Combinational IL-2/IL-15 induction does not further enhance IL-15-induced lymphokine-activated killer cell cytotoxicity against human leukemia/lymphoma cells

In vitro comparisons of induction of perforin (PFP), granzyme B (GRB), production of cytokines, and cell-mediated cytotoxicity by interleukin-2 (IL-2), interleukin-15 (IL-15), or combinational IL-2/IL-15-induced lymphokine-activated killer cells were studied in this study. Whereas IL-2-induction was associated with a decrease in cultured cell population over a 7-day period, IL-15 alone or in combination with IL-2 resulted in significant increase including cytotoxic T lymphocytes and subsets of CD56+ lymphocytes, particularly cytokine-induced killer and cytolytic natural killer-T lymphocytes. The overall PFP, GRB, and tumor necrosis factor-alpha expression in different subtypes were also significantly higher with IL-15 alone or in combination with IL-2 induction with resultant superior cytotoxicity compared to IL-2 treatment. There was no significant advantage of addition of IL-2 over IL-15 induction. These results offer further information on the cytotoxic potency of these cytokines and their mechanisms of action implicating potential use of IL-15 as part of cytokine adoptive immunotherapy.     

Perforin dependence of natural killer cell-mediated tumor control in vivo

Adaptive immune surveillance by T cells against infections and tumors depends on the presence of antigenic peptides presented by major histocompatibility complex (MHC) molecules. If antigenic tumor-specific peptides or MHC class I molecules are absent, the adaptive T cell immune response fails. Natural killer (NK) cells seem to complement the specific T cells by recognizing target cells lacking MHC class I (e.g. RMA-S). The role of perforin, which is crucially involved in T cell and NK cell-mediated target cell lysis, was evaluated in mice lacking perforin with respect to their capacity to eliminate a syngeneic lymphoid tumor. Here, we show that growth of MHC class I^? RMA-S tumor cells in unprimed mice was controlled by NK cells through perforin-dependent cytotoxicity.     

Differential expression of human granzymes A, B, and K in natural killer cells and during CD8+ T cell differentiation in peripheral blood

NK cells and cytotoxic T lymphocytes can induce apoptosis in virus-infected and transformed target cells via the granule exocytosis pathway. The key components of the cytolytic granules are perforin and several serine esterases, termed granzymes. While the cellular distribution of human granzymes A (GrA) and B (GrB) has been well characterized much less is known about the expression pattern of human granzyme K (GrK). In this study GrA, GrB, and GrK expression was analyzed in human peripheral blood lymphocytes using flow cytometry. There was a distinct population of GrK expressing CD8+ T cells with a CD27+/CD28+/CCR5high/CCR7-/perforin-/low/IFN-gamma+ memory-like phenotype, while all CD56bright NK cells were also positive for GrK. In addition, GrK was also expressed in subpopulations of CD56+ T cells, CD4+ T cells, and TCRgammadelta+ T cells. In contrast, GrB was primarily expressed in CD56dim NK cells and differentiated memory CD8+ T cells with the CD27-/low/CD28-/low/CCR5-/low/CCR7-/CD11b+/perforinhigh phenotype. Only few CD8+ T cells expressed both GrB and GrK. GrA was found to be co-expressed in all GrB- and GrK-expressing T cells. Our findings suggest that granzyme expression during the differentiation process of memory CD8+ T cells might be as follows: GrA+/GrB-/GrK+ --> GrA+/GrB+/GrK+ --> GrA+/GrB+/GrK-.     

Decreased perforin and granzyme B expression in senescent HIV-1-specific cytotoxic T lymphocytes

Cytotoxic T lymphocyte (CTL) senescence may be an important mechanism of immune failure in HIV-1 infection. We find that senescence of HIV-1-specific CTL clones causes loss of killing activity, preventable by transduction with telomerase. Furthermore, senescence is associated with reduced expression of the effector molecules granzyme and perforin, suggesting CTL "exhaustion" can result in hypofunction. These results agree with other studies showing that HIV-1-specific CTL exhibit abnormal phenotypes in vivo, and suggest the possibility that chronic turnover is an important mechanism of antiviral failure in HIV-1 infection.     

