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.

Tolerogenic probiotics Lactobacillus delbrueckii and Lactobacillus rhamnosus promote anti-inflammatory profile of macrophages-derived monocytes of newly diagnosed patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is known as an autoimmune disorder that is characterized by the breakdown of self-tolerance, resulting in disease onset and progression. Macrophages have been implicated as a factor in the development of SLE through faulty phagocytosis of dead cells or an imbalanced M1/M2 ratio. The study aimed to investigate the immunomodulatory effects of Lactobacillus delbrueckii and Lactobacillus rhamnosus on M1 and M2 macrophages in new case lupus patients. For this purpose, blood monocytes were collected from lupus patients and healthy people and were cultured for 5 days to produce macrophages. For 48 h, the macrophages were then cocultured with either probiotics or lipopolysaccharides (LPS). Flow cytometry and real-time polymerase chain reaction were then used to analyze the expression of cluster of differentiation (CD) 14, CD80, and human leukocyte antigen - DR (HLADR) markers, as well as cytokine expression (interleukin [IL]1-ß, IL-12, tumor necrosis factor α [TNF-α], IL-10, and transforming growth factor beta [TGF-ß]). The results indicated three distinct macrophage populations, M0, M1, and M2. In both control and patient-derived macrophage-derived monocytes (MDMs), the probiotic groups showed a decrease in CD14, CD80, and HLADR expression compared to the LPS group. This decrease was particularly evident in M0 and M2 macrophages from lupus patients and M1 macrophages from healthy subjects. In addition, the probiotic groups showed increased levels of IL-10 and TGF-ß and decreased levels of IL-12, IL1-ß, and TNF-α in MDMs from both healthy and lupus subjects compared to the LPS groups. Although there was a higher expression of pro-inflammatory cytokines in lupus patients, there was a higher expression of anti-inflammatory cytokines in healthy subjects. In general, L. delbrueckii and L. rhamnosus could induce anti-inflammatory effects on MDMs from both healthy and lupus subjects.

A new generation of mesenchymal stromal/stem cells differentially trained by immunoregulatory probiotics in a lupus microenvironment.

BACKGROUND: Increasing evidence suggests that multipotent mesenchymal stem/stromal cells (MSCs) are a promising intervention strategy in treating autoimmune inflammatory diseases. It should be stated that systemic immunoregulation is increasingly recognized among the beneficial effects of MSCs and probiotics in treating morbid autoimmune disorders such as lupus. This study aimed to determine if immunoregulatory probiotics L. rhamnosus or L. delbrueckii can change the immunomodulatory effects of MSCs in lupus-like disease. METHODS: Pristane-induced lupus (PIL) mice model was created via intraperitoneal injection of Pristane and then confirmed. Naïve MSCs (N-MSCs) were coincubated with two Lactobacillus strains, rhamnosus (R-MSCs) or delbrueckii (D-MSCs), and/or a combination of both (DR-MSCs) for 48 h, then administrated intravenously in separate groups. Negative (PBS-treated normal mice) and positive control groups (PBS-treated lupus mice) were also investigated. At the end of the study, flow cytometry and enzyme-linked immunosorbent assay (ELISA) analysis were used to determine the percentage of Th cell subpopulations in splenocytes and the level of their master cytokines in sera, respectively. Moreover, lupus nephritis was investigated and compared. Analysis of variance (ANOVA) was used for multiple comparisons. RESULTS: Abnormalities in serum levels of anti-dsDNA antibodies, creatinine, and urine proteinuria were significantly suppressed by MSCs transplantation, whereas engrafted MSCs coincubation with both L. strains did a lesser effect on anti-dsDNA antibodies. L. rhamnosus significantly escalated the ability of MSCs to scale down the inflammatory cytokines (IFN-É£, IL-17), while L. delbrueckii significantly elevated the capacity of MSCs to scale down the percentage of Th cell subpopulations. However, incubation with both strains induced MSCs with augmented capacity in introducing inflammatory cytokines (IFN-É£, IL-17). Strikingly, R-MSCs directly restored the serum level of TGF-ß more effectively and showed more significant improvement in disease parameters than N-MSCs. These results suggest that R-MSCs significantly attenuate lupus disease by further skew the immune phenotype of MSCs toward increased immunoregulation. CONCLUSIONS: Results demonstrated that Lactobacillus strains showed different capabilities in training/inducing new abilities in MSCs, in such a way that pretreated MSCs with L. rhamnosus might benefit the treatment of lupus-like symptoms, given their desirable properties.

