Comparative proteomics analysis in different stages of urothelial bladder cancer for identification of potential biomarkers: highlighted role for antioxidant activity.

BACKGROUND: Non-muscle-invasive bladder cancer (NMIBC) has a high recurrence rate and muscle-invasive bladder cancer (MIBC) has unfavorable outcomes in urothelial bladder cancer (UBC) patients. Complex UBC-related protein biomarkers for outcome prediction may provide a more efficient management approach with an improved clinical outcome. The aim of this study is to recognize tumor-associated proteins, which are differentially expressed in different stages of UBC patients compared non-cancerous tissues. METHODS: The proteome of tissue samples of 42 UBC patients (NMIBC n = 25 and MIBC n = 17) was subjected to two-dimensional electrophoresis (2-DE) combined with Liquid chromatography-mass spectrometry (LC-MS) system to identify differentially expressed proteins. The intensity of protein spots was quantified and compared with Prodigy SameSpots software. Functional, pathway, and interaction analyses of identified proteins were performed using geneontology (GO), PANTHER, Reactome, Gene MANIA, and STRING databases. RESULTS: Twelve proteins identified by LC-MS showed differential expression (over 1.5-fold, p < 0.05) by LC-MS, including 9 up-regulated in NMIBC and 3 up-regulated in MIBC patients. Proteins involved in the detoxification of reactive oxygen species and cellular responses to oxidative stress showed the most significant changes in UBC patients. Additionally, the most potential functions related to these detected proteins were associated with peroxidase, oxidoreductase, and antioxidant activity. CONCLUSION: We identified several alterations in protein expression involved in canonical pathways which were correlated with the clinical outcomes suggested might be useful as promising biomarkers for early detection, monitoring, and prognosis of UBC.

Association of killer-cell immunoglobulin-like receptor genes with acute myelogenous leukaemia.

Killer-cell immunoglobulin-like receptors (KIRs) are important because of their key roles in NK cell development and function. Some KIR genes have been associated with the incidence of haematological malignancies. This study was designed to determine whether the inheritance of specific KIR genes is associated with susceptibility to acute myelogenous leukaemia (AML) in Persians living in south-western Iran. KIR genes and KIR2DS4 variants were typed by polymerase chain reaction-sequence-specific primer (PCR-SSP) in 167 patients with AML and 169 healthy controls. Our results showed 10% of patients-mostly females-were classified as M3. Flt3 mutations were detected in 26% of patients, most of whom had internal tandem duplication (ITD). The frequency of activating KIRs (aKIRs)-mainly KIR3DS1-was higher in patients, whereas inhibitory KIRs (iKIRs)-particularly KIR3DL1 and KIR2DL1-were more common among controls. The incidence of the KIR2DS4fl allele was higher among patients with non-M3 AML than controls. We also found a higher frequency of 4 or more iKIR genes in the controls and a higher frequency of 4 or more aKIR genes in the patients. Individuals with more iKIR than aKIR belonged predominantly to the control group. Individuals with the telomeric AA genotype who had inherited the KIR2DS4fl allele were more frequent in the patient group. According to our results, increased frequency of aKIRs in patients with AML may lead to the hyperactivation of NK cells against malignant cells with reduced or lack of HLA class I molecules followed by NK cell exhaustion which allow malignant cells to progress.

Protective Effect of Edaravone Against Cyclosporine-Induced Chronic Nephropathy Through Antioxidant and Nitric Oxide Modulating Pathways in Rats.

BACKGROUND: Cyclosporine A (CsA) is an immunosuppressant with therapeutic indications in various immunological diseases; however, its use is associated with chronic nephropathy. Oxidative stress has a crucial role in CsA-induced nephrotoxicity. The present study evaluates the protective effect of edaravone on CsA-induced chronic nephropathy and investigates its antioxidant and nitric oxide modulating property. METHODS: Male Sprague-Dawley rats (n=66) were distributed into nine groups, including a control (group 1) (n=7). Eight groups received CsA (15 mg/kg) for 28 days while being treated. The groups were categorized as: Group 2: Vehicle (n=10)Groups 3, 4, and 5: Edaravone (1, 5, and 10 mg/kg) (n=7 each)Group 6: Diphenyliodonium chloride, a specific endothelial nitric oxide synthase (eNOS) inhibitor (n=7)Group 7: Aminoguanidine, a specific inducible nitric oxide synthase (iNOS) inhibitor (n=7)Group 8: Edaravone (10 mg/kg) plus diphenyliodonium chloride (n=7)Group 9: Edaravone (10 mg/kg) plus aminoguanidine (n=7) Blood urea nitrogen and serum creatinine levels, malondialdehyde, superoxide dismutase, and glutathione reductase enzyme activities were measured using standard kits. Renal histopathological evaluations and measurements of eNOS and iNOS gene expressions by RT-PCR were also performed. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's test (SPSS software version 18.0). RESULTS: Edaravone (10 mg/kg) significantly attenuated CsA-induced oxidative stress, renal dysfunction, and kidney tissue injury. Aminoguanidine improved the renoprotective effect of edaravone. Edaravone reduced the elevated mRNA level of iNOS, but could not alter the level of eNOS mRNA significantly. CONCLUSION: Edaravone protects against CsA-induced chronic nephropathy using antioxidant property and probably through inhibiting iNOS gene expression.

