Natural killer cell subsets and their functional molecules in peripheral blood of the patients with breast cancer.

BACKGROUND: Natural killer (NK) cells, CD3- lymphocytes, are critical players in cancer immune surveillance. This study aimed to assess two types of CD3- NK cell classifications (subsets), that is, convectional subsets (based on CD56 and CD16 expression) and new subsets (based on CD56, CD27, and CD11b expression), and their functional molecules in the peripheral blood of patients with breast cancer (BC) in comparison with healthy donors (HDs). METHODS: Thirty untreated females with BC and 20 age-matched healthy women were enrolled. Peripheral blood samples were collected and directly incubated with fluorochrome-conjugated antibodies against CD3, CD56, CD16, CD27, CD11b, CD96, NKG2C, NKG2D, NKp44, CXCR3, perforin, and granzyme B. Red blood cells were then lysed using lysing solution, and the stained cells were acquired on four-color flow cytometer. RESULT: Our results indicated 15% of lymphocytes in peripheral blood of patients with BC and HDs had NK cells phenotype. However, the frequency of total NK cells (CD3-CD56+), and NK subsets (based on conventional and new classifications) was not significantly different between patients and HDs. We observed mean fluorescent intensity (MFI) of CXCR3 in total NK cells (p = .02) and the conventional cytotoxic (CD3-CD56dim CD16+) NK cells (p = .03) were significantly elevated in the patients with BC compared to HDs. Despite this, the MFI of granzyme B expression in conventional regulatory (CD3-CD56brightCD16- /+) NK cells and CD3-CD56-CD16+ NK cells (p = .03 and p = .004, respectively) in the patients was lower than healthy subjects. CONCLUSION: The higher expression of chemokine receptor CXCR3 on total NK cells in patients with BC may be associated with increased chemotaxis-related NK cell infiltration. However, lower expression of granzyme B in conventional regulatory NK cells and CD3-CD56-CD16+ NK cells in the patients compared to HDs suggests reduced cytotoxic activity of the NK cells in BC. These results might demonstrate accumulating NK subsets with a dysfunctional phenotype in the peripheral blood of patients with BC.

Promising anticancer activity of cromolyn in colon cancer: in vitro and in vivo analysis.

PURPOSE: Colon cancer is a prevalent cancer globally, representing approximately 10% of all cancer cases and accounting for 10% of all cancer-related deaths. Therefore, finding new therapeutic methods with high efficiency will be very valuable. Cromolyn (C), a common anti-allergic and mast cell membrane stabilizing drug, has recently shown valuable anti-cancer effects in several studies. This study was designed to investigate the anti-cancer activity of cromolyn on colon cancer in vitro and in vivo and to determine values such as selectivity index and survival effect. METHODS: HT-29 (colon cancer) and MCF-10 (normal epithelial) cell lines were treated with C and Doxorubicin (DOX; Positive control). IC50 values and the effects of C and DOX on apoptosis were explored using methyl thiazole diphenyl-tetrazolium bromide (MTT) assay and Annexin V/PI Apoptosis Assay Kit. To investigate in an animal study, colon cancer was subcutaneously induced by CT26 cells (mouse colon cancer) in bulb/c mice. Mice were treated with 0.05 LD50 intraperitoneal every other day for 35 days. After the death of mice, tumor volume, tumor weight, and survival rate were evaluated. RESULTS: C selectively and significantly suppressed the proliferation of cancer cells in a dose-dependent manner. The IC50 values for the MCF-10 and HT29 cell lines were 7.33 ± 0.78 µM and 2.33 ± 0.6 µM, respectively. Notably, the selective index (SI) highlighted that C displayed greater selectivity in inhibiting cancer cell growth compared to DOX, with SI values of 3.15 and 2.60, respectively. C exhibited higher effectiveness and selectivity in inducing apoptosis in cancer cells compared to DOX, with a significant p-value (61% vs. 52%, P-value ≤ 0.0001). Also, in mice bearing colon cancer, C reduced the tumor volume (6317 ± 1685mm3) and tumor weight (9.8 ± 1.6 g) compared to the negative control group (weight 12.45 ± 0.9 g; volume 7346 ± 1077) but these values were not statistically significant (P ≤ 0.05). CONCLUSION: Our study showed that cromolyn is a selective and strong drug in inhibiting the proliferation of colon cancer cells. Based on our results, the efficacy of C in vitro analysis (MTT assays and apoptosis), as well as animal studies is competitive with the FDA-approved drug doxorubicin. C is very promising as a low-complication and good-efficacy drug for cancer drug repositioning. This requires clinical research study designs to comprehensively evaluate its anti-cancer effects.