Immune response to COVID-19 vaccines among people living with human T-cell lymphotropic virus type 1 infection: a retrospective cohort study from Iran.

The effectiveness of COVID-19 vaccination is still unclear in individuals with underlying diseases such as HTLV-1 infection. This retrospective cohort study aimed to evaluate the humoral response of COVID-19 vaccines among people living with HTLV-1 (PLHTLV) in northeastern Iran. From December 2021 to October 2022, eighty-six HTLV-1+ subjects (50 males and 36 females; 47.7 ± 11.2 years) and 90 HTLV-1 seronegative individuals (age- and sex-matched convenient samples) were enrolled. The humoral immune response was evaluated by measuring different COVID-19 Abs in serum samples at least 28 days after receiving 2nd or 3rd doses of COVID-19 vaccines. Throughout all three rounds of immunization, Sinopharm was the most commonly used COVID-19 vaccine across all three immunization rounds. Compared to the HTLV-1- group, a significantly lower frequency of all four Abs activity was observed among PLHTLV:anti-nucleocapsid (66.3% vs 86.7%, p = 0·001), anti-spike (91.9% vs 98.9%, p = 0·027), RBD (90.7% vs 97.8%, p = 0·043), and neutralizing Abs (75.6% vs 95.5%, p < 0·001). Also, the frequency of all Abs in 28 patients with HAM/TSP was higher than that of 58 asymptomatic carriers, although this difference was statistically significant only in the case of anti-spike Abs (p = 0.002). Notably, PLHTLV-vaccinated against COVID-19 demonstrated significantly lower antibody activities, indicating a reduced humoral immune response to COVID-19 vaccines.

Unraveling The Effects of DICER1 Overexpression on Immune-Related Genes Expression in Mesenchymal Stromal/Stem Cells: Insights for Therapeutic Applications.

OBJECTIVE: The immunoregulatory properties of mesenchymal stromal/stem cells (MSCs) bring a promise for the treatment of inflammatory diseases. However, their ability to suppress the immune system is unstable. To enhance their effectiveness against immune responses, it may be necessary to manipulate MSCs. Although some dsRNA transcripts come from invading viruses, the majority of dsRNA has an endogenous origin and is known as endo-siRNA. DICER1 is a ribonuclease protein that can generate small RNAs to modulate gene expression at the post-transcriptional level. We aimed to evaluate the expression of several immune-related genes at mRNA and protein levels in MSCs overexpressing DICER1 exogenously. MATERIALS AND METHODS: In this comparative transcriptomic experimental study, the adipose-derived MSCs (Ad-MSCs) were transfected using the pCAGGS-Flag-hsDicer vector for the DICER1 overexpression. Following the RNA extraction, mRNA expression level of DICER1 and several inflammatory cytokines were examined. We performed a relative real-time polymerase chain reaction (PCR) assay and transcriptome analysis between two groups including DICER1- transfected MSCs and control MSCs. Moreover, media from the transfected MSCs were evaluated for various interferon response factors by ELISA. RESULTS: The overexpression of DICER1 is associated with a significant increase in the mRNA expression level of COX-2, DDX-58, IFIH1, MYD88, RNase L, TLR3/4, and TDO2 genes and a downregulation of the TSG-6 gene in MSCs. Moreover, the expression levels of IL-1, 6, 8, 17, 18, CCL2, INF-γ, TGF-ß, and TNF-α were higher in the DICER1-transfected MSCs group. CONCLUSION: It seems that the ectopic expression of DICER1 in Ad-MSCs is linked to alterations in the expression level of immune-related genes. It is suggested that the manipulation of immune-related pathways in MSCs via the Dicer1 overexpression could facilitate the development of MSCs with distinct immunoregulatory phenotypes.

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.

Assessment of Bcl-xL, TAX, and HBZ Gene Expression in Adult T cell Leukemia/Lymphoma Patients.

