The Upregulation of hsa-mir-181b-1 and Downregulation of Its Target CYLD in the Late-Stage of Tumor Progression of Breast Cancer.

Some microRNAs are usually dysregulated in the cancers and influencing tumor behavior and progression. Hsa-miR-181b-1 and its target CYLD are involved in regulating the inflammatory pathways. This study aimed to investigate the expression levels of hsa-mir-181b-1 and CYLD in a cohort of breast tumor tissues and normal adjacent tissues to assess their association with breast cancer stages. A total number of 60 breast samples including cancerous and normal adjacent tissue specimens were collected. After pathological study, the expression of hsa-mir-181b-1 and CYLD were measured by qRT-PCR method. The hsa-mir-181b-1 expression level was significantly increased in breast tumor tissues compared to the controls. This increase was associated with the disease progression. Conversely, CYLD expression level was decreased in tumor samples compared to normal samples, significantly. ROC curve data added other prestigious information of hsa-mir-181b-1 and CYLD by defining cancer and healthy tissues with high specificity and sensitivity at a proposed cutoff point. Also, bioinformatic enrichment for the possible targets of mature sequence of "hsa-mir-181b-5p" was performed. Computational analysis showed the five most significant pathways including metabolic, cancer, calcium signaling, PI3K-Akt signaling and focal adhesion pathways which may be influenced by hsa-mir-181b-1. Thus, we suggested hsa-mir-181b-1 and CYLD might be involved in the pathogenesis of breast cancer and could be considered as two biomarkers for prediction, prognosis and diagnosis of the stages of the breast cancer.

Optimized Dose of Dendritic Cell-based Vaccination in Experimental Model of Tumor Using Artificial Neural Network.

Previous studies have demonstrated that maturation of dendritic cells (DCs) by pathogenic components through pathogen-associated molecular patterns (PAMPs) such as Listeria monocytogenes lysate (LML) or CpG DNA can improve cancer vaccination in experimental models. In this study, a mathematical model based on an artificial neural network (ANN) was used to predict several patterns and dosage of matured DC administration for improved vaccination. The ANN model predicted that repeated co-injection of tumor antigen (TA)-loaded DCs matured with CpG (CpG-DC) and LML (List-DC) results in improved antitumor immune response as well as a reduction of immunosuppression in the tumor microenvironment. In the present study, we evaluated the ANN prediction accuracy about DC-based cancer vaccines pattern in the treatment of Wehi164 fibrosarcoma cancer-bearing mice. Our results showed that the administration of the DC vaccine according to ANN predicted pattern, leads to a decrease in the rate of tumor growth and size and augments CTL effector function. Furthermore, gene expression analysis confirmed an augmented immune response in the tumor microenvironment. Experimentations justified the validity of the ANN model forecast in the tumor growth and novel optimal dosage that led to more effective treatment.

An extended mathematical model of tumor growth and its interaction with the immune system, to be used for developing an optimized immunotherapy treatment protocol.

BACKGROUND: Chemotherapy is usually known as the main modality for cancer treatment. Nevertheless, most of chronic cancers could not be treated with chemotherapy alone. Immunotherapy is a new modality for cancer treatment that is effective for early stages of cancer and it has fewer side effects compared to chemotherapy, specifically for those types of cancer that are resistant to it. METHOD: This work presents an extended mathematical model to depict interactions between cancerous and adaptive immune system in mouse. We called the model an extended model, because we embedded all those compartments that have important roles in response to tumor in one model. The model includes tumor cells, natural killers, naïve and mature cytotoxic T cells, naïve and mature helper T cells, regulatory T cells, dendritic cells and interleukin 2 cytokine. Whole cycle of cell division program of immune cells is also considered in the model. We also optimized protocol of immunotherapy with DC vaccine based on the proposed mathematical model. RESULT: Simulation results of the proposed model are in conformity with the experimental data recorded from mouse in immunology department of Tehran University of Medical Science as well as what has been explained in the literature. Our results explain dynamics of the immune cells from the first day of cancer growth and progression. Simulation result shows that reducing intervals between immunotherapy injections, efficacy of the treatment will be increased because CD8+ cells are boosted more rapidly. Optimized protocol for immunotherapy suggests that if the effect of DC vaccines on increasing number of anti-tumor immune cells be just before the maximum number of CD8+ cells, the effect of treatment will be maximized.

Improved Anti-Treg Vaccination Targeting Foxp3 Efficiently Decreases Regulatory T Cells in Mice.

INTRODUCTION: The critical role of regulatory T (Treg) cells in dampening immune responses against tumor cells is apparent. Therefore, several methods have been introduced for eliminating Treg. Among them, inducing immune responses against Treg cells expressing Foxp3 transcription factor is a hopeful approach to decrease the frequency of Tregs. In current study, we used the chimeric FoxP3-Fc(IgG) fusion construct/protein to effectively stimulate the immune responses against Treg cells. MATERIALS AND METHODS: Previously constructed FoxP3-Fc(IgG) DNA vaccine and its protein counterpart were injected into C57BL/6 mice in a prime/boost regimen. After 2 weeks, the mice were killed to measure the frequency of Tregs in their spleens, as well as analyze their specific cytokine production, T-cell proliferation, and CD8 T-cell cytotoxicity against FoxP3 protein. RESULTS: FACS analysis of FoxP3 CD4 cells in splenocytes revealed the efficiency of FoxP3 DNA-prime protein-boost strategy to decrease the Treg cells and further showed considerable superiority of Fc(IgG) fusion strategy. This significant reduction in Treg frequency was also concomitant with higher FoxP3-specific CTL and Th1 responses in FoxP3-Fc vaccinated animals. CONCLUSIONS: Prime/boost vaccination against FoxP3 in addition to enhanced antigen presentation by means of Fc fusion strategy could be successfully considered for Treg depletion studies. Validity of this approach should be experimentally tested in preclinical tumor models.

