BACKGROUND:Perforin gene(PRF1) mutations have been reported in patients with lymphoma,but the prevalence and characteristics of PRF1 mutation have not been identified in Chinese patients with lymphoma.METHODS:Seventy-...BACKGROUND:Perforin gene(PRF1) mutations have been reported in patients with lymphoma,but the prevalence and characteristics of PRF1 mutation have not been identified in Chinese patients with lymphoma.METHODS:Seventy-seven patients with lymphoma,including 6 patients with Hodgkin lymphoma and 71 patients with non-Hodgkin lymphoma,were recruited.DNA samples from peripheral blood were used for PRF1 mutation detection by the PCR-sequencing method.RESULTS:Eleven novel PRF1 mutations were found in 8 of the 77 patients with lymphoma.Biallelic or compound monoallelic missense mutations in 3 patients indicated the severe impairment of perforin function,monoallelic missense mutations in 3 patients possibly contributed a genetic predisposition to malignancies,and synonymous mutations in 2 patients showed unknown significance.CONCLUSIONS:The frequency of EBV infection was similar in lymphoma patients with PRF1 mutations and those without the mutations.The same PRF1 mutations were also found in DNA samples from nails or hair follicles from 4 patients with PRF1 mutations,suggesting that these mutations may be of germ-line origin.展开更多
The role that the immune system plays after injury of the peripheral nervous system is still not completely understood.Perforin,a natural killer cell-and T-lymphocyte-derived enzyme that mediates cytotoxicity,plays im...The role that the immune system plays after injury of the peripheral nervous system is still not completely understood.Perforin,a natural killer cell-and T-lymphocyte-derived enzyme that mediates cytotoxicity,plays important roles in autoimmune diseases,infections and central nervous system trauma,such as spinal cord injury.To dissect the roles of this single component of the immune response to injury,we tested regeneration after femoral nerve injury in perforin-deficient(Pfp^(-/-))and wild-type control mice.Single frame motion analysis showed better motor recovery in Pfp^(-/-)mice compared with control mice at 4 and 8 weeks after injury.Retrograde tracing of the motoneuron axons regrown into the motor nerve branch demonstrated more correctly projecting motoneurons in the spinal cord of Pfp^(-/-)mice compared with wild-types.Myelination of regrown axons measured by g-ratio was more extensive in Pfp^(-/-)than in wild-type mice in the motor branch of the femoral nerve.Pfp^(-/-)mice displayed more cholinergic synaptic terminals around cell bodies of spinal motoneurons after injury than the injured wild-types.We histologically analyzed lymphocyte infiltration 10 days after surgery and found that in Pfp^(-/-)mice the number of lymphocytes in the regenerating nerves was lower than in wild-types,suggesting a closed blood-nerve barrier in Pfp^(-/-)mice.We conclude that perforin restricts motor recovery after femoral nerve injury owing to decreased survival of motoneurons and reduced myelination.展开更多
Theileria parva is a protozoan parasite that causes the disease East Coast fever in cattle which results in major economic losses in Eastern, Central and Southern Africa. Efforts to generate vaccines involve measureme...Theileria parva is a protozoan parasite that causes the disease East Coast fever in cattle which results in major economic losses in Eastern, Central and Southern Africa. Efforts to generate vaccines involve measurements of cytotoxic activity since CD8 cells are believed to be responsible for protection of the animals. CTL assays are time consuming, and may require use of radioactive material and they also impose a considerable amount of in vitro work, which may skew the response compared to ex vivo assays. Hence it would be beneficial to identify other markers that correlate with the cytotoxicity. In this in vitro study we examined if the number of tetramer positive CD8 cells and the staining intensity of these correlated with lysis of the target cells. Furthermore, we investigated if the expression of the activation marker perforin correlated with the cytotoxicity. Perforin is involved in permeabilization of the cell membrane of the target cell. Bulk CTL lines and purified CD8 cell lines generated from cattle of the A18 BoLA (MHC) type were analysed for the Theileria parva specific immune responses using a peptide-MHC tetramer and antibodies for perforinin FACS analysis. Thelysis of target cells was determined by a chromium release assay. Results demonstrate that the percentage of tetramer positive cells, in six cell lines generated against the whole parasite, correlate with killing of PBMC pulsed with the peptide. The product of the percentages of perforin and tetramer double positive cells and the net MFI of perforin showed a perfect correlation with the cytotoxicity of the peptide pulsed PBMC. Likewise, the product of percentage perforin positive cells and the staining intensity had the best significant correlation with killing of the pulsed PBMC. The present results suggest that perforin could be a possible biomarker for the cytotoxicity to Theileria parva infections/immunizations.展开更多
