Comprehensive Insight into the Functional Roles of NK and NKT Cells in HTLV-1-Associated Diseases and Asymptomatic Carriers.

Human T cell leukemia virus type 1 (HTLV-1) is the first human oncogenic retrovirus to be discovered and causes two major diseases: a progressive neuro-inflammatory disease, termed HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), and an aggressive malignancy of T lymphocytes known as adult T cell leukemia (ATL). Innate and acquired immune responses play pivotal roles in controlling the status of HTLV-1-infected cells and such, the outcome of HTLV-1 infection. Natural killer cells (NKCs) are the effector cells of the innate immune system and are involved in controlling viral infections and several types of cancers. The ability of NKCs to trigger cytotoxicity to provide surveillance against viruses and cancer depends on the balance between the inhibitory and activating signals. In this review, we will discuss NKC function and the alterations in the frequency of these cells in HTLV-1 infection.

Conferone, a coumarin from Ferula flabelliloba, induced toxic effects on adult T-cell leukemia/lymphoma cells.

BACKGROUND: Adult T-cell leukemia/lymphoma (ATL) is a lymphoid malignancy caused by HTLV-1 infection, with distinct geographical distribution. Despite advances in cancer treatment, the average survival rate of ATL is low. Conferone is a natural coumarin extracted from Ferula species with a wide range of pharmaceutical effects. In search for a novel chemotherapeutic agent, we investigated the cytotoxicity of conferone on ATL cells. METHODS: To obtain conferone, the methanolic extract of the roots of F. flabelliloba was subjected to silica gel column chromatography, followed by 1H- and 13C-NMR to confirm its structure. For cytotoxicity assay, MT-2 cells were treated with different concentrations of conferone (2.5, 5, 10, 20, and 40 µM) for 24, 48, and 72 h, and viability was evaluated by a colorimetric assay using alamarBlue. Cell cycle was analyzed by PI staining and flow cytometry, and qPCR was used to study the expression of candidate genes. RESULTS AND CONCLUSION: Obtained findings indicated that conferone induced considerable cytotoxic effects on MT-2 cells in a time- and dose-dependent manner. In addition, accumulation of cells in the sub-G1 phase of the cell cycle was detected upon conferone administration. Moreover, conferone reduced the expression of CDK6, c-MYC, CFLIPL, and NF-κB (Rel-A) in MT-2 cells. Accordingly, conferone could be considered as a potent agent against ATL, although complementary investigations are required to define more precisely its mechanism of action.

Immigrating and vicinity are not risk factors in the prevalence and transmission rate of human T-lymphotropic virus type 1: A Survey in an endemic region of Iran and Afghan refugees.

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus associated with two life-threatening diseases; HAM/TSP and ATLL. Due to the slow-growing HTLV-1 infection worldwide, WHO urged for elimination. A large border with Afghanistan, northeast Iran is an endemic region for HTLV-1 infection. Historically, Afghanistan has common sociocultural similarities to Persian peoples. This study was conducted to evaluate HTLV-1 prevalence in Afghan refugees. Also, the HTLV-1 transmission rate and understanding of whether or not the Silk Road has been the route of HTLV-1 infection to Iran were investigated. This case-control study was conducted in a rural area of Fariman city, with Afghan residents who migrated around 165 years ago, from 1857, the Treaty of Paris at the end of the Anglo-Persian war, and a refugee camp in Torbat-e-Jam city. These populations in HTLV-1 endemic area were compared to a segregated population of Afghan refugees in Semnan, the centre of Iran. Blood samples of 983 volunteers were assessed with the ELISA method for the presence of HTLV-1 antibodies and then confirmed by PCR technique. All samples from Afghan refugee camps, Semnan and Torbat-e-Jam, were negative for HTLV-1 infection. However, the prevalence of HTLV-1 infection in Fariman, a rural population of Afghan origin, was approximately 2.73%. The results showed that HTLV-1 is not endemic in Afghanistan, a war-stricken region with refugees distributed worldwide. The land Silk Road has not been the route of HTLV-1 transmission to Northeastern Iran. Importantly, HTLV-1 endemicity might occur during a long time of living in an endemic area.

XCL1, a serum biomarker in neurological diseases; HTLV-1-associated myelopathy and multiple sclerosis.

