Aquaporins in colorectal cancer: exploring their role in tumorigenesis, metastasis, and drug response.

Aquaporins (AQPs) are small, integral proteins facilitating water transport across plasma cell membranes in response to osmotic gradients. This family has 13 unique members (AQP0-12), which can also transport glycerol, urea, gases, and other salute small molecules. AQPs play a crucial role in the regulation of different cellular processes, including metabolism, migration, immunity, barrier function, and angiogenesis. These proteins are found to aberrantly overexpress in various cancers, including colorectal cancer (CRC). Growing evidence has explored AQPs as a potential diagnostic biomarker and therapeutic target in different cancers. However, there is no comprehensive review compiling the available information on the crucial role of AQPs in the context of colorectal cancer. This review highlights the significance of AQPs as the biomarker and regulator of tumor cells metabolism. In addition, the proliferation, angiogenesis, and metastasis of tumor cells related to AQPs expression as well as function are discussed. Understanding the AQPs prominent role in chemotherapy resistance is of great importance clinically.

MicroRNA-124 Enhances T Cells Functions by Manipulating the Lactic Acid Metabolism of Tumor Cells.

High production of lactic acid is a common feature of various tumors. Lactic acid is an immunosuppressive molecule with crucial roles in tumor cells' immune escape, which could largely be attributed to its negative effects on the T cells present in the tumor microenvironment (TME). Strategies that decrease the glycolysis rate of tumor cells could enhance immunosurveillance and limit tumor growth. Pyruvate kinase M2 (PKM2) is a key enzyme in the glycolysis pathway, and it plays a vital role in lactic acid buildup in the TME. MicroRNA (miR)-124 has been shown to be able to decrease tumor cell lactic acid synthesis indirectly by reducing PKM2 levels. In this study, we first overexpressed miR-124 in the tumor cells and evaluated its effects on the PKM2 expression and lactic acid production of the tumor cells using quantitative real-time polymerase chain reaction (qRT-PCR) and spectrophotometry, respectively. Then, we cocultured miR-124-treated tumor cells with T cells to investigate the effects of miR-124 overexpression on T cell proliferation, cytokine production, and apoptosis. Our results demonstrated that miR-124 overexpression could significantly reduce the amount of lactic acid produced by tumor cells by manipulating their glucose metabolism, which led to the augmented proliferation and IFN-γ production of T cells. Moreover, it rescued T cells from lactic acid-induced apoptosis. Our data suggest that lactic acid is a hindering factor for T-cell-based immunotherapies; however, manipulating tumor cells' metabolism via miR-124 could be a promising way to improve antitumor responses of T cells.

Circular RNAs and tuberculosis infection.

Tuberculosis (TB) is a deadly infectious disease caused by Mycobacterium tuberculosis (Mtb) that affects the immune system chronically. Therefore, effective control and treatment of tuberculosis requires rapid and accurate diagnostic strategies. Tuberculosis has always been a global burden on health, social and economic systems due to the lack of standard curative and diagnostic (bio)markers. Accordingly, the management and monitoring of patients with active TB at the primary care level may be possible through new, rapid and cost-effective non-sputum-based diagnostic procedures. Biomarkers can help diagnose various diseases, including circular RNA (circRNA), which has recently been introduced as an endogenous, abundant and stable RNA in the cytoplasm with unique tissue specificity. There are frequent reports of circRNA involvement in many pathological and physiological processes in human beings. Recent studies have highlighted the presence of circRNAs in serum and their role as promising biomarkers in the diagnosis of the disease, potentially due to the continuous, stable, closed covalent circular structures and lack of easy degradation by nucleases. The purpose of this review article is to scrutinize the behavior of circulating plasma RNAs in relation to the pathogenesis and diagnosis of tuberculosis.

Predicting Efficacy of 5-Fluorouracil Therapy via a Mathematical Model with Fuzzy Uncertain Parameters.