Objective To prepare the monoclonal antibody against human perforin(HP). Methods Recombi- nant eukaryotic expression plasmid pCDM8-HP was extracted and purified, and the BALB/C mice were immunized with the plasmid. Th...Objective To prepare the monoclonal antibody against human perforin(HP). Methods Recombi- nant eukaryotic expression plasmid pCDM8-HP was extracted and purified, and the BALB/C mice were immunized with the plasmid. The hybridomas producing anti-HP McAbs were established by using hybridoma technique, then the specificity of the McAbs was identified by using immunocytochemical technique and Western blot. Results Three hy- bridoma cell lines secreting McAbs against human perforin were established, and the three McAbe showed positive only with LAK cells containing human perforin protein, and showed negative with inactive human peripheral blood lymphocytes (PBLs). The subclasses of the three McAbs were determined as IgG2bK. Western blot results showed that the three McAbs recognized a specific band of LAK cell lysateds with molecular weight of 70.0Kd. Conclusion The three hybridoma cell lines secreting McAbs against human perforin were established and the secreted McAbs were specific.展开更多
AIM: To analyze the level of apoptosis in different mucosal compartments and the differential expression of Fas/Fas-ligand and perforin in H pylori-associated gastric ulcer. METHODS: Antral specimens from patients wit...AIM: To analyze the level of apoptosis in different mucosal compartments and the differential expression of Fas/Fas-ligand and perforin in H pylori-associated gastric ulcer. METHODS: Antral specimens from patients with H pylori-related active gastric ulcer (GU), H pylori-related gastritis, and non-infected controls were analysed for densities and distribution of apoptotic cells determined by the TdT-mediated dUDP-biotin nick-end-labelling method. GU patients were submitted to eradication therapy with follow-up biopsy after 60 d. Fas, FasL, and perforin-expressing cells were assessed by immunoper- oxidase, and with anti-CD3, anti-CD20 and anti-CD68 by double immunofluorescence and confocal microscopy. Quantitative analysis was performed using a computer- assisted image analyser. RESULTS: H pylori-infected antrum showed greater surface epithelial apoptosis which decreased after eradi- cation therapy. In the lamina propria, higher rates of mononuclear cell apoptosis were observed in H pylori- gastritis. Co-expression of Fas with T-cell and macro- phage markers was reduced in GU. FasL- and perforin-expressing cells were increased in H pylori-infection and correlated with epithelial apoptosis. Perforin-expressing cells were also increased in GU compared with H pylori- gastritis. CONCLUSION: Epithelial apoptosis is increased in H pylori-infection and correlates to FasL- and perforin- expression by T cells. Expression of perforin is correlated with the tissue damage, and may represent the enhance- ment of a distinct cytotoxic pathway in GU. Increased expression of FasL not paralleled by Fas on T-cells and macrophages may indicate a reduced susceptibility to the Fas/FasL-mediated apoptosis of lymphoid cells in H pylori-infection.展开更多
Different types of pores ubiquitously form in cell membranes,leading to various types of cell death that profoundly influence the fate of inflammation and the disease status.However,these pores have never truly been v...Different types of pores ubiquitously form in cell membranes,leading to various types of cell death that profoundly influence the fate of inflammation and the disease status.However,these pores have never truly been visualized to date.Atomic force microscopy(AFM),which is emerging as a powerful tool to analyze the mechanical properties of biomolecules and cells,is actually an excellent imaging platform that allows biological samples to be visualized by probing surface roughness at the level of atomic resolution.Here,membrane pore structures were clearly visualized using AFM.This visualization not only describes the aperture and depth of the pore complexes but also highlights differences among the pores formed by perforin and gasdermins in tumor cell membranes and by complement in immune cell membranes.Additionally,this type of visualization also reveals the dynamic process of pore formation,fusion,and repair.展开更多