The XCL1-XCR1 axis has a potential role in the recruitment of immune cells to the site of inflammation. The present study aimed to examine the relation of XCL1 serum levels with Multiple sclerosis (MS) and HTLV-1-associated myelopathy (HAM), as chronic inflammatory diseases of the central nervous system (CNS). DNA was extracted to evaluate HTLV-1 proviral load (PVL) using real-time PCR. Serum levels of XCL1 was determined by using an ELISA assay. The serum level of XCL1 was significantly higher in patients with HAM than that of asymptomatic carriers (ACs) and healthy controls (HCs) (p < 0.001 and p < 0.0001, respectively) and was also higher in MS patients compared to HCs (p < 0.0001). Moreover, the concentration of XCL1 serum level was significantly different between the ACs and HCs group (p < 0.0001). In conclusion, increased expression of XCL1 might contribute to the migration of autoreactive T cells to the central nervous system and play a critical role in the development and pathogenesis of inflammatory neurological diseases including HAM and MS.

Galbanic Acid Improves Accumulation and Toxicity of Arsenic Trioxide in MT-2 Cells.

BACKGROUND: Galbanic acid (GBA) is a sesquiterpene coumarin with valuable pharmacological effects. Adult T-cell lymphoma (ATL) is an aggressive lymphoid malignancy with a low survival rate. Although arsenic trioxide (ATO) is a standard therapeutic agent for ATL treatment, the efficacy of chemotherapy is limited due to the chemoresistance of cells. OBJECTIVE: The present study was carried out to investigate whether GBA in combination with ATO would improve cytotoxicity against ATL cells. METHODS: GBA was isolated from the roots of Ferula szowitsiana by column chromatography on silica gel. MT-2 cells were treated with 20 µM GBA + 4 µM ATO, and viability was evaluated by alamarBlue assay. The cell cycle was analyzed by PI staining, while the activity of P-glycoprotein (P-gp) was evaluated by mitoxantrone efflux assay. To understand the molecular mechanisms of GBA effects, the expression of NF-κB (RelA), P53, CDK4, c-MYC, c-FLIPL, and c-FLIPS was evaluated using real-time PCR. RESULTS: Combinatorial use of GBA + ATO significantly reduced the viability of MT-2 cells and induced cell cycle arrest in the sub-G1 phase. GBA improved mitoxantrone accumulation in cells, indicating that this agent has inhibitory effects on the functionality of the P-gp efflux pump. Moreover, real-time PCR analysis revealed that GBA + ATO negatively regulated the expression of P53, CDK4, c-FLIPL, and c-FLIPS. CONCLUSION: Due to the interesting effects of GBA on the accumulation and toxicity of ATO, combinatorial use of these agents could be considered a new therapeutic approach for ATL treatment.

Comparing toxicity of galbanic acid, auraptene and umbelliprenin on adult T-cell leukaemia-lymphoma in normoxia and hypoxia.

Natural coumarins are valuable agents that induce anticancer effects and/or enhance sensitivity to therapeutic modalities. Galbanic acid (GBA), auraptene (AUR) and umbelliprenin (UMB) are coumarins derived from Ferula species with various pharmaceutical activities. The aim of the current research was to compare toxic effects of GBA, AUR, and UMB on human lymphoma cells in normoxia and hypoxia. In this regard, GBA and AUR were extracted from the roots of F. szowitsiana and UMB was derived from the roots of F. persica, all by thin-layer chromatography. MT-2 cells were treated with each agent for 3 consequent periods, while exposed to different O2 contents (21% and 2%). By the end of each treatment, the viability of MT-2 cells was determined by resazurin dye-based colorimetric assay. Obtained results revealed that low doses of GBA (10 and 20 µM) induced significant (p < 0.0001) toxic effects in hypoxia. However, similar toxicity was observed when cells were treated with 40 µM AUR in normoxia and hypoxia. Notably, UMB was the only coumarin that exerted cytotoxic effects in all time points (48, 72 and 96 h) in normoxia and hypoxia, although its concentration was highest (80 µM). In conclusion, this is the first report indicating GBA was the most toxic coumarin against ATL cells in hypoxia, AUR induced similar effects in normoxia and hypoxia, and low toxicity of UMB was stable during the time and different O2 contents. Future studies on other ATL cell lines are recommended to better evaluate the toxic effects of GBA, AUR and UMB in vitro.