Frequent expression of CD45RO memory T cell marker as well as low to high expression of PD-1 and PD-L1 inhibitory molecules in seminoma and dysgerminoma.

BACKGROUND: Seminoma and dysgerminoma are rare testicular and ovarian germ cell tumors characterized by a significant infiltration of immune cells in the tumor microenvironment. According to the failure of conventional treatments in some patients, it is crucial to identify novel prognostic and therapeutic biomarkers for these patients. The objectives of this study were to evaluate the expression of CD45RO and PD-1/PD-L1 and investigate their association with the clinicopathological characteristics of the patients. METHODS: Immunohistochemistry was performed to assess the expression of CD45RO, PD-1, and PD-L1 in tumor-infiltrated lymphocytes (TILs), and tumor cells in 33 seminoma and 31 dysgerminoma patients. The expression levels were evaluated using a semiquantitative approach, weighted histoscore, which considers both the intensity and extent of staining. RESULTS: All seminoma and dysgerminoma patients exhibited CD45RO expression in TILs, with 66.7 % and 90.3 % displaying high levels of expression, respectively. PD-1 expression in TILs was observed at low levels in 81.8 % and 77.4 % and at high levels in 18.2 % and 19.4 % of seminoma and dysgerminoma patients, respectively. Likewise, low expression of PD-L1 in tumor cells was detected in 63.6 % of seminoma and 61.3 % of dysgerminoma patients, while none of the patients exhibited high expression of PD-L1. In seminoma patients, a positive correlation was observed between PD-1 expression in TILs and CD45RO expression and between PD-L1 expression in tumor cells and TILs score. CONCLUSION: The frequent infiltration of CD45RO, along with variable expression of PD-1 and PD-L1 on TILs and tumor cells, could impact the effectiveness of anti-tumor responses and immunotherapy.

In vitro and in vivo anticancer activity of mebendazole in colon cancer: a promising drug repositioning.

Colon cancer is one of the most common cancers and one of the main causes of death worldwide. Therefore, new treatment methods with better efficiency and fewer risks are very necessary. Mebendazole (MBZ), a drug commonly used for helminthic infections, has recently received attention as a suitable candidate for the treatment of various cancers. This study aimed to investigate, in vitro and in vivo, anticancer activity and selectivity Index of MBZ on colon cancer. HT-29 (human colorectal adenocarcinoma) and MCF-10 (non-tumorigenic epithelial) cell lines were treated with MBZ and Doxorubicin (DOX; positive control drug). IC50 values were estimated using methyl thiazole diphenyl-tetrazolium bromide (MTT) assay. We employed flow cytometry using annexin V-FITC and propidium iodide dyes. For the animal study, colon cancer was subcutaneously induced by CT26 cells (mouse colon cancer) in Bulb/C mice. The mice were treated with 0.05 of LD50, intraperitoneal, every other day for 35 days. Finally, the survival rate, tumor volume, and tumor weight were calculated. Our results demonstrated that IC50 values after 72 h for HT29 and MCF-10 cell lines were 0.29 ± 0.04 µM and 0.80 ± 0.02 µM, respectively. MBZ was more selective than DOX in inhibiting the proliferation of cancer cells compared to normal cells (2. 75 vs. 2.45). Annexin V/PI staining demonstrated that MBZ treatment at IC50 concentrations induced (78 ± 12%) apoptosis in the HT29 cancer cell line after 48 h (P ≤ 0.0001). Also, in mice bearing colon cancer, MBZ significantly reduced the tumor volume (1177 ± 1109 mm3; P ≤ 0.001) and tumor weight (2.30 ± 1.97 g; P ≤ 0.0001) compared to the negative control group (weight 12.45 ± 2.0 g; volume 7346 ± 1077). Also, MBZ increases mean survival time (MST) and increase life span (ILS) percentage in the animal study (51.2 ± 37% vs 93%, respectively). This study suggests that mebendazole strongly and selectively inhibits proliferation and induces apoptosis in colon cancer cells. It may be, accordingly, a promising drug for clinical research and application.

Harnessing the Power of CAR-NK Cells: A Promising Off-the-Shelf Therapeutic Strategy for CD38-Positive Malignancies.

