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• W1993393670 abstract "Fibroblasts from scaffold-based three-dimensional human cultures have been demonstrated to colonize ulcer wound beds and persist for at least 6 mo without rejection. This study examines the expression in these cultures of molecules associated with activation of the immune system in acute rejection. Studies in monolayer cultures showed that fibroblasts expressed CD40 at about 10% of the surface density seen in umbilical vein endothelial cells, whereas HLA-DR was undetectable. In these cultures, both molecules were induced by γ-interferon. In scaffold-based three-dimensional cultures, however, a majority of the fibroblasts showed little induction of CD40 and HLA-DR in response to γ-interferon, although HLA class I expression was increased. Fibroblasts re-isolated from the three-dimensional cultures and cultured in monolayers recovered HLA-DR induction in response to γ-interferon. Fibroblasts cultured in an alternative three-dimensional system using collagen gels showed CD40 and HLA-DR induction by γ-interferon in the same manner as monolayer cultures. Comparison of phosphorylation of signal transducer and activator of transcription 1 on tyrosine-701 showed it to be similar in monolayer and three-dimensional culture, and phospho-signal transducer and activator of transcription 1 moved into the nucleus. Induction of the class II transcription activator was greatly reduced, however. We propose that interaction of fibroblasts with the fibroblast-derived extracellular matrix is an important modulator of γ-interferon responsiveness and that this interaction may play a role in the low immunogenicity of allogeneic fibroblasts grown on scaffolds. Fibroblasts from scaffold-based three-dimensional human cultures have been demonstrated to colonize ulcer wound beds and persist for at least 6 mo without rejection. This study examines the expression in these cultures of molecules associated with activation of the immune system in acute rejection. Studies in monolayer cultures showed that fibroblasts expressed CD40 at about 10% of the surface density seen in umbilical vein endothelial cells, whereas HLA-DR was undetectable. In these cultures, both molecules were induced by γ-interferon. In scaffold-based three-dimensional cultures, however, a majority of the fibroblasts showed little induction of CD40 and HLA-DR in response to γ-interferon, although HLA class I expression was increased. Fibroblasts re-isolated from the three-dimensional cultures and cultured in monolayers recovered HLA-DR induction in response to γ-interferon. Fibroblasts cultured in an alternative three-dimensional system using collagen gels showed CD40 and HLA-DR induction by γ-interferon in the same manner as monolayer cultures. Comparison of phosphorylation of signal transducer and activator of transcription 1 on tyrosine-701 showed it to be similar in monolayer and three-dimensional culture, and phospho-signal transducer and activator of transcription 1 moved into the nucleus. Induction of the class II transcription activator was greatly reduced, however. We propose that interaction of fibroblasts with the fibroblast-derived extracellular matrix is an important modulator of γ-interferon responsiveness and that this interaction may play a role in the low immunogenicity of allogeneic fibroblasts grown on scaffolds. class II transcription activator signal transducer and activator of transcription Acute rejection of allograft tissues is caused by activation of lymphocytes, initially by mechanisms involving interaction of HLA class II on the allogeneic, grafted cells with the T cell receptor on the host helper T lymphocytes together with engagement of CD28 on the lymphocytes by the costimulatory molecules CD80 or CD86 on the allogeneic cells. The costimulatory receptors are induced by interaction of CD40 on the allogeneic cell with CD154 on lymphocytes. Fibroblasts are poor antigen-presenting cells and typically express low amounts of CD40 (Fries et al., 1995Fries K.M. Sempowski G.D. Gaspari A.A. Blieden T. Looney R.J. Phipps R.P. CD40 expression by human fibroblasts.Clin Immunol Immunopathol. 