Differential expression analysis by gene array of cell cycle modulators in human corneal epithelial cells stimulated with epidermal growth factor (EGF), hepatocyte growth factor (HGF), or keratinocyte growth factor (KGF)

PURPOSE: To identify and differentiate cell cycle and differentiation genes that are up-regulated or down-regulated in human corneal epithelial cells in response to alternative epithelium-modulating cytokines epidermal growth factor (EGF), hepatocyte growth factor (HGF) or keratinocyte growth factor (KGF). METHODS: Primary cultures human corneal epithelial cell (HCE) were treated with 25 ng/ml of EGF, 25 ng/ml HGF, 25 ng/ml KGF, or vehicle for 8 hours. Complementary DNA (cDNA) probes were synthesized from total cellular RNA isolated from the HCE cells. The cDNA probes were hybridized to the Atlas human cell cycle/differentiation array membrane. RNAse protection assay was used to confirm up-regulation of the serine/threonine-protein kinase PITALRE gene by EGF, KGF, and HGF. RESULTS: The expression of one hundred and eleven cell cycle and differentiation genes was monitored with the gene array system. It was found that these epithelial cell-modulating cytokines shared similar effects on some of the cell cycle and differentiation genes that were monitored, but had specific effects on some cytokines. Up-regulation of PITALRE gene expression was confirmed using RNAse protection assay. CONCLUSION: EGF, HGF and KGF had differential effects on cell cycle- and differentiation-related gene expression in corneal epithelial cells. For example, all three mitogenic growth factors up-regulated the expression of cyclin D1 (BCL-1 oncogene) and serine/threonine-protein kinase PITALRE in the primary cultured human corneal epithelial cells. However, EGF and KGF, but not HGF, up-regulated expression of the E2F-1 pRB-binding protein gene. Thus, while these three epithelial mitogens have similar effects on many genes that were analyzed, important differences were noted that may relate to differing effects of these growth factors on corneal epithelial cells. Studies to analyze the significance of the identified differences among these growth factors are in progress.     

Effect of autologous platelet-rich plasma on persistent corneal epithelial defect after infectious keratitis

Purpose

Platelet-rich plasma (PRP) harbors high concentrations of growth factors related to the promotion of wound healing. We evaluated the efficacy of PRP eyedrops in the treatment of persistent epithelial defects (PEDs).

Methods

Autologous PRP and autologous serum (AS) were prepared from whole blood. The concentrations of transforming growth factor (TGF)-β1, TGF-β2, epidermal growth factor (EGF), vitamin A and fibronectin in the PRP and AS were analyzed and compared. The corneal epithelial healing efficacy of PRP was compared with that of AS in patients with PED induced by post-infectious inflammation.

Results

The concentrations of TGF-β1, TGF-β2, EGF, vitamin A and fibronectin in the PRP and AS were not statistically different. However, the concentrations of EGF in the PRP were significantly greater than in the AS. AS was used in 17 and PRP in 11 eyes of 28 patients. The healing rates of the corneal epithelia of the PRP-treated eyes were significantly higher than those treated with AS.

Conclusions

The PRP was effective in the treatment of PEDs. This may be attributable to its high concentration of platelet-contained growth factors, most notably EGF. PRP could be an effective, novel treatment option for chronic ocular surface disease.     

Reactive oxygen species (ROS) are essential mediators in epidermal growth factor (EGF)-stimulated corneal epithelial cell proliferation, adhesion, migration, and wound healing

EGF is an essential growth factor needed for epithelial cell proliferation and wound healing of the cornea, but the molecular mechanism is not understood. Although studies have shown that EGF in some non-phagocytic cells induces ROS generation, little is known about the role of ROS in corneal epithelial cells. Therefore, we examined the potential physiological role of ROS in corneal cell proliferation, adhesion and wound healing using rabbit or human corneal epithelial cells, and pig whole cornea organ culture as models. EGF (5 ng/ml)-induced ROS in serum-starved RCE or HCE cells were captured as DCFH fluorescence and detected by confocal microscopy. The elevation of ROS was eradicated when the cells were pretreated with an antioxidant N-acetylcysteine (NAC) or mannitol, or with inhibitor to NADPH oxidase (DPI), or to lipoxygenase (NDGA). EGF-induced ROS generation correlated with cell growth and activation of Akt and MAPK signaling pathways, while NAC eliminated all these effects. EGF-stimulated cell adhesion or migration in cell culture was greatly suppressed in the presence of NAC while EGF-facilitated epithelial cell wound healing in corneal organ culture was also blocked by NAC. This is the first demonstration of a novel ROS physiological function in corneal wound healing.     

