Fc receptor-like 1 (FCRL1) is a novel biomarker for prognosis and a possible therapeutic target in diffuse large B-cell lymphoma.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma, which can involve various types of mature B-cells. Considering that the incidence of DLBCL has increased, additional research is required to identify novel and effective prognostic and therapeutic molecules. Fc receptor-like 1 (FCRL1) acts as an activation co-receptor of human B-cells. Aberrant expression of this molecule has been reported in a number of B-cell-related disorders. Moreover, the clinical significance and prognosis value of FCRL1 in DLBCL are not completely identified. METHODS: In this study, the expression levels of FCRL1 were determined in thirty patients with DLBCL and 15 healthy controls (HCs). In addition, the correlation between FCRL1 expressions with clinicopathological variables of DLBCL patients were examined. Then, the potential roles of FCRL1 in proliferation, apoptosis, and cell cycle distribution of B-cells from DLBCL patients were determined using flow cytometry analysis, after knockdown of this marker using retroviral short hairpin RNA interference. Quantitative real time-PCR, western blotting, and enzyme-linked immunosorbent assay were also used to identify the possible effects of FCRL1 knockdown on the expression levels of BCL-2, BID, BAX, intracellular signaling pathway PI3K/p-Akt, and p65 nuclear factor-kappa B (NF-κB) in the B-cells of DLBCL. RESULTS: Statistical analysis revealed higher levels of FCRL1 expression in the B-cells of DLBCL patients compared to HCs at both protein and mRNA levels. A positive correlation was observed between the FCRL1 expression and some clinicopathological parameters of DLBCL patients. In addition, FCRL1 knockdown significantly decreased cell proliferation and stimulated apoptosis as well as G1 cell cycle arrest in the B-cells of DLBCL patients. The levels of p65 NF-κB and PI3K/p-Akt expressions were markedly reduced after knockdown of FCRL1 expression. CONCLUSIONS: These results suggested that FCRL1 could be a potential novel biomarker for prognosis and/or a possible effective therapeutic target for treatment of patients with DLBCL.

Epstein-Barr virus infection is associated with the nuclear factor-kappa B p65 signaling pathway in renal cell carcinoma.

BACKGROUND: There have been few studies regarding viral involvement in the pathogenesis of renal cell carcinoma (RCC). The aim of this study was to examine the possible association of Epstein-Barr virus (EBV) infection with clinicopathological features and cellular biomarkers including p53, p16INK4a, Ki-67 and nuclear factor-kappa B (NF-κB) in RCC tumors. METHODS: In this prospective study, 122 histologically confirmed Formalin-fixed Paraffin-embedded RCC tissue specimens along with 96 specimens of their corresponding peritumoral tissues and 23 samples of blunt renal injuries were subjected to nested polymerase chain reaction (nPCR) in order to amplify EBV DNA sequences. The expression of p53, p16INK4a, Ki-67 and NF-κB was investigated by immunohistochemistry (IHC) assay. Statistical analysis was employed to demonstrate the possible associations. RESULTS: Infection with EBV was found to be significantly associated with RCC. Our results indicate that p65 NF-κB signaling pathway is probably involved in EBV-mediated RCC pathogenesis. Moreover, we found p53, Ki-67 and cytoplasmic NF-κB expression to be associated with tumor nuclear grade in RCC patients. The expression of p53 and Ki-67 was associated with primary tumor category as well. In addition, p53 overexpression was significantly more frequent among nonconventional RCC tumors than the conventional histologic type. CONCLUSIONS: Infection with EBV is likely to play an important role in the development of RCC through the constitutive and permanent activation of NF-κB p65 signaling pathway. However, more experiments and supporting data are required to reach a decisive conclusion.

Phosflow assessment of PDGFRA phosphorylation state: A guide for tyrosine kinase inhibitor targeted therapy in hypereosinophilia patients.

