L. Gildea, C. Ryan, B. Hulette, R. Dearman, I. Kimber, G. Gerberick
The interaction of antigen‐specific T lymphocytes with hapten‐bearing dendritic cells (DC) and the subsequent activation and clonal expansion of specific T lymphocyte populations are critical steps in the induction of skin sensitization. Therefore, we have sought to characterize changes in gene expression in T lymphocytes stimulated by incubation with allergen‐treated DC compared with anti‐CD3‐treated T cell‐DC cocultures as a method to identify potential markers of skin sensitization. Human T cells and autologous, mature peripheral blood‐derived DC were co–cultured in the presence or absence of anti‐CD3 monoclonal antibody (mAb) for 6 hours at a 10:1 responder:stimulator ratio. In a separate experiment, autologous DC and T cells from a donor sensitized to the potent contact allergen dinitrochlorobenzene (DNCB) were isolated. T cells were cultured for 6 hours at a responder to stimulator ratio of 20:1 with mature DC that had been treated with either 1 mM 2,4‐dinitrobenzenesulfonic acid (DNBS; the water soluble analog of DNCB), or media alone for 15 minutes. Total RNA was prepared and changes in gene expression were analyzed using Affymetrix U95Av2 GeneChips®. Comparative analysis of Affymetrix mean signal values from triplicate control cultures with those from anti‐CD3‐treated samples revealed highly significant (p ≤ 0.001) changes in the expression of 344 transcripts of the total of approximately 12,000 represented on the chip. However, mean signal values for T cells cocultured with allergen‐treated DC compared to vehicle‐treated DC‐T cell co–cultures identified only 17 significant gene changes (p ≤ 0.001), 11 of which were also identified as having changed significantly in response to stimulation with anti‐CD3. In parallel assays, antigen‐specific T cell proliferative responses were assessed as a function of tritiated thymidine incorporation. Increased T cell proliferative responses were observed in the cultures that contained both DNBS‐treated DC and T cells as well as the anti‐CD3 treated cultures compared with their respective controls. These data suggest that this approach can be used to identify genes that might serve as indicators of contact allergy and may be used in an in vitro predictive assay for skin sensitization.
抗原特异性T淋巴细胞与携带半抗原的树突状细胞(DC)的相互作用以及随后特异性T淋巴细胞群的激活和克隆扩增是诱导皮肤致敏的关键步骤。因此，我们试图通过与抗CD3处理的T细胞- DC共培养物相比，表征经过敏原处理的DC孵育刺激的T淋巴细胞中基因表达的变化，作为鉴定皮肤致敏的潜在标记物的方法。在抗CD3单克隆抗体(mAb)存在或不存在的情况下，以10:1的反应:刺激比，人T细胞和自体成熟外周血源DC共培养6小时。在另一项实验中，分离了来自强效接触性过敏原二硝基氯苯(DNCB)致敏的供体的自体DC细胞和T细胞。T细胞以20:1的反应性刺激比培养6小时，成熟DC分别用1 mM 2,4‐二硝基苯磺酸(DNBS;水溶类似物DNCB)，或单独介质浸泡15分钟。制备总RNA，使用Affymetrix U95Av2 GeneChips®分析基因表达变化。对三次对照培养的Affymetrix平均信号值与抗CD3处理样本的信号值进行比较分析显示，芯片上总共约12,000个转录本中的344个转录本的表达发生了高度显著(p≤0.001)的变化。然而，与载体处理DC - T细胞共培养相比，与过敏原处理DC - T细胞共培养的T细胞的平均信号值仅鉴定出17个显著的基因变化(p≤0.001)，其中11个也被鉴定为在抗CD3刺激下发生显著变化。在平行试验中，抗原特异性T细胞增殖反应被评估为氚化胸腺嘧啶掺入的功能。在含有DNBS处理的DC和T细胞以及抗CD3处理的培养物中，与各自的对照组相比，观察到T细胞增殖反应增加。这些数据表明，该方法可用于鉴定可能作为接触性过敏指标的基因，并可用于皮肤致敏的体外预测试验。
{"title":"Transcript Profiling of T Lymphocytes and Dendritic Cells in a Co–culture System Using Anti‐CD3 and Allergen Activation","authors":"L. Gildea, C. Ryan, B. Hulette, R. Dearman, I. Kimber, G. Gerberick","doi":"10.1081/CUS-200037209","DOIUrl":"https://doi.org/10.1081/CUS-200037209","url":null,"abstract":"The interaction of antigen‐specific T lymphocytes with hapten‐bearing dendritic cells (DC) and the subsequent activation and clonal expansion of specific T lymphocyte populations are critical steps in the induction of skin sensitization. Therefore, we have sought to characterize changes in gene expression in T lymphocytes stimulated by incubation with allergen‐treated DC compared with anti‐CD3‐treated T cell‐DC cocultures as a method to identify potential markers of skin sensitization. Human T cells and autologous, mature peripheral blood‐derived DC were co–cultured in the presence or absence of anti‐CD3 monoclonal antibody (mAb) for 6 hours at a 10:1 responder:stimulator ratio. In a separate experiment, autologous DC and T cells from a donor sensitized to the potent contact allergen dinitrochlorobenzene (DNCB) were isolated. T cells were cultured for 6 hours at a responder to stimulator ratio of 20:1 with mature DC that had been treated with either 1 mM 2,4‐dinitrobenzenesulfonic acid (DNBS; the water soluble analog of DNCB), or media alone for 15 minutes. Total RNA was prepared and changes in gene expression were analyzed using Affymetrix U95Av2 GeneChips®. Comparative analysis of Affymetrix mean signal values from triplicate control cultures with those from anti‐CD3‐treated samples revealed highly significant (p ≤ 0.001) changes in the expression of 344 transcripts of the total of approximately 12,000 represented on the chip. However, mean signal values for T cells cocultured with allergen‐treated DC compared to vehicle‐treated DC‐T cell co–cultures identified only 17 significant gene changes (p ≤ 0.001), 11 of which were also identified as having changed significantly in response to stimulation with anti‐CD3. In parallel assays, antigen‐specific T cell proliferative responses were assessed as a function of tritiated thymidine incorporation. Increased T cell proliferative responses were observed in the cultures that contained both DNBS‐treated DC and T cells as well as the anti‐CD3 treated cultures compared with their respective controls. These data suggest that this approach can be used to identify genes that might serve as indicators of contact allergy and may be used in an in vitro predictive assay for skin sensitization.","PeriodicalId":17547,"journal":{"name":"Journal of Toxicology-cutaneous and Ocular Toxicology","volume":"69 1","pages":"277 - 292"},"PeriodicalIF":0.0,"publicationDate":"2004-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82380067","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