1995; 77: 42-51Crossref PubMed Scopus (150) Google Scholar) and undetectable levels of HLA class II, CD80, or CD86 on their surfaces. Several groups have reported that transplanted, cultured, allogeneic fibroblasts fail to induce acute rejection, in the presence (Bell et al., 1983Bell E. Sher S. Hull B. et al.The reconstitution of living skin.J Invest Dermatol. 1983; 81: 2s-10sCrossref PubMed Scopus (451) Google Scholar;Hull et al., 1983Hull B.E. Sher S.E. Rosen S. Church D. Bell E. Fibroblasts in isogeneic skin equivalents persist for long periods after grafting.J Invest Dermatol. 1983; 81: 436-438Crossref PubMed Scopus (29) Google Scholar;Sher et al., 1983Sher S.E. Hull B.E. Rosen S. Church D. Friedman L. Bell E. Acceptance of allogeneic fibroblasts in skin equivalent transplants.Transplantation. 1983; 36: 552-557Crossref PubMed Scopus (63) Google Scholar;Palmer et al., 1991Palmer T.D. Rosman G.J. Osborne W.R. Miller A.D. Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes.Proc Natl Acad Sci USA. 1991; 88: 1330-1334Crossref PubMed Scopus (407) Google Scholar;Rouabhia, 1996Rouabhia M. In vitro production and transplantation of immunologically active skin equivalents.Lab Invest. 1996; 75: 503-517PubMed Google Scholar;Falanga et al., 1998Falanga V. Margolis D. Alvarez O. et al.Rapid healing of venous ulcers and lack of clinical rejection with an allogeneic cultured human skin equivalent. Human Skin Equivalent Investigators Group.Arch Dermatol. 1998; 134: 293-300Crossref PubMed Scopus (572) Google Scholar;Eaglstein et al., 1999Eaglstein W.H. Alvarez O.M. Auletta M. et al.Acute excisional wounds treated with a tissue-engineered skin (Apligraf).Dermatol Surg. 1999; 25: 195-201https://doi.org/10.1046/j.1524-4725.1999.08186.xCrossref PubMed Scopus (119) Google Scholar) or absence (Hansbrough et al., 1994Hansbrough J.F. Morgan J. Greenleaf G. Underwood J. Development of a temporary living skin replacement composed of human neonatal fibroblasts cultured in Biobrane, a synthetic dressing material.Surgery. 1994; 115: 633-644PubMed Google ScholarJ. Mansbridge, unpublished data) of keratinocytes. Indeed, cultured constructs containing an allogeneic keratinocyte-derived epidermis cultured on a three-dimensional allogeneic fibroblast-containing dermal structure have been used as the nonimmunogenic basis for development of models of acute rejection by addition of immunocytes or endothelial cells (Rouabhia, 1996Rouabhia M. In vitro production and transplantation of immunologically active skin equivalents.Lab Invest. 1996; 75: 503-517PubMed Google Scholar;Strande et al., 1997Strande L.F. Foley S.T. Doolin E.J. Hewitt C.W. In vitro bioartificial skin culture model of tissue rejection and inflammatory/immune mechanisms.Transplant Proc. 1997; 29: 2118-2119https://doi.org/10.1016/s0041-1345(97)00256-xAbstract Full Text PDF PubMed Scopus (0) Google Scholar;Rose, 1998Rose M.L. Endothelial cells as antigen-presenting cells: role in human transplant rejection.Cell Mol Life Sci. 1998; 54: 965-978Crossref PubMed Scopus (74) Google Scholar). HLA class II and CD40 are induced by γ-interferon in many cell types, including fibroblasts (Saunders et al., 1994Saunders N.A. Smith R.J. Jetten A.M. Differential responsiveness of human bronchial epithelial cells, lung carcinoma cells, and bronchial fibroblasts to interferon-gamma in vitro.Am J Respir Cell Mol Biol. 1994; 11: 147-152Crossref PubMed Scopus (44) Google Scholar;Takahashi et al., 1994Takahashi K. Takigawa M. Arai H. Kurihara H. Murayama Y. The inhibition of interferon-gamma-induced upregulation of HLA-DR expression on cultured human gingival fibroblasts by interleukin-1 beta or tumor necrosis factor-alpha.J Periodontol. 1994; 65: 336-341Crossref PubMed Scopus (7) Google Scholar;Fries et al., 1995Fries K.M. Sempowski G.D. Gaspari A.A. Blieden T. Looney R.J. Phipps R.P. CD40 expression by human fibroblasts.Clin Immunol Immunopathol. 1995; 77: 42-51Crossref PubMed Scopus (150) Google Scholar;Armendariz-Borunda et al., 1996Armendariz-Borunda J. Endres R.O. Ballou L.R. Postlethwaite A.E. Transforming growth factor-beta inhibits interferon-gamma-induced HLA-DR expression by cultured human fibroblasts.Int J Biochem Cell Biol. 1996; 28: 1107-1116https://doi.org/10.1016/1357-2725(96)00067-2Crossref PubMed Scopus (8) Google Scholar;Klett et al., 1996Klett Z.G. Elner S.G. Elner V.M. Differential expression of immunoreactive HLA-DR and ICAM-1 in human cultured orbital fibroblasts and orbital tissue.Ophthal Plast Reconstr Surg. 1996; 12: 153-162Crossref PubMed Scopus (6) Google Scholar;Gruschwitz and Vieth, 1997Gruschwitz M.S. Vieth G. Up-regulation of class II major histocompatibility complex and intercellular adhesion molecule 1 expression on scleroderma fibroblasts and endothelial cells by interferon-gamma and tumor necrosis factor alpha in the early disease stage.Arthritis Rheum. 