Effects of omega-3 fatty acids on serum levels of T-helper cytokines in children with asthma.

BACKGROUND: There has been a considerable interest in the potential therapeutic value of dietary supplementation with ω-3 fatty acids in patients with asthma. OBJECTIVES: This cross-sectional study was designed to identify the effect of ω-3 fatty acids on symptom score, pulmonary function and serum T-helper cytokine concentrations in children with mild to moderate persistent asthma. METHODS: A total of 39 patients among 50 volunteers completed this 3-month study. They took a soft gel capsule containing 180mg EPA and 120mg DHA daily. Pulmonary function was evaluated in 28 eligible patients by spirometry, and serum levels of Th1, Th2, Th9, Th17 and Th22 cytokines were measured by multiplex cytometric bead assay before and after treatment. RESULTS: After treatment with ω-3, symptom score improved in 28 (72%) patients. The results of spirometry showed remarkable improvement in FEV1/FVC (P=0.044) and PEF (P<0.0001) after treatment, but considering a cut-off of 80%, real improvement was observed in 32% of patients with PEF<80% which raised above the cut-off after ω-3 treatment (P=0.004) whereas, FEV1/FVC changes were above the cut-off value in 89% of the patients. After treatment, IL-17A and TNF-α levels decreased significantly (both P=0.049). CONCLUSION: Oral administration of natural anti-inflammatory products such as ω-3 is a promising complementary approach to managing asthma.

Increased efficiency in geographic ancestry assignment and human identification by combining lineage profiles: The case of the Iranians.

OBJECTIVES: This research is a first empirical attempt to quantify the increase of the among-groups variance and the probative value of a DNA evidence when combining profiles based on markers with uniparental inheritance. METHODS: Yfiler and HVS-I panels of loci were analyzed in 130 healthy unrelated males from six Iranian native groups. RESULTS: A separate analysis of DNA profiles at the two lineage markers failed to detect a population substructure, whereas maximum levels of genetic diversity (HD = 1) and discrimination capacity (DC = 1) were obtained by combining the two profiles. CONCLUSIONS: When combined, the forensic efficiency of routinely used panels of lineage markers can be largely sufficient to resolve cases of geographic ancestry and human identification even in genetically homogeneous populations.

Attitudes of medical students and staff toward organ donation in cases of brain death: a survey at Shiraz University of Medical Sciences.

CONTEXT: Organ transplant is one of the most important management strategies for end-of-life patients. The demand for organs in patients awaiting transplant is increasing, and many of these patients die before a donor is found. OBJECTIVE: To determine the attitudes of medical students and staff at clinical institutions affiliated with a large medical university in the Eastern Mediterranean region toward organ donation in cases of brain death. PARTICIPANTS: A total of 500 medical students, physicians, and nurses recruited at hospitals and medical centers affiliated with Shiraz University of Medical Sciences in Shiraz, Iran.Design and Setting-Information about participants' demographic characteristics, knowledge of organ donation, and willingness to donate their own organs after death was collected by using self-administered questionnaires. RESULTS: Most participants (78%) had favorable attitudes toward donating their own organs after brain death. However, only about 25% of them carried an organ donation card. In addition to public media, the main sources of information about organ donation after brain death were their professors and textbooks. An association in charge of improving public awareness and facilitating the process of registration and issuance of donation cards appears to be necessary.

The Royan Public Umbilical Cord Blood Bank: Does It Cover All Ethnic Groups in Iran Based on HLA Diversity?