P. H. Nicolaysen, K. Klink, E. Shriver, G. Knutsen, A. Hubbs, G. J. Depree, P. Siegel, D. Weissman, M. Whitmer, B. Meade
Penrose drains are widely used in surgical procedures as an aid in wound healing. The studies presented here investigated the potential toxicity associated with the implantation of latex Penrose drains in BALB/c and B6C3F1 mice. Animals were implanted subcutaneously in the dorsal surface of the neck with 100, 150, or 200 mg of Perry latex drain or 200 mg of Bard (comparative control) latex drain for up to 36 hours. High‐dose (200 mg) exposure to the Perry drain induced severe local and systemic toxicity, resulting in mortality within 24 hours. Time‐ and dose‐responsive effects included decreased response to stimulus, inflammation at the implantation site, epaxial myositis, lesions consistent with hepatic glycogen depletion, apoptotic necrosis of the adrenal “X zone,” and massive thymic apoptosis and atrophy. Negligible levels of endotoxin were quantified from Perry drain samples using the Limulus Amebocyte Lysate Assay. Extraction studies revealed the presence of zinc diethyldithiocarbamate (ZDEC) in the Perry drains but not in the control drains. No other differences were noted from gas chromatography mass spectrometry (GCMS) analyses. Quantitation studies measured ZDEC levels at 2.22 ± 0.04 µg/mg in Perry samples. When ZDEC was eluted from Perry drains prior to implantation, animals exhibited no signs of toxicity. Although FDA regulations limit accelerators to 1.5% of rubber medical products, these studies indicate that the presence of ZDEC in concentrations lower than 0.25% of the drain weight may induce local toxicity and delayed wound healing.
{"title":"Local and Systemic Toxicity in Mice Following Subcutaneous Implantation of Latex Penrose Drains","authors":"P. H. Nicolaysen, K. Klink, E. Shriver, G. Knutsen, A. Hubbs, G. J. Depree, P. Siegel, D. Weissman, M. Whitmer, B. Meade","doi":"10.1081/CUS-200036691","DOIUrl":"https://doi.org/10.1081/CUS-200036691","url":null,"abstract":"Penrose drains are widely used in surgical procedures as an aid in wound healing. The studies presented here investigated the potential toxicity associated with the implantation of latex Penrose drains in BALB/c and B6C3F1 mice. Animals were implanted subcutaneously in the dorsal surface of the neck with 100, 150, or 200 mg of Perry latex drain or 200 mg of Bard (comparative control) latex drain for up to 36 hours. High‐dose (200 mg) exposure to the Perry drain induced severe local and systemic toxicity, resulting in mortality within 24 hours. Time‐ and dose‐responsive effects included decreased response to stimulus, inflammation at the implantation site, epaxial myositis, lesions consistent with hepatic glycogen depletion, apoptotic necrosis of the adrenal “X zone,” and massive thymic apoptosis and atrophy. Negligible levels of endotoxin were quantified from Perry drain samples using the Limulus Amebocyte Lysate Assay. Extraction studies revealed the presence of zinc diethyldithiocarbamate (ZDEC) in the Perry drains but not in the control drains. No other differences were noted from gas chromatography mass spectrometry (GCMS) analyses. Quantitation studies measured ZDEC levels at 2.22 ± 0.04 µg/mg in Perry samples. When ZDEC was eluted from Perry drains prior to implantation, animals exhibited no signs of toxicity. Although FDA regulations limit accelerators to 1.5% of rubber medical products, these studies indicate that the presence of ZDEC in concentrations lower than 0.25% of the drain weight may induce local toxicity and delayed wound healing.","PeriodicalId":17547,"journal":{"name":"Journal of Toxicology-cutaneous and Ocular Toxicology","volume":"41 1","pages":"233 - 248"},"PeriodicalIF":0.0,"publicationDate":"2004-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76235888","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Background: Sulfur mustard has been a popular chemical warfare agent in the twentieth century. This agent was used in the Iraqi–Iranian conflict in 1983–88. The casualties exposed to sulfur mustard have exhibited acute and chronic complications in the eye, lungs, and skin. Around 15 years post exposure to sulfur mustard, we performed this study to evaluate the severity of the delayed complications in the eyes and the respiratory system. Material and Methods: In this descriptive study, we evaluated 500 male subjects in the age range of 30–50 years, in 2001. These soldiers' first in toxification to sulfur mustard was confirmed by the Department of Medical Consultation of the Janbazan Organization (the Iranian Veterans' Affairs Agency). Complete ocular and pulmonary examinations were performed. The complications were divided into three grades of mild, moderate, and severe. In order to be classified in a certain grade of severity, the patient must have exhibited at least 50% of the designated signs and symptoms in that category. The frequencies of the patients in each grade were reported and chi‐square analysis was performed. Results: The distribution of the subjects within each grade of the complications was as follows: mild ocular, 80%; moderate ocular, 13.2%; severe ocular, 6.8%; mild pulmonary, 69.2%; moderate pulmonary, 19.6%; and severe pulmonary, 11.6%. The distribution of the patients within the different grades of the ocular and pulmonary complications concurrently was as follows: mild ocular and pulmonary complications, 57.2%; moderate ocular and pulmonary complications, 2%; and severe ocular and pulmonary complications, 