Evaluation of Th17 and Treg cytokines in patients with unexplained recurrent pregnancy loss.

Background and Aim: The Th17/Treg balance in peripheral blood and reproductive tissues may have a role in the etiology of unexplained recurrent pregnancy loss (URPL). In this study, we evaluated the major cytokine of Treg cells transforming growth factor-beta (TGF-ß), and their specific transcription factor Forkhead box P3 (FOXP3), as well as the most highlighted cytokine of Th17 cells (interleukin [IL]-17A) in both URPL patients and healthy women. Methods: Samples were extracted from the peripheral blood, endocervix, endometrium, and vagina of 14 patients with URPL and 12 normal non-pregnant women as a control (normal) group. Quantitative reverse transcription polymerase chain reaction was used to determine gene expression. Enzyme-linked immunosorbent assay was used to determine the levels of cytokines in the serum and cervicovaginal fluid. Results: We found that in the URPL group, FOXP3 gene expression was considerably higher in peripheral blood than in the normal group (P=0.043). TGF-ß levels in the cervicovaginal fluid were different in the URPL and normal groups (P=0.015). In comparison to the control group, women with URPL had significantly greater IL-17 gene expression in the peripheral blood (P=0.01). Conclusion: Lower TGF-ß levels in the cervicovaginal fluid of patients compared to controls may be related with increased IL-17 and FOXP3 mRNA levels in patients with URPL. Dysregulation of local immune responses in reproductive tissues may represent dysregulation of systemic regulatory immunological responses in the pathogenesis of URPL. Relevance for Patients: Dysregulation of immune responses may play a role in the pathogenesis of URPL at least in some patients with URPL. We conclude that the breakdown of tolerance in the local immune responses is more critical than the breakdown of tolerance in systemic tolerance in the pathogenesis of URPL. Therefore, modulating immune responses in the endometrium and decidua may be the focus of future therapeutic approaches in URPL. The impact of seminal plasma on the expansion of Tregs may provide a novel therapeutic intervention that has already been used in assisted reproductive technologies. Therefore, we suggest that transvaginal TGF-ß in women with URPL may induce maternal tolerance which leads to the successful pregnancy.

Association Between IL-17A, FOXP3, and CTLA4 Genes Expression and Severity of Lupus Nephritis

INTRODUCTION: Elevated levels of interleukin 17A (IL-17A) have been found in systemic lupus erythematosus (SLE). Forkhead box protein P3 (FOXP3) activates T-regulation lymphocytes and is a master regulator cell function. The cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene plays a similar role. We investigated the role of these expressions in SLE patients with/without nephritis. METHODS: The present study was a case-controlled study including 49 patients with SLE and 26 healthy controls. The genes expression of IL-17A, FOXP3, and CTLA4 were measured by quantitative Real-Time PCR. The relation between lupus nephritis and disease activity with IL-17A, FOXP3, and CTLA4 genes expression was evaluated. RESULTS: IL-17A, FOXP3, and CTLA4 expressions in T-cells were significantly higher in SLE patients than controls (P < .0001). When comparing the nephritis group and no nephritis group to the control group individually, the expression of mentioned genes is also higher (P < .05). There was no significant difference regarding IL-17A, FOXP3, and CTLA4 genes expression in the nephritis group and no nephritis group (P > .05). But there was a low expression of FOXP3 and IL-17A in patients with the higher stage of nephritis (P < .05). CONCLUSION: Our findings elevated IL-17A, FOXP3, and CTLA4 expressions significantly contribute to SLE pathophysiology. This study provides new insight into the function of IL-17A, FOXP3, and CTLA4 in disease setting. The heterogeneity of SLE patients is reflected in the multiple abnormalities found in the immune system. Finding such variations can provide targets for better manipulation of the immune system. DOI Code: DOI: 10.52547/ijkd.6537

Upregulation of Cytotoxic T-Lymphocyte-Associated Protein 4 and Forkhead Box P3 Transcripts in Peripheral Blood of Patients with Bladder Cancer.