1997; 40: 540-550Crossref PubMed Scopus (64) Google Scholar;Sempowski et al., 1997Sempowski G.D. Chess P.R. Phipps R.P. CD40 is a functional activation antigen and B7-independent T cell costimulatory molecule on normal human lung fibroblasts.J Immunol. 1997; 158: 4670-4677PubMed Google Scholar), and might be expected to be activated in an inflammatory environment to a point that might induce immunologic attack (Shimabukuro et al., 1996Shimabukuro Y. Murakami S. Okada H. Antigen-presenting-cell function of interferon gamma-treated human gingival fibroblasts.J Periodontal Res. 1996; 31: 217-228Crossref PubMed Scopus (37) Google Scholar;Sempowski et al., 1997Sempowski G.D. Chess P.R. Phipps R.P. CD40 is a functional activation antigen and B7-independent T cell costimulatory molecule on normal human lung fibroblasts.J Immunol. 1997; 158: 4670-4677PubMed Google Scholar), leading to rejection. In contrast to this hypothesis,Wu et al., 1995Wu J. Barisoni D. Armato U. An investigation into the mechanisms by which human dermis does not significantly contribute to the rejection of allo-skin grafts.Burns. 1995; 21: 11-16https://doi.org/10.1016/0305-4179(95)90774-tAbstract Full Text PDF PubMed Google Scholar reported that allogeneic fibroblasts in a dermal structure did not significantly contribute to lymphocyte activation, whereas monolayer cultures of fibroblasts were more effective. This result suggested that the three-dimensional structure of the dermis or interaction of the fibroblasts with the extracellular matrix might affect lymphocyte activation. To investigate this further, we compared the induction of HLA class II and CD40 by γ-interferon in scaffold-based three-dimensional fibroblast cultures with the same fibroblasts in monolayer cultures and collagen gel three-dimensional cultures. All fibroblast cultures were prepared using a neonatal dermal fibroblast strain, derived from a single donor, and maintained by Advanced Tissue Sciences. Three types of fibroblast culture were employed in these studies: monolayer, collagen-gel-based three-dimensional cultures and scaffold-based three-dimensional cultures. Fibroblasts were cultured in high glucose (4 g per liter) Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Grand Island, NY) supplemented with 10% bovine serum (Hyclone, Irvine, CA), nonessential amino acids, and 2 mM L-glutamine (growth medium). All incubations were at 37°C in a humidified 5% CO2 atmosphere and all cultures were at eighth passage (about 30–35 population doublings). Where appropriate, cultures were incubated with 500 U per ml γ-interferon (Boehringer Mannheim, Indianapolis, IN) for 7 d in growth medium, the medium being changed every 3 d. Monolayer cultures were grown to confluence. Collagen-gel-based, three-dimensional cultures were prepared by seeding fibroblasts into Vitrogen (Cohesion, Palo Alto, CA), which was neutralized, then brought to 1 × DMEM by addition of 10 × concentrated DMEM prior to cell seeding. The gels were cultured with attachment to the tissue culture plate (stressed gel conditions) (Nakagawa et al., 1989Nakagawa S. Pawelek P. Grinnell F. Long-term culture of fibroblasts in contracted collagen gels: effects on cell growth and biosynthetic activity.J Invest Dermatol. 1989; 93: 792-798Abstract Full Text PDF PubMed Google Scholar). To obtain scaffold-based three-dimensional cultures, fibroblasts were seeded onto knitted lactate-glycollate copolymer scaffolds and cultured in growth medium supplemented with 50 mg per liter ascorbic acid, with periodic changes of medium for about 14 d. At harvest they contained about 1.3 × 106 cells per cm2 and about 1 mg per cm2 collagen (Mansbridge et al., 1999Mansbridge J.N. Liu K. Pinney R.E. Patch R. Ratcliffe A. Naughton G.K. Growth factors secreted by fibroblasts: role in healing diabetic foot ulcers.Diabetes, Obesity Metab. 