Background: CD38 is highly expressed on multiple myeloma (MM) cells and has been successfully targeted by different target therapy methods. This molecule is a critical prognostic marker in both diffuse large B-cell lymphoma and chronic lymphocytic leukemia. Objective: We have designed and generated an anti-CD38 CAR-NK cell applying NK 92 cell line. The approach has potential application as an off-the-shelf strategy for treatment of CD38 positive malignancies. Methods: A second generation of anti-CD38 CAR-NK cell was designed and generated, and their efficacy against CD38-positive cell lines was assessed in vitro. The PE-Annexin V and 7-AAD methods were used to determine the percentage of apoptotic target cells. Flow cytometry was used to measure IFN-γ, Perforin, and Granzyme-B production following intracellular staining. Using in silico analyses, the binding capacity and interaction interface were evaluated. Results: Using Lentivirus, cells were transduced with anti-CD38 construct and were expanded. The expression of anti-CD38 CAR on the surface of NK 92 cells was approximately 25%. As we expected from in silico analysis, our designed CD38-chimeric antigen receptor was bound appropriately to the CD38 protein. NK 92 cells that transduced with the CD38 chimeric antigen receptor, generated significantly more IFN-γ, perforin, and granzyme than Mock cells, and successfully lysed Daudi and Jurkat malignant cells in a CD38-dependent manner. Conclusion: The in vitro findings indicated that the anti-CD38 CAR-NK cells have the potential to be used as an off-the-shelf therapeutic strategy against CD38-positive malignancies. It is recommended that the present engineered NK cells undergo additional preclinical investigations before they can be considered for subsequent clinical trial studies.

Simultaneous Expression of PD-1 and PD-L1 in Peripheral and Central Immune Cells and Tumor Cells in the Benign and Malignant Salivary Gland Tumors Microenvironment.

BACKGROUND: To investigate the differential expression of PD-1 and PD-L1 in salivary gland tumors (SGTs, malignant and benign subtypes) and determine their association with the clinicopathological characterization of the patients. METHODS: The immunohistochemistry was used to examine PD-1 and PD-L1 expression in specimens from 83 patients with primary SGTs including salivary ductal carcinoma (SDC), adenoid cystic carcinoma (AdCC), acinic cell carcinoma (ACC), mucoepidermoid carcinoma (MEC), warthin's tumors (WT), poleomorphic adenoma (PA) and other subtypes. RESULTS: The expression of PD-1 in peripheral and central immune cells (ICs) of MEC, and peripheral ICs of ACC was significantly higher than those with AdCC (P = 0.02, P = 0.02, P = 0.03, respectively). Interestingly, the expression of PD-1 was also observed in peripheral and central malignant tumor cells (TCs), particularly in SDC and ACC. Despite no significant difference in PD-L1 expression of TCs among malignant subtypes, the peripheral and central ICs of ACC and MEC were revealed to express PDL-1 significantly more than those with AdCC (P < 0.05). WTs were rich in PD-1/PD-L1 expressing ICs. However, the tumor microenvironment of PA generally had low levels of PD-1/PD-L1 expression. In general, the expression of PD-1 in peripheral and central TCs was found to be significantly higher in malignant tumors than in benign ones (P = 0.002 and P = 0.003, respectively). CONCLUSION: The simultaneous presentation of PD-1 and PD-L1 in TCs and ICs of SGTs, their significant association with disease severity as well as the positive correlation between these immune checkpoints may suggest the therapeutic potential of anti-PD-1 and anti-PDL-1 combinational immunotherapy for SGTs.

IFNγ-IL-17-IL-22+CD4+ subset and IL-22-producing cells in tumor draining lymph nodes of patients with breast cancer.

