PERFORIN gene (PRF1) mutations have been reported in 20-30% of patients with familial hemophagocytic lymphohistiocytosis (FHL), an immune disorder of infancy and early childhood. Cytotoxic T and natural killer (NK) cell activities are remarkably reduced or absent in FHL patients. We report the first cases of familial hemophagocytic lymphohistiocytosis in an Iranian family with two siblings. Exons 2 and 3 of the PRF1 gene were analyzed by polymerase chain reaction (PCR) amplification and direct sequencing. PERFORIN gene mutation(s) were detected in none of the cases. The result of our study indicates that not much evidence is present concerning a correlation between PERFORIN gene defects and familial hemophagocytic lymphohistiocytosis etiology in these cases.

Background: PERFORIN is known to be important in cytolytic activity mediated by natural killer (NK) cells. Objective: To study the relationship between the efficiency of NK and lymphokine-activated killer (LAK) cells activity and the expression of PERFORIN and HLAclass I molecules. Methods: LAK cells were generated by in vitro culturing of human peripheral blood lymphocytes (PBLs) in the presence of human recombinant interleukin-2 (rIL-2). Cytotoxic activity was measured at different intervals of activation by MTTcolorimetric assay using different human tumor cell lines. Immunocytochemical staining of molecules was performed on LAK/NK cells using specific monoclonal antibodies and Biotin-conjugated anti-immunoglobulin.Results: LAK/NK killing against Fen and two other cell lines, KB and Scaber showed that at day 9 and 15 of activation, 57% to 60% and 45.5% to 92.5% of Fen cells were killed at different E/Tratios. At the same time, the maximum percent killing against Scaber and KB cell lines was 47.3 and 54.3 at 5/1 ratio, respectively, showing that Fen cells were more sensitive than the two other cells. Time-course experiments using Fen cell line demonstrated 60.0, 83.9 and 34.8 percent killing at days 9, 15 and 22 at 10/1 E/Tratios. When other E/Tratios were investigated, a similar profile was observed. The maximum activity was at day 15 and 5/1 E/Tratio (92.5%). In immunocytochem-ical staining of activated LAK cells, 75.9% to 86.3% of LAK cells expressed HLA class I molecules. PERFORIN expression changed from 30.3% at day 7 to 42.7% at day 17 followed by a decrease to 27.9% at day 24. Conclusion: These data indicate that PERFORIN expression is closely correlated with NK/LAK killing activity.

Background: Chronic hepatitis B is one of the most common causes of cirrhosis and hepatocellular toxicity in many countries, including Iran. Cytotoxic T lymphocyte (CTL) and Natural killer (NK) cells are the two of main cell populations considered as cytotoxic cells. One of the distinct pathways CTL and NK cells exert cytotoxicity is PERFORIN/granzyme. After the cytotoxic cell/target cell junction, PERFORIN is released from granules by exocytosis. Once it is anchored, PERFORIN forms cylindrical pores through which granzymes and granulysin enter and induce apoptosis.Objectives: Large controlled trials have demonstrated the efficacy of PEG-IFN-a-2a in treatment of chronic hepatitis B. This study was aimed to examine whether the enhancement of cytotoxicity by PEG-IFN- a-2a is mainly due to the PERFORIN pathway.Patients and Methods: This research work was performed on 50 patients and five healthy people. Patients with chronic hepatitis B were further subdivided into two groups: patients with inactive chronic hepatitis B (carriers, n = 30), and those with active chronic hepatitis B who were under treatment with PEG-IFN-alfa-2a (n = 20) for minimum six and maximum 12 months. Serum PERFORIN level was measured using ELISA method (CUSABIO Company), HBV viral load was assessed using COBAS Taq-man, and we used Elecsys hepatitis B surface antigen (HBs Ag) II quantitative assay method for HBs Ag determination. HBeAg was evaluated by ELISA method, and AST and ALT were measured by routine laboratorymethods.Results: Based on the results obtained serum PERFORIN level in healthy group was 0.64 ng/mL, the mean of serum PERFORIN level in inactive HBs Ag carriers was 2.63ng/mL, and 4.63 ng/mL in patients with active chronic hepatitis B under treatment with PEG-IFN- a -2a. The mean of serum PERFORIN level in patients with and without virologic response to treatment were 5.45 ng/mL, and 3.4 ng/mL respectively. Finally in patients with virologic response and seroconverted serum PERFORIN level was 7.23 ng/mL.Conclusions: Based on our results higher PERFORIN level in patients under treatment with PEG-IFN- a-2a, could be an indication of elevated cytotoxicity via PERFORIN/granzyme pathway.

Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and potentially life-threatening disease and has to be considered in the differential diagnosis of many conditions. HLH comprises two different conditions that are difficult to differentiate, Familial hemophagocytic lymphohistiocytosis (FHLH) or familial erythrophagocytic lymphohistiocytosis (FEL), and Secondary hemophagocytic syndromes (secondary HLH, sHLH). Herein, we report a case series of Iranian children with HLH and describe the symptoms and outcome of this disease in Iran.