1.4%. Of all the patients, 14.8% had more severe ocular complications and 24.6% had more severe pulmonary complications. The pulmonary complications were present in higher severity than the ocular complications and statistically the relationship was significant (P < 0.005). Conclusion: In this study, all of the subjects exhibited at least mild ocular and pulmonary complications, around 15 years post exposure to sulfur mustard. The severity of the pulmonary complications is higher than the ocular complications. We conclude that these chronic complications are rising. The subjects with no or mild complications in earlier years might develop more severe complications in the future. From the previous cytological studies, it is apparent that the changes induced due to exposure to sulfur mustard are at the cellular structural levels. These changes are not easily reversible or treatable. The proper management of these individuals, with the possible rise in the severity of their complications, remains a major health concern.
{"title":"The Delayed Ocular and Pulmonary Complications of Mustard Gas","authors":"M. Ghassemi‐Broumand, K. Agin, Haleh Kangari","doi":"10.1081/CUS-200037213","DOIUrl":"https://doi.org/10.1081/CUS-200037213","url":null,"abstract":"Background: Sulfur mustard has been a popular chemical warfare agent in the twentieth century. This agent was used in the Iraqi–Iranian conflict in 1983–88. The casualties exposed to sulfur mustard have exhibited acute and chronic complications in the eye, lungs, and skin. Around 15 years post exposure to sulfur mustard, we performed this study to evaluate the severity of the delayed complications in the eyes and the respiratory system. Material and Methods: In this descriptive study, we evaluated 500 male subjects in the age range of 30–50 years, in 2001. These soldiers' first in toxification to sulfur mustard was confirmed by the Department of Medical Consultation of the Janbazan Organization (the Iranian Veterans' Affairs Agency). Complete ocular and pulmonary examinations were performed. The complications were divided into three grades of mild, moderate, and severe. In order to be classified in a certain grade of severity, the patient must have exhibited at least 50% of the designated signs and symptoms in that category. The frequencies of the patients in each grade were reported and chi‐square analysis was performed. Results: The distribution of the subjects within each grade of the complications was as follows: mild ocular, 80%; moderate ocular, 13.2%; severe ocular, 6.8%; mild pulmonary, 69.2%; moderate pulmonary, 19.6%; and severe pulmonary, 11.6%. The distribution of the patients within the different grades of the ocular and pulmonary complications concurrently was as follows: mild ocular and pulmonary complications, 57.2%; moderate ocular and pulmonary complications, 2%; and severe ocular and pulmonary complications, 1.4%. Of all the patients, 14.8% had more severe ocular complications and 24.6% had more severe pulmonary complications. The pulmonary complications were present in higher severity than the ocular complications and statistically the relationship was significant (P < 0.005). Conclusion: In this study, all of the subjects exhibited at least mild ocular and pulmonary complications, around 15 years post exposure to sulfur mustard. The severity of the pulmonary complications is higher than the ocular complications. We conclude that these chronic complications are rising. The subjects with no or mild complications in earlier years might develop more severe complications in the future. From the previous cytological studies, it is apparent that the changes induced due to exposure to sulfur mustard are at the cellular structural levels. These changes are not easily reversible or treatable. The proper management of these individuals, with the possible rise in the severity of their complications, remains a major health concern.","PeriodicalId":17547,"journal":{"name":"Journal of Toxicology-cutaneous and Ocular Toxicology","volume":"1 1","pages":"293 - 302"},"PeriodicalIF":0.0,"publicationDate":"2004-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78455568","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sodium dodecyl sulphate (SDS) is the most commonly studied irritant. Beside local skin effects, there are data that suggest effects of SDS in the context of the systemic microenvironment. The aim of this study was to investigate whether there are quantitative and qualitative changes in peripheral blood granulocytes following one‐time open epicutaneous application of SDS in rats. An increase in total leukocyte numbers with a shift toward granulocytes was noted following application of 0.4% SDS, while the metabolic activity of isolated peripheral blood granulocytes was increased after application of both 0.2% and 0.4% SDS. Differences were not noted in both spontaneous cell activation [evaluated by cytochemical nitroblue tetrazolium (NBT) reduction assay] and adhesion to plastic. Examination of granulocyte activity following 0.4% SDS application (when both quantitative and changes in metabolic activity were observed) demonstrated an increase in phorbol myristate acetate (PMA)‐stimulated activation and adhesion of granulocytes compared to responses of cells from control animals, suggesting their primed state. An increase in metabolic granulocyte activity was noted in overnight cultures supplemented with autologous plasma of granulocytes from the 0.4% SDS group, pointing to the role of systemic factors in observed increase in functional activity. As presented in this study, changes in peripheral blood granulocytes illustrate systemic effects of topical SDS application.