Background: Regulatory T cells (Tregs) play a key role in the progression of tumors. These cells express forkhead box P3 (FOXP3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4), which are the potential targets for cancer immunotherapy. The present study aimed to evaluate FOXP3 and CTLA4 transcripts in patients with bladder cancer (BC) compared with healthy individuals. Methods: Transcripts of CTLA4 and FOXP3 genes in the peripheral blood mononuclear cells (PBMCs) of 50 patients with histologically confirmed BC and 50 healthy individuals were assessed at the Institute for Cancer Research, Shiraz University of Medical Sciences (Shiraz, Iran) during 2014-2016. RNA was extracted from PBMCs, then cDNA was synthesized and subjected to quantitative real-time PCR (qRT-PCR) using appropriate primers. Statistical analysis was performed using SPSS software (version 21.0). Results: Significantly higher amounts of CTLA4 and FOXP3 gene transcripts were found in the peripheral blood of BC patients compared with healthy individuals. The expression of both genes was significantly higher in patients with non-invasive and grade I/II BC. The median of CTLA4 and FOXP3 transcript expressions was 3.74 and 5.39, respectively, in non-invasive BC patients, which was significant compared with the control group (P=0.0016 and P=0.009, respectively). The median of target gene mRNA expression in grade I/II BC patients was 2.9 for CTLA4 and 6.61 for FOXP3, which was significant compared with the controls (P=0.013 and P=0.0037, respectively). Conclusion: This study highlights the functional activity of Tregs in early stages of bladder cancer and showed the importance of CTLA4 and FOXP3, when it comes to screening BC.

Expression Pattern of Telomerase Reverse Transcriptase (hTERT) Variants and Bcl-2 in Peripheral Lymphocytes of Systemic Lupus Erythematosus Patients.

BACKGROUND & OBJECTIVE: It is not clear whether activated lymphocytes of patients with systemic lupus erythematosus (SLE) are more proliferative or less apoptotic. We aimed to delineate potential differences between B and T cells of SLE patients compared to healthy controls regarding the telomerase activity and apoptosis status. METHODS: In this cross-sectional case control study, Blood samples were taken from 10 SLE patients and 10 healthy controls. B and T cells were separated using magnetic cell sorting system. Telomeric repeat amplification protocol (TRAP) assay and real-time PCR were used to determine the telomerase activity and the expression of alternatively spliced variants. RESULTS: Four patients under treatment showed significant telomerase activity in their T cells. Four of the newly diagnosed patients showed telomerase activity in their B cells (20% of all patients and 40% of new onset patients). There was no specific pattern of human telomerase reverse transcriptase variant expression within the patients' lymphocytes. A significantly reduced expression of Bcl-2 was detected in B cells (P=0.018) and a trend toward lower Bcl-2 expression in T cells was seen in SLE patients compared to healthy controls. CONCLUSION: Although not definitive, our results may suggest that B cells may have more active roles during the earlier phases of the disease attack, while T cells take over when the disease reaches its chronic stages.

Helios, CD73 and CD39 Induction in Regulatory T Cells Exposed to Adipose Derived Mesenchymal Stem Cells.

OBJECTIVE: Mesenchymal stem cells (MSCs) have prominent immunomodulatory roles in the tumor microenvironment. The current study intended to elucidate Treg subsets and their cytokines after exposing naïve T lymphocytes to adiposederived MSCs (ASCs). MATERIALS AND METHODS: In this experimental study, to obtain ASCs, breast adipose tissues of a breast cancer patient and a normal individual were used. Magnetic cell sorting (MACS) was employed for purifying naïve CD4+ T cells from peripheral blood of five healthy donors. Naïve CD4+ T cells were then co-cultured with ASCs for five days. The phenotype of regulatory T cells (Tregs) and production of interleukine-10 (IL-10), transforming growth factor beta (TGF-ß) and IL-17 were assessed using flow cytometry and ELISPOT assays, respectively. RESULT: CD4+CD25-FOXP3+CD45RA+ Tregs were expanded in the presence of cancer ASCs but CD4+CD25+Foxp3+CD45RA+ regulatory T cells were up-regulated in the presence of both cancer- and normal-ASCs. This up-regulation was statistically significant in breast cancer-ASCs compared to the cells cultured without ASCs (P=0.002). CD4+CD25+ FOXP3+Helios+, CD4+CD25- FOXP3+Helios+ and CD25+ FOXP3+CD73+CD39+ Tregs were expanded after co-culturing of T cells with both cancer-ASCs and normal-ASCs, while they were statistically significant only in the presence of cancer-ASCs (P<0.05). Production of IL-10, IL-17 and TGF-ß by T cells was increased in the presence of either normal- or cancer-ASCs; however, significant effect was only observed in the IL-10 and TGF-ß of cancer-ASCs (P<0.05). CONCLUSION: The results further confirm the immunosuppressive impacts of ASCs on T lymphocytes and direct them to specific regulatory phenotypes which may support immune evasion and tumor growth.