Epidermal growth factor stimulates phospholipase cgamma1 in cultured rabbit corneal epithelial cells

Phospholipase Cgamma1 (PLCgamma1) catalyses hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate (IP(3)), two second messengers which play important roles in cell proliferation and differentiation. The purpose of the current study was to identify PLCgamma1 in corneal epithelial cells and investigate whether epidermal growth factor (EGF) stimulates the activity of this enzyme. Addition of EGF to [(3)H]myo-inositol-labeled, cultured corneal epithelial cells stimulated production of IP(3), indicating activation of PLC. Western immunoblot analysis and an in vitro assay of PLC activity revealed that EGF activates gamma1 isoform of PLC, which is localized predominantly in the cytosolic fraction of the epithelial cells. EGF receptors were detected in the epithelial cells by EGF receptor antibody. Addition of EGF to the cells caused tyrosine phosphorylation of the receptors, translocation of PLCgamma1 from cytosol to plasma membrane, and phosphorylation of the enzyme at tyrosine residues. Addition of tyrphostin A-25, an inhibitor of receptor tyrosine kinase, attenuated the tyrosine phosphorylation of PLCgamma1 as well as its enzyme activity. These findings suggest that EGF stimulates PLCgamma1 in rabbit corneal epithelial cells, and that this effect is probably mediated by tyrosine phosphorylation of the enzyme.     

Differential effects of epidermal growth factor and interleukin 6 on corneal epithelial cells and vascular endothelial cells.

PURPOSE: Epidermal growth factor (EGF) and interleukin 6 (IL-6) stimulate corneal epithelial wound healing. When applied to the cornea, these cytokines act on various types of cells and therefore may induce corneal neovascularization. We investigated the effects of EGF and IL-6 on cell proliferation and cell migration in rabbit corneal epithelial cells and human umbilical vein endothelial cells (HUVECs). METHODS: Corneal epithelial cells or HUVECs were cultured with EGF or IL-6 in the presence of 1% fetal bovine serum, and the number of cells were counted, or the radioactivity of [3H]thymidine-incorporated cells was measured. Monolayered cultured corneal epithelial cells or HUVECs were mechanically wounded, and then the cells were cultured with serum-free basal medium containing EGF or IL-6. After 12 or 24 h, the wounded area was measured. Corneal blocks were cultured with serum-free TC-199 medium containing EGF or IL-6 for 24 h, and then the length of the path of the corneal epithelium was measured. RESULTS: Estimated cell count and [3H]thymidine uptake showed that EGF stimulated cell proliferation in both corneal epithelial cells and HUVECs in a dose-dependent manner. In contrast, IL-6 did not affect cell proliferation in either cell type. Furthermore, EGF also stimulated cell migration by increasing the monolayer and organ-culture system in both cells in a dose-dependent fashion. However, IL-6 stimulated cell migration only in corneal epithelial cells and not in HUVECs. CONCLUSION: These results demonstrated that EGF stimulated cell proliferation and migration in both corneal epithelial cells and HUVECs. In contrast, IL-6 stimulated only corneal epithelial cell migration and did not affect cell proliferation in either cell type or cell migration in HUVECs. These results suggest that, when applied to the cornea, EGF might induce corneal neovascularization, and IL-6 probably would not.     

A serum-free clonal growth assay for limbal, peripheral, and central corneal epithelium.

