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Gamma-interferon-inducible lysosomal thiol reductase is upregulated in human melanoma
1286
936
Etanercept treatment is able to modulate apoptosis process in patients with moderate to severe psoriasis Serena Lembo, MD, PhD, Department of Dermatology, University of Naples Federico II, Naples, Italy; Matteo Megna, MD, Department of Dermatology, University of Naples Federico II, Naples, Italy; Giuseppina Caiazzo, PhD, Department of Dermatology, University of Naples Federico II, Naples, Italy; Fabio Ayala, MD, PhD, Department of Dermatology, University of Naples Federico II, Naples, Italy; Nicola Balato, MD, Department of dermatology, University of Naples Federico II, Naples, Italy; Anna Balato, MD, PhD, Department of Dermatology, University of Naples Federico II, Naples, Italy
Growth on compliant substrates reduces TGFb-induced epithelial to mesencymal transition (EMT) Jose Rapanan, MS, Midwestern University- Arizona College of Osteopathic Medicine, Glendale, AZ, United States; Agnes Pascual, Midwestern University, Glendale, AZ, United States; Elizabeth Hull, PhD, Midwestern University, Glendale, AZ, United States
Psoriasis is a chronic inflammatory skin disorder characterized by genetic predisposition in which the occurrence of environmental factors may lead to the complex inflammatory pathway and the well-known epidermal hyperproliferation. In this context, other than increased proliferation, psoriatic keratinocytes show differentiation impairment and significant resistance to apoptosis process. Markers of proliferation (proliferating cell nuclear antigen: PCNA), apoptosis (caspase 3) and survival (survivin) have been investigated in psoriasis, but little is known on the effects of anti-TNF-a therapy on such mechanisms. The p60 protein subunit of chromatin assembly factor-1 (CAF-1), which is specifically active during cell replication and DNA-repair processes, represents a reliable prognostic index in multiple types of cancer, including malignant melanoma. The aim of our study was to investigate the effects of etanercept treatment on the expression of proliferation, apoptosis, survival and replication markers in patients with moderate to severe psoriasis. Twenty moderate to severe psoriatic subjects were enrolled in the study. BSA, DLQI, PASI and PGA were recorded at baseline and after 2, 8, 12 and 24 weeks of therapy. In addition, 3 mm skin biopsies were performed from lesional and nonlesional skin at baseline and week 12. CAF-1/p60, caspase 3, PCNA, and survivin were assessed through gene expression as well as protein analysis. Skin samples obtained from healthy subjects undergoing abdominoplasty were used as controls. Etanercept confirmed its efficacy with PASI75 and PASI90 achieved by 77% and 38.5% of subjects at week 24, respectively. Gene expression of CAF-1/p60, as well as PCNA, was increased in lesional skin compared to nonlesional skin and healthy controls, at baseline and after 12 weeks of treatment, showing correlation with PGA values. Interestingly, survivin and caspase 3 were enhanced in psoriatic patients compared to controls, and etanercept treatment was able to still increase these markers, indicating that this treatment may interfere with apoptosis process. In conclusion, we can assess that the antiproliferative effect of etanercept might be based on the modulation of apoptosis and cell survival mechanisms.