Tumour suppressive effects of WEE1 gene silencing in neuroblastomas.

AIM OF STUDY: WEE1, a member of serine/threonine protein kinase family is the master inhibitor of cyclin-dependent kinase 1 in cell cycle. Over-expression of WEE1 in glioblastomas (GBMs) and some other cancers has been shown. Here, we investigated the expression of WEE1 in 13 brain samples from GBM patients and two GBM cell lines. Further to that, we asked whether if knocking down WEE1 expression in the cell lines change tumor cells' reaction. MATERIALS AND METHODS: All brain tumor samples were collected after confirmed pathological diagnosis. Western blotting was used to screen the expression of WEE1 and a panel of tumor markers. As a model of WEE1 gene silencing with small hairpin RNA (shRNA) technology in GBMs, A172, and U373GM cell lines were transfected with four WEE1 specific shRNAs. The growth characteristics of the cells and the expression of a panel of downstream genes were investigated after gene suppression. RESULTS: All GBMs and both cell lines over-expressed WEE1. Transduction of the cell lines with different shRNAs suppressed WEE1 expression with different extent and pooling of four shRNAs together resulted in additive effect. Suppression of WEE1 not only repressed cellular growth but also changed the profile of gene expression of the cells. Quantitative real-time polymerase chain reaction showed also reduced expression of genes such as hypoxia-inducible factor-1, B-cell lymphoma-2, vascular endothelial growth factor, and p53 with crucial roles in tumor survival and invasiveness. CONCLUSION: These results highlight the key role of WEE1 suppression to combat GBMs. Moreover, it showed beneficial possibilities of WEE1 suppression with different anticancer approaches for neurological malignancies.

The effect of amniotic membrane extract on umbilical cord blood mesenchymal stem cell expansion: is there any need to save the amniotic membrane besides the umbilical cord blood?

OBJECTIVES: Umbilical cord blood is a good source of the mesenchymal stem cells that can be banked, expanded and used in regenerative medicine. The objective of this study was to test whether amniotic membrane extract, as a rich source of growth factors such as basic-fibroblast growth factor, can promote the proliferation potential of the umbilical cord mesenchymal stem cells. MATERIALS AND METHODS: The study design was interventional. Umbilical cord mesenchymal stem cells were isolated from voluntary healthy infants from hospitals in Shiraz, Iran, cultured in the presence of basic-fibroblast growth factor and amniotic membrane extracts (from pooled - samples), and compared with control cultures. Proliferation assay was performed and duplication number and time were calculated. The expression of stem cell's specific markers and the differentiation capacity toward osteogenic and adipogenic lineages were evaluated. RESULTS: Amniotic membrane extract led to a significant increase in the proliferation rate and duplication number and a decrease in the duplication time without any change in the cell morphology. Both amniotic membrane extract and basic-fibroblast growth factor altered the expressing of CD44 and CD105 in cell population. Treating basic-fibroblast growth factor but not the amniotic membrane extract favored the differentiation potential of the stem cells toward osteogenic lineage. CONCLUSION: The amniotic membrane extract administration accelerated cell proliferation and modified the CD marker characteristics which may be due to the induction of differentiation toward a specific lineage. Amniotic membrane extract may enhance the proliferation rate and duplication number of the stem cell through changing the duplication time.

No Definite Association between Human Parvovirus B19 Infection and Behçet Disease.