BACKGROUND: A recently introduced CD4+ T subset that mainly secretes interleukin (IL-) 22 has been reported to be associated with a variety of tumors, including colon, gastric, hepatocellular, and small- and large-cell lung carcinoma. Both tumor-promoting and - suppressing roles have been suggested for these cells. In the present study, we aimed to investigate the frequency of IL-22-producing subsets in tumor-draining lymph nodes (TDLNs) of the patients with breast cancer and determine their association with the clinicopathological characterizations of the disease. METHODS: Thirty untreated women diagnosed with breast cancer were enrolled and their axillary lymph nodes were dissected during surgery. Mononuclear cells were isolated using Ficoll density gradient, activated, permeabilized, and stained by fluorochrome-conjugated antibodies against CD4, IL-22, IL-17, and IFNγ. The cells were then acquired on the FACSCalibur flow cytometer, and raw data was analyzed by the FlowJo software package (V10). RESULTS: Our results demonstrated that 2.39% ± 0.39 of CD4+ lymphocytes in TDLNs of patients with breast cancer produced IL-22. Among them, 0.64% ± 0.8 just produced IL-22 but were negative for IFNγ and IL-17. Statistical analysis indicated that the frequency of CD4+IL-22+ cells was significantly higher in the patients with stage III and the ones with 3-9 tumor involved lymph nodes (N2) compared to those with stage II and those having 1-3 tumor involved lymph nodes (N1) (P = 0.008 and P = 0.004, respectively). CONCLUSION: The higher frequency of IL-22-producing cells in draining lymph nodes of patients with more advanced tumors (higher stage (stage III) and more involved lymph nodes) suggests a role for IL-22-producing cells in the tumor progression and invasion. However, further studies with larger sample size and more functional studies are needed to clarify the role of IL-22-producing cells in breast cancer pathogenesis.

Reference Ranges of Lymphocyte Subsets in Healthy Individuals from Southern Iran.

BACKGROUND: Lymphocyte immune-phenotyping is considered as a useful tool in diagnosis and monitoring different medical conditions. This study aimed to establish a reference value of different lymphocyte subsets in healthy individuals from southern Iran. METHODS: Flow cytometric method was used to determine the frequency of T cells, helper T cells, cytotoxic T cells, activated T cells, NK cells, NKT cells, and B cells in peripheral blood of 86 healthy subjects from southern Iran. RESULTS: Regardless of gender, the mean percentage of the lymphocyte subsets have been observed as T cells (69.2 ± 8.84%), B cells (11.88 ± 4.14%), T helper cells (39.85 ± 7.75%), T cytotoxic cells (33.97 ± 6.64%), activated T cells (6.67 ± 3.60%), NK cells (12.56 ± 7.32%), and NKT cells (5.07 ± 2.83%). The ratio of CD4+/CD8+ was found to be 1.22 ± 0.4. An insignificant difference was found between lymphocytes of male and female. The percentage of helper T-cells has demonstrated a positive correlation and the percentage of cytotoxic T-cells and NK cells have il-lustrated a negative correlation with age. CONCLUSIONS: The results provide normal reference values of peripheral blood lymphocyte subsets in healthy individuals from southern Iran with the age range of 15 - 55 years old. Moreover, the results support this approach that reference values of lymphocyte subsets should be provided for each population. The reference range value of circulating lymphocytes is a powerful tool for monitoring and/or clinical management of immune related disorders.

Proteomics Study of Mesenchymal Stem Cell-Like Cells Obtained from Tumor Microenvironment of Patients with Malignant and Benign Salivary Gland Tumors.

Objective: Salivary gland tumors (SGTs) show some aggressive and peculiar clinicopathological behaviors that might be related to the components of the tumor microenvironment, especially mesenchymal stem cells (MSCs)-associated proteins. However, the role of MSCs-related proteins in SGTs tumorigenesis is poorly understood. This study aimed to isolate and characterize MSCs from malignant and benign tumor tissues and to identify differentially expressed proteins between these two types of MSCs. Materials and Methods: In this experimental study, MSC-like cells derived from benign (pleomorphic adenoma, n=5) and malignant (mucoepidermoid carcinoma, n=5) tumor tissues were verified by fluorochrome antibodies and flow cytometric analysis. Differentially expressed proteins were identified using two-dimensional polyacrylamide gel electrophoresis (2DE) and Mass spectrometry. Results: Results showed that isolated cells strongly expressed characteristic MSCs markers such as CD44, CD73, CD90, CD105, and CD166, but they did not express or weakly expressed CD14, CD34, CD45 markers. Furthermore, the expression of CD24 and CD133 was absent or near absent in both isolated cells. Results also discovered overexpression of Annexin A4 (Anxa4), elongation factor 1-delta (EF1-D), FK506 binding protein 9 (FKBP9), cytosolic platelet-activating factor acetylhydrolase type IB subunit beta (PAFAH1B), type II transglutaminase (TG2), and s-formylglutathione hydrolase (FGH) in MSCs isolated from the malignant tissues. Additionally, heat shock protein 70 (Hsp70), as well as keratin, type II cytoskeletal 7 (CK-7), were found to be overexpressed in MSCs derived from the benign ones. Conclusion: Malignant and benign SGTs probably exhibit a distinct pattern of tissue proteins that are most likely related to the metabolic pathway. However, further studies in a large number of patients are required to determine the applicability of identified proteins as new targets for cancer therapy.