{"title":"Peripheral Blood Granulocyte Activity Following Epicutaneous Application of Sodium Dodecyl Sulphate (SDS) in Rats","authors":"Kataranovski Milena, V. Marija, K. Dragan","doi":"10.1081/CUS-200037205","DOIUrl":"https://doi.org/10.1081/CUS-200037205","url":null,"abstract":"Sodium dodecyl sulphate (SDS) is the most commonly studied irritant. Beside local skin effects, there are data that suggest effects of SDS in the context of the systemic microenvironment. The aim of this study was to investigate whether there are quantitative and qualitative changes in peripheral blood granulocytes following one‐time open epicutaneous application of SDS in rats. An increase in total leukocyte numbers with a shift toward granulocytes was noted following application of 0.4% SDS, while the metabolic activity of isolated peripheral blood granulocytes was increased after application of both 0.2% and 0.4% SDS. Differences were not noted in both spontaneous cell activation [evaluated by cytochemical nitroblue tetrazolium (NBT) reduction assay] and adhesion to plastic. Examination of granulocyte activity following 0.4% SDS application (when both quantitative and changes in metabolic activity were observed) demonstrated an increase in phorbol myristate acetate (PMA)‐stimulated activation and adhesion of granulocytes compared to responses of cells from control animals, suggesting their primed state. An increase in metabolic granulocyte activity was noted in overnight cultures supplemented with autologous plasma of granulocytes from the 0.4% SDS group, pointing to the role of systemic factors in observed increase in functional activity. As presented in this study, changes in peripheral blood granulocytes illustrate systemic effects of topical SDS application.","PeriodicalId":17547,"journal":{"name":"Journal of Toxicology-cutaneous and Ocular Toxicology","volume":"90 1","pages":"263 - 275"},"PeriodicalIF":0.0,"publicationDate":"2004-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88953754","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
1. The present study investigated the use of drugs that affect calcium (Ca2 +) levels and thus reduction of triamcinolone (TA)‐induced cytotoxicity on human retinal epithelial (ARPE19) cells. 2. Four groups were compared: ARPE19 cells alone, cells exposed to TA (0.1 mg/mL), cells that have been pretreated with one of the testing agents for 30 min before the addition of TA, and cells that have only been treated with one of the testing agents. Pinacidil (PIN) and its analogue, P1060, were used to test the effect of potassium (K+) channel opening on TA‐induced toxicity. Verapamil (VP) and diltiazem (DZ) were used to test their Ca2 + channel blocking effect. The cell viability under different settings was assessed using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Ca2 +‐imaging was used to determine the changes in intracellular Ca2 + levels [(Ca2 +)i] upon different treatments. 3. Both PIN and P1060 reduced TA‐induced toxicity. Verapamil and DZ increased the viability of cells treated with TA significantly, suggesting that the excessive influx of Ca2 + was one of the main contributory factors to the TA‐induced toxicity. 4. The results suggest that the prevention of Ca2 + entry may be effective in the reduction of cell necrosis in the presence of TA.