1999; 1: 265-279Crossref PubMed Scopus (142) Google Scholar). These allogeneic three-dimensional fibroblast cultures (Dermagraft, Advanced Tissue Sciences) are marketed or in clinical trials in various countries for the treatment of diabetic foot ulcers, and for this purpose are supplied in a cryopreserved state. For the studies described here, both cryopreserved cultures and cultures prior to cryopreservation were used. Human dermal microvascular endothelial cells were obtained from Cascade Biologics (Portland, OR) and cultured according to the supplier's instructions in medium 131 supplemented with appropriate growth supplement. Fibroblasts and endothelial cells were suspended from cultures of each type by incubation for 3 h at 37°C in 5 mg per ml bacterial collagenase B (Boehringer Mannheim) in DMEM supplemented with 5% bovine serum with agitation and were collected by centrifugation. All cell preparations were treated in the same way to equalize damage to the surface markers. The cells were then incubated in suspension at 37°C for 1 h in growth medium with 10 mM ethylenediamine tetraacetic acid (EDTA). Control experiments demonstrated that these conditions allowed re-expression of CD40 and HLA-DR on the cell surface. Cells were stained in phosphate-buffered saline (PBS) containing 5% bovine serum using anti-HLA A, B, C (clone G46-2.6), anti-HLA-DR (clone 46–6), and anti-CD86 (FUN-1) from Pharmingen (San Diego, CA), anti-CD40 (EA-5, Calbiochem, San Diego, CA), and anti-CD80 (MAB104, BioDesign, Kennebunk, ME) antibodies. Mouse IgG1 κ from Pharmingen was used as a control antibody. The second antibody was fluorescein-conjugated goat antimouse IgG from Pharmingen. Cell surface marker densities were determined by flow cytometry using a FACSTAR (Becton Dickinson, San Jose, CA). The cells were generally fixed with 2% paraformaldehyde for storage between staining and flow cytometry analysis. The analyses illustrated were representative of at least three independent experiments. In experiments requiring selection of viable cells, the suspended cells were stained with antibodies and propidium iodide prior to flow cytometry. Cells were sorted aseptically on a fluorescence-activated cell sorter (CONSORT; Becton Dickinson) in the absence of paraformaldehyde. The experiments reported here have been performed at least five times. STAT-1 was detected by immunoblot following sodium dodecyl sulfate electrophoresis on 10% gels by means of a specific antibody for STAT-1 either unphosphorylated or phosphorylated on tyrosine-701 (Cell Signaling, Beverly, MA). Fibroblasts in monolayer or three-dimensional culture were incubated with 500 U per ml γ-interferon, and the reactions were terminated at times up to 3 h by extraction with 1% Triton X-100 in PBS containing 2 µg per ml aprotinin, 1 µg per ml leupeptin, 1 µg per ml pepstatin, 1 mM EDTA, and 1 mM sodium vanadate. Triton-insoluble proteins were removed by centrifugation at 4°C. The supernatant was boiled with an equal volume of 4.6% sodium dodecyl sulfate and 10% 2-mercaptoethanol in 50 mM Tris chloride, pH 6.8. After electrophoresis and immunoblotting onto Immobilon p (Millepore, Burlington, MA), the bands were identified by incubation with a rabbit antibody appropriate for detecting unphosphorylated STAT or STAT phosphorylated on tyrosine-701 and visualized by means of a second peroxidase-conjugated goat antirabbit antibody and ECL+ according to the manufacturer's instructions. CIITA mRNA was determined by real-time quantitative polymerase chain reaction (PCR) of reverse transcribed (Heid et al., 1996Heid C.A. Stevens J. Livak K.J. Williams P.M. Real time quantitative PCR.Genome Res. 1996; 6: 986-994Crossref PubMed Scopus (5010) Google Scholar) mRNA. Cells in monolayer and three-dimensional culture were incubated with 500 U per ml γ-interferon for times up to 48 h. The mRNA was prepared using either QIAGEN (Santa Clarita, CA) or Amresco (Solon, OH) kits according to the manufacturer's instructions. The PCR primers and probes were as follows: forward primer, 5′-CCC TCAATCTGTCCCAGAACA-3′; reverse primer, 5′-CCACGTCGC AGATGCAGTT-3′; probe, 5′-FAM-TGACCTGGGTGCCTACAA ACTCGCC-3′-TAMRA; the standard was derived as a PCR fragment. The results were taken from four experiments, containing five time-courses