BACKGROUND: The etiology of the Behçet disease (BD) has remained obscured. There have been studies to show the association of BD to infections like herpes simplex, hepatitis, and parvovirus B19 however, the findings are rather controversial. MATERIALS AND METHODS: We selected 55 patients with the best matched symptoms of BD and measured the loads of B19 DNA in their plasma by quantitative real time PCR and verified their seropositivity by ELISA. All findings were compared to the results from 42 healthy persons. RESULTS: Patients showed a wide spectrum of BD symptoms. Serologic studies showed high prevalence of B19 IgG among the tested patients which was not statistically different with the healthy population (72.7% vs. 85.7%, respectively). Similarly, the prevalence of B19 IgM between patients and controls was not different (18% vs. 11.9%, respectively). No correlation was found between the presence of anti-B19 antibodies and the clinical observations. Only one person from the patient and control groups had detectable levels of B19 DNA without any difference or correlation with the disease symptoms. CONCLUSION: Our data could not establish an association between B19 parvovirus infection and Behçet disease, although there have been reports of such correlation. Nevertheless, there might be indirect relation in genetically susceptible individuals after viral infections. More studies on designed animal models and surveys on patients should be done to resolve this controversy.

Patients with Active Rheumatoid Arthritis Have Lower Frequency of nTregs in Peripheral Blood.

BACKGROUND: Patients with rheumatoid arthritis (RA) suffer from wide ranges of autoimmune reactions in joints. The mechanism of which is generally unknown and maybe associated with Treg deregulation. OBJECTIVE: To compare the frequency of nTregs in peripheral blood of patients with active rheumatoid disease with healthy individuals. METHODS: Twenty five newly diagnosed patients with active RA disease were selected based on the clinical and laboratory criteria before starting their therapies. Treg cells in peripheral blood samples were enumerated by immune staining and flowcytometry analysis. RESULTS: Clinical and laboratory results were in favor of active disease in all the studied patients although they showed variations in Disease Activity Score-28 (DAS-28). Compared to the healthy controls, RA patients had significantly lower frequency of CD4+CD25hi or CD4+CD25+FoxP3+ natural regulatory T cells. In spite of that, there were no significant differences between patients and healthy controls in respect to the CD4/CD8 ratio. Interestingly, more CD4+CD25-FoxP3+ cells were found in peripheral blood of patients. The frequencies of the Tregs did not show strong associations with the DAS-28. CONCLUSION: We showed lower abundance of nTregs in peripheral blood of RA patients which highlights the significance of these cells in RA.

Absence of Correlation between Changes in the Number of Endothelial Progenitor Cell Subsets.

BACKGROUND AND OBJECTIVES: Previously, various methodologies were used to enumerate the endothelial progenitor cells (EPCs). We now know that these methodologies enumerate at least three different EPC subsets: circulating angiogenic cells (CACs), colony-forming unit endothelial cells (CFU-ECs), and endothelial colony-forming cells (ECFCs). It is not clear whether there is a correlation between changes in the number of these subsets. The aim of the current study is to find an answer to this question. MATERIALS AND METHODS: The number of all EPC subsets was quantified in the peripheral blood of nine pregnant women in their first and third trimesters of pregnancy. We enumerated 14 cell populations by quantitative flow-cytometry using various combinations of the markers, CD34, CD133, CD309, and CD45, to cover most of the reported phenotypes of CACs and ECFCs. Culturing technique was used to enumerate the CFU-ECs. Changes in the number of cells were calculated by subtracting the number of cells in the first trimester peripheral blood from the number of cells in the third trimester peripheral blood, and correlations between these changes were analyzed. RESULTS: The number of CFU-ECs did not correlate with the number of ECFCs and CACs. Also, CACs and ECFCs showed independent behaviors. However, the number of CACs showed a strong correlation with the number of CD133(+)CD309(+) cells (p=0.001) and a moderate correlation with the number of CD34(+)CD309(+) cells (p=0.042). Also, the number of ECFCs was correlated with the number of CD309(+)CD45(-) cells (p=0.029) and CD34(+)CD45(-) cells (p=0.03). CONCLUSION: Our study showed that the three commonly used methods for quantifying EPC subsets represent different cells with independent behaviors. Also, any study that measured the number of EPCs using the flow cytometry method with a marker combination that lacks CD309 may be inaccurate.