The stem cells and transient amplifying cells of the corneal epithelium are thought to be localized in the limbal and corneal basal epithelium, respectively. To study the differential regulation of proliferation of these progenitor cells, a defined, serum-free, clonal growth assay was developed for central (CC) and peripheral (PC) corneal and limbal (L) epithelial cells. After incubation in Dispase II (1.2 U/ml; 1 hr for PC and CC and 3 hr for L) and subsequent brief trypsin-ethylenediaminetetraacetic acid digestion, 18 or 180 single cells/cm2 were seeded in MCDB 151 medium supplemented with insulin, transferrin, selenium, hydrocortisone, epidermal growth factor (EGF), phosphoethanolamine, ethanolamine, and calcium. During the first week of culture, the cells gradually developed an increasing number of colonies, and the mean colony-forming efficiency on day 6 for L was 4.2 +/- 2.4%, significantly lower than 11.4 +/- 5.9% for PC and 12.8 +/- 7.6% for CC (P less than 0.003). Colony morphology was identical in L, PC, and CC with small, elongated cells more cohesive in the center but more migratory in the periphery. There were no differences in immunofluorescent staining with monoclonal antibody AE-5, indicating the corneal derivation of all colonies. Cultures could be passaged on day 14 and grown for more than 3 weeks with increasing desquamation. Addition of a mixture of trace metals to yield MCDB 153 did not enhance growth; increased selenium concentrations were inhibitory. Elimination of EGF from the supplement abolished most of the clonal growth. The lower rate of L proliferation might be explained by the absence of serum and stromal mitogens. This culture system seems preferentially to support transient amplifying cells and allows investigation of the differentiation of isolated corneal stem cells to transient amplifying cells or the proliferation and differentiation of transient amplifying cells by various factors without the interaction of undefined serum components or paracrine influences from other cells.     

Growth factor changes in ex vivo expansion of human limbal epithelial cells on human amniotic membrane.

PURPOSE: Human limbal epithelial cells cultured on human amniotic membrane have been used for transplantation to treat corneal surface injuries. We determined whether the amniotic basement membrane affects the growth of human limbal epithelial cells through the production of growth factors. METHODS: The epithelial cells grown out from limbal basal epithelium were placed on conventional culture plastic or on the epithelial side of denuded amniotic membrane under serum-free conditions. Culture supernatant was assayed for growth factor release at 24, 48, and 96 hours. RESULTS: The cells grown on both substrata produced similar levels of epidermal growth factor (EGF). Cells grown on amniotic membrane showed enhanced secretion of tissue inhibitor of metalloproteinase type 1 (TIMP1) and reduced production of transforming growth factor beta1 and beta2. Depletion of EGF and TIMPI in cell culture slowed down cell growth and reduced EGF receptor expression, respectively. CONCLUSION: Increased TIMPI influences the proteolytic system in the cell and extracellular matrix interaction, and decreased transforming growth factor beta1 and beta2 may stimulate corneal cell proliferation. We show that the amniotic membrane leads to differential expression of cytokines of limbal epithelial cells cultured on its surface. Such effects may be favorable to the growth and differentiation of the cells when used for ocular surface reconstruction.     

Transforming growth factor-beta modulates effects of epidermal growth factor on corneal epithelial cells.

In order to understand the mechanisms that bring about maintenance and restoration of the integrity of corneal epithelium, we investigated independent and combined effects of transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) on rabbit corneal epithelial cells in cell and organ culture. Specifically, we determined whether incubation with these factors influenced 1) cellular proliferation, 2) ability of cells to attach to a fibronectin matrix, and 3) the rate of epithelial migration over corneal stroma. Incubation with TGF-beta caused a dose-related decrease in the incorporation of 3H-thymidine by the epithelial cells. EGF increased 3H-thymidine incorporation, but this effect was antagonized by the addition of TGF-beta into the incubation medium. Incubation with EGF increased the numbers of cells that attached to a fibronectin matrix. TGF-beta itself did not affect the number of attached cells but, again, it antagonized the stimulatory effect of EGF. Similarly, when corneal blocks were cultured with EGF, epithelial migration increased in a dose-related manner. TGF-beta itself did not affect epithelial migration at any of the concentrations tested (0.1-10 ng/ml), but it antagonized EGF-stimulated epithelial migration. These findings suggest that the proliferation and the migration of corneal epithelial cells are regulated by different mechanisms, and that TGF-beta serves as a modulator of the effects of EGF.     