BACKGROUND: Umbilical cord blood (UCB) stem cells allow the transplantation of partially human leukocyte antigen (HLA)-matched grafts and are a valuable resource for the treatment of hematologic malignancies and heritable hematologic, immunologic and metabolic diseases, especially when a compatible bone marrow donor is unavailable. The aim of this study was to determine how many ethnic groups in Iran are covered by the available UCB units based on HLA diversity. METHODS: From 2009 until mid-2013, 4,981 (30.3%) of the 16,437 UCB samples collected met the storage criteria and were cryopreserved at a public cord blood bank (CBB) in Tehran, Iran. HLA-A, -B and -DRB1 were typed in 1,793 samples. RESULTS: The mean volume of the cryopreserved samples was 81.25 ± 20.3 ml. The range of total nucleated cells per unit was 51 × 10(7)-107 × 10(7). The most common HLA alleles were HLA-A*2 (17%) and HLA-A*24 (15.6%), HLA-B*35 (16.8%) and HLA-B*51 (13.9%), and HLA-DRB1*11 (20%) and HLA-DRB1*15 (14%). The predominant haplotypes were HLA-A*24-B*35-DRB1*11 (2%), HLA-A*02-B*50-DR*07 (1.8%), and HLA-A*02-B*51-DRB1*11 (1.5%). CONCLUSIONS: Based on the HLA-DRB1 profiles, the UCB units available at the Royan public UCB bank are a potentially adequate resource for hematopoietic stem cell transplantation for Iranian recipients belonging to particular ethnic groups. Regular educational programs to improve the public knowledge of UCB for transplantation can enhance the public CBB stocks for all Iranian ethnic groups in the future.

Activating KIR/HLA-I combinations as a risk factor of adult B-ALL.

B-cell acute lymphoblastic leukemia (B-ALL), the most predominant type of ALL, is less common and incurable among adults. Regarding the pivotal role of NK cells in immune surveillance against hematological malignancies, studying the effective factors in regulating their function, particularly KIRs as the most important NK cell receptors and HLA-I molecules as their main ligands, is of importance. Since NK responses against malignant lymphoblasts are influenced by KIR signals, we did a case-control study on 154 adult patients with B-ALL and 181 healthy controls to investigate the correlation of KIR/HLA-I combinations with susceptibility to B-ALL in Iranians. The genotyping of KIR genes and HLA-I alleles was performed by PCR-SSP with 11 and 9 primer pairs, respectively. Our data revealed an increased frequency of activating (a)KIRs and aKIR/HLA-I combinations in our patients: KIR3DS1 (p = 0.009, OR = 1.81), Bx genotype (p = 0.038, OR = 1.81), KIR3DS1(+)/HLA-Bw4Thr80(+) (p = 0.004, OR = 3.61), and KIR3DS1(+)/HLA-B Bw4(+) (p = 0.037, OR = 1.76). The presence of inhibitory (i)KIRs in the absence of their cognate HLA-I ligands was also more frequent among the patients. However, the frequency of inhibitory combinations was more common in controls: KIR2DL1(+)/HLA-C2(+) (p = 0.027, OR = 0.57), KIR2DL2/3(+)/HLA-C1(+) (p = 0.004, OR = 0.5), and KIR3DL2(+)/HLA-A3/A11(+) (p = 0.0012, OR = 0.46). To sum up, the less inherited iKIR/HLA-I combinations might make individuals more susceptible to B-ALL because of inefficient education of NK cells.

Sequential radiation exposure: uncovering the potential of low dose ionizing radiation in mitigating high dose effects on immune cells.

PURPOSE: The radioadaptive response refers to a phenomenon wherein exposure to a low dose of ionizing radiation (LDIR) can induce a protective response in cells or organisms, reducing the adverse effects of a subsequent higher dose of ionizing radiation (HDIR). However, it is possible to administer the low dose after the challenge dose. This study was conducted to determine the potential mitigating effect of LDIR administered after HDIR on mice immune cells. MATERIALS AND METHODS: Alongside the conventional adaptive response setting, one group of mice was initially exposed to HDIR and subsequently treated with LDIR. Neutrophil activation was done using DHR-reducing assay and cell proliferation was evaluated through CFSE-dilution assay in helper (CD4+) and cytotoxic (CD8+) T cells. Cytokine production by these T cell subsets was also assessed by intracellular staining using flow cytometry. RESULTS: The results of this study revealed no change in neutrophil function between any of the mice groups compared to the untreated control group. Although significant changes were not detected in the proliferation of CD4+ T cells, decreased proliferation was observed in stimulated CD8+ T cells in the HDIR group. In contrast to IFN-É£, which showed no evident change in either of the T cell subsets after stimulation, IL-4 was rigorously dropped in stimulated CD4+ T cells in the HDIR group. CONCLUSIONS: In summary, the results of this study indicated that the administration of LDIR to mice before HDIR was not able to reduce the detrimental effects of HDIR in our experimental setting. Instead, we observed a mitigating effect of LDIR when administered after the challenge dose. This suggests that not only the dose and duration but also the order of LDIR relative to HDIR affects its efficacy.