慢性乙型肝炎患者外周血淋巴细胞穿孔素的表达

目的：了解慢性乙型肝炎患者外周血淋巴细胞（PBL）穿孔素（PFP）的表达情况及其与疾病的关系。方法：用抗人PFP单克隆抗体，采用免疫组化染色法，观察慢性乙型肝炎患者PBL中总的PFP阳性细胞的百分率，结果：慢性乙型肝炎PBL中PFP表达与正常对照组之间存在明显差异，结论：乙型肝炎病毒感染慢性化与机体PBL中PFP的表达水平低下密切相关。     

Clinical and Genetic Studies of Familial Hemophagocytic Lymphohistiocytosis in Oman: Need for Early Treatment

Hemophagocytic lymphohistiocytosis (HLH) embraces the frequently indistinguishable conditions of familial hemophagocytic lymphohistiocytosis (FHL) and virus-associated hemophagocytic syndrome (VAHS). Without therapy FHL is invariably fatal, but successful therapy, including chemotherapy and immunotherapy followed by bone marrow transplantation (BMT), has been presented. To clarify the outcome of HLH in a developing country, with regard to clinical, laboratory, and genetic features, a nationwide study on all patients diagnosed with HLH in Oman during the 5-year period 1997-2001 was performed. In 5 patients and their families, mutational analysis was made. Thirteen patients with HLH were identified, 5 of whom had clinical manifestations of central nervous system involvement at presentation. In none of the patients could an infectious cause be identified. Ten children were referred late in the disease course, and the concern about starting chemotherapy before exclusion of an acute viral infection resulted in delayed treatment in some patients. Two children were started early on the HLH-94-therapy followed by successful BMT in one child. In the successfully transplanted child, the response to intrathecal hydrocortisone appeared to be better than standard therapy with intrathecal methotrexate. Finally, a novel missense mutation in the perforin gene was identified in 2 patients and their family members, causing a transition of proline to threonine at codon 89. Early diagnosis and treatment is important to improve outcome. Intrathecal corticosteroids may be considered, in addition to intrathecal methotrexate, in certain patients. Since the novel perforin mutation has been reported in only 2 patients from Oman, and since similar polymorphism in the sequencing data of the members of their families has been identified, a founder effect is possible in this population.     

Co-expression of perforin and granzyme B genes induces apoptosis and inhibits the tumorigenicity of laryngeal cancer cell line Hep-2

Granzyme B and perforin, two of the most important components, have shown anticancer properties in various cancers, but their effects in laryngeal cancer remain unexplored. Here we decided to examine the effects of Granzyme B and perforin in Hep-2 cells and clarify the role of perforin and granzyme B in the tumorigenicity of laryngeal cancer cell line. Hep-2 cells were transfected with pVAX1-PIG co-expression vector (comprising perforin and granzyme B genes), and then the growth and apoptosis of these Hep-2 cells were evaluated. The tumorigenicity of Hep-2 cell line co-expressing perforin and granzyme B genes was tested in BALB/c nu/nu mice. We found that the co-expression of perforin and granzyme B genes could obviously inhibit cell focus formation and induce cell apoptosis in Hep-2 cells. Furthermore, after subcutaneous injection of Hep-2 cells transfected with pVAX1-PIG, an extensive delay in tumor growth was observed in BALB/c-nu/nu mice. Moreover, our studies demonstrated that the anticancer activity of perforin and granzyme B was sustainable in vivo as tumor development by inducing cell apoptosis. Taken together, our data indicate that the co-expression of perforin and granzyme B genes exhibits anticancer potential, and hopefully provide potential therapeutic applications in laryngeal cancer.     