Adult T cell leukemia/lymphoma is a malignancy with a poor prognosis caused by human T lymphocyte virus type 1 (HTLV-1) infection. Tax and HBZ are two major viral proteins that may be involved in oncogenesis by disrupting apoptosis. Because Bcl-xL plays an integral role in the anti-apoptotic pathway, this study examines the interaction between host apoptosis and oncoproteins. We investigated 37 HTLV-1-infected individuals, including 18 asymptomatic and 19 adult T cell leukemia/lymphoma (ATLL) subjects. mRNA was extracted and converted to cDNA from peripheral blood mononuclear cells, and then gene expression was determined using TaqMan q-PCR. Moreover, the HTLV-1 proviral load (PVL) was also measured using a commercial absolute quantification kit (Novin Gene, Iran). Data analysis revealed that the mean of TAX, HBZ, and PVL was significantly higher among the study groups (ATLL and carrier groups p = .003, p = .000, and p = .002 respectively). There was no statistical difference in Bcl-xL gene expression between the study groups (p = .323). It is proposed that this anti-apoptotic pathway may not be directly involved in the development of ATLL lymphoma. Bcl-xL, TAX, HBZ gene expression, and PVL can be utilized as prognostic markers.

Expression pattern of glucocorticoid receptor α gene and associations with clinicolaboratory features in patients with systemic lupus erythematosus.

Background: Glucocorticoid receptor α (GRα) gene is a transcription factor with clinically significant immune-modulating properties in various autoimmune diseases. However, the expression pattern of the GRα gene and associations with clinical features in patients with systemic lupus erythematosus (SLE) is controversial. This study aimed to assess the correlation between the GRα expression and different clinical and laboratory-related parameters in SLE patients. Methods: A total of 45 women with newly diagnosed SLE and 31 gender and age-matched healthy controls were enrolled in this cross-sectional study. The real-time quantitative PCR (qRT-PCT) method evaluated the differences in GRα expression in peripheral blood mononuclear cells from cases and controls. The correlation between the GRα gene expression levels, clinicolaboratory features, and potential prognostic application was also analyzed. Results: Compared to the healthy individuals, the GRα gene expression in newly diagnosed SLE patients who did not receive any treatment was numerically reduced, but this reduction did not achieve statistical significance (P=0.87). No significant correlation was also found with the activity and severity of SLE according to SLEDAI2K (P=0.41). The GRα gene expression showed a negative correlation with CRP (P=0.034) and a positive correlation with lupus anticoagulant (P=0.039) levels in SLE. The receiver operating characteristic (ROC) curve analysis indicated that the GRα expression level might be a predictor biomarker for low CRP and positive lupus anticoagulant in SLE, respectively. Conclusion: This study proposed that expression of the GRα in newly diagnosed lupus patients has no statistically significant difference with healthy age and sex-matched controls. Besides, its expression does not correlate with lupus disease activity according to SLEDAI2k. However, further studies in this area are required.

Rigosertib is more potent than wortmannin and rapamycin against adult T-cell leukemia-lymphoma.

Human T lymphotropic virus type 1 (HTLV-1) infection can cause adult T-cell lymphoblastic leukemia (ATLL), an incurable, chemotherapy-resistant malignancy. In a quest for new therapeutic targets, our study sought to determine the levels of AKT, mTOR, and PI3K in ATLL MT-2 cells, HTLV-1 infected NIH/3T3 cells (Inf-3T3), and HTLV-1 infected patients (Carrier, HAM/TSP, and ATLL). Furthermore, the effects of rigosertib, wortmannin, and rapamycin on the PI3K/Akt/mTOR pathway to inhibit the proliferation of ATLL cells were examined. The results showed that mRNA expression of Akt/PI3K/mTOR was down-regulated in carrier, HAM/TSP, and ATLL patients, as well as MT-2, and Inf-3T3 cells, compared to the healthy individuals and untreated MT-2 and Inf-3T3 as controls. However, western blotting revealed an increase in the phosphorylated and activated forms of AKT and mTOR. Treating the cells with rapamycin, wortmannin, and rigosertib decreased the phosphorylated forms of Akt and mTOR and restored their mRNA expression levels. Using these inhibitors also significantly boosted the expression of the pro-apoptotic genes, Bax/Bcl-2 ratio as well as the expression of the tumor suppressor gene p53 in the MT-2 and Inf-3T3cells. Rigosertib was more potent than wortmannin and rapamycin in inducing sub-G1 and G2-M cell cycle arrest, as well as late apoptosis in the Inf-3T3 and MT-2 cells. It also synergized the cytotoxic effects of vincristine. These findings demonstrate that HTLV-1 downregulation of the mRNA level may occur as a negative feedback response to increased PI3K-Akt-mTOR phosphorylation by HTLV-1. Therefore, using rigosertib alone or in combination with common chemotherapy drugs may be beneficial in ATLL patients.