Indoleamine 2, 3-Dioxygenase: A Professional Immunomodulator and Its Potential Functions in Immune Related Diseases.

Indoleamine 2, 3-dioxygenase (IDO) as an intracellular cytosolic enzyme converts tryptophan (Trp) to N-formyl kynurenine which leads to proinflammatory T-cell apoptosis and prevention of immune cells maturation via decreasing the level of cellular energy. Trp catabolism products such as kynurenine increase the recruitment of regulatory T cells and induce immune tolerance in dendritic cells. IDO expression can locally suppress immunity in the tumor microenvironment and tumor progression actively recruits IDO expressing cells in tumor-draining lymph nodes. Also, tumor infiltrating Tregs' activity leads to IDO expression in the tumor microenvironment. In this review, we described the immunomodulatory function of IDO and IDO-based therapeutic strategies for immune related diseases. According to positive-feedback loop between Tregs and IDO in the tumor microenvironment, IDO can be targeted as a promising immunostimulatory approach for immunotherapy of cancer. However, several studies revealed controversial consequences for influences of IDO in immunity. Considering the common concept, IDO1 and also IDO2 repress the function of T lymphocytes, while inactivation of IDO results in aggravation of some autoimmune diseases. Eventually, the extensive evaluation of IDO function in immunomodulatory procedure can help achieve IDO inhibitors as optimal drugs to inhibit tumor growth without motivating autoimmunity.

Two new cytotoxic ursane triterpenoids from the aerial parts of Salvia urmiensis Bunge.

Bioassay-guided fractionation of a dichloromethane extract of the aerial parts of Salvia urmiensis, an endemic plant species of Iran, led to the isolation of two new cytotoxic ursane triterpenoids, Salvurmin A and Salvurmin B. Their structures were elucidated by a combination of 1D and 2D NMR, HR-ESI-MS, IR and UV analysis. Cytotoxicity of the above-mentioned compounds were evaluated against two human cancerous cell lines (SW1116, MCF-7). IC50 values for Salvurmin A and Salvurmin B on colon cancer cell line (SW1116) were 41.6 ± 2.6 and 23.2 ± 0.4 µM respectively, in comparison to cisplatin as control positive. In addition, these two compounds exhibited cytotoxic activity on breast cancer cell line (MCF-7) with an IC50 of 54.2 ± 5.3 and 40.2 ± 3.1 µM for Salvurmin A and Salvurmin B, respectively. The cytotoxic activities of these two compounds present a promising potential for the future investigation on this endemic species of Salvia.

Effects of indoleamine 2, 3-dioxygenase (IDO) silencing on immunomodulatory function and cancer-promoting characteristic of adipose-derived mesenchymal stem cells (ASCs).

Indoleamine 2, 3-dioxygenase (IDO) catabolizes tryptophan, mediates immunomodulatory functions, and is released by stromal cells such as mesenchymal stem cells. The aims of this study were to investigate the effects of IDO silencing on immunosuppressive function of adipose-derived mesenchymal stem cells (ASCs), T cells phenotype, and the proliferation/migration of tumor cells. ASCs isolated from adipose tissues of healthy women were transfected with IDO-siRNA. Galectin-3, transforming growth factor-ß1, hepatocyte growth factor, and interleukin-10 as immunomodulators were measured in ASCs using qRT-PCR. T cells phenotype, interferon-γ, and interleukin-17 expression were evaluated in peripheral blood lymphocytes (PBLs) cocultured with IDO silenced-ASCs by flow cytometry and qRT-PCR, respectively. Scratch assay was applied to assess the proliferation/migration of MDA-MB-231 cell line. Galectin-3 was upregulated (p ˂ 0.05) while hepatocyte growth factor was downregulated (p ˂ 0.05) in IDO-silenced ASCs compared to control groups. Regulatory T cells were inhibited in PBLs cocultured with IDO-silenced ASCs; also T helper2 was decreased in PBLs cocultured with IDO-silenced ASCs relative to the scramble group. IDO-silenced ASCs caused interferon-γ overexpression but interleukin-17 downregulation in PBLs. The proliferation/migration of MDA-MB-231 was suppressed after exposing to condition media of IDO-silenced ASCs compared with condition media of untransfected (p < 0.01) and scramble-transfected ASCs (p < 0.05). The results exhibited the weakened capacity of IDO-silenced ASCs for suppressing the immune cells and promoting the tumor cells' proliferation/migration. IDO suppression may be utilized as a strategy for cancer treatment. Simultaneous blocking of immunomodulators along with IDO inhibitors may show more effects on boosting the efficiency of immune-based cancer therapies.