Association between serotonin transporter gene polymorphisms and childhood asthma.

BACKGROUND: Asthma is a common chronic inflammatory disease of the airways in which genetic factors play a major role in its pathogenesis. High serotonin serum levels in patients with asthma suggest that serotonin is involved in the pathophysiology of the disease. Serotonin clearance is mediated by the serotonin reuptake transporter, and functional polymorphisms in this gene lead to altered serotonin reuptake efficiency. OBJECTIVE: The aim of this study was to investigate the relationship between serotonin transporter gene polymorphisms and asthma. METHODS: Serotonin transporter gene polymorphisms (5-HTTLPR, rs35521 and STin2.VNTR) were assessed by PCR-based methods in 100 children with mild to moderate persistent asthma and compared with 100 healthy controls. RESULTS: There were no significant differences in allele, genotype or haplotype frequencies between patients and controls. No association was observed between SERT gene polymorphisms after stratification of patients for sex, age, spirometry indices, family history, passive smoking behavior and concomitant allergic rhinitis. Significant differences were observed in the distribution of 5-HTTLPR alleles (p = 0.025) and genotypes (p = 0.021) between patients with and without atopic dermatitis. CONCLUSIONS: Despite strong evidence suggesting the role of serotonin in the pathophysiology of asthma, we found no association between serotonin transporter gene polymorphisms and mild to moderate persistent asthma. Further serotonin transporter gene analyses in patients with severe asthma may open up new horizons in the utilization of common serotonin regulators to treat asthma, based on their pharmacogenetic effects. However, serotonin may also be indirectly influenced by emotional stress during asthma attacks.

Association of killer cell immunoglobulin-like receptors and their cognate HLA class I ligands with susceptibility to acute myeloid leukemia in Iranian patients.

Acute myeloid leukemia (AML) is one of the most prevalent leukemia in adults. Among the various NK receptors, killer immunoglobulin-like receptors (KIRs) carry out indispensable roles in NK cell development and function through engaging with class I human leukocyte antigens (HLA-I) as their ligands. Besides divergent KIR and HLA loci, KIR/HLA-I combinations have a significant effect on NK cell response. In this case-control study, we aimed to verify the association of KIR/HLA-I combinations with susceptibility to AML in the Southwestern Iranian population. KIR and HLA genotyping was performed with PCR-SSP by some novel primers for 181 patients with AML and 181 healthy controls. According to our results, the frequencies of KIR3DS1 (p = 0.0001, OR = 2.32, 95% CI 1.51-3.58), KIR2DS4fl (p = 0.02, OR = 1.53, 95% CI 1.05-2.21), CxT4 genotypes (p = 0.03, OR = 2.0, 95% CI 1.05-3.82), and T4 gene cluster (p = 0.01, OR = 1.99, 95% CI 1.17-3.41) were significantly higher in patients than controls, while C1/C2 genotype (p = 0.00002, OR = 0.39, 95% CI 0.25-0.61), HLA-A Bw4 (p = 0.02, OR = 0.6, 95% CI 0.38-0.94), and HLA-A*11 (p = 0.03, OR = 0.57, 95% CI 0.34-0.95) alleles were more frequent in controls. In addition, inhibitory (i)KIR/HLA-I combinations analysis revealed higher frequencies of KIR2DL1( +)/HLA-C2( +), KIR2DL2/3( +)/HLA-C1( +), KIR3DL1( +)/HLA-A Bw4( +), and KIR3DL2( +)/HLA-A*03/11( +) in the control group (p = 0.002, OR = 0.49, 95% CI 0.3-0.78; p = 0.04, OR = 0.62, 95% CI 0.39-0.99; p = 0.04, OR = 0.63, 95% CI 0.4-0.99; and p = 0.03, OR = 0.62, 95% CI 0.4-0.95, respectively). Overall, the number of iKIR/HLA-I combinations was more in the control group. Moreover, KIR3DS1( +)/HLA-B Bw4Ile80( +) and the sum of HLA-B Bw4/A Bw4 combined with KIR3DS1 as activating KIR/HLA-I combinations were more frequent among patients than controls (p = 0.01, OR = 1.99, 95% CI 1.14-3.49 and p = 0.005, OR = 1.97, 95% CI 1.22-3.19, respectively). In conclusion, our results postulate that inhibitory combinations play a protective role against AML by developing potent NK cells during education. It is noteworthy that KIR/HLA-I combination studies can be applicable in donor selection for allogeneic NK cell therapy in hematological malignancies.