Megavoltage X-ray Dose Enhancement with Gold Nanoparticles in Tumor Bearing Mice.

One of the applications of gold nanoparticles (GNPs) in medicine is radiation dose-enhancing effect. Although there are many simulations, in vitro and in vivo evidence that GNPs can enhance significantly the radiation dose effect of orthovoltage beams. These beams compared with megavoltage (MV) beams, have limited applications in radiotherapy. In order to evaluate GNPs radiosensitization performance with MV beams in-vivo, we used two most clinically used X-ray beams (6 and 18 MV) with the dose of 20 Gy for each mouse. Intratumoral injection of 50 nm GNPs with the concentration of 5 mg ml(-1) was applied to melanoma tumor growing in the left leg of 7 to 8 mice in 4 control and treatment groups of C57BL/6 mice. Albeit, using 10 cm plexiglass jig phantom in the beam path improved the radiation - treatments, the statistical differences between groups were not significant. According to the results, it is concluded that mice can be treated with smaller tumors and higher concentrations of GNPs in MV radiation beams.

Production of recombinant adenovirus containing human interlukin-4 gene.

OBJECTIVES: Recombinant adenoviruses are currently used for a variety of purposes, including in vitro gene transfer, in vivo vaccination, and gene therapy. Ability to infect many cell types, high efficiency in gene transfer, entering both dividing and non dividing cells, and growing to high titers make this virus a good choice for using in various experiments. In the present experiment, a recombinant adenovirus containing human IL-4 coding sequence was made. IL-4 has several characteristics that made it a good choice for using in cancer gene therapy, controlling inflammatory diseases, and studies on autoimmune diseases. MATERIALS AND METHODS: In brief, IL-4 coding sequence was amplified by and cloned in pAd-Track-CMV. Then, by means of homologous recombination between recombinant pAd-Track-CMV and Adeasy-1 plasmid in bacteria, recombinant adenovirus complete genome was made and IL-4 containing shuttle vector was incorporated into the viral backbone. After linearization, for virus packaging, viral genome was transfected into HEK-293 cell line. Viral production was conveniently followed with the aid of green fluorescent protein. RESULTS: Recombinant adenovirus produced here, was capable to infecting cell lines and express interlukin-4 in cell. CONCLUSION: This system can be used as a powerful, easy, and cost benefit tool in various studies on cancer gene therapy and also studies on immunogenetics.

The efficient generation of immunocompetent dendritic cells from leukemic blasts in acute myeloid leukemia: a local experience.

Dendritic cells (DCs) are the most important antigen presenting cells with potentially useful applications in cancer immunotherapy. Leukemic cells of patients with acute myeloid leukemia (AML) could be differentiated to DC-like cells possessing the ability of stimulating anti-leukemic immune response. Despite obvious progress in DC-based immunotherapy, some discrepancies were reported in differentiation potential of AML blasts from all patients toward DC like cells. The present study, as a local experience, was set up to generate DCs from AML blasts of various subtypes. Leukemic Blasts from 16 Iranian AML patients were differentiated into functional DCs by culturing in the presence of rhGM-CSF, rhIL-4 and TNF-alpha for 8 days. The morphology, expression of key surface molecules and allostimulatory activity of resultant DCs were compared with primary blasts and cultured but cytokine untreated control groups. The pattern of angiotensin-converting enzyme (ACE) expression was used to approve the leukemic origin of generated DCs. Neo-expression or upregulation of DC-associated markers were occurred during culturing period in cytokine treated cells compared with primary blasts and cultured but cytokine untreated control groups: CD1a (63.22% vs. 3.22% and 11.79%), CD83 (41.27% vs. 0.11% and 0.70%), CD40 (15.17% vs. 0.00% and 0.04%), CD80 (49.96 vs. 0.02% and 0.32%), CD86 (56.49% vs. 0.50% and 5.71%) and HLA-DR (52.52% vs. 14.32% and 2.49%) respectively. The potency of generated DCs to induce allogeneic T cell proliferation increased significantly compared to pre and post culture control groups (27,533.4 +/- 2,548.3, 8,820.4 +/- 1,639.4 and 3,200.35 +/- 976 respectively). The expression pattern of ACE in AML-DCs, blast cells and DCs derived from normal monocytes (7.93%, 1.28% and 74.97% respectively) confirmed the leukemic origin of DCs. Our data confirmed the generation of sufficient AML-derived cells with the properties of DCs in all cases. This potency of AML blasts, offers a useful route for active immunotherapy of AML patients.