Objective(s): Human T cell leukaemia virus type 1 (HTLV-1) is associated with adult T cell leukaemia (ATL), a malignant lymphoproliferative disease that infects CD4 T cells. It is not clear why the majority of HTLV-1-infected individuals remain asymptomatic carries (ACs) and a minority develop ATL. Cellular immune response has a critical role in ATL and destroys malignant and HTLV-1-infected cells. PERFORIN and granzyme have important functional roles in apoptosis and destruction of infected cells. In the present study we examined the role of PERFORIN and granzyme in ATL patients and ACs. Materials and Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from ATL patients and ACs by using Ficoll-hypaque density centrifugation. RNA was extracted and cDNA was synthesized. A real-time PCR TaqMan method was designed and optimized for evaluation of PERFORIN, granzyme, tax, and HBZ gene expression. HTLV-1 proviral load (PVL) was quantified in patients with ATL and ACs. Results: The mRNA expression of tax and HBZ was significantly higher in ATL patients than ACs (P=0. 011 and P=0. 0001, respectively). The HTLV-1 PVL was higher in ATL patients compared to with AC group (P=0. 015). There was a significant increase in PERFORIN gene expression in ACs compared with ATL patients (P=0. 002). Furthermore, the expression of granzyme was also higher in ACs compared with ATL patients, and significant differences were observed between the two groups (P=0. 036). Conclusion: Low expression of PERFORIN and granzyme in ATL patients seems to influence the efficiency of CTL function and destruction of HTLV-1-infected cells, which might contribute to the disease pathogenesis.

Cytotoxic CD4+ T cells eliminate human cytomegalovirus (HCMV)-infected cells through direct cytotoxic granules exocytosis. We aimed to evaluate the functional cytotoxic gene profile of CD4+ T cells alongside the frequency of the cytotoxic phenotype in renal transplant recipients with cytomegalovirus reactivation. Blood samples were collected from twenty renal recipients with and without HCMV reactivation (HCMV+ and HCMV- groups) and ten healthy adults (control group). CD4+ T cells were isolated to assess the frequency of cytotoxic CD4+ T cells via CD107a surface staining using flow cytometry and to evaluate gene expression of PERFORIN, granzyme B, Runt-related transcription factor 3 (RUNX3), and Eomesodermin (Eomes) by quantitative PCR. The frequency of CD4+ CD107a+ T cells was higher in the HCMV+ group compared to the HCMV- group and significantly higher than in the control group (22.69 ± 3.47 vs 16.41 ± 2.24 and 11.60 ± 1.17, respectively). PERFORIN gene levels were reduced in the HCMV+ group compared to the other two groups, while granzyme B gene levels were similar between HCMV+ and HCMV- groups but lower than in the control group (0.63 ± 1.24 vs 0.67 ± 2.27 and 1.00 ± 0.00, respectively). This study demonstrated an increased frequency of cytotoxic CD4+ T cells with potentially reduced functionality in kidney transplant patients with HCMV infection. It also suggests that these cells might employ other mechanisms, such as death receptor-mediated killing, or the production of other granules.

Background: The prognostic value of peripheral natural killer (pNK) cells, as a screening test in women with recurrentpregnancy loss (RPL) and unexplained infertility, is still a matter for discussion. The purpose of this study was tocompare the percentage of circulating CD56+NK cells, CD69 and PERFORIN markers between women with unexplainedinfertility and RPL with the healthy control group. Materials and Methods: In this case-control study, the percentage of CD56+NK cells and activation markers (CD69and PERFORIN levels) in the peripheral blood were measured in 25 women with unexplained infertility, 24 women withidiopathic RPL and 26 women from the healthy control group, using specific monoclonal antibodies by flow cytometry. Results: The percentage of CD56+NK cells was significantly higher in patients with infertility in comparison withthe healthy control group (P=0. 007). There were not significant differences either in the total number of CD56+ cellsbetween the RPL group and the control group (P=0. 2) or between the RPL group and the infertile group (P=0. 36). The percentage of CD69+ lymphocytes in RPL group was significantly higher than in the infertility group (P=0. 004). There was a statistically significant difference in PERFORIN levels between RLP and control (P=0. 001) as well as RPLand infertile (P=0. 002) groups. Conclusion: An increased percentage of CD56+NK cells in patients with unexplained infertility, an elevated expressionof CD69 on NK cells in patients with RPL and infertility and a high level of PERFORIN on CD56+ cells in the RPL groupmight be considered as immunological risk factors in these women.

Background: Natural killer (NK) cells play a role in the pathogenesis of various metabolic diseases related to obesity. While our initial findings have indicated a potential involvement of NK cells in the pathogenesis of type 2 diabetes mellitus, the precise mechanism underlying NK cell-mediated development of this form of diabetes remains inadequately comprehended.Objective: To investigate the impact and the underlying mechanism of high glucose and elevated levels of free fatty acids (FFAs) on immune and inflammatory responses and oxidative stress in NK92 cells.Methods: In this experiment, the CCK8 cytotoxicity assay was used to select the 44.4 mM and 1.5 mM concentrations of high glucose and high FFAs, respectively, to treat NK92 cells for 4 days. The concentrations of superoxide dismutase (SOD) and glutathione (GSH) were determined using a biochemical analyzer. Intracellular reactive oxygen species (ROS) levels, cytokines concentrations (TNF-α, IFN-γ, IL-6, and IL-10), and the expression levels of intracellular molecules (PERFORIN and granzyme B) were assessed by flow cytometry.Results: The number of NK92 cell clumps was significantly reduced in the high-FFA (HF) group. In addition, the production of ROS and levels of cytokines (TNF-α, IFN-γ, IL-6, and IL-10) significantly decreased in the HF group but showed no significant change in the high-glucose (HG) group. This observation was consistent with the expression levels of PERFORIN and granzyme B that decreased in the HF group.Conclusion: High FFAs induced morphological changes and serious damage to oxidative stress and inflammatory response in NK92 cells.