Background: Due to imprecise/missing data used for parameterization of ordinary differential equations (ODEs), model parameters are uncertain. Uncertainty of parameters has hindered the application of ODEs that require accurate parameters. Methods: We extended an available ODE model of tumor-immune system interactions via fuzzy logic to illustrate the fuzzification procedure of an ODE model. The fuzzy ODE (FODE) model assigns a fuzzy number to the parameters, to capture parametric uncertainty. We used the FODE model to predict tumor and immune cell dynamics and to assess the efficacy of 5-fluorouracil (5-FU) chemotherapy. Result: FODE model investigates how parametric uncertainty affects the uncertainty band of cell dynamics in the presence and absence of 5-FU treatment. In silico experiments revealed that the frequent 5-FU injection created a beneficial tumor microenvironment that exerted detrimental effects on tumor cells by enhancing the infiltration of CD8+ T cells, and natural killer cells, and decreasing that of myeloid-derived suppressor cells. The global sensitivity analysis was proved model robustness against random perturbation to parameters. Conclusion: ODE models with fuzzy uncertain kinetic parameters cope with insufficient/imprecise experimental data in the field of mathematical oncology and can predict cell dynamics uncertainty band.

Vulvovaginal candidiasis: An overview of mycological, clinical, and immunological aspects.

AIM: To provide an overview of clinical, immunological, and mycological aspects of vulvovaginal candidiasis (VVC). METHODS: A literature search was conducted to find relevant articles about different aspects of VVC. Related data from retrieved articles were summarized in different headings. RESULTS: VVC has a global distribution and Candida albicans is the leading cause of infection except for specific patient groups like postmenopausal, diabetic, or immunocompromised women. VVC has a range of clinical presentations, accordingly, its diagnosis should be based on clinical examination coupled with laboratory investigations. The best therapeutic regimen depends on the patient's conditions and the causative agent. Moreover, factors like drug resistance of the causative agents and different mutations in the immunity-related genes could affect the treatment outcome. CONCLUSION: As a globally distributed disease, VVC needs further attention, especially in areas related to the treatment failure and recurrence of the disease.

Restricting tumor lactic acid metabolism using dichloroacetate improves T cell functions.

BACKGROUND: Lactic acid produced by tumors has been shown to overcome immune surveillance, by suppressing the activation and function of T cells in the tumor microenvironment. The strategies employed to impair tumor cell glycolysis could improve immunosurveillance and tumor growth regulation. Dichloroacetate (DCA) limits the tumor-derived lactic acid by altering the cancer cell metabolism. In this study, the effects of lactic acid on the activation and function of T cells, were analyzed by assessing T cell proliferation, cytokine production and the cellular redox state of T cells. We examined the redox system in T cells by analyzing the intracellular level of reactive oxygen species (ROS), superoxide and glutathione and gene expression of some proteins that have a role in the redox system. Then we co-cultured DCA-treated tumor cells with T cells to examine the effect of reduced tumor-derived lactic acid on proliferative response, cytokine secretion and viability of T cells. RESULT: We found that lactic acid could dampen T cell function through suppression of T cell proliferation and cytokine production as well as restrain the redox system of T cells by decreasing the production of oxidant and antioxidant molecules. DCA decreased the concentration of tumor lactic acid by manipulating glucose metabolism in tumor cells. This led to increases in T cell proliferation and cytokine production and also rescued the T cells from apoptosis. CONCLUSION: Taken together, our results suggest accumulation of lactic acid in the tumor microenvironment restricts T cell responses and could prevent the success of T cell therapy. DCA supports anti-tumor responses of T cells by metabolic reprogramming of tumor cells.

The application of immune checkpoint blockade in breast cancer and the emerging role of nanoparticle.