Growth factors: importance in wound healing and maintenance of transparency of the cornea

The mechanism of corneal wound healing has not been clarified yet. However, evidence has accumulated that various kinds of growth factor such as epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF) play a key role in corneal wound healing. For example, these growth factors are expressed in the corneal epithelial cells, keratocytes and endothelial cells, and their receptors are expressed in the corneal cells. Furthermore, these growth factors promote the proliferation of corneal cells and induce the migration of corneal cells. In addition to the growth factors, inflammatory cytokines such as interleukin (IL)-1, IL-6 and TNF-alpha are involved in corneal wound healing. These cytokines are expressed in the normal and inflammatory cornea after infections, alkaliburn, etc. where they control the growth of corneal cells and induce the migration of corneal cells. Thus, a number of growth factors and cytokines function in the regulation of corneal cell proliferation and in the maintenance of corneal transparency.     

表皮生长因子及其受体在眼表创伤愈合中的作用

表皮生长因子及其受体家族是细胞生长分化的重要调节因素。目前已证实表皮生长因子家族成员可持续表达于泪液中,其受体可表达于眼表上皮,对维持眼表的完整和促进角膜组织的创伤愈合具有重要意义。本文就表皮生长因子及其受体家族在眼表创伤愈合中的作用进行综述。     

EGF联合KGF促进人角膜上皮细胞生长

目的:寻找促进角膜上皮损伤修复,治疗持续性角膜上皮缺损的有效药物方法:用~3H—胸腺嘧啶核苷(~3H—TDR)掺入及液体闪烁技术,观察外源性表皮生长因子(EGF)联合角蛋白细胞生长因子(KGF)对体外培养的角膜上皮细胞DNA合成的影响,并计算细胞倍增时间。结果:10ng/mlEGF,10ng/mlKGF单独或联合应用均有明显促进人角膜上皮细胞DNA合成的作用(与对照组比较 P<0.01),联合用药,作用更强(P<0.05)。应用EGF与KGF明显缩短了细胞倍增时间。结论:外源性EGF与KGF对体外培养的人角膜上皮细胞有明显的促细胞增生作用,联合用药,效果更佳。表明EGF与KGF具有应用于临床,促进角膜上皮损伤修复的可能性。眼科学报1996; 12:107-109。     

Development of genetically engineered tet HPV16-E6/E7 transduced human corneal epithelial clones having tight regulation of proliferation and normal differentiation

Lack of an optimal in vitro model of human corneal epithelial (HCE) cells is a major limitation in studying normal functions and gene regulations in HCE. Moreover, availability of a multi-layered HCE culture can reduce the usage of animals in the toxicity testing of consumer products. We have developed tetracycline-responsive human papilloma virus (HPV) 16-E6/E7 transduced HCE clones showing tight regulation of proliferation and normal differentiation. Expression of HPV16-E6/E7 mRNA and HPV16-E7 and keratin K3 proteins was examined by RNase protection assay and western blotting, respectively, in presence and absence (+/-) of Dox in identified clones. Localization of cornea-specific keratin k3 in +/- of Dox was evaluated by immunocytochemistry. The response of growth factors such as hepatocyte growth factor (HGF) and epidermal growth factor to the cellular proliferation in +/- of Dox in the newly identified clones was measured by cell counting. Cellular morphology, formation of multi-layered cultures at air-liquid interface and ultrastructural features were evaluated by light and transmission electron microscopy. The physical barrier established by the newly developed clones was determined by the transepithelial permeability to sodium fluorescein and transepithelial electrical resistance assays in the airlifted-stratified cultures.     

Differential regulation of fibronectin synthesis in three different types of corneal cells

The production of fibronectin (FN) and its response to serum or epidermal growth factor (EGF) were investigated in three different types of rabbit corneal cells cultured in vitro. The corneal epithelial cells, stromal fibroblasts (keratocytes) and endothelial cells were separately cultured in different media: basic medium containing minimal serum (0.5%), basic medium with supplementary serum at a final concentration of 10% and basic medium with 100 ng/ml EGF, respectively. FN production by each type of cell was examined either by the immunofluorescent staining method or by the metabolic labeling method followed by immunoprecipitation of FN in the culture medium. Each type of corneal cell produced and secreted FN. FN secretion into the culture medium by keratocytes and by endothelial cells was enhanced by the addition of EGF. However, FN secretion by epithelial cells was lowered by the additional serum or EGF. Furthermore, when the epithelial cells were cultured in the basic medium, DNA synthesis was low but FN secretion was high. These results suggest that the control mechanism of FN production differs between epithelial cells and keratocytes or endothelial cells.     