Specific Targeting of Recombinant Human Pancreatic Ribonuclease 1 using Gonadotropin-Releasing Hormone Targeting Peptide toward Gonadotropin-Releasing Hormone Receptor-Positive Cancer Cells.

Background: Targeted drug delivery is a novel method to specifically deliver anticancer therapeutics to tumor sites. Gonadotropin-releasing hormone (GnRH) is a decapeptide, and its target binding property has attracted attention as a means of targeted drug delivery. Human pancreatic ribonuclease 1 (hpRNase1) has been shown to exert anticancer properties, when fused to a targeting moiety. The goal of the present study was to add a GnRH targeting peptide to the N-terminus of hpRNase1 to specifically target GnRH receptor (GnRH-R) expressing cells. Methods: This in vitro study was conducted at Shiraz Institute for Cancer Research (Shiraz, Iran) in 2019. The coding sequence of GnRH and hpRNase1 were fused, and the chimeric protein together with non-fused hpRNase1 were produced in E. coli (BL21). The recombinant proteins were purified, and their biological activity was evaluated using MTT and apoptosis assays. Non-parametric Kruskal-Wallis tests with Dunn's post hoc tests were performed to determine the significant differences between the study groups. Results: GnRH-hpRNase1 chimeric protein specifically inhibited the proliferation of PC-3 (P=0.021), LNCaP (P=0.034), and AD-Gn (P=0.041) cells, while the growth of negative cells (AD-293) was not significantly affected (P=0.081). GnRH-hpRNase1 decreased the IC50 values more than non-fused hpRNase1, by approximately 26.5-fold (P=0.036) for PC-3 cells, and exerted its growth inhibitory effects through apoptosis induction. Conclusion: Fusion of GnRH to hpRNase1 structure produced an enzyme, which could specifically target tumor cells. This approach can be used to eliminate tumors that harbor GnRH-R.

Tumor infiltrating NK cell (TINK) subsets and functional molecules in patients with breast cancer.

INTRODUCTION: NK cells have been introduced as the main innate arm of immunity against malignancies. Recent advances introduced new subsets of, and new effector molecules on NK cells suggesting new paradigms for NK cell functions in tumor immunity. Considering these new paradigms, in the current research we investigated the frequency of tumor infiltrating NK cell (TINK) subsets and their functional molecules in breast tumor tissues by flowcytometry method. METHODS: Breast tumor tissues were obtained from 32 untreated patients with breast cancer. The tissues were then minced mechanically to acquire a single cell suspension and surface-stained with monoclonal antibodies against CD3, CD56, CD11b, CD27, NKG2A, NKG2D and CXCR3. For intracellular staining (ICS), the surface-stained cells were then fixed, permeabilized and stained with anti-Perforin and anti-Granzyme B antibodies. The samples were run and the data were acquired on a four-color flowcytometer. RESULTS: The cell suspension derived from tumor tissue encompassed 3.10 ± 0.52 % CD3-CD56+(bright/dim) total NK cells. Based on the conventional classification the percentages of cytotoxic (CD3- CD56dim) and regulatory (CD3- CD56bright) NK cells were respectively 1.74 ± 0.24 % and 1.36 ± 0.48 %. According to the new classification the percentages of cytotoxic (CD3- CD56+ CD11b+ CD27-), regulatory (CD3-CD56+ CD11b+/- CD27+) and tolerant (CD3-CD56+ CD27- CD11b-) NK cells were respectively 0.48 ± 0.07, 1.55 ± 0.34 and 1.15 ± 0.51. A significant higher frequency of total NK cells (CD3-CD56+ (bright/dim)) in the breast tumor tissues of the patients whose tumor draining lymph nodes (TDLNs) has not been yet involved by tumor cells (LN- patients) compared with the ones with lymph nodes involvement (LN+) (5.91 ± 1.79 % Vs. 2.20 ± 0.20 %, P < 0.004). Furthermore, NK cells with overexpressed activating receptor; NKGD2 (CD3- CD56+(bright/dim) NKG2D+ NK cells) was observed to be elevated in LN- patients compared with the LN+ ones (70.01 ± 7.96 Vs. 42.5 ± 4.81, P < 0.011). Correlation analysis revealed the percentages of conventional regulatory NK cells (CD3- CD56bright) in breast tumor tissue to be in positive correlation with the tumor size (R = 0.380, P < 0.04). The mean percentage of this cell subset was also observed to be higher in patients with T3 tumor size compared with smaller T1 tumor size (1.61 ± 0.20 % vs. 0.75 ± 0.15 %, P < 0.023. CONCLUSION: Our observations suggest that accumulation of NK cells as well as the expression of activating NKG2D receptor by TINKs may play roles in breast tumor regression especially in the LN- patients. As the tumor growths and the size of tumor increases the accumulation of regulatory NK cells may facilitate the tumor improvement. These observations may have implications in cancer NK cell-based immunotherapy.

