Human Gyrovirus Apoptin as a Potential Selective Anticancer Agent: An In Vitro Study

Background: Selective therapy has always been the main challenge in cancer treatments. Recently, it has been shown that Human Gyrovirus-derived protein apoptin (HGV-Apoptin) has selective cytotoxic effects on cancer cells similar to its homologue, Chicken Anemia Virus-derived Apoptin (CAV-Apoptin). However, apoptotic effects of Human Gyrovirus apoptin have been only evaluated on a few cancerous cell lines and need to be further investigated. In this study, we have evaluated the apoptotic effects of HGV-Apoptin and CAV-Apoptin expression on lung cancer (A549) and normal (HEK-293) cell lines, in order to provide more information about the specificity of these proteins on cancerous cells. Methods: Target cells were transfected by the calcium-phosphate precipitation method with constructed plasmids expressing HGV-Apoptin and CAV-Apoptin proteins as well as the control plasmid. Transfection efficiency was followed and imaged by fluorescence microscopy. Quantification of apoptosis was performed by flow cytometry. Measurements were compared by paired Student t-test. Results: Cells were successfully transfected with control and constructed plasmids. Flowcytometry analysis showed that A549 cells transfected with HGV-Apoptin and CAV-Apoptin expressing plasmids, undergone the apoptosis compared to A549 cells transfected with control plasmid (P<0.001). None of the plasmids could induce apoptosis in HEK-293 cells. Conclusion: Human Gyrovirus-derived apoptin (HGV-Apoptin) similar to its homologue, chicken anemia virus derived Apoptin (CAV-Apoptin) can induce apoptosis in Non-small-cell lung carcinoma cell line A549, but not in normal human embryonic kidney cell line HEK-293, which can be introduced as a promising novel specific antitumor agent

Evaluation of Flow Cytometry and Kleihauer Techniques for Quantification of Fetomaternal Hemorrhage: A Prospective Cohort Study in Southwestern Iran

Background: Quantification of fetal red blood cells (RBCs) in maternal blood is of great importance to calculate appropriate dose of post-deliver anti D immunoglobulin in a rhesus D (RhD)-negative woman. Objective: The aim of this study is to evaluate a direct immunofluorescence flow cytometry technique in artificial and clinical samples and compared it to the Kleihauer-Betke test (KBT). Methods: This study was a prospective cohort design. Blood samples from 26 pregnant women who gave birth to RhD positive babies were tested using direct immunofluorescence flow cytometry and KBT techniques to determine the amount of FMH in the maternal circulation. The zone of D-positive cells was identified employing artificial samples including 0.3%, 0.6%, 1%, 1.5%, 2%, 5%, 10%, and 50% of D-positive fetal cells in D-negative maternal cells. Results: Analysis of 26 clinical samples for FMH showed consistent quantification with the flow cytometry and Kleihauer techniques. Although a good correlation was found between the KBT and flow cytometry results, in artificial samples containing more than 2% of fetal RhD positive cells, the flow cytometry results were closer to theoretical percentages. In a patient with FMH >4 mL, the FMH and consequently the required vial of Ig were overestimated using KBT. Conclusion: Most of the FMH calculated could have been neutralized by doses less than 625 IU, whereas the routine dose in Iran is more than double that amount (1500 IU). This achievement demonstrates that adjusting between the RhD immune globulin (RhDIg) dose and FMH size is inevitable

Human herpesvirus-6 viral load and antibody titer in serum samples of patients with multiple sclerosis