Clonal eosinophilia is a hematologic disorder caused by translocation in growth factor receptor (GFR) genes. Despite the identified molecular mechanisms underlying clonal hypereosinophilia, the distinction between clonal and reactive eosinophilia has remained challenging due to the diversity of partner genes for translocated GFRs. This study aimed to examine the feasibility of phosphoflow cytometry in the diagnosis of clonal hypereosinophilia through evaluating the level of platelet-derived growth factor receptor alpha (PDGFRA) phosphorylation and its correlation with PDGFRA genetic aberration. Blood samples were collected from 45 hypereosinophilia patients and 10 healthy controls. Using phosphoflow cytometry method, the phosphorylation state of PDGFRA was assessed. The specificity of phosflow results was confirmed by western blotting and eventually compared with qRT-PCR expression analysis of 3'-region of PDGFRA. To detect the genetic aberration of PDGFRA, 5'-rapid amplification of cDNA ends (5'-RACE) was performed. Phosflow analysis illustrated that 9 of 45 hypereosinophilic patients had higher level of PDGFRA phosphorylation while sequence analysis of 5'-RACE-PCR fragments confirmed that in seven cases of them, there was a PDGFRA-FIP1L1 fusion. We also verified that two of nine patients with hyperposphorylated PDGFRA hold ETV6-PDGFRA and STRN-PDGFRA rearrangements. Importantly, nine cases also had significantly higher levels of PDGFRA mRNA expression when compared with healthy controls, and cases with no PDGFRA rearrangement. These findings highlight a robust correlation between hyperphosphorylation state of PDGFRA and aberrant PDGFRA gene fusions. This implicates phosflow as an efficient and reliable technique raising an intriguing possibility that it could replace other genomic and cDNA-amplification-based diagnostic approaches with limited effectiveness.

Skewed intracellular cytokine production of iNKT cells toward Th2-related responses in alloimmunized thalassemia patients.

INTRODUCTION: Red blood cell alloimmunization is a challenging issue in thalassemia patients. Several studies have investigated the role of different immune system compartment in alloimmunization, but the exact mechanism remains unclear. Considering the immunoregulatory function of iNKT cells and their subsets, in this study, we evaluated the possible role of these cells in alloimmunization status of thalassemia patients. METHODS: 78 ß-thalassemia major patients (41 alloimmunized and 37 non-alloimmunized) and 17 healthy controls were engaged in this study. Mononuclear cells were isolated from peripheral blood samples and stimulated for cytokine production. Samples were subjected to flow cytometry for enumeration of iNKT cells and characterized based on their cytokine production pattern. Finally, the results correlated with alloimmunization status, clinical and laboratory data. RESULTS: Results demonstrated that the number of iNKT, iNKT+IFN-ɤ+, and iNKT+IL-4+ cells in thalassemia group was significantly higher than healthy controls while no significant change was observed in the number of these cells between alloimmunized and non-alloimmunized thalassemia patients. Interestingly, the ratio of iNKT+IL-4+: iNKT+IFN-γ+ cells in alloimmunized thalassemia group represent a considerable increase in comparison to both non-alloimmunized thalassemia group and healthy controls. However, evaluating this value in non-alloimmunized group represents an approximately equal ratio of 0.94, which was almost similar to this ratio in the control group (0.99). CONCLUSION: Our results illustrated a noteworthy imbalance in the ratio of iNKT cell subsets in favour of IL-4 producing iNKT cells in alloimmunized thalassemia patients. Regarding the role of IL-4 in stimulating the Th2-related immune responses, this imbalance could consider as a possible mechanism in alloantibody responses of thalassemia patients.

Expression of the immune checkpoint receptors CTLA-4, LAG-3, and TIM-3 in ß-thalassemia major patients: correlation with alloantibody production and regulatory T cells (Tregs) phenotype.