Expression Pattern of Alternative Splicing Variants of Human Telomerase Reverse Transcriptase (hTERT) in Cancer Cell Lines Was not Associated with the Origin of the Cells.

Telomerase and systems controlling their activity have been of great attention. There are controversies regarding the role of the alternative splicing forms of the human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase. Therefore, the correlation between telomerase enzyme activity, the abundance of alternatively spliced variants of hTERT and doubling time of a series of cancer cell lines originated from hematopoietic, breast, colorectal, neural, ovarian, lung, kidney, bladder, prostate and head and neck cancers were investigated. Expression levels of four different variants of hTERT (the full length, α-deletion, ß-deletion and α/ß-deletion) were quantitatively measured by real time PCR. Telomerase activity was determined by the telomerase repeat amplification protocol (TRAP) while doubling time of the cells measured by plotting growth curves. Results showed high diversity in the relative proportions of hTERT transcripts while the majority of the cells expressed the full length variant as the main transcript. Telomerase activity could not be detected in all cells. Relative assessment of hTERT expression showed greater expression of the α-deleted variant in the telomerase negative cells (P= 0.04). Those cells possessed the α/ß-deleted variant to a smaller extent when compared to the cells with telomerase activity. Greater association between full length spliced variant and ß-variant expression was observed in cells presenting telomerase activity (P= 0.0007, r= 0.74). High degrees of variation among the studied cells regarding the pattern of hTERT expression were present. In spite that, the regulatory roles of hTERT on telomerase activity is still a potential to be utilized as targets for cancer therapies.

Changes in endothelial progenitor cell subsets in normal pregnancy compared with preeclampsia.

BACKGROUND: The results of studies measuring the number of endothelial progenitor cells (EPCs) in normal pregnancies and in preeclampsia have been highly controversial or even contradictory because of cross-sectional designs and different methodologies enumerating three distinct subsets of EPCs: circulating angiogenic cells (CAC), colony-forming unit endothelial cell (CFU-ECs), and endothelial colony forming cells (ECFCs). To provide a clear explanation for these underlying controversies, we designed a prospective study to compare the number of all EPC subsets between three trimesters of normal gestation and a case-control study to compare these values as preeclampsia occurs with those from gestational age (GA) matched normal pregnancy. METHODS: Samples from peripheral blood of nine women were taken during their three consecutive trimesters of normal pregnancy, and from eight women with preeclampsia. To cover most of the reported phenotypes for CACs and ECFCs in the literature, we enumerated 13 cell populations by quantitative flow cytometry using various combinations of the markers CD34, CD133, CD309, and CD45. We used routine culturing techniques to enumerate CFU-ECs. RESULTS: The numbers of CACs and ECFCs were higher in women with preeclampsia (p = 0.014). By contrast, preeclampsia was associated with a reduced number of CFU-ECs (p = 0.039). The CAC number rose with the increase in GA (p = 0.016) during normal pregnancy, while the number of CFU-ECs and ECFCs did not differ during the trimesters. CONCLUSION: Although we did demonstrate an increase in absolute counts of CACs and ECFCs in preeclampsia, fewer colony formation capacities indicated a loss in their functional capabilities. By contrast, the number of CACs increased without alterations in colony formation ability in normal pregnancy with the growth of the fetus. Here, by comparing different methodologies to calculate the number of EPC subsets, we could imitate the existing controversy in the literature for such calculations, which may help to elucidate clearer explanations.

Increased interleukin-17 transcripts in peripheral blood mononuclear cells, a link between T-helper 17 and proinflammatory responses in bladder cancer.