Breast cancer is the most common malignancy in the female population with a high mortality rate. Despite the satisfying depth of studies evaluating the contributory role of immune checkpoints in this malignancy, few articles have reviewed the pros and cons of immune checkpoint blockades (ICBs). In the current review, we provide an overview of immune-related inhibitory molecules and also discuss the original data obtained from international research laboratories on the aberrant expression of T and non-T cell-associated immune checkpoints in breast cancer. Then, we especially focus on recent studies that utilized ICBs as the treatment strategy in breast cancer and provide their efficiency reports. As there are always costs and benefits, we discuss the limitations and challenges toward ICB therapy such as adverse events and drug resistance. In the last section, we allocate an overview of the recent data concerning the application of nanoparticle systems for cancer immunotherapy and propose that nano-based ICB approaches may overcome the challenges related to ICB therapy in breast cancer. In conclusion, it seems it is time for nanoscience to more rapidly move forward into clinical trials and illuminates the breast cancer treatment area with its potent features for the target delivery of ICBs.

CRISPR/Cas9-mediated TGFßRII disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells in vitro.

BACKGROUND: CAR T-cell therapy has been recently unveiled as one of the most promising cancer therapies in hematological malignancies. However, solid tumors mount a profound line of defense to escape immunosurveillance by CAR T-cells. Among them, cytokines with an inhibitory impact on the immune system such as IL-10 and TGFß are of great importance: TGFß is a pleiotropic cytokine, which potently suppresses the immune system and is secreted by a couple of TME resident and tumor cells. METHODS: In this study, we hypothesized that knocking out the TGFß receptor II gene, could improve CAR T-cell functions in vitro and in vivo. Hereby, we used the CRISPR/Cas9 system, to knockout the TGFßRII gene in T-cells and could monitor the efficient gene knock out by genome analysis techniques. Next, Mesothelin or Claudin 6 specific CAR constructs were overexpressed via IVT-RNA electroporation or retroviral transduction and the poly-functionality of these TGFßRII KO CAR T-cells in terms of proliferation, cytokine secretion and cytotoxicity were assessed and compared with parental CAR T-cells. RESULTS: Our experiments demonstrated that TGFßRII KO CAR T-cells fully retained their capabilities in killing tumor antigen positive target cells and more intriguingly, could resist the anti-proliferative effect of exogenous TGFß in vitro outperforming wild type CAR T-cells. Noteworthy, no antigen or growth factor-independent proliferation of these TGFßRII KO CAR T-cells has been recorded. TGFßRII KO CAR T-cells also resisted the suppressive effect of induced regulatory T-cells in vitro to a larger extent. Repetitive antigen stimulation demonstrated that these TGFßRII KO CAR T-cells will experience less activation induced exhaustion in comparison to the WT counterpart. CONCLUSION: The TGFßRII KO approach may become an indispensable tool in immunotherapy of solid tumors, as it may surmount one of the key negative regulatory signaling pathways in T-cells.

Targeted knockdown of Tim3 by short hairpin RNAs improves the function of anti-mesothelin CAR T cells.

T-cell immunoglobulin mucin 3 (Tim3) is an immune checkpoint receptor that plays a central role in chimeric antigen receptor (CAR) T cell exhaustion within the tumor microenvironment. This study was aimed to evaluate the effects of targeted-knockdown of Tim3 on the antitumor function of anti-mesothelin (MSLN)-CAR T cells. To knockdown Tim3 expression, three different shRNA sequences specific to different segments of the human Tim3 gene were designed and co-inserted with an anti-MSLN-CAR transgene into lentiviral vectors. To investigate the efficacy of Tim3 targeting in T cells, expression of Tim3 was assessed before and after antigen stimulation. Afterwards, cytotoxic effects, proliferative response and cytokine production of MSLN-CAR T cells and Tim3-targeted MSLN-CAR T cells were analyzed. Our results showed that activation of T cells and MSLN-CAR T cells led to up-regulation of Tim3. Tim3 knockdown significantly decreased its expression in different groups of MSLN-CAR T cells. Tim3 knockdown significantly improved cytotoxic function, cytokine production and proliferation capacity of MSLN-CAR T cells. Our findings indicate that targeted knockdown of Tim3 allows tumor-infiltrating CAR T cells that would otherwise be inactivated to continue to expand and carry out effector functions, thereby altering the tumor microenvironment from immunosuppressive to immunosupportive via mitigated Tim3 signaling.

