AIM:To validate methods for determining mast cell density,extracellular major basic protein content,and presence of fibrosis in esophageal eosinophilia.METHODS:Twenty specimens with > 20 eosinophils/high-power fiel...AIM:To validate methods for determining mast cell density,extracellular major basic protein content,and presence of fibrosis in esophageal eosinophilia.METHODS:Twenty specimens with > 20 eosinophils/high-power field(hpf) classified as high eosinophil density(HE) and 20 specimens with < 5 eosinophils/hpf classified as low esophageal density(LE) were identified.All 40 specimens underwent immunohistochemical staining and trichrome staining.Mast cell density,extracellular major basic protein(MBP) density,and presence of subepithelial fibrosis were assessed in a standardized manner.All specimens were evaluated by two separate observers and by a single observer on two separate occasions to evaluate reproducibility of the methods.RESULTS:A strong inter-observer correlation was noted for both peak and mean mast cell counts(r = 0.725,P < 0.0001 and r = 0.823,P < 0.0001).A strong intraobserver correlation also was noted for both peak and mean mast cell counts(r = 0.752,P < 0.0001 and r =0.878,P < 0.0001).A very strong inter-observer correlation was noted for both peak(τ = 0.867,P < 0.0001)and mean extracellular MBP densities(r = 0.925,P <0.0001).A very strong intra-observer correlation was noted for both peak(τ = 0.875;P < 0.0001) and mean extracellular MBP densities(r = 0.956,P < 0.0001).Excellent inter-rater reliability was found for fibrosis(κ= 0.887).Mast cell and MBP densities,as well as presence of fibrosis,were significantly increased in HE vs LE.The HE group had significantly higher intraepithelial mast cell peak(29.35 ± 21.61 vs 12.45 ± 8.26,P =0.002) and mean(19.84 ± 15.81 vs 6.35 ± 4.5,P =0.001) densities than the LE group.The HE group had significantly higher peak extracellular MBP(2.35 ± 0.67vs 0.45 ± 0.61,P < 0.001) and mean extracellular MBP(1.95 ± 0.76 vs 0.20 ± 0.29,P < 0.0001) densities than the LE group.Seventy-three percent of patients with HE(11/15) had fibrosis,whereas only 10% of patients with LE(1/10) had fibrosis(P < 0.01).MBP performed the best in predicting classification of HE vs LE,with mean MBP demonstrating 100% sensitivity and95% specificity at the optimal cut point.CONCLUSION:This study provides methodology and proof-of-concept for future evaluation of these biomarkers for differentiating esophageal eosinophilic diseases such as reflux esophagitis and eosinophilic esophagitis.展开更多
Prostate cancer is the second leading cause of cancer deaths in the United States and remains a significant health concern for men throughout the world. Despite the discovery of promising immunotherapeutic strategies,...Prostate cancer is the second leading cause of cancer deaths in the United States and remains a significant health concern for men throughout the world. Despite the discovery of promising immunotherapeutic strategies, curative outcomes remain elusive. We have investigated eosinophils as potential anti-cancer effector cells, and have reported the ability of their toxic granular proteins (MBP, EPO, ECP, EDN) to inhibit prostate tumor cell growth?in vitro. This study investigates the effect of eosinophil MBP extract on the expression of oncogenes p53, bcl-xl, bax, and c-myc, which modulate tumor growth, proliferation, and apoptosis. Briefly, granular proteins were differentially extracted from GRC.014.22 and GRC.014.24, eosinophilic cell lines established in our laboratory from a patient with moderate asthma. Protein extracts were fractionated on Sephadex G-50 columns, and prostate tumor cell lines DU-145, LNCaP, PC-3, and HPC8L (established in our laboratory from a tumor resected from an African American patient) were treated with MBP extracts from the pooled third peaks. Colony formation and monolayer cell growth inhibition assays were used to evaluate the protein’s growth inhibitory activity against prostate tumor cells;and gene expression analyses, to determine p53, bcl-xl, bax, and c-myc oncogene expression. We show that the granular proteins were potent in their action on HPC8L, inhibiting colony formation in a dose-dependent manner. Treated prostate tumor cell lines trended toward apoptosis-induction, as evident in bcl-xl/bax ratios < 1, increased p53 expression, and up or downregulation of c-myc. These preliminary results demonstrate the growth inhibitory potential of eosinophil granular proteins and strongly support the hypothesis that eosinophils modulate the expression of oncogenes associated with prostate tumor proliferation and apoptosis. More importantly, this study offers insights into possible applications of eosinophilic mediators in oncogenic-targeted prostate cancer treatment strategies and demonstrates the potential therapeutic implications of enhancing eosinophilic activity in prostate cancer.展开更多