Alloimmunization is a serious complication in ß-thalassemia major patients as a result of repeated blood transfusion. The immune checkpoint receptors play an important role in regulating immune system homeostasis and the function of the immune cells. This study aimed to evaluate the expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene 3 (LAG-3), and T-cell immunoglobulin and mucin domain-containing protein-3 (TIM-3) immune checkpoint molecules in ß-thalassemia major patients with and without alloantibody. For this purpose, 68 ß-thalassemia major patients with (34 patients) and without (34 patients) alloantibody as well as 20 healthy controls were enrolled. The expression of these genes was evaluated in different groups of patients by SYBR Green real-time PCR method. Our results showed that the mean expression of LAG-3 was significantly increased in thalassemia patients compared to the control group (*P < 0.001). However, there was no significant difference in expression of the CTLA-4 and TIM-3 as well as LAG-3 genes between patients with and without alloantibody (P > 0.05). A positive correlation was observed between the level of LAG-3 expression with markers associated with Treg function including FOXP3 and GDF-15 genes in ß-thalassemia major patients. Taken together, the LAG-3 molecule might have a more prominent role in the abnormality of the immune system in thalassemia patients especially the function of regulatory T cells (Tregs), prior to the CTLA-4 and TIM-3 genes.

Evaluation of regulatory T cells frequency and FoxP3/GDF-15 gene expression in ß-thalassemia major patients with and without alloantibody; correlation with serum ferritin and folate levels.

ß-thalassemia major is one of the most common hematologic disorders in the world. It causes severe anemia and patients require regular blood transfusions, which causes different complications such as iron overload and alloimmunization. Regulatory T cells (Tregs) have an important role in regulation of immune responses. FoxP3 is the major marker of Tregs and its expression can be influenced by different factors. GDF-15 is another gene that plays a role in iron homeostasis and regulation of immune system in different diseases. The aim of this study was to assess the frequency of Tregs and FoxP3/GDF-15 gene expression in ß-thalassemia major patients with and without alloantibody as well as its correlation with different factors such as serum ferritin and folate levels. This study was conducted on 68 ß-thalassemia major patients with and without alloantibodies in comparison with 20 healthy individuals with matched age and sex as control group. Enzyme-linked immunosorbent assay (ELISA), flow cytometry, and real-time PCR were performed in order to evaluate serum ferritin and folate levels, frequency of Tregs, and the expression of FoxP3 and GDF-15 genes, respectively. The percentage and absolute count of Tregs were increased in patients compared with controls (P = 0.0003), but there was no difference between responders and non-responders (P > 0.05). The Tregs count correlated positively with serum ferritin. No correlation was observed between target genes and serum ferritin and folate, but there was a positive significant correlation between the expression of FoxP3 and GDF-15 genes, which shows the immunosuppressive role of GDF-15.

In vitro cytotoxic effect of Trastuzumab in combination with Pertuzumab in breast cancer cells is improved by interleukin-2 activated NK cells.

Targeting erb-b2 receptor tyrosine kinase 2 (ERBB2) using the combination of Trastuzumab and Pertuzumab has demonstrated promising results in breast cancer therapy. It has further been revealed that interleukin-2 (IL-2) can activate Natural Killer cells (NK cells) and elevate their cytotoxic potency against tumor cells. In this study, we explored the cytotoxic effect of recombinant human IL-2 in combination with Trastuzumab and Pertuzumab on the ERBB2 positive (SK-BR-3) and negative (MDA-MB-231) breast cancer cell lines. The cytotoxicity level of IL-2 activated NK cells (approximately 75%) were significantly higher than untreated cells (approximately 55%) in the presence of Trastuzumab and Pertuzumab against SK-BR-3 cells, while no difference was observed in the case of MDA-MB-231 cells (about 15%).

Engineering, expression and purification of a chimeric fibrin-specific streptokinase.