Autologous Natural Killer Cell-enrichment for Immune Cell Therapy: Preclinical Setting Phase, Shiraz Experience.

Natural killer (NK) cell therapy has proven to be a promising approach for the treatment of malignancies. Osaki method for ex-vivo autologous NK cell expansion has been recently introduced in Japan. To start clinical trial phase I at Shiraz University of Medical Sciences in collaboration with the Japanese group, this preclinical setting study aimed to evaluate the proliferative efficacy of the method, the activation status of expanded autologous NK cells, and the likely unwanted contamination of the final cell product. Peripheral blood mononuclear cells (PBMCs) were isolated from 5 healthy individuals' peripheral blood and transferred directly to the specified initial culture bag containing anti-CD52 and anti-CD3 and Interleukin (IL)-2. The cells were cultured for 14-17 days in an incubator, during which the cells received condition media, and underwent several passages into bigger culture bags. All the procedures were carried out in a cleanroom and associated facilities. Before and after activation PBMCs were analyzed for their phenotype and cytotoxic activity; using flow cytometry and cytokine release assay. Our results indicated that NK (CD3-CD16+/-CD56+) cells were expanded 510-fold on average (range 200-1100 fold), and the purity of NK cells per whole lymphocytes exceeded 68%. The expanded cells were highly lytic as indicated by in-vitro cytotoxic assay, with a strong expression of Natural killer group 2 member D (NKG2D) and CD16. The prepared final cell products were negative for HCV, HBV, HIV, mycoplasma, and endotoxin. In the preclinical phase, large numbers of activated and un-contaminated NK cells from healthy individuals' peripheral blood were successfully generated. The method seems to provide ample clean cell product with no contamination and has the potential to be used for NK cell therapy in future clinical trials, suitable to be infused back to the donors in phase I clinical trial.

Investigation of olfactory receptor family 51 subfamily j member 1 (OR51J1) gene susceptibility as a potential breast cancer-associated biomarker.

Among cancer treatment methods, targeted therapy using cancer-associated biomarkers has minimum side effects. Recently olfactory receptor (OR) family attracts the researcher's attention as a favorable biomarker of cancer. Here, a statistical approach using complete data from the human protein atlas database was used to evaluate the potential of OR51J1 gene as a cancer-associated biomarker. To confirm the findings of statistical analysis, the OR51J1 mRNA and protein expression levels in breast tumor and normal tissue were measured using quantitative Real Time PCR (qRT-PCR) and immunohistochemistry (IHC) techniques. The association with clinicopathological factors was analyzed. Statistical analysis revealed that OR51J1 has a high expression level in more than 20 types of cancer tissues without any expression in 44 normal tissues. In 15 cancer types, including breast cancer, expression score was more than 90%. The qRT-PCR analysis in breast cancer showed OR51J1 have significantly higher expression level in tumors than normal tissues (2.91 fold). The IHC results showed OR51J1 expression on other cellular subtypes than tumor and normal cells, including myoepithelium, fibroblast, and lymphocytes. OR51J1 protein expression in invasive cells, as well as its overall score, showed a significant correlation with ER and PR expression and breast cancer (BC) subtypes. Results revealed the potential of OR51J1 as a cancer-associated biomarker for the diagnosis of breast cancer at the mRNA level.

Production and Immunological Evaluation of Epitope-based Preventative Pneumococcal Candidate Vaccine Comprising Immunodominant Epitopes from PspA, CbpA, PhtD and PiuA Antigens.