{"title":"Lowering Intracellular Calcium Concentration May Reduce the Cytotoxicity of Triamcinolone on Human Retinal Pigment Epithelial (ARPE19) Cells","authors":"C. Yeung, S. Chiang, K. Chan, C. Pang, D. Lam","doi":"10.1081/CUS-200036699","DOIUrl":"https://doi.org/10.1081/CUS-200036699","url":null,"abstract":"1. The present study investigated the use of drugs that affect calcium (Ca2 +) levels and thus reduction of triamcinolone (TA)‐induced cytotoxicity on human retinal epithelial (ARPE19) cells. 2. Four groups were compared: ARPE19 cells alone, cells exposed to TA (0.1 mg/mL), cells that have been pretreated with one of the testing agents for 30 min before the addition of TA, and cells that have only been treated with one of the testing agents. Pinacidil (PIN) and its analogue, P1060, were used to test the effect of potassium (K+) channel opening on TA‐induced toxicity. Verapamil (VP) and diltiazem (DZ) were used to test their Ca2 + channel blocking effect. The cell viability under different settings was assessed using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Ca2 +‐imaging was used to determine the changes in intracellular Ca2 + levels [(Ca2 +)i] upon different treatments. 3. Both PIN and P1060 reduced TA‐induced toxicity. Verapamil and DZ increased the viability of cells treated with TA significantly, suggesting that the excessive influx of Ca2 + was one of the main contributory factors to the TA‐induced toxicity. 4. The results suggest that the prevention of Ca2 + entry may be effective in the reduction of cell necrosis in the presence of TA.","PeriodicalId":17547,"journal":{"name":"Journal of Toxicology-cutaneous and Ocular Toxicology","volume":"1 1","pages":"249 - 261"},"PeriodicalIF":0.0,"publicationDate":"2004-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91231342","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
We reported previously that prolonged (13 day) topical exposure to chemical contact and respiratory allergens such as 2,4‐dinitrochlorobenzene (DNCB) and trimellitic anhydride (TMA) results in cytokine phenotypes consistent with the selective activation of type 1 and type 2 cells, respectively. In the current experiments, the role of the type 1 inducing heterodimeric cytokine interleukin (IL)‐12 in the development of these divergent cytokine secretion phenotypes has been examined by using an enzyme‐linked immunosorbant assay and neutralizing antibody specific for the p40 subunit of the dimer. After acute exposure, when a mixed Th0 phenotype is observed following treatment with allergen, both DNCB‐ and TMA‐activated lymph node cells (LNC) secrete similar levels of interferon‐γ (IFN‐γ) and IL‐12 p40, and neutralizing anti‐IL‐12 p40 antibody profoundly inhibits expression of IFN‐γ by both DNCB‐ and TMA‐stimulated LNC. After more prolonged treatment, divergent IFN‐γ and IL‐12 p40 production is recorded, with DNCB treatment inducing much higher levels of both cytokines than did identical exposure to TMA. Macrophage depletion studies have shown that the cellular source of IL‐12 p40 is primarily nonphagocytic cells. Given that DNCB‐ and TMA‐activated LNC are expressing similar levels of IL‐12 p40 and equivalent functional activity of IL‐12 with respect to IFN‐γ production following acute exposure, it seems unlikely that the divergent cytokine profiles observed following chronic exposure to allergen are being driven by differential IL‐12 production. As the response polarizes, a divergent capacity to express p40 IL‐12 is apparent, as is the functional activity of this cytokine, with IFN‐γ secretion by DNCB‐activated T helper 1 (Th1) and T cytotoxic 1 (Tc1) cells inhibited by neutralizing antibody, whereas TMA‐stimulated Tc1 cells are refractory to anti‐IL‐12 p40 antibody. These results show that the phenotype of mature DNCB‐ and TMA‐activated LNC is even more polarized than demonstrated previously, with exposure to TMA, but not to DNCB, resulting in a loss of functional responsiveness to IL‐12 p40, presumably as a result of loss of functional IL‐12 receptor expression.
{"title":"Cytokine Responses Induced by Skin Exposure of Mice to Chemical Allergens: Role of Interleukin 12","authors":"R. Dearman, I. Kimber","doi":"10.1081/CUS-200036689","DOIUrl":"https://doi.org/10.1081/CUS-200036689","url":null,"abstract":"We reported previously that prolonged (13 day) topical exposure to chemical contact and respiratory allergens such as 2,4‐dinitrochlorobenzene (DNCB) and trimellitic anhydride (TMA) results in cytokine phenotypes consistent with the selective activation of type 1 and type 2 cells, respectively. In the current experiments, the role of the type 1 inducing heterodimeric cytokine interleukin (IL)‐12 in the development of these divergent cytokine secretion phenotypes has been examined by using an enzyme‐linked immunosorbant assay and neutralizing antibody specific for the p40 subunit of the dimer. After acute exposure, when a mixed Th0 phenotype is observed following treatment with allergen, both DNCB‐ and TMA‐activated lymph node cells (LNC) secrete similar levels of interferon‐γ (IFN‐γ) and IL‐12 p40, and neutralizing anti‐IL‐12 p40 antibody profoundly inhibits expression of IFN‐γ by both DNCB‐ and TMA‐stimulated LNC. After more prolonged treatment, divergent IFN‐γ and IL‐12 p40 production is recorded, with DNCB treatment inducing much higher levels of both cytokines than did identical exposure to TMA. Macrophage depletion studies have shown that the cellular source of IL‐12 p40 is primarily nonphagocytic cells. Given that DNCB‐ and TMA‐activated LNC are expressing similar levels of IL‐12 p40 and equivalent functional activity of IL‐12 with respect to IFN‐γ production following acute exposure, it seems unlikely that the divergent cytokine profiles observed following chronic exposure to allergen are being driven by differential IL‐12 production. As the response polarizes, a divergent capacity to express p40 IL‐12 is apparent, as is the functional activity of this cytokine, with IFN‐γ secretion by DNCB‐activated T helper 1 (Th1) and T cytotoxic 1 (Tc1) cells inhibited by neutralizing antibody, whereas TMA‐stimulated Tc1 cells are refractory to anti‐IL‐12 p40 antibody. These results show that the phenotype of mature DNCB‐ and TMA‐activated LNC is even more polarized than demonstrated previously, with exposure to TMA, but not to DNCB, resulting in a loss of functional responsiveness to IL‐12 p40, presumably as a result of loss of functional IL‐12 receptor expression.","PeriodicalId":17547,"journal":{"name":"Journal of Toxicology-cutaneous and Ocular Toxicology","volume":"25 1","pages":"215 - 232"},"PeriodicalIF":0.0,"publicationDate":"2004-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83205237","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Purpose. In order to test irritancy levels of commercial products, the Draize animal test has been the universally accepted choice. However, since this test uses live animals, many researchers have been trying to develop an alternative organotypic culture. These organotypic cultures, however, utilize animal cells, and it is our belief that a model using human cells would be more predictive for determining human irritancy levels. Methods. Primary human corneal epithelial cells and fibroblasts were separately isolated from a limbal rim and grown in culture. SV40-transformed mouse corneal endothelial cells were seeded onto a membrane and grown to confluence. A homogenous fibroblast/collagen mixture was then added and allowed to gel. After about a week, the epithelial cells were seeded on top of the gel and the constructs were kept submerged in culture for 3-7 days. The constructs were then airlifted to allow for the stratification of the epithelial cells. Following this, the constructs were either fixed and processed for methacrylate sectioning to study morphology, or they were frozen and sectioned for indirect immunofluorescence. Indirect immunofluorescence was performed with ZO-1, a marker of tight junctions; keratins 3 and 12, markers for differentiation; and laminin, a marker of basement membrane components. Initially, a SV40-transformed human endothelial cell line was to be used; however, it did not grow well in this culture system. Results. In these experiments, we tested two methods of isolating epithelial cells. One method, the explant technique-a method we previously used to isolate rabbit epithelial cells-could not be maintained beyond two passages, and when added to the construct only stratified to 2-3 layers of flattened cells. However, with the second method, the dispase technique, the epithelial cells obtained grew rapidly and could be maintained beyond two passages. These cells, when added to the construct, stratified to 5-7 layers and exhibited a more epithelium-like morphology. Staining with ZO-1 indicated that tight junctions in the superficial cells were formed. Staining with keratins 3 and 12 indicated that the epithelial cells were differentiating, and staining with laminin indicated that basement membrane components were being synthesized. Conclusion. Constructing an organotypic culture with human corneal cells is possible, and the morphology of the construct appears to be equivalent to a construct already created with bovine cells that has been used for irritancy studies.
{"title":"Human Corneal Organotypic Cultures","authors":"J. Zieske, E. Chung, Xiaoqing Q. Guo, A. Hutcheon","doi":"10.1081/CUS-120027484","DOIUrl":"https://doi.org/10.1081/CUS-120027484","url":null,"abstract":"Purpose. In order to test irritancy levels of commercial products, the Draize animal test has been the universally accepted choice. However, since this test uses live animals, many researchers have been trying to develop an alternative organotypic culture. These organotypic cultures, however, utilize animal cells, and it is our belief that a model using human cells would be more predictive for determining human irritancy levels. Methods. Primary human corneal epithelial cells and fibroblasts were separately isolated from a limbal rim and grown in culture. SV40-transformed mouse corneal endothelial cells were seeded onto a membrane and grown to confluence. A homogenous fibroblast/collagen mixture was then added and allowed to gel. After about a week, the epithelial cells were seeded on top of the gel and the constructs were kept submerged in culture for 3-7 days. The constructs were then airlifted to allow for the stratification of the epithelial cells. Following this, the constructs were either fixed and processed for methacrylate sectioning to study morphology, or they were frozen and sectioned for indirect immunofluorescence. Indirect immunofluorescence was performed with ZO-1, a marker of tight junctions; keratins 3 and 12, markers for differentiation; and laminin, a marker of basement membrane components. Initially, a SV40-transformed human endothelial cell line was to be used; however, it did not grow well in this culture system. Results. In these experiments, we tested two methods of isolating epithelial cells. One method, the explant technique-a method we previously used to isolate rabbit epithelial cells-could not be maintained beyond two passages, and when added to the construct only stratified to 2-3 layers of flattened cells. However, with the second method, the dispase technique, the epithelial cells obtained grew rapidly and could be maintained beyond two passages. These cells, when added to the construct, stratified to 5-7 layers and exhibited a more epithelium-like morphology. Staining with ZO-1 indicated that tight junctions in the superficial cells were formed. Staining with keratins 3 and 12 indicated that the epithelial cells were differentiating, and staining with laminin indicated that basement membrane components were being synthesized. Conclusion. Constructing an organotypic culture with human corneal cells is possible, and the morphology of the construct appears to be equivalent to a construct already created with bovine cells that has been used for irritancy studies.","PeriodicalId":17547,"journal":{"name":"Journal of Toxicology-cutaneous and Ocular Toxicology","volume":"228 1","pages":"19-28"},"PeriodicalIF":0.0,"publicationDate":"2004-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77222152","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
