2019.1.6.
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At the beginning of the XXI century the Countering Improvised Explosive Devices (CIED) tasks became very important in NATO missions. Every C-IED related mission is connected to the three essential C-IED pillars (Attack the Networks, Defeat the Device and Prepare the Force). Since 2010; when the CIED COE was established in Madrid, Spain; CIED COE has taken on a significant role in the NATO’s C-IED Education & Training activities. One of its main tasks is to prepare, provide, run and lead different C-IED courses. The C-IED staff course is a very important part in the C-IED education and training landscape.This kind of staff course evolution and development is always based on the current IED threat and the NATO requirements. Updating the existing course materials and lectures or developing brand new courses are significant issues for which, over the past few years, the C-IED COE has been recognized, even to the point of taking over these responsibilities to support NATO, Allied and partner nations.
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Wiseman, T. P., and Ella Hermon. "Pouvoir et "imperium" (IIIe AV. J.-C.-Ier AP. J.-C.)." Phoenix 51, no. 3/4 (1997): 426. http://dx.doi.org/10.2307/1192557.
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Fremont, Daved H., Shaodong Dai, Herbert Chiang, Frances Crawford, Philippa Marrack, and John Kappler. "Structural Basis of Cytochrome c Presentation by IEk." Journal of Experimental Medicine 195, no. 8 (April 15, 2002): 1043–52. http://dx.doi.org/10.1084/jem.20011971.
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The COOH-terminal peptides of pigeon and moth cytochrome c, bound to mouse IEk, are two of the most thoroughly studied T cell antigens. We have solved the crystal structures of the moth peptide and a weak agonist–antagonist variant of the pigeon peptide bound to IEk. The moth peptide and all other peptides whose structures have been solved bound to IEk, have a lysine filling the p9 pocket of IEk. However, the pigeon peptide has an alanine at p9 shifting the lysine to p10. Rather than kinking to place the lysine in the anchor pocket, the pigeon peptide takes the extended course through the binding groove, which is characteristic of all other peptides bound to major histocompatibility complex (MHC) class II. Thus, unlike MHC class I, in which peptides often kink to place optimally anchoring side chains, MHC class II imposes an extended peptide conformation even at the cost of a highly conserved anchor residue. The substitution of Ser for Thr at p8 in the variant pigeon peptide induces no detectable surface change other than the loss of the side chain methyl group, despite the dramatic change in recognition by T cells. Finally, these structures can be used to interpret the many published mutational studies of these ligands and the T cell receptors that recognize them.
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Wells, Michelle, Brian Brown, and Judith Hall. "Pepsinogen C Expression in Intestinal IEC-6 Cells." Cellular Physiology and Biochemistry 13, no. 5 (2003): 301–8. http://dx.doi.org/10.1159/000074545.
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Bakos, Tamás. "NATO-követelmények a rögtönzött robbanóeszközök elleni tevékenység (C-IED) kiképzésében." Hadtudomány 33, no. 2 (August 4, 2023): 17–29. http://dx.doi.org/10.17047/hadtud.2023.33.2.17.
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Napjainkban, az aszimmetrikus hadviselés térnyerésével a terrorista csoportok legelterjedtebb fegyverei a rögtönzött robbanóeszközök (IED1). Az IED-k elleni védekezés, azalkalmazásuk elleni tevékenység (C-IED2) kiemelt feladattá vált a katonai műveletek tervezése és végrehajtása során. A NATO NSA3 2012-ben adta ki egységesítési irányelveit a C-IED kiképzési követelményeire vonatkozóan.4 A Magyar Honvédség még ugyanabban az évben elfogadta és bevezette az egyezményt, majd az ahhoz kapcsolódódokumentum5 a kiadás óta több alkalommal is felülvizsgálatra került és módosult. Célom egy, a NATO C-IED-képzésre, kiképzésre vonatkozó követelményekről szólóösszefoglaló készítése annak érdekében, hogy a tanulmány irányt mutasson a Magyar Honvédség C-IED-képzési, kiképzési rendszerének felülvizsgálatához és fejlesztéséhez.