BACKGROUND: Interleukin (IL)-17-producing CD4+ T helper (Th17) cells thatare known by producing IL-17 have recently been defined as a unique subset of proinflammatory helper cells. IL-17 is an inflammatory cytokine with robust effect on many cells and it can play important roles in pathogenesis of diverse groups of immune-mediated diseases. OBJECTIVES: The aim of this case-control study was to determine the gene expression of IL-6, IL-17, and transforming growth factor beta (TGF-ß) in Iranian patients with bladder cancer. PATIENTS AND METHODS: Blood samples were collected from 37 patients with bladder cancer and 37 healthy individuals with no history of malignancies or autoimmune disorders, based of simple sampling. The expression of IL-6, IL-17, and TGF-ß were measured by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The mean of IL-17 transcripts was significantly higher in patients with bladder cancer compared with healthy individuals (0.33 ± 0.06 vs. 0.42 ± 0.14, ) (P = 0.04), but their TGF-ß was lower (12.53 ± 8.41 vs. 54.94 ± 17.95, ) (P = 0.04). However, the IL-6 transcripts level was similar in both groups (5.34 ± 2.40 vs. 8.07 ± 3.28, ) (P > 0.05) and there was not any significant difference between the noted cytokines expressions among patients with different stages and grades. CONCLUSIONS: As most of the cases studied in this investigation were in stages I and II, IL-17 as a prominent proinflammatory cytokine may play an important role in recruiting and infiltrating of antitumor immune responses in early stages of bladder cancer. Furthermore, it can be used as predictor for the clinical stage and prognosis of cancers such as bladder carcinoma.

Tumor suppressive effects of WEE1 gene silencing could not enhance immunopotentiation effects of CD80 and 4-1BBL co-stimulation in human T cells.

BACKGROUND: Activation of T cells against tumors by recruiting co-stimulatory molecules has been an attractive approach for cancer immunotherapy. Reports suggested that targeting different genes in tumors might also boost T cell-mediated tumor destruction. AIMS: We investigated whether in vitro WEE1 gene silencing in MDA-MB-468 and MCF7 breast cancer cell lines could enhance immunopotentiating effects of CD80 and 4-1BBL co-stimulation in human T cells. MATERIALS AND METHODS: WEE1 gene was specifically silenced in the cancer cells using shRNA technology. The co-stimulatory molecules were over-expressed on the surface of the cancer cells by recombinant non-replicative adenoviruses. The immune reaction of T cells in the co-culture with tumor cells was studied. IFN-g production was assessed by intracellular staining of T cells. To assess cytotoxic activity of CD8+ T cells, the CD107a mobilization-degranulation assay was performed. Expression of granzyme B, perforin and fasl were examined by real time PCR. RESULTS: T cell dual co-stimulation led to a significant increase in the frequency of IFN-g producing cells and higher percentages of degranulation in CD8+ T cells. It also resulted in higher expression levels of the cytotoxicity-related genes. WEE1 gene silencing in the target cells alone however, could not produce significant immune reactivation in the cultured T cells. Likewise, the immune responses of T cells neither improved nor suppressed when dually co-stimulated PBMCs were exposed to the cancer cells with silenced WEE1. CONCLUSIONS: In spite of antitumor effects of WEE1 silencing, combination of this approach with immune co-stimulation could not boost the reactivity of cultured T cells against the tested breast cancer cells.

Natural Killer Cell Functional Activity After 4-1BB Costimulation.

Reports show enhancement of CD8 T cells' activity through CD137 (4-1BB) signal; however, not all data proved similar effect in natural killer (NK) cells. Here, the impact of 4-1BB signal on NK cells' function was assessed during short term cultures. To that end, cytokine-activated NK cells were cocultured with adenovirally transduced MCF-7 stimulator cells expressing 4-1BB ligand. Cellular cytotoxicity, cytokine production, and expression of cytotoxicity related genes were assessed after overnight cultures. Sharp decrease of CD56+ and CD56bright NK cells was demonstrated. 4-1BB neither enhanced cellular degranulation nor improved IFN-γ production although it promoted granzyme B, perforin, and FasL gene expression. 4-1BB signal stimulated higher proportions of CD56bright population to degranulate and express CD107a; however, it could not recover killing activity against K562 targets. Our data could not show major promotion in activity of all NK subpopulations. Due to great heterogeneity of NK cells, more investigation is needed to draw a comprehensive conclusion.