A. Amir, T. Kadar, S. Chapman, J. Turetz, A. Levy, Michael C. Babin, K. M. Ricketts, J. Brozetti, T. Logan, M. Ross
Sulfur mustard (2,2‐dichlorodiethyl sulfide; HD), is a potent alkylating agent which in liquid or vapor form is capable of causing severe injuries to skin and respiratory tract, and was shown to cause short‐ and long‐term ocular injuries. N‐Acetylcysteine (NAC) may act as a mucolytic agent, changing the “wetting” and scavenging properties of the cornea and thus the adhesion of HD. Moreover, NAC is a scavenger of HD, an antioxidant and a glutathione precursor, which was shown to reduce HD toxicity in various systems. The ocular distribution of 14C, after topical application of liquid 14C‐sulfur mustard (14C‐HD) to the rabbit cornea, and the role of NAC in reducing HD retention and toxicity are presented in this study. Groups of rabbits were exposed to 0.4 µL of liquid 14C‐HD, placed at the center of the cornea, with or without NAC treatment. Fifty µL NAC (10% aqueous solution) was topically applied, 10 minutes before and 10 minutes after HD exposure. Three time points were evaluated: 1, 6, and 24 hr after HD exposure, six rabbits per time point. Evaluation consisted of clinical observation, measurement of biochemical parameters in aqueous humor (AQ), counting radioactivity concentration in ocular tissues, and histology of corneal sections. One hour after corneal exposure to liquid 14C‐HD, approximately 2% of total applied radioactivity was recovered. The highest 14C concentration was found in the cornea, followed by the tarsal section of eyelid, aqueous humor, nictitating membrane, and the frontal sclera (including conjunctiva). The rate of radioactivity decrease varied from one ocular tissue to the other, the highest rate was found in aqueous and vitreous humors, also in accordance with their higher turnover rates. The NAC treatment reduced the radioactivity in most ocular tissues. The HD exposure caused typical clinical and histological signs of HD intoxication, and increased the aqueous protein and prostaglandin (PGE) content. The NAC treatment lowered eyelid edema but had no effect on AQ protein or PGE content; however, there was some aggravating effect of the NAC treatment on corneal epithelial cells, seen at 1 and 6 hr after exposure.
{"title":"The Distribution Kinetics of Topical 14C‐Sulfur Mustard in Rabbit Ocular Tissues and the Effect of Acetylcysteine","authors":"A. Amir, T. Kadar, S. Chapman, J. Turetz, A. Levy, Michael C. Babin, K. M. Ricketts, J. Brozetti, T. Logan, M. Ross","doi":"10.1081/CUS-120026300","DOIUrl":"https://doi.org/10.1081/CUS-120026300","url":null,"abstract":"Sulfur mustard (2,2‐dichlorodiethyl sulfide; HD), is a potent alkylating agent which in liquid or vapor form is capable of causing severe injuries to skin and respiratory tract, and was shown to cause short‐ and long‐term ocular injuries. N‐Acetylcysteine (NAC) may act as a mucolytic agent, changing the “wetting” and scavenging properties of the cornea and thus the adhesion of HD. Moreover, NAC is a scavenger of HD, an antioxidant and a glutathione precursor, which was shown to reduce HD toxicity in various systems. The ocular distribution of 14C, after topical application of liquid 14C‐sulfur mustard (14C‐HD) to the rabbit cornea, and the role of NAC in reducing HD retention and toxicity are presented in this study. Groups of rabbits were exposed to 0.4 µL of liquid 14C‐HD, placed at the center of the cornea, with or without NAC treatment. Fifty µL NAC (10% aqueous solution) was topically applied, 10 minutes before and 10 minutes after HD exposure. Three time points were evaluated: 1, 6, and 24 hr after HD exposure, six rabbits per time point. Evaluation consisted of clinical observation, measurement of biochemical parameters in aqueous humor (AQ), counting radioactivity concentration in ocular tissues, and histology of corneal sections. One hour after corneal exposure to liquid 14C‐HD, approximately 2% of total applied radioactivity was recovered. The highest 14C concentration was found in the cornea, followed by the tarsal section of eyelid, aqueous humor, nictitating membrane, and the frontal sclera (including conjunctiva). The rate of radioactivity decrease varied from one ocular tissue to the other, the highest rate was found in aqueous and vitreous humors, also in accordance with their higher turnover rates. The NAC treatment reduced the radioactivity in most ocular tissues. The HD exposure caused typical clinical and histological signs of HD intoxication, and increased the aqueous protein and prostaglandin (PGE) content. The NAC treatment lowered eyelid edema but had no effect on AQ protein or PGE content; however, there was some aggravating effect of the NAC treatment on corneal epithelial cells, seen at 1 and 6 hr after exposure.","PeriodicalId":17547,"journal":{"name":"Journal of Toxicology-cutaneous and Ocular Toxicology","volume":"92 1","pages":"201 - 214"},"PeriodicalIF":0.0,"publicationDate":"2003-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79801278","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
E. Emmanouil-Nikoloussi, M. Goret-Nicaise, A. Manthos, C. Foroglou
The purpose of this study is to describe the eye malformations associated with exencephaly observed after retinoic acid (R.A.) administration during gestation in white rat embryos. R.A. suspended in corn oil was given to 13 pregnant rats by gastric intubation. They received various oral doses of R.A. (from 0 to 30 mg/kg b.w. a day) from day 7.5 to 11.5 with an additional dose on day 14.5. No abnormalities have been found in the control groups. The embryos from the treated groups showing anophthalmia and exencephaly were chosen for histological study. The microscopic examination points out dose-dependent abnormalities from absence of eye structures to hypoplastic and atypical more or less compact lens vesicle.