Streptokinase is a valuable fibrinolytic agent used to cope with myocardial infarction and brain stroke. Despite its high efficiency in dissolving blood clots, streptokinase (SK) has no specificity in binding fibrin, causing some problems such as internal bleedings following its administration. To make streptokinase fibrin specific and limit the fibrinolytic process to the clot location, we engineered a chimeric streptokinase by fusing the fibrin binding Kringle 2 domain of tissue plasminogen activator (TPA) to the streptokinase N-terminal end. The chimeric SK construct (KSK) with inserted Kringle 2 domain was cloned into pET28a expression vector. The expression of recombinant protein was carried out in Escherichia coli origami (DE3) and confirmed by SDS-PAGE and Western blotting analyses. We used the chromogenic substrate S-2251 method to assess the specific activities of the chimeric and control wild-type proteins. Then, the two proteins were added in amounts with equal activity to fibrin clots of identical size. Finally, the supernatant above the fibrin clots was collected and subjected to the chromogenic assay to analyze the specificity of the chimeric protein. The specific activities of the chimeric and wild-type proteins were found to be 0.06 U/mg and 0.07 U/mg, respectively. Because of the binding of the chimeric protein to fibrin, the mean specific activity was significantly lower in the KSK supernatant (0.01) compared with the control (approximately 0.06) (p < 0.05). Our in vitro results indicate that the chimeric streptokinase protein has strong fibrin-specific activity compared to the wild-type protein. However, further in vivo studies are needed to evaluate its potential fibrinolytic effects.

High-risk human papillomavirus infection in different histological subtypes of renal cell carcinoma.

Limited data exist regarding whether a high-risk human papillomavirus (HR-HPV) infection increases the risk of developing renal cell carcinoma. The aim of this study was to investigate whether HPV infection has a role in the pathogenesis or development of a certain histological subtype of renal cell carcinoma. Formalin-fixed paraffin-embedded (FFPE) specimens of 122 patients with histopathologically proven renal cell carcinoma and their respective peritumoral tissues were examined. The presence of HPV-DNA was determined by a combination of MY/GP+ consensus primers and HPV-16/18 type specific nested PCRs followed by direct sequencing. Catalyzed signal-amplified colorimetric in situ hybridization (CSAC-ISH) technique was applied to determine the physical status of viral genome. The expression of p16INK4a and HPV L1 capsid proteins was evaluated using immunohistochemistry. HPV genome was detected in 37 (30.3%) tumor specimens and their four (4.1%) corresponding peritumoral tissues. HPV-18 was the most common viral type identified followed by HPV-16 and 58. Immunoexpression of p16INK4a was detected in 24 (20.3%) cases. Data analysis showed a significant correlation between p16INK4a expression and the presence of HR-HPV DNA (P < 0.001). CSAC-ISH analysis confirmed HR-HPV infection in 45% of tumors, which were previously tested positive for HPV-DNA. Diffuse signal pattern was identified in 15 (83.3%) samples whereas a mixed pattern of diffuse and punctate signals was only detectable in three cases. The results indicate an association of HR-HPV types with renal cell carcinoma. It is proposed that HPV infection in high-grade tumors might precede disease progression in a number of tumors, particularly of the papillary subtype.

Evaluation of red cell membrane cytoskeletal disorders using a flow cytometric method in South iran.

OBJECTIVE: The diagnosis of hereditary red blood cell (RBC) membrane disorders, and in particular hereditary spherocytosis (HS) and Southeast Asian ovalocytosis (SAO), is based on clinical history, RBC morphology, and other conventional tests such as osmotic fragility. However, there are some milder cases of these disorders that are difficult to diagnose. The application of eosin-5'-maleimide (EMA) was evaluated for screening of RBC membrane defects along with some other anemias. We used EMA dye, which binds mostly to band 3 protein and to a lesser extent some other membrane proteins, for screening of some membrane defects such as HS. MATERIALS AND METHODS: Fresh RBCs from hematologically normal controls and patients with HS, SAO, hereditary elliptocytosis, hereditary spherocytosis with pincered cells, severe iron deficiency, thalassemia minor, and autoimmune hemolytic anemia were stained with EMA dye and analyzed for mean fluorescent intensity (MFI) using a flow cytometer. RESULTS: RBCs from patients with HS and iron deficiency showed a significant reduction in MFI compared to those from normal controls (p<0.0001 and p<0.001, respectively), while macrocytic RBCs showed a significant increase in MFI (p<0.01). A significant correlation was shown between mean corpuscular volume and MFI, with the exceptions of HS and thalassemia minor. CONCLUSION: Our results showed that the flow cytometric method could be a reliable diagnostic method for screening and confirmation, with higher sensitivity and specificity (95% and 93%, respectively) than conventional routine tests for HS patients prior to further specific membrane protein molecular tests.