Pentraxin 3, a serum biomarker in human T-cell lymphotropic virus type-1-associated myelopathy patients and asymptomatic carriers.

Human T-cell lymphotropic virus type 1 (HTLV-1) can induce a neuroinflammatory condition that leads to myelopathy. Pentraxin 3 (PTX3) is an acute-phase protein that its plasma concentration increases during inflammation. We aimed to determine whether PTX3 serum level is elevated in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients and HTLV-1 asymptomatic carriers (ACs) and evaluate its association with proviral load and clinical features. The serum level of PTX3 was measured using an enzyme-linked immunosorbent assay in 30 HAM patients, 30 HTLV-1 ACs, and 30 healthy controls. Also, the HTLV-1 proviral load was determined via real-time PCR technique. The findings showed that PTX3 serum level was significantly higher in HAM patients than in both asymptomatic carriers and healthy controls (p values < 0.0001). No correlation between PTX3 and the proviral load was observed in HAM patients and asymptomatic carriers (r = - 0.238, p = 0.205 and r = - 0.078, p = 0.681, respectively). The findings showed that there was no significant correlation between PTX3 and motor disability grading (MDG) (r = - 0.155, p = 0.41) nor urinary disturbance score (UDS) (r = - 0.238, p = 0.20). Higher levels of PTX3 are associated with HTLV-1-associated myelopathy compared to asymptomatic carriers. This finding may support the idea that PTX3 has the potential as a diagnostic biomarker.

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.

Alteration of immunoregulatory genes expression in mesenchymal stromal cells upon priming with B18R as an interferon binding protein.

Objectives: The B18R protein encoded by the Vaccinia virus decoys Type 1 interferons and inhibits the activity of several type I IFN members. In vitro transcription protocols benefit from this molecule's involvement in enhancing cell viability by inhibiting interferon signal transduction. As a result of their immunomodulatory properties and potential to regenerate, mesenchymal stromal cells (MSCs) are increasingly considered an alternative treatment for a wide range of immune disorders. In this study, we investigated the modification of expression of several genes involved in immune-related pathways after preconditioning MSCs with two immune stimuli, including poly(I:C) and LPS. Materials and Methods: ASCs were isolated and primed with B18R, and after exposure to poly(I:C) and LPS, the expression of the same sets of genes as in the previous experiment was evaluated. Following total RNA isolation from primed cells and cDNA preparation, real-time quantitative PCR was performed for several immunomodulatory and immune-related genes, including IDO1, TDO2, COX-2, TGF- ß 1, TNF- α, IL-1 ß , IL-6, TLR3, TLR4, and MCP-1. Results: Pretreatment of MSCs with poly(I:C) and LPS significantly increased the expression of all mentioned genes, while upon the B18R challenge followed by poly(I:C) and LPS treatment, they were down-regulated. Finally, it was observed that the relative expression level of IFN -ß has significantly decreased in MSCs+B18R+poly(I:C) and LPS in comparison with these groups without B18R. Conclusion: The data indicated that the presence of B18R prevents the overexpression of several immune-related genes, which are overexpressed in the in vitro inflammatory environment.

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.

Genetic Association of VDR gene Apa1 and Taq1 Variants with Scleroderma in an Iranian Northeast Population.

BACKGROUND: Despite vigorous research efforts, the etiology of scleroderma (systemic sclerosis (SSc)) remains still unclear and both genetic and environmental factors clearly contribute to the pathogenesis of scleroderma. Reports of aberrant vitamin D status in scleroderma patients suggest a need for considering the genotype and allele frequencies of VDR gene polymorphisms. This case-control study aimed to investigate the possible association of two common polymorphisms of the VDR gene (ApaI, and TaqI) with susceptibility to scleroderma in an Iranian population. METHODS: Using polymerase chain reaction and restriction fragment length polymorphism (PCRRFLP), ApaI and TaqI polymorphisms in the VDR region were genotyped in 51 patients with scleroderma and 50 healthy controls. Logistic regression analysis was performed to calculate the genotypes odds ratios (ORs) as a measure of association with the presence of scleroderma. Haplotype and linkage disequilibrium analyses were also performed on the detected genotypes. RESULTS: No significant differences were found for the allelic and genotype distributions of ApaI and TaqI polymorphisms between patients with scleroderma and healthy controls (p>0.05). In haplotype analysis, three haplotypes TA, CA, and TC, with a frequency greater than 1% were identified. However, none of them was associated with the risk of scleroderma. CONCLUSION: Our preliminary study showed no evidence of an association between ApaI and TaqI polymorphisms and scleroderma. As the association between VDR polymorphisms and autoimmune diseases varies across the different ethnic populations, further large cohort studies are necessary to confirm the results.