for monolayer fibroblast cultures and six for three-dimensional cultures. Monolayer cultures of dermal fibroblasts express HLA class I but not HLA-DR (Figure 1a, b, fine line). Following incubation for 7 d with 500 U per ml γ-interferon, however, monolayer cultures showed an increase in the expression of HLA-class I of 40% Figure 1a and strong induction of HLA-DR Figure 1b. When cryopreserved and thawed scaffold-based three-dimensional cultures were incubated with γ-interferon in the same manner as the monolayer cultures, a bimodal distribution of HLA-DR expression was observed, including both a responsive and a nonresponsive population Figure 1d. As this material had been cryopreserved, the question arose whether the nonresponsive population might result from reduced cell viability following the freezing process. This was addressed by examining the distribution of HLA-DR in response to γ-interferon in cells selected for viability by their ability to exclude propidium iodide. The viable cell population also showed a bimodal distribution, indicating that lack of responsiveness was a property of live cells (not shown). As cryopreservation has been shown to cause a profound inhibition of protein synthesis (Mansbridge et al., 1998Mansbridge J. Liu K. Patch R. Symons K. Pinney E. Three-dimensional fibroblast culture implant for the treatment of diabetic foot ulcers: metabolic activity and therapeutic range.Tissue Eng. 1998; 4: 403-414Crossref PubMed Scopus (117) Google Scholar), lack of HLA-DR induction by γ-interferon might still be a result of the cryopreservation process even in viable cells in the tissue. To answer this question, the responsiveness of scaffold-based three-dimensional cultures that had not been cryopreserved was tested. Expression of HLA class I and HLA-DR in the three-dimensional cultures in the absence of γ-interferon did not differ from that of the monolayer cultures (Figure 1c, d, fine line). After 7 d exposure to γ-interferon, however, noncryopreserved scaffold-based three-dimensional cultures (21% positive cells) gave the same bimodal distribution of HLA-DR induction as did the cryopreserved tissue (27% positive cells, Figure 1d). One possible explanation for this observation is that the fibroblast population in the scaffold-based three-dimensional culture was a selected subpopulation with low γ-interferon responsiveness. To test this, cells were re-isolated from the three-dimensional cultures and exposed to 500 U per ml γ-interferon as a monolayer for 7 d. Under these conditions, all cells responded with high expression of HLA-DR Figure 2b. The possibility that the γ-interferon nonresponsive cells in the three-dimensional culture failed to proliferate in monolayer culture was tested by isolating the nonresponsive cells by fluorescence-activated cell sorting and culturing them in monolayer. When subsequently treated with γ-interferon in monolayer culture, these previously unresponsive cells Figure 3a showed unimodal induction of HLA-DR Figure 3b or CD40 Table I comparable to cells from monolayer culture. From this result it was concluded that the presence of a subpopulation nonresponsive to γ-interferon in terms of HLA-DR expression was a reversible consequence of the conditions of culture in the scaffold-based system.Figure 3HLA-DR expression in fibroblasts derived from three-dimensional culture. Fibroblasts were isolated from scaffold-based three-dimensional culture after treatment with γ-interferon and were sorted for the expression of HLA-DR (A). Cells negative for HLA-DR (1) or positive for HLA-DR (2) were treated with γ-interferon and analyzed for the expression of HLA-DR by flow cytometry. Cells negative for HLA-DR expression in three-dimensional culture express high levels of this epitope when grown in the presence of γ-interferon in monolayer (B) in the same way as cells positive for HLA-DR in three-dimensional culture (C).View Large Image Figure ViewerDownload (PPT)Table IExpression of HLA-DR and CD40 in the presence of γ-interferonaThree-dimensional scaffold-based fibroblast cultures were exposed to γ-interferon. After isolation, HLA-DR positive and negative cells were isolated by sorting and re-exposed to γ-interferon in monolayer culture. Numbers represent the percentage of cells positive for the