The epithelial to mesenchymal transition (EMT) is a transcriptional program which is activated in epithelial wound healing and in metastasis. Understanding the control of the EMT process is important as EMT is necessary for healing skin but can also contribute to the development of metastasis. Specifically, TGFbR signaling 1) plays a role in activating the transcription factors which are necessary for EMT and 2) is a key cytokine pathway controlling the tumor cell environment to allow for successful metastasis. Upon both wounding and ex vivo culture, cells are initially exposed to serum which contains significant amounts of TGFb. As TGFb is present in the culture medium, TGFbR signaling may be responsible (at least in part) for the transcriptional changes which occur within hours of removal of tissue from the skin. However, work is often carried out on traditional culture surfaces including glass and tissue culture plastic which are not an accurate representation of the compliance of tissues within the body. Therefore, we have investigated the ability of TGFbR signaling to initiate and sustain EMT on elastic substrates as well as glass with traditional tissue culture and matrigel surfaces. As we are interested in events which are triggered within minutes of removal of epithelial cells from the skin, we have used the zebrafish keratocyte explant system, a characterized wound healing model which undergoes EMT. This system avoids the changes in gene expression which continue during the routine culture of primary cells and each culture is initiated by wounding. Confirming published data, our results suggest that on hard surfaces 1) addition of TGFbR signaling appears to be sufficient to trigger EMT and that 2) inhibiting TGFßR signaling halts EMT. However, on compliant surfaces, even when levels of TGFb are increased, EMT is not initiated. In addition, ß-catenin (member of the interacting wnt signaling pathway) shows altered localization and Rho associated kinase (ROCK) appears to be less active as evidenced by alterations in actin stress fibers. These results suggest that the role of TGFbR signaling in EMT needs to be reassessed immediately after establishment of ex vivo cultures using compliant substrata. Commercial support: None identified.
Research study was Supported by Pfizer.
976 HLA associations in patients with pemphigus vulgaris: A Canadian perspective Ilya Shoimer, MD, University of Calgary, Calgary, Alberta, Canada; Sunil Kalia, MD, University of British Columbia, Vancouver, British Columbia, Canada; Dafna Gladman, MD, University of Toronto, Toronto Western Hospital, Toronto, Ontario, Canada; Fawnda Pellett, Centre for Prognosis Studies in the Rheumatic Diseases, Toronto Western Research Institute, Toronto, Ontario, Canada; Regine Mydlarski, MD, University of Calgary, Calgary, Alberta, Canada
2003 Gamma-interferon-inducible lysosomal thiol reductase is upregulated in human melanoma K. Taraszka Hastings, MD, PhD, University of Arizona College of Medicine Phoenix, Phoenix, AZ, United States; Jennifer Nguyen, MD, University of Arizona College of Medicine Phoenix, Phoenix, AZ, United States; Richard Bernert, MD, Arizona Dermatopathology, Scottsdale, AZ, United States; Christine Ko, MD, Yale University School of Medicine, New Haven, CT, United States; Noemi Sebastiao, Ventana Medical Systems, Tucson, AZ, United States; Chengcheng Hu, PhD, University of Arizona Mel and Enid Zuckerman College of Public Health, Phoenix, AZ, United States T cell-mediated immunity has the ability to produce durable antimelanoma responses resulting in improved survival of melanoma patients. Antigen presentation in the tumor microenvironment likely influences anti-melanoma T cell responses. Gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for efficient MHC class II-restricted presentation of multiple melanoma antigens to CD4+ T cells. Therefore, we evaluated GILT and MHC class II expression in melanocytes, antigen presenting cells and keratinocytes of human primary and metastatic melanomas and nevi using immunohistochemistry. Both GILT and MHC class II expression were increased in the melanocytes of primary and metastatic melanomas compared with nevi. GILT expression was increased in antigen presenting cells of primary and metastatic melanomas compared to nevi, whereas MHC class II had equivalent high expression in antigen presenting cells of all melanocytic lesions. Keratinocytes in primary melanoma lesions had increased GILT and MHC class II expression compared with nevi. Increased GILT expression in melanocytes, antigen presenting cells and keratinocytes of melanoma demonstrates that changes in antigen processing pathways occur in the tumor microenvironment. GILT expression in melanoma is anticipated to improve antigen presentation and T cell recognition of melanoma and may be an indicator of immune recognition of melanoma. Employees of Ventana Medical Systems provided technical assistance with immunohistochemistry as part of a collaborative research agreement with no payment to either Ventana or academic investigators.