{"title":"Histological Study of Anophthalmia Observed in Exencephalic Rat Embryos After All-Trans-Retinoic Acid Administration","authors":"E. Emmanouil-Nikoloussi, M. Goret-Nicaise, A. Manthos, C. Foroglou","doi":"10.1081/CUS-120019328","DOIUrl":"https://doi.org/10.1081/CUS-120019328","url":null,"abstract":"The purpose of this study is to describe the eye malformations associated with exencephaly observed after retinoic acid (R.A.) administration during gestation in white rat embryos. R.A. suspended in corn oil was given to 13 pregnant rats by gastric intubation. They received various oral doses of R.A. (from 0 to 30 mg/kg b.w. a day) from day 7.5 to 11.5 with an additional dose on day 14.5. No abnormalities have been found in the control groups. The embryos from the treated groups showing anophthalmia and exencephaly were chosen for histological study. The microscopic examination points out dose-dependent abnormalities from absence of eye structures to hypoplastic and atypical more or less compact lens vesicle.","PeriodicalId":17547,"journal":{"name":"Journal of Toxicology-cutaneous and Ocular Toxicology","volume":"1 1","pages":"33 - 46"},"PeriodicalIF":0.0,"publicationDate":"2003-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90472524","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
M. Yağmur, Okan Okay, T. R. Ersöz, A. Özcan, A. Bozkurt
In order to evaluate the clinical and confocal microscopic features of amiodarone keratopathy, we examined 12 eyes of six patients receiving amiodarone and 12 eyes of six healthy control subjects. The duration of amiodarone therapy ranged from 6 to 24 months (16 ± 5.9 months). According to slit‐lamp findings, seven eyes were graded as grade 1 and five as having grade 2 amiodarone keratopathy. In the basal cell layer there were highly reflective, bright, intracellular inclusions in the epithelial layer of all patients on amiodarone. In advanced cases, there were bright intracellular inclusions even in the endothelial cell layer, as well as the anterior and posterior stroma. The keratocyte density of the anterior stroma was reduced in cases with amiodarone keratopathy compared to the control group, and irregularity in stromal nerve fibers was significant in advanced cases (p < 0.001). Ultrasonic pachimetry showed a corneal thickness of 538 ± 17.9 µm in the amiodarone group and 519 ± 16.7 µm in the controls (ns). We suggest that in amiodarone‐induced keratopathy, confocal microscopy is a useful, noninvasive technique for detecting deposits in the epithelial basal membrane early on, and which later appear in deeper layers.
{"title":"Confocal Microscopic Features of Amiodarone Keratopathy","authors":"M. Yağmur, Okan Okay, T. R. Ersöz, A. Özcan, A. Bozkurt","doi":"10.1081/CUS-120026303","DOIUrl":"https://doi.org/10.1081/CUS-120026303","url":null,"abstract":"In order to evaluate the clinical and confocal microscopic features of amiodarone keratopathy, we examined 12 eyes of six patients receiving amiodarone and 12 eyes of six healthy control subjects. The duration of amiodarone therapy ranged from 6 to 24 months (16 ± 5.9 months). According to slit‐lamp findings, seven eyes were graded as grade 1 and five as having grade 2 amiodarone keratopathy. In the basal cell layer there were highly reflective, bright, intracellular inclusions in the epithelial layer of all patients on amiodarone. In advanced cases, there were bright intracellular inclusions even in the endothelial cell layer, as well as the anterior and posterior stroma. The keratocyte density of the anterior stroma was reduced in cases with amiodarone keratopathy compared to the control group, and irregularity in stromal nerve fibers was significant in advanced cases (p < 0.001). Ultrasonic pachimetry showed a corneal thickness of 538 ± 17.9 µm in the amiodarone group and 519 ± 16.7 µm in the controls (ns). We suggest that in amiodarone‐induced keratopathy, confocal microscopy is a useful, noninvasive technique for detecting deposits in the epithelial basal membrane early on, and which later appear in deeper layers.","PeriodicalId":17547,"journal":{"name":"Journal of Toxicology-cutaneous and Ocular Toxicology","volume":"14 1","pages":"243 - 253"},"PeriodicalIF":0.0,"publicationDate":"2003-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89249911","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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