expression of HLA-DR or CD40.IgG CoHLA-DRCD40Cells from 3D culture before sort2.127.317.4HLA-DR negative cells0.798.662.8HLA-DR positive cells0.899.367.7a Three-dimensional scaffold-based fibroblast cultures were exposed to γ-interferon. After isolation, HLA-DR positive and negative cells were isolated by sorting and re-exposed to γ-interferon in monolayer culture. Numbers represent the percentage of cells positive for the expression of HLA-DR or CD40. Open table in a new tab The scaffold-based cultures not only are three-dimensional but also contain the cells embedded within extracellular matrix that they themselves have deposited. The lack of responsiveness to γ-interferon might thus be a consequence of three-dimensionality per se or a property of the extracellular matrix. To address this point, the response of fibroblasts in collagen-gel-based, three-dimensional culture was tested under the standard conditions of 7 d exposure to 500 U per ml γ-interferon. The resulting distribution of HLA-DR was unimodal and high, similar to monolayer culture Figure 1f. We concluded that the nonresponsive population was a reversible consequence of interaction with naturally deposited, fibroblast extracellular matrix, which was not reproduced by the three-dimensional collagen gel. In time-course studies, HLA class II was fully induced by γ-interferon in a unimodal manner within 48 h of the addition of the cytokine in monolayer cultures, whereas the bimodal pattern was established in three-dimensional cultures in a similar time and remained stable until at least 96 h Figure 4. We concluded that the bimodal pattern reflects stable lack of responsiveness by a subpopulation rather than slow induction of all the cells. CD40 was present on the cultured fibroblasts Figure 5a, but at only about 10% of the surface expression observed on endothelial cells Figure 5b. It was induced by γ-interferon, and showed a similar pattern of responses in monolayer, in scaffold-based three-dimensional culture Figure 5c, and in collagen gels Figure 5d as did HLA-DR. This induction was unimodal in monolayer and collagen-based culture, but biphasic in scaffold-based culture, with 50%-80% of the cells showing no response. The possibility that the bimodal distribution of HLA-DR expression was caused by uneven distribution of γ-interferon in the three-dimensional cultures was approached through examination of HLA class I expression. HLA class I was expressed constitutively in all three types of culture, but could be induced further by γ-interferon Figure 1a, c, e. In γ-interferon-treated scaffold-based three-dimensional cultures, by contrast to HLA-DR and CD40, the induction of HLA class I was unimodal. All the cells responded similarly, which argues against an explanation for the bimodal distributions based on nonuniform penetration by the cytokine. Furthermore, collagen gel cultures, in which the thickness of the three-dimensional structure is greater than in the scaffold-based cultures, showed unimodal HLA-DR induction, supporting the view that penetration of γ-interferon was uniform. A similar result was obtained for ICAM-1, which was induced in a similar unimodal manner in both monolayer and three-dimensional culture. We also tested other molecules required for T cell activation. Cells from all culture systems were analyzed for the expression of CD80 and CD86. These molecules were barely detectable on fibroblasts regardless of how they were cultured. A major pathway in signal transduction from the γ-interferon receptor is through JAK and STAT-1. We therefore compared the phosphorylation of STAT-1 in both detergent-soluble and detergent-insoluble cellular fractions of monolayer and three-dimensional fibroblast cultures, as shown in Figure 6. STAT-1 was phosphorylated on tyrosine-701 in both types of culture to a similar extent. The slight delay in three-dimensional culture can be explained by the increased time needed for the penetration of the cytokine into the tissue. In both cases, the STAT-1 content of the soluble fraction rose and fell, whereas the content in the insoluble fraction increased as the soluble fraction fell. We interpret this observation as reflecting movement of phosphorylated STAT-1 into the nucleus. The overall conclusion is that the response to γ-interferon is indistinguishable in monolayer and three-dimensional culture in terms of STAT-1 phosphorylation. The activation of the HLA-DR promoter is exerted through the transcription factor CIITA (Steimle et al., 1993Steimle V. Otten L.A. Zufferey M. Mach B. Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or bare lymphocyte syndrome).Cell. 1993; 75: 135-146Abstract Full Text PDF PubMed Scopus (762) Google Scholar;Chang et al., 1994Chang C.-H. Fontes J.D. Peterlin M. Flavell R.A. Class II transactivator (CIITA) is sufficient for the inducible expression of major compatibility complex class II genes.J Exp Med. 1994; 180: 1367-1374Crossref PubMed Scopus (299) Google Scholar;Rigaud et al., 1996Rigaud G. De Lerma Barbaro A. Nicolis M. Cestari T. Ramarli D. Riviera A.P. Accolla R.S. Induction of CIITA and modification of in vivo HLA-DR promoter occupancy in normal thymic epithelial cells treated with IFN-gamma: similarities and distinctions with respect to HLA-DR-constitutive B cells.J Immunol. 1996; 156: 4254-4258PubMed Google Scholar;Bradley et al., 1997Bradley M.B. Fernandez J.M. Ungers G. et al.Correction of defective expression in MHC class II deficiency (bare lymphocyte syndrome) cells by retroviral transduction of CIITA.J Immunol. 1997; 159: 1086-1095PubMed Google Scholar), and the presence of this factor has been shown to be sufficient to induce it. We compared its induction in monolayer and three-dimensional culture. It was strongly induced in monolayer cultures whereas its increase was much reduced in three-dimensional cultures Figure 7. The small amount of CIITA induction in the three-dimensional cultures could be accounted for by the minority of cells that showed HLA-DR induction. It was concluded that three-dimensional culture inhibited the induction of CIITA, thus inhibiting HLA-DR induction. Numerous cases of variable survival of allograft cells in a host without immunosuppression have been reported but the mechanism is not clear (Hefton et al., 1983Hefton J.M. Madden M.R. Finkelstein J.L. Shires G.T. Grafting of burn patients with allografts of cultured epidermal cells.Lancet. 1983; 2: 428-430Abstract PubMed Scopus (219) Google Scholar;Hull et al., 1983Hull B.E. Sher S.E. Rosen S. Church D. Bell E. Fibroblasts in isogeneic skin equivalents persist for long periods after grafting.J Invest Dermatol. 1983; 81: 436-438Crossref PubMed Scopus (29) Google Scholar;Sher et al., 1983Sher S.E. Hull B.E. Rosen S. Church D. Friedman L. Bell E. Acceptance of allogeneic fibroblasts in skin equivalent transplants.Transplantation. 1983; 36: 552-557Crossref PubMed Scopus (63) Google Scholar;Thivolet et al., 1986Thivolet J. Fauré M. Demidem A. Mauduit G. Long-term survival and immunological tolerance of human epidermal allografts produced in culture.Transplantation. 1986; 42: 274-280Crossref PubMed Scopus (120) Google Scholar;Palmer et al., 1991Palmer T.D. Rosman G.J. Osborne W.R. Miller A.D. Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes.Proc Natl Acad Sci USA. 1991; 88: 1330-1334Crossref PubMed Scopus (407) Google Scholar;Otto et al., 1995Otto W.R. Nanchahal J. Lu Q.L. Boddy N. Dover R. Survival of allogeneic cells in cultured organotypic skin grafts.Plast Reconstr Surg. 1995; 96: 166-176Crossref PubMed Scopus (32) Google Scholar). Human split skin allografts show rejection within 2 wk, strongly related to major histocompatibility complex (MHC) incompatibility, the prime target lying within the epidermis. The most important cells expressing HLA class II in the epidermis are the Langerhans cells, which are professional antigen-presenting cells, normally also expressing costimulatory molecules such as CD40, CD80, and CD86, and able to stimulate T lymphocytes directly. These interactions, together with activation through endothelial cells, are thought to be the major factor in the rejection of ski" @default.
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• W1993393670 date "2001-07-01" @default.
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• W1993393670 title "Modification of Fibroblast γ-Interferon Responses by Extracellular Matrix" @default.
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• W1993393670 doi "https://doi.org/10.1046/j.0022-202x.2001.01386.x" @default.
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