Evaluation of interleukin-32 and cyclooxygenase-2 expression in HAM/TSP patients and HTLV-1 asymptomatic carriers.

OBJECTIVES: HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neuroinflammatory disorder associated with HTLV-1. Cytokines and inflammatory mediators have a major role in forming inflammation in HAM/TSP patients. This study aimed to measure the levels of IL-32, a proinflammatory cytokine associated with autoinflammatory disorders, and also cyclooxygenase -2 (COX-2) as a key mediator of inflammatory pathways in HAM/TSP patients and HTLV-1 asymptomatic carriers (ACs). MATERIALS AND METHODS: Peripheral blood monocyte cells (PBMCs) were isolated from HAM/TSP patients, ACs, and healthy controls (HCs), and DNA and RNA were extracted to evaluate HTLV-1 proviral load (PVL) and expression of IL-32 and COX-2, using real-time PCR. Serum levels of IL-32 were determined by using an ELISA assay. RESULTS: The expression level of IL-32 was significantly higher in ACs compared with HAM/TSP patients and HCs (P<0.0001 and P>0.05, respectively). There were no statistically significant differences in the expression levels of Cox-2 and protein levels of IL-32 between the study groups. HTLV-1 PVL was higher in HAM/TSP patients compared with ACs. CONCLUSION: Results showed increased mRNA levels of IL-32 in ACs. Since HTLV-1 PVL in ACs is lower than in HAM/TSP patients, it could be concluded that IL-32 might be an HTLV-1 inhibitor that seems to control virus replication. Despite the difference in IL-32 mRNA levels between study groups, no statistically significant differences were observed in IL-32 serum levels. Also, there were no significant differences in COX-2 expression.

Status of humoral and cellular immune markers in human T-cell lymphotropic virus type 1 (HTLV-1) asymptomatic carriers in northeastern Iran, Mashhad.

It is estimated that about 10-20 million peoples are infected with human T-cell leukemia virus type 1 (HTLV-1) around the world and suffered from HTLV-related diseases. The present study was aimed to evaluate the cellular immunity, T-cell activation, humoral immunity, and inflammatory response hallmarks which affect HTLV-1-associated disease progression. A total of 78 participants were included in the study, comprising 39 HTLV-1 asymptomatic careers (ACs) and 39 healthy controls. The HTLV-proviral load (PVL) was determined via real-time PCR technique, and anti-HTLV antibody, sIL2R, sCD30, Neoptrin, hs-CRP, IgE, anti-VCA, anti-EBNA, and anti-EA were assessed by ELISA method. Mean PVL in ACs was 352.7 ± 418.7 copies/104 PBMCs. A significant higher level of sIL-2R was observed in ACs (P < 0.0001). Anti-VCA antibody titer in ACs and healthy controls was 80.72 ± 105.95 and 156.05 ± 130.71, respectively (P = 0.007). Intriguingly, suppression in ACs immune response was not observed. Resultantly, HTLV-1 infection has no effect on the humoral immune response in ACs but greater T-cell activation and function cellular responses were detected. Finally, more studies on various immune markers in adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients are greatly needed to illuminate the association of ACs' immune status with the development of the related diseases.

Interaction of perforin and granzyme B and HTLV-1 viral factors is associated with Adult T cell Leukemia development.

OBJECTIVES: 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.

The Role of Chemokines in the Pathogenesis of HTLV-1.

Human T cell leukemia virus type 1 (HTLV-1) is a human retrovirus that is associated with two main diseases: HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T cell leukemia/lymphoma (ATL). Chemokines are highly specialized groups of cytokines that play important roles in organizing, trafficking, homing, and in the migration of immune cells to the bone marrow, lymphoid organs and sites of infection and inflammation. Aberrant expression or function of chemokines, or their receptors, has been linked to the protection against or susceptibility to specific infectious diseases, as well as increased the risk of autoimmune diseases and malignancy. Chemokines and their receptors participate in pathogenesis of HTLV-1 associated diseases from inflammation in the central nervous system (CNS) which occurs in cases of HAM/TSP to T cell immortalization and tissue infiltration observed in ATL patients. Chemokines represent viable effective prognostic biomarkers for HTLV-1-associated diseases which provide the early identification of high-risk, treatment possibilities and high-yielding clinical trials. This review focuses on the emerging roles of these molecules in the outcome of HTLV-1-associated diseases.