Synergistic anti-tumor effects of nitroreductase mutants and p53.

INTRODUCTION: The p53 gene therapy showed promising results for treatment of numerous cancers particularly in combination with chemotherapy or radiotherapy. Gene therapy combining two or more treatment options may lead to the synergistic effects between diverse therapies and provide many opportunities in our fight against cancer. AIM: This study focused on the effects of p53 combining with the suicide gene therapy, nitroreductase (NTR)/5-(aziridin-1-yl)-2,4 dinitrobenzamide, on different cancer cell lines. MATERIALS AND METHODS: Effects of adenoviral expressing p53 alone or in combination with wild type (WT) NTR, NTR single mutant, F124N and two NTR double mutants, T41L/N71S and T41L/F70A on survival rate of A549, QU-DB, MCF-7, MDA-MB-468 and DU145 cancer cell lines were determined by MTT assay. Expressions of MDM2 and TP53 transcripts were then assessed by quantitative real-time polymerase chain reaction in p53, NTR and combination of p53 with NTR infected cell lines. RESULTS: According to the results, combination of p53 with NTR double mutant, T41L/F70A or NTR single mutant F124N, showed statistically significant decrease in vitality of all cancer cell lines studied compared with status of IC 50 from p53 or WT NTR and other NTR mutants alone (P < 0.05). Expressions of TP53 and MDM2 were downregulated in all T41L/F70A infected cells except for MCF-7. CONCLUSION: Combination of T41L/F70A NTR with p53 may have more advantages for treatment of different types of cancers compared to the other NTRs and p53 alone. The present study results may open new windows for getting desired outcome in gene therapy of different types of cancer.

IL-23/IL-27 Ratio in Peripheral Blood of Patients with Breast Cancer.

BACKGROUND: Interleukin (IL)-23 and IL-27 are two IL-12-related cytokines which their function may dramatically influence the inflammatory response to tumor development. IL-12 and IL-27 seem to have antagonistic roles with IL-23 in tumor site. In this study, IL-23 and IL-27 mRNA expressions were analyzed in peripheral blood of patients with breast cancer and healthy volunteers using quantitative real-time PCR. METHODS: Peripheral blood samples were collected from 50 women with breast cancer and 50 healthy ones. The total RNA was extracted from peripheral blood after lysis with ammonium chloride and TRizol reagent and the cDNA was synthesized. The expression of IL-23 and IL-27 gene transcripts was determined with real-time polymerase chain reaction (qRT-PCR) using Syber Green PCR Master Mix. RESULTS: It is found that IL-23 and IL-27 transcripts had significantly higher expression in peripheral blood of patients compared with the healthy controls. The ratio of IL-23 transcript expression to IL-27 was 3.4 fold lower in the studied patients compared with the normal individuals. CONCLUSION: It is concluded that the over expression of IL-23 and IL-27 gene transcript in peripheral blood of breast cancer patients may be an immune response against tumor development and the inflammatory response plays a critical role in tumor development via up regulating the corresponding cytokines. However, the IL-23/IL-27 ratio may play an important role in cytokine-based immunotherapy against cancer. Further research should be carried out to assess these cytokines in a larger sample size. .

Tumour suppressive effects of WEE1 gene silencing in breast cancer cells.