Growth factor-induced proliferation in corneal epithelial cells is mediated by 12(S)-HETE

PURPOSE: Previous studies in our laboratory have shown that 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), a product of 12-lipoxygenase (12-LOX) activity, is the predominant metabolite formed in rabbit corneas after injury. The present study was undertaken to investigate the effects of epidermal growth factor (EGF), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF) on 12-LOX expression and activity. We also investigated whether 12(S)-HETE mediated the growth factor-induced proliferation of corneal epithelial cells. METHODS: Rabbit corneas were stimulated with EGF, HGF, and KGF (10 ng ml(-1)) for different times. 12-LOX activity was assayed by incubating corneal microsomal preparations with radiolabeled arachidonic acid (AA) as substrate. For inhibitor studies, the microsomes were pretreated with 12-LOX-specific inhibitors baicalein (BC) or cinnamyl 3,4-dihydroxy-(alpha)-cyanocinnamate (CDC). Lipid extracts were injected onto an Ultramex 5 microm C(18) column and radioactivity was monitored online by a Radiomatic Flo-One Beta detector. Stereochemical analysis of 12-HETE product was determined by chiral-phase HPLC. To evaluate the effects of growth factors on 12-LOX mRNA expression, mRNA was extracted at several time points (12, 24, 36, 48 hr) and subjected to real-time PCR. For 12-LOX protein expression, microsomal preparations from 24- and 48-hr incubations were analyzed by Western blot. In cell-proliferation studies, epithelial cells treated with EGF, HGF, or KGF for 24, 48, and 72 hr were measured with a CyQUANT cell-proliferation assay kit. To determine the role of growth factor-induced 12(S)-HETE synthesis on corneal epithelial cell proliferation, cells were pretreated with 12-LOX-specific inhibitors BC or CDC prior to growth-factor supplementation. RESULTS: Stimulation with EGF, HGF, or KGF for 12 hr induced 12-LOX mRNA expression in rabbit corneal epithelial cells. This gene induction was followed by an increase in protein expression at 24 and 48 hr and a marked increase in 12(S)-HETE synthesis when compared to untreated controls. At 24-hr incubations, KGF showed a greater capacity than did EGF and HGF to stimulate microsomal 12-LOX activity, while at 48 hr 12(S)-HETE synthesis was significantly greater in EGF-treated cells as compared to that of HGF- and KGF-treated cells. Pretreatment with 12-LOX inhibitors blocked the growth factor-induced increase in 12(S)-HETE synthesis. Stimulation with growth factors or 12(S)-HETE for 24, 48, and 72hr produced a significant increase in corneal epithelial proliferation, which was partially inhibited by pretreatment of cells with 12-LOX-specific inhibitors. CONCLUSION: These findings suggest that EGF, HGF, and KGF stimulate 12(S)-HETE production in rabbit corneal epithelial cells through gene induction of 12-LOX. Furthermore, 12(S)-HETE may play a role in regulating epithelial cell proliferation and the rate of corneal re-epithelialization following an injury.     

Topische EGF-Applikation bei therapierefraktärer Erosio corneae unter Cetuximab-Behandlung

BACKGROUND: Cetuximab (Erbitux), a monoclonal epidermal growth factor receptor (EGFR) antibody, has been used for the treatment of advanced colorectal carcinoma over the last two years. Inhibition of EGFR also influences corneal wound healing as EGF stimulates the proliferation of epithelial cells. METHOD: Extensive corneal erosion was seen in both eyes of a 62-year-old patient under treatment with cetuximab for a metastasized colorectal carcinoma. Progression was fast despite vigorous conservative treatment. The application of autologous serum could not be considered because of the antibody treatment. Human EGF was applied topically several times daily in order to utilize the proliferative effect on corneal epithelial cells and to antagonize the inhibition of EGF receptors. RESULTS: Improvement was seen shortly after the onset of therapy with EGF eye drops.. The epithelial defect was closed 7 days (left eye) and 19 days (right eye), respectively, after the onset of therapy. During this time treatment with cetuximab was continued. CONCLUSIONS: Cetuximab (Erbitux) can cause persisting epithelial defects. Patients with an impairment of corneal wound healing under cetuximab treatment can benefit from the topical application of human EGF. Consequently, surgical measures or complications such as infections can be avoided.     