组织芯片检测淋巴瘤中活化的细胞毒性细胞

目的：运用组织芯片及常规病理切片检测活化的细胞毒性细胞在各型淋巴瘤中的表达分布情况，为临床治疗和判断预后提供依据。方法：运用免疫组化方法检测60份淋巴瘤标本制成的组织芯片中穿孔素、颗粒酶B的表达和分布情况，同时选择10份鼻自然杀伤细胞（NK）／T细胞淋巴瘤常规标本与组织芯片进行比较研究，10份淋巴组织反应性增生标本作为对照。结果：在60份淋巴瘤组织芯片中，发生在淋巴结内者48份，淋巴结外者12份。B细胞淋巴瘤42份，T细胞淋巴瘤16份（外周T细胞淋巴瘤10份、NK／T细胞淋巴瘤2份、T淋巴母细胞瘤2份及问变性大细胞淋巴瘤2份），2份霍奇金淋巴瘤。42份B细胞淋巴瘤的瘤细胞均不表达穿孔素和颗粒酶B。10份外周T细胞淋巴瘤中8份表达穿孔素，9份表达颗粒酶B，阳性表达细胞均为非肿瘤细胞。芯片中的2份和常规10份鼻NK／T细胞淋巴瘤中均见穿孔素和颗粒酶B过度表达。T、B细胞淋巴瘤与NK／T细胞淋巴瘤比较有显著性差异（P〈0．01）。结论：穿孔素和颗粒酶B是鉴定活化的细胞毒性细胞的免疫标志物，可作为NK／T细胞淋巴瘤的诊断性标志物；其在T细胞淋巴瘤中的表达反映了机体的抗肿瘤免疫反应机制。组织芯片技术具有高信息量、简捷、高效、实验误差小、重复性好等优点，可作为研究淋巴瘤的有用工具。     

外周血穿孔素和颗粒酶B mRNA检测对胰腺移植急性排斥反应早期诊断的价值

目的 研究胰腺移植术后外周血穿孔素和颗粒酶B的基因表达对急性排斥反应早期诊断的价值.方法 实验动物为杂种长白猪,分为3组(n=8):对照组,移植组.免疫抑制剂治疗组.移植手术为同种异体门静脉回流、肠内引流全胰十二指肠移植.免疫抑制剂治疗组在行同种异体胰十二指肠移植的同时应用环孢A 骁悉 甲强龙三联免疫抑制治疗.分别于术前1 d和术后1、3、5、7 d采集受者锁骨下静脉血,抽提总RNA,RT.PCR方法检测静脉血淋巴细胞中穿孔素和颗粒酶B的mRNA表达,检测受者血糖、胰岛素、胰高血糖素水平.在术后1、3、5、7d,开腹取胰腺组织进行常规病理学检查,与血液中穿孔素和颗粒酶B的mRNA表达水平进行比较分析.结果 ①对照组、移植组、免疫抑制剂组之间受体的血糖、胰岛素、胰高血糖素水平变化差异无统计学意义.②与移植组比较,免疫抑制剂组静脉血淋巴细胞中穿孔索和颗粒酶B的mRNA表达水平下降(P<0.05).③胰腺移植术后静脉血穿孔素、颗粒酶B mRNA表达变化比急性排斥反应病理学改变早2～d.结论 监测外周血淋巴细胞中穿孔素和颗粒酶B mRNA表达的变化,可以对胰腺急性排斥反应做出早期诊断.     

Clinical and genetic studies of familial hemophagocytic lymphohistiocytosis in Oman: need for early treatment

Hemophagocytic lymphohistiocytosis (HLH) embraces the frequently indistinguishable conditions of familial hemophagocytic lymphohistiocytosis (FHL) and virus-associated hemophagocytic syndrome (VAHS). Without therapy FHL is invariably fatal, but successful therapy, including chemotherapy and immunotherapy followed by bone marrow transplantation (BMT), has been presented. To clarify the outcome of HLH in a developing country, with regard to clinical, laboratory, and genetic features, a nationwide study on all patients diagnosed with HLH in Oman during the 5-year period 1997-2001 was performed. In 5 patients and their families, mutational analysis was made. Thirteen patients with HLH were identified, 5 of whom had clinical manifestations of central nervous system involvement at presentation. In none of the patients could an infectious cause be identified. Ten children were referred late in the disease course, and the concern about starting chemotherapy before exclusion of an acute viral infection resulted in delayed treatment in some patients. Two children were started early on the HLH-94-therapy followed by successful BMT in one child. In the successfully transplanted child, the response to intrathecal hydrocortisone appeared to be better than standard therapy with intrathecal methotrexate. Finally, a novel missense mutation in the perforin gene was identified in 2 patients and their family members, causing a transition of proline to threonine at codon 89. Early diagnosis and treatment is important to improve outcome. Intrathecal corticosteroids may be considered, in addition to intrathecal methotrexate, in certain patients. Since the novel perforin mutation has been reported in only 2 patients from Oman, and since similar polymorphism in the sequencing data of the members of their families has been identified, a founder effect is possible in this population.