AB30
J AM ACAD DERMATOL
Background: Pemphigus vulgaris (PV) is an autoimmune disorder mediated by IgG antibodies against desmogleins (Dsg) 1 and 3. The human leukocyte antigen (HLA) haplotypes encode for the major histocompatibility complex proteins, which play an integral role in autoimmunity. Previous studies have shown an association between PV and HLA alleles, but the results show ethnic variations. There have been no studies to date examining HLA alleles in Canadian patients with PV. Methods: An observational, prospective, controlled study was carried out between January 2000 and July 2006 involving a Canadian population. Fifty-two patients with PV were enrolled in the study (39 Caucasians, 13 non-Caucasians). The Caucasian PV cases were compared with 610 healthy Caucasian controls. Standardized methods using phenol extraction were employed to extract genomic DNA from peripheral blood leukocytes. Low resolution typing of HLA class I and II alleles was performed using the reverse line blot method. Additionally, sequence-specific oligonucleotide probe hybridization techniques and sequence-specific primers were used for high resolution typing of the DRB1 and DQB1 alleles. Descriptive statistical analysis was performed and significance was determined using a chi-squared test. Results: The HLA class I alleles identified HLA-A*26, HLA-B*38, and HLA-Cw*12 (P \ .00001) as risk factors for PV, whereas HLA-Cw*7 conferred protection (P ¼.0036). Among HLA class II alleles, HLA-DRB1*04 and DRB1*14 were more prevalent in PV patients (P \.00001), while HLA-DRB1*07 was protective (P ¼.0008). Additionally, high resolution typing identified DRB1*0102 (P ¼.0141), DRB1*0402 (P \.00001), DRB1*1401 (P \.00001), DRB1*1404 (P \.00001), DQB1*0302 (P \.00001), and DQB1*0503 (P \ .00001) as susceptibility alleles. In contrast, DRB1*0401 (P ¼ .0117), DQB1*02 (P ¼ .0004), and DQB1*0602 (P ¼ .0157) appeared to be protective. When comparing the PV patients, HLA-B*38 was only increased in the Caucasian patients. Additionally, DRB1*04 was twice as prevalent in Caucasians with PV, while the opposite trend was seen with DRB1*14. Conclusions: Our study confirms that specific HLA alleles may render patients genetically susceptible to PV, and demonstrates ethnic variability. Moreover, we report the HLA-Cw*12 allele as a novel risk factor for PV. Commercial support: None identified.
MAY 2015
936
Etanercept treatment is able to modulate apoptosis process in patients with moderate to severe psoriasis Serena Lembo, MD, PhD, Department of Dermatology, University of Naples Federico II, Naples, Italy; Matteo Megna, MD, Department of Dermatology, University of Naples Federico II, Naples, Italy; Giuseppina Caiazzo, PhD, Department of Dermatology, University of Naples Federico II, Naples, Italy; Fabio Ayala, MD, PhD, Department of Dermatology, University of Naples Federico II, Naples, Italy; Nicola Balato, MD, Department of dermatology, University of Naples Federico II, Naples, Italy; Anna Balato, MD, PhD, Department of Dermatology, University of Naples Federico II, Naples, Italy
Growth on compliant substrates reduces TGFb-induced epithelial to mesencymal transition (EMT) Jose Rapanan, MS, Midwestern University- Arizona College of Osteopathic Medicine, Glendale, AZ, United States; Agnes Pascual, Midwestern University, Glendale, AZ, United States; Elizabeth Hull, PhD, Midwestern University, Glendale, AZ, United States
Psoriasis is a chronic inflammatory skin disorder characterized by genetic predisposition in which the occurrence of environmental factors may lead to the complex inflammatory pathway and the well-known epidermal hyperproliferation. In this context, other than increased proliferation, psoriatic keratinocytes show differentiation impairment and significant resistance to apoptosis process. Markers of proliferation (proliferating cell nuclear antigen: PCNA), apoptosis (caspase 3) and survival (survivin) have been investigated in psoriasis, but little is known on the effects of anti-TNF-a therapy on such mechanisms. The p60 protein subunit of chromatin assembly factor-1 (CAF-1), which is specifically active during cell replication and DNA-repair processes, represents a reliable prognostic index in multiple types of cancer, including malignant melanoma. The aim of our study was to investigate the effects of etanercept treatment on the expression of proliferation, apoptosis, survival and replication markers in patients with moderate to severe psoriasis. Twenty moderate to severe psoriatic subjects were enrolled in the study. BSA, DLQI, PASI and PGA were recorded at baseline and after 2, 8, 12 and 24 weeks of therapy. In addition, 3 mm skin biopsies were performed from lesional and nonlesional skin at baseline and week 12. CAF-1/p60, caspase 3, PCNA, and survivin were assessed through gene expression as well as protein analysis. Skin samples obtained from healthy subjects undergoing abdominoplasty were used as controls. Etanercept confirmed its efficacy with PASI75 and PASI90 achieved by 77% and 38.5% of subjects at week 24, respectively. Gene expression of CAF-1/p60, as well as PCNA, was increased in lesional skin compared to nonlesional skin and healthy controls, at baseline and after 12 weeks of treatment, showing correlation with PGA values. Interestingly, survivin and caspase 3 were enhanced in psoriatic patients compared to controls, and etanercept treatment was able to still increase these markers, indicating that this treatment may interfere with apoptosis process. In conclusion, we can assess that the antiproliferative effect of etanercept might be based on the modulation of apoptosis and cell survival mechanisms.