BACKGROUND: WEE1 is a G2/M checkpoint regulator protein. Various studies have indicated that WEE1 could be a good target for cancer therapy. The main aim of this study was to asssess the tumor suppressive potential of WEE1 silencing in two different breast cancer cell lines, MCF7 which carries the wild-type p53 and MDA-MB468 which contains a mutant type. MATERIALS AND METHODS: After WEE1 knockdown with specific shRNAs downstream effects on cell viability and cell cycle progression were determined using MTT and flow cytometry analyses, respectively. Real-time PCR and Western blotting were conducted to assess the effect of WEE1 inhibition on the expression of apoptotic (p53) and anti-apoptotic (Bcl2) factors and also a growth marker (VEGF). RESULTS: The results showed that WEE1 inhibition could cause a significant decrease in the viability of both MCF7 and MDA-MB-468 breast cancer cell lines by more than 50%. Interestingly, DNA content assays showed a significant increase in apoptotic cells following WEE1 silencing. WEE1 inhibition also induced up- regulation of the apoptotic marker, p53, in breast cancer cells. A significant decrease in the expression of VEGF and Bcl-2 was observed following WEE1 inhibition in both cell lines. CONCLUSIONS: In concordance with previous studies, our data showed that WEE1 inhibition could induce G2 arrest abrogation and consequent cell death in breast cancer cells. Moreover, in this study, the observed interactions between the pro- and anti-apoptotic proteins and decrease in the angiogenesis marker expression confirm the susceptibility to apoptosis and validate the tumor suppressive effect of WEE1 inhibition in breast cancer cells. Interestingly, the levels of the sensitivity to WEE1 silencing in breast cancer cells, MCF7 and MDA-MB468, seem to be in concordance with the level of p53 expression.

Determining the Latent Tuberculosis Infection by IFN - γ Elispot Assay in Healthcare Workers From University Hospitals of Shiraz, South West of Iran.

BACKGROUND: Classical screening methods are incapable to properly detect LTBI (Latent TB Infection) and HCWs (Healthcare Workers) are at the high risk of exposure. Only few reports estimated the prevalence of LTBI among Iranian HCWs and they mostly used the TST (Tuberculin Skin Test), rather than assessing the response against TB-specific antigens. OBJECTIVES: The current study aimed to determine the frequency of IFN - γ producing blood cells of microbiology and radiology ward technicians by an in-house IFN - γ ELISPOT assay in the University hospitals of Shiraz University of Medical Sciences (SUMS) against recombinant ESAT - 6 and PPD antigens. MATERIALS AND METHODS: 89 HCWs from medical laboratory and radiology departments of Shiraz University of Medical Sciences' hospitals, South of Iran, were screened for LTBI. To achieve the goal, an in-house IFN - γ (Interferon - gamma) ELISPOT (Enzyme Linked ImmunoSpot) assay was used to detect the reactivity against ESAT - 6 (Early Secreted Antigen Target protein - 6) and the PPD (Purified Protein Derivate). RESULTS: Almost 8% of the personnel showed positive TST (over 10 mm) reaction while 29% of them had considerable T - cell reactivity against PPD in ELISPOT assays. However, the ESAT - 6 reactivity was found only in one case of HCWs. No correlation was found between the patterns of the reactions and the age or the duration of the employment or previous vaccination history of the participants. The ELISPOT results were not correlated with the TST results. CONCLUSIONS: Considering the hindrance of TST, the IFN - γ ESAT - 6 ELISPOT assay, even in forms of in-house tests, could replace traditional methods to properly spot the LTBI among the high risk groups from Iran's health system.

Combined 4-1BB and CD28 costimulation could unleash lymphocytes from immunosuppression induced by adipose derived stem cell soluble products.

Adipose derived stem cells (ASCs) have the potential to differentiate into multiple cell lineages with the capacity to suppress immune cells. However, the exact mechanism of this suppression is not fully understood. We hypothesized that supplying additional lymphocyte costimulation through CD28 and 4-1BB could overturn the inhibitory effect of ASCs. To that end, PHA-activated human PBMCs were cocultured with ASCs or with conditioned media (CM) prepared from cultured ASCs. Growth was analyzed in the presence or absence of anti-CD28 and anti-4-1BB antibodies. Results from CFSE dilution analysis with flow cytometry showed that significant and dose-dependent suppression of PHA-activated lymphocytes occurred in the presence of ASC-like cells or ASC's CM. However, additional costimulation of T cells through CD28 and 4-1BB was able to fully recover lymphocyte proliferative capacity in the presence of ASC's CM. Neither of the costimulatory antibodies could fully recover lymphocyte proliferation following coculture with ASCs. Reversal of ASC's immunosuppression through costimulation suggests that further investigation of ASC suppression mechanisms is warranted, since many clinical applications of ASCs are based on this feature. Moreover, such findings have the potential to boost the usefulness of ASCs in the treatment of autoimmune disease.