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.

Development of a Novel HTLV-1 Protease: Human Fcγ1 Recombinant Fusion Molecule in the CHO Eukaryotic Expression System.

Human T-cell leukaemia virus type 1 (HTLV-1) is the causative agent of two life-threatening diseases, adult T cell leukaemia/lymphoma (ATLL), and HTLV-1-associated myelopathy/tropical spastic (HAM/TSP). HTLV-1 protease (HTLV-1-PR) is an aspartic protease that represents a promising target for therapeutic purposes like human immunodeficiency virus-PR inhibitors (HIV-PR). Therefore, in this study, the human Fc fusion recombinant-PR (HTLV-1-PR:hFcγ1) was designed and expressed for two applications, finding a blocking substrate as a potential therapeutic or a potential subunit peptide vaccine. The PCR amplified DNA sequences encoding the HTLV-1-PR from the MT2-cell line using specific primers with restriction enzyme sites of Not1 and Xba1. The construct was then cloned to pTZ57R/T TA plasmid and, after confirming the PR sequence, subcloned into the pDR2ΔEF1α Fc-expression vector to create pDR2ΔEF1α.HTLV-1-PR:hFcγ1. The integrity of recombinant DNA was confirmed by sequencing to ensure that the engineered construct was in the frame. The recombinant fusion protein was then produced in the Chinese hamster ovary cell (CHO) system and was purified from its supernatant using HiTrap-rPA column affinity chromatography. Then, the immunofluorescence assay (IFA) co-localisation method showed that HTLV-1-PR:hFc recombinant fusion protein has appropriate folding as it binds to the anti-Fcγ antibody; the Fcγ1 tag participates to have HTLV-1-PR:hFcγ1 as a dimeric secretory protein. The development and production of HTLV-1-PR can be used to find a blocking substrate as a potential therapeutic molecule and apply it in an animal model to assess its immunogenicity and potential protection against HTLV-1 infection.

Molecular insight into the study of adult T-cell leukemia/lymphoma (ATLL): Ten-year studies on HTLV-1 associated diseases in an endemic region.

The outcome of successful infection, including human T-cell leukemia virus type 1 (HTLV-1), is determined by the interactions between the host and the infectious agent. Ten years of work on HTLV-1-associated diseases in an endemic region of Iran have been critically compared in the present study. The outstanding findings of RNA-seq, system biology analysis, and gene expression measurements on adult T-cell leukemia/lymphoma (ATLL) and enzootic bovine leukosis(EBL) in our lab encouraged us to investigate the significant role of oncogenes in the ATLL malignancy. Most studies assessed such interactions by the proviral load (PVL), Tax, and HBZ regulatory proteins in HTLV-1 and the host's immunological and cell cycle factors. The current study is a comprehensive comparing view of our previously published and unpublished results investigating the HTLV-1-host interactions leading to the transformation of the infected cell. The main focus has been on the essential proteins implicated in the virus dissemination, cell survival, and proliferation of infected cells toward leukemia development and progression. Similar to its homolog BLV-AS-1-2 in EBL, the HTLV-1-HBZ is a pivotal factor in the maintenance and progression of the ATLL. In addition, the inappropriate activities of the PI3K/Akt pathway, BRCAs, and RAD51 in the DNA repair system, which are orchestrating many other immortalization pathways, might be the central factors in the manifestation of ATLL. HTLV-1-HBZ and the host PI3K/Akt pathway, BCAs, and RAD51 could be suggested as influential targets for the prognosis and proper therapy of ATLL.

Radiation Response of Human Leukemia/Lymphoma Cells was Improved by 7-Geranyloxycoumarin.