Biomarkers for predicting the outcome of various cancer immunotherapies.

Cancer immunotherapy has appeared as a well-known therapeutic modality for different cancers. Yet, only a subset of patients derive clinical benefit. It is thus critical to understand the determinants driving response, resistance and adverse effects.Predictive biomarkers in different modalities of cancer immunotherapy offer novel information about the effect of a therapeutic intervention. These biomarkers and their patterns may not only operate similarly across different tumor types that are amenable to these therapies but also assist to identify patients who will benefit from the treatment and subsequently leading to tailored immunotherapy with the wider-successfully-targeted patient population. In this review, we will outline a variety of predictive biomarkers in various cancer immunotherapies and their clinical utility. It is anticipated that the incorporation of biomarker studies in the clinical practice will help optimize therapeutic decision making and realize the potential clinical benefit of biomarker-guided therapy.

Construction and Functional Characterization of a Fully Human Anti-mesothelin Chimeric Antigen Receptor (CAR) Expressing T Cell.

Chimeric antigen receptor (CAR) T cell therapy is considered as an encouraging approach for the treatment of hematological malignancies. However, its efficacy in solid tumors has not been satisfying, mainly in the immunosuppressive network of the tumor microenvironment and paucity of appropriate target antigens. Mesothelin (MSLN) is a tumor-associated antigen (TAA) expressed in numerous types of solid tumors such as gastrointestinal, ovarian, and pancreatic tumors. Owing to high expression in tumor cells and low expression in normal tissues, MSLN-targeted therapies like monoclonal antibodies have been previously developed. In the present study, a CAR T cell harboring the second-generation of a fully human anti-MSLN-CAR construct containing CD3ζ and 4-1BB signaling domains was produced and it was functionally evaluated against an MSLN-expressing cell line. The findings showed potent, specific proliferation, cytotoxic activity, and interleukin (IL)-2, Tumor necrosis factor-(TNF) α, and Interferon-(IFN) γ production in an antigen-dependent manner. Cytotoxic activity was shown in effector-to-target ratio from 1:1 to 20:1, but the most adequate efficacy was observed in the ratio of 10:1. Non-specific activity against MSLN negative cell line was not observed. Our data demonstrated that primary human T cells expressing fully human MSLN-CAR construct are effective against MSLN-expressing cell lines in vitro, suggesting this MSLN-CAR construct as a potential therapeutic tool in a clinical setting.

Prolonged Persistence of Chimeric Antigen Receptor (CAR) T Cell in Adoptive Cancer Immunotherapy: Challenges and Ways Forward.

CAR T cell qualities, such as persistence and functionality play important roles in determining the outcome of cancer immunotherapy. In spite of full functionality, it has been shown that poor persistence of CAR T cells can limit an effective antitumor immune response. Here, we outline specific strategies that can be employed to overcome intrinsic and extrinsic barriers to CAR T cell persistence. We also offer our viewpoint on how growing use of CAR T cells in various cancers may require modifications in the intrinsic and extrinsic survival signals of CAR T cells. We anticipate these amendments will additionally provide the rationales for generation of more persistent, and thereby, more effective CAR T cell treatments. CAR T cell qualities, such as persistence and functionality play important roles in determining the outcome of cancer immunotherapy. In spite of full functionality, it has been shown that poor persistence of CAR T cells can limit an effective antitumor immune response. Here, we outline specific strategies that can be employed to overcome intrinsic and extrinsic barriers to CAR T cell persistence. We also offer our viewpoint on how growing use of CAR T cells in various cancers may require modifications in the intrinsic and extrinsic survival signals of CAR T cells. We anticipate these amendments will additionally provide the rationales for generation of more persistent, and thereby, more effective CAR T cell treatments.

Pharmacological targeting of immune checkpoint A2aR improves function of anti-CD19 CAR T cells in vitro.