Intra-fractional dose rate effect in continuous and interrupted irradiation of the MCF-7 cell line: Possible radiobiological implications for breath-hold techniques in breast radiotherapy?

PURPOSE: To investigate the effects of different dose rates (DRs) in continuous and interrupted irradiation on in-vitro survival of the MCF-7 cell line, towards finding possible radiobiological effects of breath-hold techniques in breast radiotherapy (RT), in which intra-fractional beam interruptions and delivery prolongation can occur. MATERIALS AND METHODS: MCF-7 cells were irradiated continuously or with regular interruptions using 6 MV x-rays at different accelerator DRs (50-400 cGy/min) to deliver a 2 Gy dose. The interrupted irradiation was delivered in a 10 s on, 10 s off manner. Then, cell survival and viability were studied using colony and MTT assays, respectively. RESULTS: Survival and viability with continuous and interrupted irradiation were similar (P > 0.5). A significant increase in survival at 50, 100, and 400 cGy/min compared to 200 and 300 cGy/min was observed, also a significant decreasing and then increasing trend from 50 to 200 cGy/min and 200 to 400 cGy/min, respectively (P < 0.04). Relative to 200 cGy/min, the survival fractions at 50, 100, 300, and 400 cGy/min were 1.24, 1.23, 1.05, and 1.20 times greater, respectively. Cell viability did not show significant differences between the DRs, despite following the same trend as cell survival. CONCLUSION: Our results suggest that for continuous irradiation of in-vitro MCF-7 cells, with increasing DR within the 50-400 cGy/min range, sensitivity increases and then decreases (inverse effect), also that up to doubling of treatment time in breath-hold techniques does not affect in-vitro radiobiological efficacy with 200-400 cGy/min accelerator DRs. Further confirmatory studies are required.

Analysis of ß/α globin ratio by using relative qRT-PCR for diagnosis of beta-thalassemia carriers.

BACKGROUND: Current routine tests for premarital screening of ß-thalassemia carriers are not applicable for diagnosis of rare atypical minor ß-thalassemia cases. A more specialized laboratory evaluation for them is the measurement of ß/α chain synthesis ratio with the assistance of radioactive amino acids. This method is also no longer routinely accessible. Consequently it is required to establish a rapid, trouble-free, and reliable method that encompasses all the cases of ß-thalassemia carriers. Therefore we have determined ß/α-globin mRNA ratio by applying relative qRT-PCR in various ß-thalassemia patients. METHODS: Reticulocytes RNA extraction and subsequent cDNA synthesis were performed, followed by relative qRT-PCR for α- and ß-globin chain genes and ß-actin gene as an endogenous reference. ß/α-Globin gene ratio was then evaluated with the Pfaffl method. RESULTS: The mean of ß/α ratio was 0.99, 0.81, 0.69, and 0.69 for normal population, minor, intermediate, and major ß-thalassemia, respectively. Approximately 6% of cases with minor thalassemia RBC index and normal HbA2 and having a decreased ß/α ratio were located in the minor ß-thalassemia group. The mean of ß/α mRNA ratio in normal individuals and minor ß-thalassemia was significantly different with all other groups (P-value < 0.05). Nevertheless, there was no such association between ß/α mRNA ratio in major and intermediate ß-thalassemia. CONCLUSION: According to the significant differences achieved, no overlapping between minor ß-thalassemia and normal group, capability of diagnosing atypical minor ß-thalassemia, and accessibility of this technique, we can declare that this method could be suggested as a routine premarital screening test for ß-thalassemia carriers.

Resveratrol plus low-dose hydroxyurea compared to high-dose hydroxyurea alone is more effective in γ-globin gene expression and ROS reduction in K562 cells.