The epithelial to mesenchymal transition (EMT) is a transcriptional program which is activated in epithelial wound healing and in metastasis. Understanding the control of the EMT process is important as EMT is necessary for healing skin but can also contribute to the development of metastasis. Specifically, TGFbR signaling 1) plays a role in activating the transcription factors which are necessary for EMT and 2) is a key cytokine pathway controlling the tumor cell environment to allow for successful metastasis. Upon both wounding and ex vivo culture, cells are initially exposed to serum which contains significant amounts of TGFb. As TGFb is present in the culture medium, TGFbR signaling may be responsible (at least in part) for the transcriptional changes which occur within hours of removal of tissue from the skin. However, work is often carried out on traditional culture surfaces including glass and tissue culture plastic which are not an accurate representation of the compliance of tissues within the body. Therefore, we have investigated the ability of TGFbR signaling to initiate and sustain EMT on elastic substrates as well as glass with traditional tissue culture and matrigel surfaces. As we are interested in events which are triggered within minutes of removal of epithelial cells from the skin, we have used the zebrafish keratocyte explant system, a characterized wound healing model which undergoes EMT. This system avoids the changes in gene expression which continue during the routine culture of primary cells and each culture is initiated by wounding. Confirming published data, our results suggest that on hard surfaces 1) addition of TGFbR signaling appears to be sufficient to trigger EMT and that 2) inhibiting TGFßR signaling halts EMT. However, on compliant surfaces, even when levels of TGFb are increased, EMT is not initiated. In addition, ß-catenin (member of the interacting wnt signaling pathway) shows altered localization and Rho associated kinase (ROCK) appears to be less active as evidenced by alterations in actin stress fibers. These results suggest that the role of TGFbR signaling in EMT needs to be reassessed immediately after establishment of ex vivo cultures using compliant substrata. Commercial support: None identified.
Research study was Supported by Pfizer.