BACKGROUND: Streptococcus pneumoniae is the leading cause of pneumonia, mostly in children less than five years and elderly people. Although the Pneumoniae Polysaccharide Vaccine (PPV) and Pneumonia Conjugate Vaccines (PCV) are the efficient pneumococcal vaccine in adults and children, the serotype replacement of S. pneumoniae strains causes the reduction in the efficacy of PPV and PCV vaccines. Epitope-based vaccines are a promising alternative to the present capsular antigen vaccines. METHODS: In this study, we evaluated cellular and humoral immune responses induced by our novel designed multi-epitope vaccine in BALB/c mice. CD8+ Cytolytic T Lymphocytes (CTLs) epitopes were selected from PspA and CbpA antigens, and CD4+ Helper T Lymphocytes (HTLs) epitopes were chosen from PhtD and PiuA antigens. PorB, the TLR2 agonist, as an adjuvant, was employed to increase the immunogenicity of the vaccine. RESULTS AND CONCLUSION: The high levels of specific anti-peptide vaccine IgG and an increase in the level of IgG2 in the vaccinated group demonstrated our vaccine could elicit robust antibody production. The significant increase in IFN-γ, IL-2, TNF-α, IL-4, IL-6, and decrease in IL-10 showed that the designed vaccine could be proposed as the efficient preventative pneumococcal vaccine in the mouse model.

Prognostic significance of CD4-positive regulatory T cells in tumor draining lymph nodes from patients with bladder cancer.

INTRODUCTION AND METHODS: To clarify the role of CD4+ regulatory T cells in bladder cancer, we investigated the frequency of these cells in tumor draining lymph nodes of 50 patients with bladder cancer who underwent radical cystectomy using flow cytometry method. We also assessed their association with prognosis and survival. RESULTS: On average, 30.13 ± 2.17% of lymphocytes in draining lymph nodes from patients with bladder cancer were positive for both CD4 and FOXP3 molecules. Analyses also showed that 9.92 ± 0.8% of CD4+ lymphocytes had a regulatory phenotype (CD4+CD25+FOXP3+CD127low/neg). The frequency of total CD4+FOXP3+ lymphocytes as well as regulatory T cells was significantly greater in patients with at least one tumor-involved lymph node compared to those with tumor-free nodes (P = 0.026 and P = 0.036, respectively). Mean FOXP3 expression in CD4+ lymphocytes was greater in patients with stage IV compared with those in stage III (P = 0.046). No other significant associations were found between the frequency of regulatory T cells and other clinicopathological characteristics or patient survival. CONCLUSIONS: The increased frequency of regulatory T cells in patients with involved lymph nodes suggests that these cells may negatively regulate antitumor immune responses in draining lymph nodes. Our findings may have implications for immunotherapy-based treatments for bladder cancer.

NADPH oxidase 5 activation; a novel approach to human sperm cryoinjury.

Sperm cryopreservation leads to various structural and functional damages, some of which induce by oxidative stress. The reactive oxygen species (ROS) generates by mitochondria and membrane NADPH oxidases (NOXs). Among the NOXs, only NOX5 has been identified in the cell membrane of human sperm. This study was designed to clarify the possible role of NOX5 on sperm cryoinjury. Forty human semen samples were washed and randomly divided into fresh and cryopreserved groups. Each group was divided into 4 subgroups containing Ham's F10 (control), 0.1% DMSO (vehicle), 100 nM of PMA (phorbol 12-myristate 13-acetate) and 1 µM of DPI (diphenyleneiodonium), as NOX5 activator and inhibitor. The samples of cryopreserved groups were preserved in liquid nitrogen for 1 month. The sperm kinematics, membrane integrity, ROS production, apoptosis rate, mitochondrial membrane potential (MMP), intracellular ATP and calcium concentration [Ca2+]i were evaluated. The percent of sperm with intact membrane and motile sperm reduced significantly after thawing (p ≤ 0.01). The ROS production (p ≤ 0.01) and the apoptotic rate increased, MMP dissipated, and the percentage of live cells with high [Ca2+]i decreased significantly in the cryopreserved control group relative to the fresh control group. DPI, in contrast to PMA, improved sperm progressive motility (p ≤ 0.01), membrane integrity in fresh and cryopreserved groups and reduced the ROS amount in cryopreserved group (p ≤ 0.01). Apoptotic rate, [Ca2+]i, ATP, and MMP did not change with DPI and PMA in cryopreserved groups. We conclude that NOX5 activity in fresh sperm is low, and it increases during cryopreservation. NOX5 inhibition improves the cryopreserved sperm quality.