Hydroxyurea (HU) is an anti-cancer drug that is used for the treatment of hemoglobinopathies as a γ-globin inducer. However, its dose-dependent effects have hampered its clinical reliability. Resveratrol (RSV) is an antioxidant and γ-globin inducer. The present study aimed to assess their combined effects on the γ-globin gene expression and reactive oxygen species (ROS) level of K562 cells. The results indicated that the γ-globin gene expression was approximately two folds higher in the group treated with RSV 50 µM + HU 25 µM in comparison to HU 100 µM alone (***p < 0.001). However, there was an inverse relationship between the expression of γ-globin gene and HU concentration in the combined groups. Furthermore, the combinations of RSV and HU significantly reduced ROS levels compared to single drugs. Overall, the combination of these compounds was an appropriate strategy for increasing γ-globin expression, reducing oxidant levels, and alleviating the adverse effects of HU.

The mRNA Expression of PTEN, LEF1, JAK3, LC3 and p62/SQSTM1 Genes in Patients with Chronic Myeloid Leukemia.

INTRODUCTION: Chronic myeloid leukemia (CML) is a progressive myeloproliferative disorder resulting from forming a chimeric BCR-ABL gene. The proteins derived from this gene can affect some genes from various signaling pathways such as PI3K/AKT/Wnt/catenin/JAK/Stat involved in proliferation, differentiation, cell death, and genes related to autophagy. Imatinib is the first-line treatment for CML patients, with durable and proper responses in Iranian children and adult CML patients. Hence, we aimed to evaluate the mRNA expression of some selected key genes from those pathways in patients with CML before and under treatment. METHODS: In the case-control study, the mRNA expression of PTEN, LEF1, JAK3, LC3 and p62 genes were measured in 51 CML patients (6 patients before treatment and 45 patients under treatment with imatinib mesylate) and 40 healthy controls using the Real-time PCR method. RESULTS: The mRNA expression of PTEN and P62 were significantly higher in newly diagnosed patients than in controls (P<0.0001 and P = 0.0183, respectively), while the expression of the LC3 gene was significantly lower in the untreated newly diagnosed group than in control subjects (P = 0.0191). The expression level of PTEN, LEF1, JAK3 and P62 genes were significantly decreased in patients under treatment than in the group before treatment (P = 0.0172, P = 0.0002, P = 0.0047 and P = 0.0038, respectively). A positive correlation was seen between the gene expression of P62 and BCR-ABL in the patients under treatment (r 0529, P = 0.016). CONCLUSION: Our findings showed that the changes in expression of these genes were related to the patient's treatment. Due to the key role of these genes in proliferation, differentiation and tumor suppression, it is proposed that these genes may be helpful for follow-up of treatment in CML patients.

Philadelphia Chromosome Positive Chronic Myelogenous Leukemia Blastic Crisis in A Patient with Unusual Primary Myelofibrosis Characteristics; A Case Report.

INTRODUCTION: Myeloproliferative neoplasms (MPNs) are divided into BCR-ABL positive Chronic myeloid leukemia (CML) and BCR-ABL negative MPNs including Polycythemia vera (PV), Essential Thrombocythemia (ET) and Primary myelofibrosis (PMF). Evaluation of the Philadelphia chromosome in MPNs is a diagnostic requirement for classic CML. CASE REPORT: In 2020, a 37-year-old woman with negative cytogenetic testing for Janus kinase2 (JAK2), Calreticulin (CALR), myeloproliferative leukemia virus oncogene (MPL), and positive for BCR-ABL1 mutation with reticular fibrosis in bone marrow was diagnosed as CML. Some years ago, the patient had been diagnosed with PMF with evidence of histiocytic necrotizing lymphadenitis or Kikuchi-Fujimoto disease (KFD). The BCR-ABL fusion gene was initially evaluated which was negative. Then, Cutaneous squamous cell carcinoma (cSCC) was confirmed by Dermatopathologist with palpable splenomegaly and high white blood cell (WBC) count with basophilia. Finally, BCR-ABL was detected positive by the fluorescence in situ hybridization (FISH) and quantitative real-time polymerase chain reaction (qRT-PCR). In fact, the co-occurrence of PMF with CML was identified. CONCLUSION: This case study highlighted the importance of some cytogenetic methods in the detection and classification of MPNs. It is recommended that physicians pay more attention to it and be aware of the planning treatment.