Correlation of proliferative and anti-apoptotic effects of HGF, insulin, IGF-1, IGF-2, and EGF in SV40-transformed human corneal epithelial cells

The effects of various growth factors on the proliferation and apoptosis of human corneal epithelial cells were investigated. Simian virus 40-transformed human corneal epithelial cells were thus incubated separately with eight different growth factors, after which cell proliferation was evaluated by measurement of [(3)H]thymidine incorporation or with the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay and apoptosis was quantified by the terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick-end labeling assay. Phosphorylation of the protein kinase Akt, which plays an important role in anti-apoptotic signaling, was also assessed by immunoblot analysis. The growth factors examined could be classified into three groups on the basis of their effects on the proliferation and apoptosis of human corneal epithelial cells: hepatocyte growth factor (HGF), insulin, insulin-like growth factor (IGF)-1, IGF-2, and epidermal growth factor (EGF) each increased cell proliferation, inhibited the induction of apoptosis by sodium nitroprusside, and elicited the activation of Akt; transforming growth factor-beta1 and -beta2 inhibited [3H]thymidine incorporation but had no effect on sodium nitroprusside-induced apoptosis or on Akt activity; and platelet-derived growth factor-BB had no effects on the measured parameters. HGF, insulin, IGF-1, IGF-2, and EGF may thus contribute to maintenance of the corneal epithelium and coordinate the proliferative and apoptotic responses of this tissue.     

新型VEGF靶向抗体眼局部应用的安全性评价

目的：通过体外细胞实验和在体动物实验初步评价MIL60在眼局部应用的安全性。

方法：常规培养人角膜上皮细胞,CCK8法体外检测MIL60抗体对角膜上皮细胞毒副作用。流式细胞仪检测MIL60对角膜上皮细胞的凋亡影响。正常SD大鼠结膜下注射MIL60抗体,观察眼部反应情况,通过裂隙灯显微镜检查、Draize的评分系统和病理切片等,分析结膜和角膜病理改变。建立SD大鼠角膜上皮缺损模型,结膜下注射MIL60抗体,观察角膜上皮愈合情况。

结果：MIL60不影响角膜上皮细胞增殖,不促进角膜上皮细胞的凋亡。结膜下注射MIL60抗体后,大鼠角结膜和眼部其他各组织形态正常,组织学检查显示结构正常,未见炎症细胞浸润。结膜下注射MIL60不影响角膜上皮愈合。

结论：MIL60结膜下应用安全性较好,不影响角膜上皮细胞正常功能。     

Regulation of HLA class II antigen expression on cultured corneal epithelium by interferon-gamma.

The effect of recombinant human interferon gamma (IFN-gamma) on the induction of HLA class II (HLA-DR, -DP, -DQ) antigen expression on human corneal epithelial (HCE) cells was examined in different stages of culture. Primary cultures were established with limbal explants without endothelium. HCE cells in Stage 1 and Stage 2, with cells negative and positive for the 64K corneal keratin (the marker for advanced corneal epithelial differentiation), respectively, were prepared. HCE cells in both stages were treated with IFN-gamma at a concentration of 0 to 1000 U/ml for two to six days and were stained by the avidin-biotin peroxidase complex method. Class II antigens were not detected on HCE cells in either stage without IFN-gamma treatment. IFN-gamma induced three class II antigens on HCE cells in both stages in a dose- and time-dependent manner but at different levels for each antigen (DR greater than DP greater than DQ). In addition, DQ expression was related to cell differentiation, with DQ extremely rare at Stage 1 and more frequent at Stage 2 (5% vs. 20%). These findings indicate that the induction of class II antigens on HCE cells may be regulated by IFN-gamma independently for each of the antigens and that DQ induction may depend upon the differentiation of HCE cells in culture.