BackgroundDespite the number of cases with definite diagnosis of multiple sclerosis (MS) being on increase, the role of human herpesvirus-6 (HHV-6) infection as a trigger for MS disease still is deliberated. Based on antibody detection and quantitative HHV-6 polymerase chain reaction assay, this study was achieved to find out the possible association between infection with HHV-6 and clinical progression of MS disease.MethodsA total of 108 serum samples were obtained from 30 MS patients followed prospectively for a 6-month period. These samples were analyzed for the presence of HHV-6 DNA by nested polymerase chain reaction enzyme-linked immunosorbent assay and for anti-HHV-6 IgG titer. Activation of the disease was determined by either magnetic resonance imaging or by clinical status of the patients. Control groups were also included.ResultsThe average antibody index for the MS patients in the first sample collection was higher than both control groups (p = 0.001). HHV-6 DNA was detected in the serum samples of 10 of 30 MS patients. The mean HHV-6 viral load in patients with relapsing-remitting multiple sclerosis (RRMS) with and without relapse was 973 and 714, respectively. Seven patients showed an exacerbation during the study period. Of those, four patients had HHV-6 DNA in their collected samples. The prevalence of HHV-6 DNA was significantly higher in patients with MS as compared with control groups (p = 0.001).ConclusionsThe results indicate that HHV-6 is implicated somehow in MS disease. Over time, rising HHV-6 IgG antibody titers together with an exacerbation and detection of HHV-6 DNA in serum samples of some MS patients suggests possible association between the reactivation of the virus and disease progression

Expression and purification of TAT-NDRG2 recombinant protein and evaluation of its anti-proliferative effect on LNCaP cell line

N-myc downstream regulated gene2 (NDRG2) belongs to tumor suppressor protein family of NDRG. Anti proliferative and anti-metastasis of NDRG2 overexpression has been demonstrated in a number of tumors. The aim of this study was to fuse the gene of Trans Activator of Transcription (TAT) protein transduction domain with NDRG2 gene and express and purify TAT-NDRG2 fusion protein in order to investigate the effects of TAT-NDRG2 protein on proliferation and apoptosis of LNCaP prostate carcinoma cell line. pET28a-TAT-NDRG2 and pET28a-NDRG2 plasmids were constructed and transformed into E. coli-BL21(DE3). TAT-NDRG2 and NDRG2 proteins were expressed in the bacteria, purified using affinity chromatography and verified using western blotting. The effects of TAT-NDRG2 and NDRG2 protein treatment on LNCaP cells proliferation and apoptosis were evaluated using MIT assay and AnnexinV, 7-AAD flow cytometry assay, respectively. Western blot analysis confirmed the expression and purification of TAT-NDRG2 and NDRG2 proteins. Treatment of LNCaP cells with TAT-NDRG2 protein increased cell death and induced apoptosis significantly (P < 0.05) compared to control and NDRG2 protein-treated cells. These results suggest that TAT-NDRG2 protein can be considered as a therapeutic modality for the treatment of tumors. (c) 2017 Elsevier Inc. All rights reserved

Monitoring wound healing of burn in rat model using human Wharton’s jelly mesenchymal stem cells containing cGFP integrated by lentiviral vectors

Objective(s): Human Wharton’s Jelly mesenchymal stem cells (hWMSCs) are undifferentiated cells commonly used in regenerative medicine. The aim of this study was to develop a reliable tool for tracking hWMSCs when utilized as therapeutics in burnt disorders and also to optimize the cell-based treatment procedure. Materials and Methods: The hWMSCs were first isolated from fresh umbilical cord Wharton’s jelly and cultured. The 293LTV cell line was transfected by cGFP containing lentiviral vector and the helper plasmids for production of the viral particle. The viral particles were collected to transduce the hWMSCs. The transduced cells were finally selected based on resistance to puromycin. The burned rats (n=24) were treated with cGFP expressing hWMSCs using the cell spray method, with the cells being tracked 7, 14 and 21 days later. The rats were sacrificed 7, 14 and 21 days following treatment and paraffin embedded sections prepared from the burned area for downstream pathological analyses. Results: The lentiviral particles carrying the cGFP gene were generated and the hWMSCs were transduced. The cGFP-expressing hWMSCs were detected in the burned tissue and the burned injuries were improved dramatically as compared to control. Conclusion: Because of the establishment of stably transduced cGFP expressing cells and the ability to detect cGFP for a relatively long-time interval, the method was found to be quite efficient for the purpose of cell tracking. The combination of hWMSC-based cell therapy and sterile Gauze Vaseline (GV) as covering was proven much more efficient than the traditional methods based on GV alone

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

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. 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