976 HLA associations in patients with pemphigus vulgaris: A Canadian perspective Ilya Shoimer, MD, University of Calgary, Calgary, Alberta, Canada; Sunil Kalia, MD, University of British Columbia, Vancouver, British Columbia, Canada; Dafna Gladman, MD, University of Toronto, Toronto Western Hospital, Toronto, Ontario, Canada; Fawnda Pellett, Centre for Prognosis Studies in the Rheumatic Diseases, Toronto Western Research Institute, Toronto, Ontario, Canada; Regine Mydlarski, MD, University of Calgary, Calgary, Alberta, Canada
2003 Gamma-interferon-inducible lysosomal thiol reductase is upregulated in human melanoma K. Taraszka Hastings, MD, PhD, University of Arizona College of Medicine Phoenix, Phoenix, AZ, United States; Jennifer Nguyen, MD, University of Arizona College of Medicine Phoenix, Phoenix, AZ, United States; Richard Bernert, MD, Arizona Dermatopathology, Scottsdale, AZ, United States; Christine Ko, MD, Yale University School of Medicine, New Haven, CT, United States; Noemi Sebastiao, Ventana Medical Systems, Tucson, AZ, United States; Chengcheng Hu, PhD, University of Arizona Mel and Enid Zuckerman College of Public Health, Phoenix, AZ, United States T cell-mediated immunity has the ability to produce durable antimelanoma responses resulting in improved survival of melanoma patients. Antigen presentation in the tumor microenvironment likely influences anti-melanoma T cell responses. Gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for efficient MHC class II-restricted presentation of multiple melanoma antigens to CD4+ T cells. Therefore, we evaluated GILT and MHC class II expression in melanocytes, antigen presenting cells and keratinocytes of human primary and metastatic melanomas and nevi using immunohistochemistry. Both GILT and MHC class II expression were increased in the melanocytes of primary and metastatic melanomas compared with nevi. GILT expression was increased in antigen presenting cells of primary and metastatic melanomas compared to nevi, whereas MHC class II had equivalent high expression in antigen presenting cells of all melanocytic lesions. Keratinocytes in primary melanoma lesions had increased GILT and MHC class II expression compared with nevi. Increased GILT expression in melanocytes, antigen presenting cells and keratinocytes of melanoma demonstrates that changes in antigen processing pathways occur in the tumor microenvironment. GILT expression in melanoma is anticipated to improve antigen presentation and T cell recognition of melanoma and may be an indicator of immune recognition of melanoma. Employees of Ventana Medical Systems provided technical assistance with immunohistochemistry as part of a collaborative research agreement with no payment to either Ventana or academic investigators.
AB30
J AM ACAD DERMATOL
Background: Pemphigus vulgaris (PV) is an autoimmune disorder mediated by IgG antibodies against desmogleins (Dsg) 1 and 3. The human leukocyte antigen (HLA) haplotypes encode for the major histocompatibility complex proteins, which play an integral role in autoimmunity. Previous studies have shown an association between PV and HLA alleles, but the results show ethnic variations. There have been no studies to date examining HLA alleles in Canadian patients with PV. Methods: An observational, prospective, controlled study was carried out between January 2000 and July 2006 involving a Canadian population. Fifty-two patients with PV were enrolled in the study (39 Caucasians, 13 non-Caucasians). The Caucasian PV cases were compared with 610 healthy Caucasian controls. Standardized methods using phenol extraction were employed to extract genomic DNA from peripheral blood leukocytes. Low resolution typing of HLA class I and II alleles was performed using the reverse line blot method. Additionally, sequence-specific oligonucleotide probe hybridization techniques and sequence-specific primers were used for high resolution typing of the DRB1 and DQB1 alleles. Descriptive statistical analysis was performed and significance was determined using a chi-squared test. Results: The HLA class I alleles identified HLA-A*26, HLA-B*38, and HLA-Cw*12 (P \ .00001) as risk factors for PV, whereas HLA-Cw*7 conferred protection (P ¼.0036). Among HLA class II alleles, HLA-DRB1*04 and DRB1*14 were more prevalent in PV patients (P \.00001), while HLA-DRB1*07 was protective (P ¼.0008). Additionally, high resolution typing identified DRB1*0102 (P ¼.0141), DRB1*0402 (P \.00001), DRB1*1401 (P \.00001), DRB1*1404 (P \.00001), DQB1*0302 (P \.00001), and DQB1*0503 (P \ .00001) as susceptibility alleles. In contrast, DRB1*0401 (P ¼ .0117), DQB1*02 (P ¼ .0004), and DQB1*0602 (P ¼ .0157) appeared to be protective. When comparing the PV patients, HLA-B*38 was only increased in the Caucasian patients. Additionally, DRB1*04 was twice as prevalent in Caucasians with PV, while the opposite trend was seen with DRB1*14. Conclusions: Our study confirms that specific HLA alleles may render patients genetically susceptible to PV, and demonstrates ethnic variability. Moreover, we report the HLA-Cw*12 allele as a novel risk factor for PV. Commercial support: None identified.
MAY 2015