Awareness of Obesity-Related Cancers: A Complex Issue.

Cancer rates are on the rise across the world, making the illness a public health crisis, particularly in developed countries where cancer has become a leading cause of death [...].

Cholesterol: An important actor on the cancer immune scene.

Based on the structural and signaling roles of cholesterol, which are necessary for immune cell activity, high concentrations of cholesterol and its metabolites not only trigger malignant cell activities but also impede immune responses against cancer cells. To proliferate and evade immune responses, tumor cells overcome environmental restrictions by changing their metabolic and signaling pathways. Overexpression of mevalonate pathway enzymes and low-density lipoprotein receptor cause elevated cholesterol synthesis and uptake, respectively. Accordingly, cholesterol can be considered as both a cause and an effect of cancer. Variations in the effects of blood cholesterol levels on the outcome of different types of cancer may depend on the stage of cancer. However, positive effects of cholesterol-lowering drugs have been reported in the treatment of patients with some malignancies.

Allergenome profiling of Vespa orientalis venom by serum IgE in patients with anaphylactic reaction to this hornet sting.

BACKGROUND: Hymenoptera stings are one of the most common causes of anaphylaxis. Vespa orientalis (red hornet) is a common and very aggressive hymenopteran endemic in central and southern areas of Iran. Allergy testing and venom immunotherapies are carried out with venom components which are expensive, have limited commercial availability, and often lack specificity. Although proteomic analysis of hymenopteran venom has been shown to be a powerful technique to identify allergens, data on the protein components of V. orientalis venom are lacking. AIM: This study was designed to characterize the allergenome profile (proteome of allergenic proteins) of this local hornet venom. METHODS: Venom was extracted from V. orientalis worker venom sacs. The venom constituents were separated by two-dimensional gel electrophoresis (2DE). Protein components were blotted and probed with serum from 10 allergic patients by immunoblotting. Reactive spots were isolated and characterized by liquid chromatography with tandem mass spectrometry. RESULTS: A total of 195 protein spots were detected on the 2DE gels. Fifteen distinct venom proteins showed reactivity to IgE in patients' sera. Four major allergens in order of allergenicity in patients were identified as hyaluronidase, arginine kinase, phospholipase A1 (PLA1) and PLA1 magnifin. CONCLUSIONS: Broadening our knowledge of V. orientalis venom constituents can contribute to improvements in diagnostic and immunotherapeutic techniques, both of which are dependent on the major allergens in venom extract. This information is also potentially helpful to develop medical uses of major allergens in this venom to improve the diagnostic specificity and the efficacy of immunotherapy.

Immunomodulatory Effects of Omega-3 Fatty Acids in Patients with Differentiated Thyroid Cancer Before or After Radioiodine Ablation.

BACKGROUND: Thyroid cancer and radioactive iodine (RAI) ablation for postsurgical management may lead to uncontrolled inflammation. OBJECTIVE: This study was intended to assess the prophylactic and therapeutic immunomodulatory effects of omega-3 fatty acids in patients with differentiated thyroid cancer (DTC). METHODS: A total of 85 patients with DTC were allocated into two groups based on RAI dosage after thyroidectomy. Patients in each group were randomly distributed into three subgroups: G1 with RAI ablation only, G2 treated with omega-3 for 30 days before RAI ablation, and G3 treated with omega-3 for 30 days after RAI ablation. Fifteen healthy individuals were included as controls. Serum cytokine levels including IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, TNF-α and IFN-γ were determined by cytometric bead assay. RESULTS: IL-4, IL-6, IL-21 and IL-22 levels in patients with DTC were higher than in the healthy controls. Regardless of RAI dosage, IL-6 showed an increasing trend after RAI ablation. IL-4, IL-22, and IL-17A remained at considerably higher levels than in the healthy controls after RAI ablation. Within-group comparisons showed a significant reduction in Th1+Th17/Th2+Th22 ratio in G2 patients 1 week after RAI ablation. Between-group comparisons showed increased IL-10 levels in G3 compared with G1 patients one week after high-dose RAI ablation. In G3, Th1+Th17/Th2+Th22 and Th1+Th17/Th2+Th9+Th22 ratios were remarkably lesser than in G2 patients 1 month after intermediate-dose RAI ablation. CONCLUSION: Our results showed better anti-inflammatory effects of omega-3 when it was used therapeutically after RAI ablation in patients with DTC than when it was used prophylactically before RAI.