Obejctive To study the lymphocytes, perforin protein and mRNA expression in endometrium of different phases in the menstrual cycle and to explore the regularity and role of the lymphocytes and perforin in the reproduc...Obejctive To study the lymphocytes, perforin protein and mRNA expression in endometrium of different phases in the menstrual cycle and to explore the regularity and role of the lymphocytes and perforin in the reproductive system Methods Immunohistochemical and in situ hybridization techniques were employed to demonstrate the population, phenotype and mediator (perforin) of lymphocytes in the proliferative and secretory endometrium Results In different phases of the endometrium, there were very few CD 3 + typical T lymphocytes, CD 4 + helper lymphocytes and CD 8 + suppressor/cytotoxic T lymphocytes, while CD 56 + lymphocyte (also named NK like cell) were abundant CD 56 + lymphocytes were absent in postmenopausal endometrium A double immunohistochemical labelling method demonstrated the co expression of perforin protein and CD 56 antigen of cells in different phases of the endometrium Quantitative analysis of CD 56 + lymphocytes and perforin positive cells increased in number from the proliferative to the secretory endometrium ( P <0 05) In situ hybridization analysis showed that perforin mRNA positive cells formed islands in proliferative endometrial stroma, but were scattered in secretory endometrium There was difference between perforin gene expression and protein expression in the proliferative endometrium Conclusions Lymphocytes and perforin expression in the endometrium at different phases of the menstrual cycle are specific and may play an important role in the reproductive system They may exert a positive influence on embryo implantation and be involved in endometrial stroma breakdown during展开更多
CD134, a member of the tumor necrosis factor receptor superfamily, plays a crucial role in T cell survival. In this study, peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin (PHA, 50 μg/ml)...CD134, a member of the tumor necrosis factor receptor superfamily, plays a crucial role in T cell survival. In this study, peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin (PHA, 50 μg/ml) were treated by CD134 mAb (1 μg/ml, 5 μg/ml, 10 μg/ml) for 6 h, 12 h, 24 h and 48 h. The level of perforin mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR) technique. Our data showed that the expression of perforin mRNA in PBMCs was down-regulated by CD134 mAb in a dose-dependent manner in range of 1 μg/ml to 5 μg/ml and dropped down to its minimum on 24 h (p ＜ 0.05). The level of perforin mRNA reached a plateau when the concentration of CD134 mAb exceeded 5 μg/ml. In conclusion, CD134 mAb can inhibit perforin expression, which may enhance the ability of T cells for survival.展开更多
基金supported by the grants from the International S&T Cooperation Project from Ministry of Science&Technology of China(2006DFB31430)National 863 High-Tech Research and Development Program of China(2008AA02503)
文摘BACKGROUND:Perforin gene(PRF1) mutations have been reported in patients with lymphoma,but the prevalence and characteristics of PRF1 mutation have not been identified in Chinese patients with lymphoma.METHODS:Seventy-seven patients with lymphoma,including 6 patients with Hodgkin lymphoma and 71 patients with non-Hodgkin lymphoma,were recruited.DNA samples from peripheral blood were used for PRF1 mutation detection by the PCR-sequencing method.RESULTS:Eleven novel PRF1 mutations were found in 8 of the 77 patients with lymphoma.Biallelic or compound monoallelic missense mutations in 3 patients indicated the severe impairment of perforin function,monoallelic missense mutations in 3 patients possibly contributed a genetic predisposition to malignancies,and synonymous mutations in 2 patients showed unknown significance.CONCLUSIONS:The frequency of EBV infection was similar in lymphoma patients with PRF1 mutations and those without the mutations.The same PRF1 mutations were also found in DNA samples from nails or hair follicles from 4 patients with PRF1 mutations,suggesting that these mutations may be of germ-line origin.