文摘AIM:To validate methods for determining mast cell density,extracellular major basic protein content,and presence of fibrosis in esophageal eosinophilia.METHODS:Twenty specimens with > 20 eosinophils/high-power field(hpf) classified as high eosinophil density(HE) and 20 specimens with < 5 eosinophils/hpf classified as low esophageal density(LE) were identified.All 40 specimens underwent immunohistochemical staining and trichrome staining.Mast cell density,extracellular major basic protein(MBP) density,and presence of subepithelial fibrosis were assessed in a standardized manner.All specimens were evaluated by two separate observers and by a single observer on two separate occasions to evaluate reproducibility of the methods.RESULTS:A strong inter-observer correlation was noted for both peak and mean mast cell counts(r = 0.725,P < 0.0001 and r = 0.823,P < 0.0001).A strong intraobserver correlation also was noted for both peak and mean mast cell counts(r = 0.752,P < 0.0001 and r =0.878,P < 0.0001).A very strong inter-observer correlation was noted for both peak(τ = 0.867,P < 0.0001)and mean extracellular MBP densities(r = 0.925,P <0.0001).A very strong intra-observer correlation was noted for both peak(τ = 0.875;P < 0.0001) and mean extracellular MBP densities(r = 0.956,P < 0.0001).Excellent inter-rater reliability was found for fibrosis(κ= 0.887).Mast cell and MBP densities,as well as presence of fibrosis,were significantly increased in HE vs LE.The HE group had significantly higher intraepithelial mast cell peak(29.35 ± 21.61 vs 12.45 ± 8.26,P =0.002) and mean(19.84 ± 15.81 vs 6.35 ± 4.5,P =0.001) densities than the LE group.The HE group had significantly higher peak extracellular MBP(2.35 ± 0.67vs 0.45 ± 0.61,P < 0.001) and mean extracellular MBP(1.95 ± 0.76 vs 0.20 ± 0.29,P < 0.0001) densities than the LE group.Seventy-three percent of patients with HE(11/15) had fibrosis,whereas only 10% of patients with LE(1/10) had fibrosis(P < 0.01).MBP performed the best in predicting classification of HE vs LE,with mean MBP demonstrating 100% sensitivity and95% specificity at the optimal cut point.CONCLUSION:This study provides methodology and proof-of-concept for future evaluation of these biomarkers for differentiating esophageal eosinophilic diseases such as reflux esophagitis and eosinophilic esophagitis.
文摘Prostate cancer is the second leading cause of cancer deaths in the United States and remains a significant health concern for men throughout the world. Despite the discovery of promising immunotherapeutic strategies, curative outcomes remain elusive. We have investigated eosinophils as potential anti-cancer effector cells, and have reported the ability of their toxic granular proteins (MBP, EPO, ECP, EDN) to inhibit prostate tumor cell growth?in vitro. This study investigates the effect of eosinophil MBP extract on the expression of oncogenes p53, bcl-xl, bax, and c-myc, which modulate tumor growth, proliferation, and apoptosis. Briefly, granular proteins were differentially extracted from GRC.014.22 and GRC.014.24, eosinophilic cell lines established in our laboratory from a patient with moderate asthma. Protein extracts were fractionated on Sephadex G-50 columns, and prostate tumor cell lines DU-145, LNCaP, PC-3, and HPC8L (established in our laboratory from a tumor resected from an African American patient) were treated with MBP extracts from the pooled third peaks. Colony formation and monolayer cell growth inhibition assays were used to evaluate the protein’s growth inhibitory activity against prostate tumor cells;and gene expression analyses, to determine p53, bcl-xl, bax, and c-myc oncogene expression. We show that the granular proteins were potent in their action on HPC8L, inhibiting colony formation in a dose-dependent manner. Treated prostate tumor cell lines trended toward apoptosis-induction, as evident in bcl-xl/bax ratios < 1, increased p53 expression, and up or downregulation of c-myc. These preliminary results demonstrate the growth inhibitory potential of eosinophil granular proteins and strongly support the hypothesis that eosinophils modulate the expression of oncogenes associated with prostate tumor proliferation and apoptosis. More importantly, this study offers insights into possible applications of eosinophilic mediators in oncogenic-targeted prostate cancer treatment strategies and demonstrates the potential therapeutic implications of enhancing eosinophilic activity in prostate cancer.