Objectives: Adult T-cell leukemia/lymphoma (ATLL) is a blood neoplasm with specific geographic distribution. Although radiotherapy is a palliative treatment that provides long-term local control, single use of radiation leads to complications for patients. To introduce a novel multimodal approach against ATLL, we investigated combinatorial effects of 7-geranyloxycoumarin and radiation in vitro. Methods: Viability of MT-2 cells was determined by resazurin assay upon administration of 7-geranyloxycoumarin alone and followed by radiation. Then, apoptosis was detected by annexin V and propidium iodide, and the expression of candidate genes was analyzed by qPCR. Results: Findings revealed significant (P<.0001) improvement in radiation effects upon 7-geranyloxycoumarin pretreatment, most notably when cells were pretreated with 5 µg/ml 7-geranyloxycoumarin for 96 h, exposed to 6 Gy radiation and recovered for 48 h. These results were confirmed by flow cytometry, as the percentage of early and late apoptotic cells was increased after combinatorial treatment. In addition, significant (P< .0001) changes in CD44, c-MYC, cFLIPL, BMI-1, NF-κB (Rel A), and P53 expression was induced by 7-geranyloxycoumarin and radiation. Conclusions: Current research indicated, for the first time, that combinatorial use of 7-geranyloxycoumarin and ionizing radiation could be considered as an effective therapeutic modality for ATLL.

Potential protective effects of chrysin against immunotoxicity induced by diazinon.

Acute intoxication with diazinon (DZN) as a pesticide causes mortality and morbidity annually. This study shows the impact of sub-acute toxicity of DZN 20 mg/kg and the protective activities of chrysin (CH) as a flavone under the flavonoids family (12.5, 25 and 50 mg/kg) were assessed on BALB/c mouse immune system. The changes in morphological and functional properties of the immune system on thymus, spleen and liver histopathology, sub-populations of T lymphocytes, cytokines levels, transcription factors, complement function, phagocytosis, specific and total antibody productions were considered. The histopathological effects of DZN on the spleen and thymus were not significant, but the liver was damaged remarkably. In the presence of CH, the toxic effect of DZN is suppressed. DZN significantly decreased the number of whole blood TCD4+, TCD8+ and NK cells and suppressed the phagocytosis, delayed-type hypersensitivity (DTH) responses to sheep red blood cell (SRBC). Furthermore, it suppressed specific anti-SRBC-Ab, total IgG and IgM production, T-bet expression, and IFN-γ production. In contrast, DZN did not significantly affect complement function and the number of NK cells, TCD4+ and TCD8+ splenocytes. However, it potentiated the expression of GATA-3, ROR-γt and FOXP3 gene expression and consequently produced IL-4, IL-10, IL-17 and TGF-ß in whole blood. CH not only significantly increased the variables mentioned above at 12.5, 25 and 50 mg/kg but also could overcome the toxic effects of DZN on whole blood lymphocyte sub-populations and specific and total Ab production in 25 and 50 mg/kg concentrations, phagocytosis and DTH responses in 50 mg/kg, and modulation of the transcription factors and cytokine production, mainly in 25 and 50 mg/kg. In conclusion, DZN in sub-acute doses could remarkably deteriorate immune responses. However, CH can overcome the toxic effects of DZN on the immune components and functions of the immune system.

TAX and HBZ: hFc Ɣ 1 proteins as targets for passive immunotherapy.

Objectives: Human T leukemia virus type one (HTLV-1) causes two life-threatening diseases in around five percent of infected subjects, a T cell malignancy and a neurodegenerative disease. TAX and HBZ are the main virulence agents implicated in the manifestation of HTLV-1-associated diseases. Therefore, this study aims to produce these HTLV-1 factors as recombinant Fc fusion proteins to study the structures, their immunogenic properties as vaccines, and their capability to produce specific neutralization antibodies. Materials and Methods: TAX and HBZ sequences were chosen from the NCBI-nucleotide database, then designed as human Fc chimers and cloned into Pichia pastoris. Produced proteins were purified by HiTrap affinity chromatography and subcutaneously injected into rabbits. Rabbit Abs were purified by batch chromatography, and their neutralization activities for the HTLV-1-infected MT-2 cell line were assessed. Furthermore, the protective abilities of recombinant proteins were evaluated in Tax or HBZ immunized rabbits by MT-2 cell line inoculation and measurement of HTLV-1-proviral load. Results: Specific Abs against Tax and HBZ can eliminate 2 million MT-2 cells in 1/1000 dilution in vitro. In challenging assays, the immunization of the animals using Tax or HBZ had no protective activity as HTLV-1 PVL was still positive. Conclusion: The result suggests that recombinant TAX and HBZ: hFcγ1 proteins can produce a proper humoral immune response. Therefore, they could be considered a passive immunotherapy source for HTLV-1-associated diseases, while total TAX and HBZ proteins are unsuitable as HTLV-1 vaccine candidates.