基金supported by the Li Kashing Foundation(to MS)the FoRUM grant F957N-2019 of the Ruhr-University Bochum(to DL)the Heinrich und Alma Vogelsang Stiftung(to IJ).
文摘The role that the immune system plays after injury of the peripheral nervous system is still not completely understood.Perforin,a natural killer cell-and T-lymphocyte-derived enzyme that mediates cytotoxicity,plays important roles in autoimmune diseases,infections and central nervous system trauma,such as spinal cord injury.To dissect the roles of this single component of the immune response to injury,we tested regeneration after femoral nerve injury in perforin-deficient(Pfp^(-/-))and wild-type control mice.Single frame motion analysis showed better motor recovery in Pfp^(-/-)mice compared with control mice at 4 and 8 weeks after injury.Retrograde tracing of the motoneuron axons regrown into the motor nerve branch demonstrated more correctly projecting motoneurons in the spinal cord of Pfp^(-/-)mice compared with wild-types.Myelination of regrown axons measured by g-ratio was more extensive in Pfp^(-/-)than in wild-type mice in the motor branch of the femoral nerve.Pfp^(-/-)mice displayed more cholinergic synaptic terminals around cell bodies of spinal motoneurons after injury than the injured wild-types.We histologically analyzed lymphocyte infiltration 10 days after surgery and found that in Pfp^(-/-)mice the number of lymphocytes in the regenerating nerves was lower than in wild-types,suggesting a closed blood-nerve barrier in Pfp^(-/-)mice.We conclude that perforin restricts motor recovery after femoral nerve injury owing to decreased survival of motoneurons and reduced myelination.
文摘Theileria parva is a protozoan parasite that causes the disease East Coast fever in cattle which results in major economic losses in Eastern, Central and Southern Africa. Efforts to generate vaccines involve measurements of cytotoxic activity since CD8 cells are believed to be responsible for protection of the animals. CTL assays are time consuming, and may require use of radioactive material and they also impose a considerable amount of in vitro work, which may skew the response compared to ex vivo assays. Hence it would be beneficial to identify other markers that correlate with the cytotoxicity. In this in vitro study we examined if the number of tetramer positive CD8 cells and the staining intensity of these correlated with lysis of the target cells. Furthermore, we investigated if the expression of the activation marker perforin correlated with the cytotoxicity. Perforin is involved in permeabilization of the cell membrane of the target cell. Bulk CTL lines and purified CD8 cell lines generated from cattle of the A18 BoLA (MHC) type were analysed for the Theileria parva specific immune responses using a peptide-MHC tetramer and antibodies for perforinin FACS analysis. Thelysis of target cells was determined by a chromium release assay. Results demonstrate that the percentage of tetramer positive cells, in six cell lines generated against the whole parasite, correlate with killing of PBMC pulsed with the peptide. The product of the percentages of perforin and tetramer double positive cells and the net MFI of perforin showed a perfect correlation with the cytotoxicity of the peptide pulsed PBMC. Likewise, the product of percentage perforin positive cells and the staining intensity had the best significant correlation with killing of the pulsed PBMC. The present results suggest that perforin could be a possible biomarker for the cytotoxicity to Theileria parva infections/immunizations.
基金This research was supported by Natural Science Foundation of China(No. 39200045).