Identification of the Most Common Allergens of Acer velutinum Pollen.

Pollens have been identified as potent inducers of allergic diseases worldwide. Acer velutinum (Persian maple) tree is an important source of allergic pollens in Iran. This study aimed to identify the immunoglobulin E (IgE)-reactive components of A. velutinum pollen extract in patients with maple allergy. We aimed to evaluate its allergenic components; using IgE in the serum of patients with maple allergy. Twenty-two patients with a clinical history of reaction and a positive skin-prick test to maple pollen extract were included in this study. Identification of IgE-binding proteins in A. velutinum pollen extract was performed by immunoblotting using sera from sensitive patients. A protein band with a molecular weight of around 70 kDa was the most IgE-reactive allergen in A. velutinum pollen extract detected by this method. Identification of a protein with a molecular weight of about 70kDa, as the most reactive allergen of A. velutinum pollen extract, can be considered as a potential allergen for designing diagnostic kits or as a target for immunotherapy of allergic patients with maple allergy.

Evaluation of T Cell Proliferation Using CFSE Dilution Assay: A Comparison between Stimulation with PHA and Anti-CD3/Anti-CD28 Coated Beads.

A decrease in T cell count or reduced T cell function can be indicative of T cell immunodeficiency. In the present study, T-cell function was assessed using Carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution test after stimulation with commonly used Phytohaemagglutinin (PHA) or anti-CD3/anti-CD28 coated beads in pediatric patients with recurrent infections. Seven infants with recurrent infections and seven sex/age-matched healthy infants were included in this study. A blood cell count, immunophenotyping, and serum immunoglobulin level were performed. The proliferation of T cells was also assessed with CFSE dilution after stimulation with PHA or anti-CD3/anti-CD28 coated beads.  This study showed increased IgA, IgG, and IgM levels in patients compared to the controls. In contrast to the controls, the immunophenotyping results showed a significant decline in the number of CD4+ T cells in patients. Although there was no difference in CD3+ T cell proliferation between patients and controls, the CD4+ and CD8+ T cell proliferation rates were significantly decreased in patients when stimulated with PHA. As a mitogen with the potential for maximum proliferation of T cells, PHA is better able to distinguish between patients with recurrent infections and controls than anti-CD3/anti-CD28, which mimics only the TCR pathway for stimulation of T cells.

Eugenol: A New Option in Combination Therapy with Sorafenib for the Treatment of Undifferentiated Thyroid Cancer.

Thyroid cancer (TC) is the most common endocrine malignancy. Thyroidectomy and radiotherapy are common treatment modalities for patients with undifferentiated TC (UTC), and sorafenib is usually recommended to prevent a recurrence. However, malignant cells may evade chemotherapy-induced apoptosis, and combination therapy was developed to achieve better outcomes. This study investigated whether eugenol in combination with sorafenib was more effective than either substance individually in triggering apoptosis in the UTC. The IC50 of sorafenib and eugenol was determined in a UTC cell line (8305C) by MTT assay, and their synergistic effect in combination therapy was investigated. Flow cytometry was used to evaluate the rate of apoptosis in treated cells. To confirm that cell death occurred through apoptosis, immunoblotting was used to determine the relative cleavage of caspase-8 and caspase-9. The IC50 of sorafenib was 20 µM, and that of eugenol was 2100 µM. The sorafenib-eugenol combination (1:105) showed synergistic effects at concentrations equal to or less than their IC50. The rate of apoptosis induction was higher in cells treated with eugenol or the eugenol-sorafenib combination compared to sorafenib-treated cells. The relative intensity of cleaved/un cleaved forms of caspase-8 increased in eugenol-treated cells compared to sorafenib-treated cells.Sorafenib and eugenol at concentrations equal to or less than their IC50 had a synergistic effect in 8305C cells. The most potent apoptotic effect was achieved with sorafenib and eugenol at their IC50. Lower doses of sorafenib could be used with eugenol to improve its efficacy while reducing its side effects.

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.