Evaluation of the relationship between γ-H2AX biomarker levels and dose received after radiation exposure in abdominal-pelvic and chest CT scans.

Background: As one of the most informative diagnostic radiation instruments, computed tomography (CT) has seen considerable improvement since its implementation in the 1970s; however, the possibility of low-dose radiation risk after CT procedures is still challenging and little is known about the biological effects of CT exposure on patients. As a result, this research aimed to look at the biological and cytogenetic effects of low-dose abdominal-pelvic and chest CT scans on adults, focusing on the number of γ-H2AX foci formation. Materials and Methods: Blood tests were taken before and 10 min after CT exams on patients aged 25-55 who were undergoing abdominal-pelvic and chest CT exams with very low-ionizing radiation exposure (TLD doses of 15.67-63.45 mGy). Blood lymphocytes that had been isolated, fixed, and stained were dyed with γ-H2AX antibodies. Finally, the percentage of phosphorylation of histone H2AX as an indicator of double-strand breaks was determined using a cytometry technique. Results: Our findings showed that after CT examination, the mean value of γ-H2AX foci in patients increased (P < 0.0001). A statistically significant correlation between dose radiation and the number of γ-H2AX foci was also found (P = 0.047, r = 0.4731). The current study also found a pattern of elevated γ-H2AX foci in patients over 40 years of age relative to younger patients. Conclusion: A Significant activation of γ-H2AX foci was found in lymphocytes of peripheral blood samples of patients after CT compared to before CT scan. This increase in γ-H2AX foci levels in blood cells may be a useful quantitative biomarker of low-level radiation exposure in humans.

Expression of the Hepatitis C Virus core-NS3 Fusion Protein on the Surface of Bacterial Ghosts: Prospects for Vaccine Production.

Background: Antigen presentation using bacterial surface display systems, on one hand, has the benefits of bacterial carriers, including low-cost production and ease of manipulation. On the other hand, the bacteria can help in stimulating the immune system as an adjuvant. For example, using bacterial surface display technology, we developed a hepatitis C virus (HCV) multiple antigens displaying bacteria's surface and then turned it into a bacterial ghost. Methods: The HCV core and NS3 proteins' conserved epitopes were cloned into the AIDA gene plasmid as an auto transporter. The recombinant plasmid was then transformed into Escherichia coli (E. coli) Bl21 (DE3). Recombinant bacteria were then turned into a bacterial ghost, an empty cell envelope. Whole-cell ELISA, flow cytometry, and Western blot techniques were used for monitoring the expression of proteins on the surface of bacteria. Results: A fusion protein of HCV core-NS3-AIDA was successfully expressed on the E. coli Bl21 (DE3) surface and confirmed by western blotting, Enzyme-Linked Immunosorbent Assay (ELISA), and flow cytometry detection techniques. Conclusion: The presence of HCV antigens on non-pathogen bacteria surfaces holds promise for developing safe and cost-benefit-accessible vaccines with optimal intrinsic adjuvant effects and exposure of heterologous antigens to the immune system.

Efficacy of Mesenchymal Stem Cells Therapy in Parasitic Infections: Are Anti-parasitic Drugs Combined with MSCs More Effective?

PURPOSE: Mesenchymal stem cells (MSCs) are mesodermal-origin postnatal stem cells that are able to self-renew and differentiate into several cell lineages. MSCs possess anti-inflammatory and anti-apoptotic activity, immunomodulatory action, as well as regenerative properties. Since MSCs also have antimicrobial properties, it has been suggested that they should be utilized for treating infectious diseases. In this study, the last pre-clinical advances in the efficacy of MSCs' therapy against parasitic diseases were reviewed. METHODS: Data about the effects of MSCs' therapy on experimental and pre-clinical parasitic infections were collected by searching relevant articles and reviewing them. RESULTS: In the present study, empirical findings on the impacts of MSCs' therapy against parasitic diseases were recapitulated. Studies have reported that the administration of MSCs reduces the burden of the parasite and modulates the levels of inflammatory and anti-inflammatory cytokines in parasitic diseases, including schistosomiasis, malaria, cystic echinococcosis, toxocariasis, leishmaniasis, and trypanosomiasis. Also, the administration of MSCs combined with anti-parasitic drugs enhanced anti-parasitic effects and immunomodulatory actions. CONCLUSION: Based on this review, empirical studies have revealed the beneficial effects of MSCs against some parasitic infections. This new therapeutic strategy showed both anti-parasitic and immunomodulatory effects. Also, the combination of anti-parasitic drugs with MSCs' therapy promoted anti-parasitic and immunomodulatory activities against parasitic infections.