文摘Objective To prepare the monoclonal antibody against human perforin(HP). Methods Recombi- nant eukaryotic expression plasmid pCDM8-HP was extracted and purified, and the BALB/C mice were immunized with the plasmid. The hybridomas producing anti-HP McAbs were established by using hybridoma technique, then the specificity of the McAbs was identified by using immunocytochemical technique and Western blot. Results Three hy- bridoma cell lines secreting McAbs against human perforin were established, and the three McAbe showed positive only with LAK cells containing human perforin protein, and showed negative with inactive human peripheral blood lymphocytes (PBLs). The subclasses of the three McAbs were determined as IgG2bK. Western blot results showed that the three McAbs recognized a specific band of LAK cell lysateds with molecular weight of 70.0Kd. Conclusion The three hybridoma cell lines secreting McAbs against human perforin were established and the secreted McAbs were specific.
基金Acknowledgments We thank Drs Hua Gu （Columbia University, USA）, Weiguo Zhang （Duke University Medical Center, USA）, and Youhai H Chen （University of Pennsylvania, USA） for reviewing the manuscript and for suggestions, and Dr Ilia Voskoboinik （Peter MacCallum Cancer Centre, Australia） for providing the mouse perforin cDNA in pKS（＋） Bluescript. Ragl^-/- mice were gifts from Xiaolong Liu （Shanghai Institutes for Biological Sciences, China）. This work was supported by grants from the National Natural Science Foundation of China （30325018, 30530700, 30623003, and 30421005） and CAS project （KSCX1-YW-R-43）, grants from the National Key Project 973 （2006CB504300 and 2007CB512404）, grants from the Technology Commission of Shanghai Municipality （04DZ14902, 04DZ19108, 06DZ22032, 04DZ19112, 07XD14033, and 07DZ22916）, 863 key project （2006AA02A247）, and a grant from the E-institutes of Shanghai Universities Immunology Division.
文摘Perforin 是主要从事调停的形成毛孔的蛋白质目标 T 房间死亡并且被细胞毒素的 T 淋巴细胞(CTL ) 和自然漂亮房间采用。然而，它是否也在常规 CD4+ T 房间功能起一个作用，仍然保持不清楚。这里，我们报导那在 perforin 缺乏(PKO ) 老鼠， CD4+ T 房间是响应 T 的 hyperproliferative 房间受体(TCR ) 刺激。hyperproliferation 的这个特征被改进在房间分割并且在 IL-2 分泌物伴随。看起来， perforin 缺乏不在胸腺怒气和淋巴节点影响 T 房间开发。在 vivo， perforin 缺乏导致增加的抗原特定的 T 房间增长和抗体生产。而且， PKO 老鼠更产生试验性的自体免疫的眼色素层炎。探讨分子的机制，我们发现在 TCR 刺激以后，从 PKO 老鼠的 CD4+ T 房间显示增加的细胞内部的钙流动并且随后提高抄写因素 NFAT1 的激活。我们的结果显示 perforin 在由影响 TCR 依赖的 Ca2+ 发信号调整 CD4+ T 房间激活和有免疫力的反应起一个否定作用。
基金Supported by grants from the Brazilian Research Council/CNPq, FAPERJ, and Fundao José Bonifácio/FUJB
文摘AIM: To analyze the level of apoptosis in different mucosal compartments and the differential expression of Fas/Fas-ligand and perforin in H pylori-associated gastric ulcer. METHODS: Antral specimens from patients with H pylori-related active gastric ulcer (GU), H pylori-related gastritis, and non-infected controls were analysed for densities and distribution of apoptotic cells determined by the TdT-mediated dUDP-biotin nick-end-labelling method. GU patients were submitted to eradication therapy with follow-up biopsy after 60 d. Fas, FasL, and perforin-expressing cells were assessed by immunoper- oxidase, and with anti-CD3, anti-CD20 and anti-CD68 by double immunofluorescence and confocal microscopy. Quantitative analysis was performed using a computer- assisted image analyser. RESULTS: H pylori-infected antrum showed greater surface epithelial apoptosis which decreased after eradi- cation therapy. In the lamina propria, higher rates of mononuclear cell apoptosis were observed in H pylori- gastritis. Co-expression of Fas with T-cell and macro- phage markers was reduced in GU. FasL- and perforin-expressing cells were increased in H pylori-infection and correlated with epithelial apoptosis. Perforin-expressing cells were also increased in GU compared with H pylori- gastritis. CONCLUSION: Epithelial apoptosis is increased in H pylori-infection and correlates to FasL- and perforin- expression by T cells. Expression of perforin is correlated with the tissue damage, and may represent the enhance- ment of a distinct cytotoxic pathway in GU. Increased expression of FasL not paralleled by Fas on T-cells and macrophages may indicate a reduced susceptibility to the Fas/FasL-mediated apoptosis of lymphoid cells in H pylori-infection.