In spite of impressive results in the treatment of acute lymphoblastic B cell leukemia (B-ALL) with chimeric antigen receptor (CAR) T cells, the clinical outcome of some hematological cancers like follicular lymphoma (FL) and chronic lymphocytic leukemia (CLL) has not been very promising likely due to immunosuppressive networks within tumor microenvironment. Hypoxia in the microenvironment of hematological malignancies and consequently generation of adenosine molecule is appeared to be correlated with immunosuppression, tumor progression, and relapse. Herein, we hypothesized that whether pharmacological targeting of adenosine 2a receptor (A2aR) can enhance antitumor activity of anti-CD19 CAR T cells in vitro. Prior to functional assays, A2aR expression was assessed in CAR-expressing T cells. Our results showed that A2aR was not only up-regulated in the fully human anti-CD19 CAR T cells (hereafter referred to as huCAR19 T cells) but also was further overexpressed following re-stimulation with target cells. Although pharmacological inhibition of A2aR could significantly increase proliferation capacity and cytokine production of huCAR19 T cells following treatment with an adenosine analog, cytotoxic activity of huCAR19 T cells was not significantly improved. Considering A2aR overexpression in huCAR19 T cells in the tumor microenvironment, our results indicated that pharmacological targeting of A2aR could not only improve huCAR19 T cells functionality in a hostile tumor microenvironment but also could have a therapeutic advantage, and sought to assess the possibility in a pre-clinical setting.

Genetic and pharmacological targeting of A2a receptor improves function of anti-mesothelin CAR T cells.

BACKGROUND: CAR T cell-based therapies have shown promising results in hematological malignancies. Results of CAR T cell projects in solid tumors have been less impressive, and factors including lack of targetable antigens and immunosuppressive tumor microenvironment (TME) have been suggested as culprits. Adenosine, a metabolite which is highly produced in TME, is known to mediate the suppression of anti-tumor T cell responses via binding and signaling through adenosine 2a receptor (A2aR). METHODS: In this study, the expression of A2aR and the effects of its activation on the function of fully human anti-mesothelin CAR T cells (MSLN-CAR T), were analyzed. Afterwards, the molecular and pharmacological means to overcome the inhibitory effects of A2aR signaling on CAR T cell function were used. This was performed by targeting A2aR expression in MSLN-CAR T cells using various anti-A2aR shRNA sequences embedded in the CAR vector and an A2aR pharmacological antagonist, SCH-58261. Statistical analyses were performed Prism 7 software. RESULTS: Our experiments showed significant A2aR upregulation on T cells during the CAR T cell production procedure (cell activation and transduction). Activation of adenosine signaling using adenosine analog led to the suppression of all major anti-tumor functions in MSLN-CAR T cells. Interestingly, CAR T cells that carried the anti-A2aR shRNA sequences were resistant to the inhibitory effects of adenosine signaling. Pharmacological inhibition of A2aR reversed the reduction in CAR T cell proliferation and cytokine response caused by the adenosine analog; however, it failed to rescue the cytotoxic function of the cells. CONCLUSION: Altogether, our results indicate that mitigating A2aR signaling by genetic targeting of the receptor might be a promising approach in improving CAR T cell function in an unreceptive microenvironment and could potentially improve the outcome of treatment in clinical settings.

Construction and functional characterization of a fully human anti-CD19 chimeric antigen receptor (huCAR)-expressing primary human T cells.