The Pathogenic Aspects of Human Parvovirus B19 NS1 Protein in Chronic and Inflammatory Diseases.

Background: The nonstructural protein (NS1) of human parvovirus B19 (hPVB19) is considered to be a double-edged sword in its pathogenesis. NS1 protein promotes cell death by apoptosis in erythroid-lineage cells and is also implicated in triggering and the progression of various inflammation and autoimmune disorders. Objectives: We investigated the possible role of hPVB19 NS1 in the modulation of proinflammatory cytokines in nonpermissive HEK-293T cells. Methods: A plasmid containing the fully sequenced NS1 gene (pCMV6-AC-GFP-NS1) was transfected into HEK-293T cells. Transfection efficiency was assessed by fluorescent microscopy over time. Mock (pCMV6-AC-GFP) transfected cells were used as controls. The percentage of apoptotic cells was measured by flow cytometry at 24, 48, and 72 h posttransfection. Interleukin 6 (IL-6) mRNA, as a pleiotropic cytokine, was measured by real-time PCR. Furthermore, cellular supernatants were collected to determine the type and quantity of cytokines produced by mock- and NS1-transfected cells using flow cytometry. Results: Fold change in the expression level of IL-6 mRNA in transfected cells after 72 hr of incubation was found to be 3.01 when compared with mock-transfected cells; however, cell apoptosis did not happen over time. Also, the concentration of cytokines such as IL-2, IL-6, IL-9, IL-17A, IL-21, IL-22, interferon (IFN)-γ, and tumor necrosis factor α (TNF-α) increased in NS1-transfected cells. Conclusions: Overall, our results indicated that proinflammatory cytokine levels had increased following the expression of hPVB19 NS1 in HEK-293T cells, consistent with a role for NS1 expression facilitating the upregulation of inflammatory reactions. Therefore, hPVB19 NS1 function may play a role in the progression of some chronic and inflammatory diseases.

The Evaluation of tLyP-1-Bound Mda-7/IL-24 Killing Activity on a Liver Tumor Cell Line.

Background: The melanoma differentiation-associated gene-7 (Mda-7)/interleukin-24 (IL-24) is a tumor killing cytokine, the bystander effect of which can be enhanced through tethering to tumor homing peptides (THPs). Materials and Methods: After fusing tLyP-1, RGR, and buforin as THPs to Mda-7/IL-24, enzyme-linked immunosorbent assay (ELISA) was used to determine the secretion potency of the recombinant proteins. The killing potency of plasmids expressing IL-24, IL-24.tLyP1, IL-24.RGR, and buf.IL-24 were assessed, using MTT, Annexin/PI staining assays, as well as measuring the expression level of GADD-153 and BCL2-associated X (BAX) on Huh-7 cells. Three-dimensional structural analysis and protein-receptor interaction were also evaluated by modeling. Results: The ELISA result showed that contrary to IL-24.RGR and buf.IL-24, IL-24.tLyP-1 retained the secretion potency, similar to the native form. The viability assessments showed that IL-24 and IL-24.tLyP-1 had the most growth suppressive effects in comparison with the control group (p < 0.0001). Furthermore, IL-24 and IL-24.tLyP-1 had the highest apoptotic activity and significant upregulatory effect on the GADD-153 and BAX genes (p < 0.0003). The modeling showed that peptide modifications left no detrimental effect on IL-24 attachment to the cognate receptor. Conclusion: IL-24 can tolerate tLyP-1 peptide modification by retaining its secretion potency. Tethering tLyP-1 to IL-24 can induce more apoptosis than its modified versions by RGR or buforin.