基金supported by grants from Chinese Academy of Medical Sciences(CAMS)Initiative for Innovative Medicine(CAMS-I2M)2017-I2M-1-001,the National Natural Science Foundation of China(81788101,81661128007,81530080,and 81773062)the CAMS Initiative for Innovative Medicine(2016-I2M-1–007).
文摘Different types of pores ubiquitously form in cell membranes,leading to various types of cell death that profoundly influence the fate of inflammation and the disease status.However,these pores have never truly been visualized to date.Atomic force microscopy(AFM),which is emerging as a powerful tool to analyze the mechanical properties of biomolecules and cells,is actually an excellent imaging platform that allows biological samples to be visualized by probing surface roughness at the level of atomic resolution.Here,membrane pore structures were clearly visualized using AFM.This visualization not only describes the aperture and depth of the pore complexes but also highlights differences among the pores formed by perforin and gasdermins in tumor cell membranes and by complement in immune cell membranes.Additionally,this type of visualization also reveals the dynamic process of pore formation,fusion,and repair.
文摘Obejctive To study the lymphocytes, perforin protein and mRNA expression in endometrium of different phases in the menstrual cycle and to explore the regularity and role of the lymphocytes and perforin in the reproductive system Methods Immunohistochemical and in situ hybridization techniques were employed to demonstrate the population, phenotype and mediator (perforin) of lymphocytes in the proliferative and secretory endometrium Results In different phases of the endometrium, there were very few CD 3 + typical T lymphocytes, CD 4 + helper lymphocytes and CD 8 + suppressor/cytotoxic T lymphocytes, while CD 56 + lymphocyte (also named NK like cell) were abundant CD 56 + lymphocytes were absent in postmenopausal endometrium A double immunohistochemical labelling method demonstrated the co expression of perforin protein and CD 56 antigen of cells in different phases of the endometrium Quantitative analysis of CD 56 + lymphocytes and perforin positive cells increased in number from the proliferative to the secretory endometrium ( P <0 05) In situ hybridization analysis showed that perforin mRNA positive cells formed islands in proliferative endometrial stroma, but were scattered in secretory endometrium There was difference between perforin gene expression and protein expression in the proliferative endometrium Conclusions Lymphocytes and perforin expression in the endometrium at different phases of the menstrual cycle are specific and may play an important role in the reproductive system They may exert a positive influence on embryo implantation and be involved in endometrial stroma breakdown during
文摘CD134, a member of the tumor necrosis factor receptor superfamily, plays a crucial role in T cell survival. In this study, peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin (PHA, 50 μg/ml) were treated by CD134 mAb (1 μg/ml, 5 μg/ml, 10 μg/ml) for 6 h, 12 h, 24 h and 48 h. The level of perforin mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR) technique. Our data showed that the expression of perforin mRNA in PBMCs was down-regulated by CD134 mAb in a dose-dependent manner in range of 1 μg/ml to 5 μg/ml and dropped down to its minimum on 24 h (p ＜ 0.05). The level of perforin mRNA reached a plateau when the concentration of CD134 mAb exceeded 5 μg/ml. In conclusion, CD134 mAb can inhibit perforin expression, which may enhance the ability of T cells for survival.