Although remarkable results have been attained by adoptively transferring T cells expressing fully murine and/or humanized anti-CD19 chimeric antigen receptors (CARs) to treat B cell malignancies, evidence of human anti-mouse immune responses against CARs provides a rationale for the development of less immunogenic CARs. By developing a fully human CAR (huCAR), these human anti-mouse immune responses are likely eliminated. This, perhaps, not only increases the persistence of anti-CD19 CAR T cells-thereby reducing the risk of tumor relapse-but also facilitates administration of multiple, temporally separated doses of CAR T cells to the same recipient. To these ends, we have designed and constructed a second-generation fully human anti-CD19 CAR (or huCAR19) containing a fully human single-chain variable fragment (ScFv) fused with a CD8a hinge, a 4-1BB transmembrane domain and intracellular T cell signaling domains of 4-1BB and CD3z. T cells expressing this CAR specifically recognized and lysed CD19+ target cells produced cytokines and proliferated in vitro. Moreover, cell volume data revealed that our huCAR construct cannot induce antigen-independent tonic signaling in the absence of cognate antigen. Considering our results, our anti-CD19 huCAR may overcome issues of transgene immunogenicity that plague trials utilizing CARs containing mouse-derived ScFvs. These results suggest that this huCAR19 be safely and effectively applied for adaptive T cell immunotherapy in clinical practice.

Presence of autoantibody against two placental proteins, peroxiredoxin 3 and peroxiredoxin 4, in sera of recurrent pregnancy loss patients.

PROBLEM: Recurrent pregnancy loss (RPL) is defined as two or more consecutive abortion before the 20th week of gestation. Autoimmune diseases increase the risk and accounts for at least 20% of RPL. Placenta is a pregnancy unique tissue, and proper formation of placenta is key phenomenal for success of a pregnancy. The aim of this study was to investigate the placental proteins that may act as antibody targets in RPL patients. METHOD OF STUDY: Total placental proteins were extracted and separated using 2D-PAGE technique. Separated protein spots were transferred on PVDF membrane and blotted with sera from 20 RPL patients and compared with the protein spots that membrane blotted with sera from 20 normal women. Differentially blotted spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry mass spectrometry technique. The results of the mass analysis also were confirmed by western blot using mAb and reverse transcriptase polymerase chain reaction technique. RESULTS: The results indicated that RPL women may produce antibody against Peroxiredoxin 3 (Prx3) and Peroxiredoxin 4 (Prx4). CONCLUSION: Our results indicate that two placental proteins, Prx3 and Prx4, may act as new placental immune targets. Considering the role of antioxidant defense in the protection of placenta from oxidative stress, production of antibodies against peroxiredoxins 3 and 4 may introduce a new autoimmune hypothesis in RPL, which is needed to be tested in the future works.

Soluble CD30 in normal pregnancy, pre-eclampsia and recurrent pregnancy loss.

BACKGROUND: Normal pregnancy is thought to be dependent on Th2 deviation, while Recurrent Pregnancy Loss (RPL) and Pre-eclampsia (PE) appear to be biased toward the Th1 immune response. It is believed that the soluble form of CD30 (sCD30) is an index of Th2 immune response or modulator of Th1/Th2 responses. OBJECTIVE: The aim of this study was determination of the sCD30 level in RPL and PE patients. METHODS: The sCD30 level was measured in sera of a group of normal non-pregnant women (N=43) and compared with normal pregnancy at the first (N=42) and third (N=42) trimester. Furthermore, the level of sCD30 in the normal first and third trimester pregnancies were compared with that of RPL (N=38) and severe pre-eclamptic (N=41) patients, respectively. sCD30 levels were measured by ELISA method and student t-test was used for statistical analysis. RESULTS: The mean level of sCD30 at the first trimester in normal pregnancy was significantly elevated as compared with normal non-pregnant women (21.4 vs. 15.2 ng/ml, p<0.0001). A significant difference between sCD30 concentration at the first and third trimester of normal pregnancies was also observed (21.4 vs. 14.3 ng/ml, p<0.0001). Interestingly, the sCD30 concentration did not show any significant changes at the first trimester of normal pregnancy as compared with RPL (21.4 vs. 20.9 ng/ml) and third trimester of normal pregnancy as compared with PE (14.3 vs. 13.1 ng/ml). CONCLUSION: The data of this study indicated that the concentration of sCD30 in serum during pregnancy period is not associated with RPL or PE and serum sCD30 is not a good correlate of Th2 immune responses in pregnancy.