Intestinal Microbiota: Novel Personalized Cancer Immunotherapy in Colorectal Cancer.

The human colon harbors a diverse array of microorganisms that play fundamental roles in colorectal cancer (CRC). Increasing evidence indicates that dysbiosis of the intestinal microbiome has been associated with the development of CRC. Interaction between host genetics, intestinal microbiota, and lifestyle is well-indicated in the influence, prevention, and treatment of CRC. Various microbiome compositions have reported anticancer and/or anti-inflammatory properties. The presence of our microbiota is integral to our development, but a change in its composition can often lead to adverse effects, increasing the propensity for serious diseases like cancers. Recently, molecular detection and metabolomic techniques have increased our knowledge of the role of microbiota in promoting tumorigenesis. Dietary interventions may be appropriate to regulate the growth of beneficial microbiota in the gut. Metagenomic approaches along with immunology and metabolomics will obvious a new path for the treatment of CRC. In this study, we summarized recent advances in understanding the mechanisms involved in microbiota-related colorectal carcinoma, based on evidence from immunotherapy studies.

Modulation of Colorectal Tumor Behavior via lncRNA TP53TG1-Lipidic Nanosystem.

Long non-coding RNAs (lncRNAs) are an emerging group of RNAs with a crucial role in cancer pathogenesis. In gastrointestinal cancers, TP53 target 1 (TP53TG1) is an epigenetically regulated lncRNA that represents a promising therapeutic target due to its tumor suppressor properties regulating the p53-mediated DNA damage and the intracellular localization of the oncogenic YBX1 protein. However, to translate this finding into the clinic as a gene therapy, it is important to develop effective carriers able to deliver exogenous lncRNAs to the targeted cancer cells. Here, we propose the use of biocompatible sphingomyelin nanosystems comprising DOTAP (DSNs) to carry and deliver a plasmid vector encoding for TP53TG1 (pc(TP53TG1)-DSNs) to a colorectal cancer cell line (HCT-116). DSNs presented a high association capacity and convenient physicochemical properties. In addition, pc(TP53TG1)-DSNs showed anti-tumor activities in vitro, specifically a decrease in the proliferation rate, a diminished colony-forming capacity, and hampered migration and invasiveness of the treated cancer cells. Consequently, the proposed strategy displays a high potential as a therapeutic approach for colorectal cancer.

Malat1 long noncoding RNA regulates inflammation and leukocyte differentiation in experimental autoimmune encephalomyelitis.

In this study, we investigated the contributions of the MALAT1 long noncoding RNA to autoimmune neuroinflammation in central nervous system tissues from patients with multiple sclerosis (MS) and mice with experimental autoimmune encephalomyelitis (EAE). Expression of MALAT1 was decreased in the spinal cords of EAE mice as well as in stimulated splenocytes and primary macrophages. MALAT1 downregulation by specific siRNAs enhanced the polarization of macrophages towards the M1 phenotype. Interestingly, siRNA-mediated MALAT1 downregulation shifted the pattern of T-cell differentiation towards a Th1/Th17 cell profile and decreased differentiation towards a Tregs phenotype. Proliferation of T-cells was also increased following MALAT1 downregulation. These data point to a potential anti-inflammatory effect for MALAT1 in the context of autoimmune neuroinflammation.

The Profile of Toll-like Receptor 2 (TLR2), TLR4 and Their Cytosolic Downstream Signaling Pathway in Common Variable Immunodeficiency (CVID) Patients.

Common variable immunodeficiency (CVID) is the most common clinical primary antibody deficiency, characterized by increased susceptibility to recurrent bacterial infections. Since Toll-like receptors (TLRs) play an important role in the maturation and differentiation of B-cells, TLRs' defect can be involved in the pathogenesis of CVID. Therefore, we evaluated the expression of TLR2 and TLR4 and their signaling pathway; also their association with autoimmunity, B-cell subtypes and response to pneumovax-23 were assessed in CVID patients. Sixteen CVID patients were enrolled in the study. Flow cytometry was used for assessing the protein expression of TLR2 and TLR4, and real-time PCR was used for gene expression of myeloid differentiation primary response 88 (MyD88) and toll interacting protein (Tollip). We found a higher protein expression of TLR2 in CVID patients which was associated with lower number of end stage B-cells and hyporesponse to pneumovax-23 vaccination. We showed a lower mRNA expression of MyD88 and an almost equal Tollip mRNA expression in CVID patients compared with controls. There was a profound association between MyD88 gene expression and autoimmunity in CVID patients. According to the presence of the lower number of end stage B-cells and poor vaccine response in CVID patients and their correlation with the higher expression of TLR2, we hypothesized that there is a functional defect in this receptor and/or its downstream in the peripheral blood mononuclear cells (PBMCs) of CVID patients.

Evaluation of regulatory T lymphocytes and IL2Ra and FOXP3 gene expression in peripheral mononuclear cells from patients with amyotrophic lateral sclerosis.

BACKGROUND: Loss of neuroprotective role of CD4+ helper T cells, regulatory T cells, and M2 microglia constitutively results in the rapid neural death in the "rapidly progressive phase" of amyotrophic lateral sclerosis (ALS). AIM: We aimed to investigate relative count of CD4+ and regulatory T lymphocytes and expression level of IL2Ra and FOXP3 genes in peripheral blood mononuclear cells (PBMCs) from patients with ALS. METHOD: We performed a flow cytometric analysis on PBMC from 38 patients with ALS and 32 controls to determine the count of CD4+ and CD4+CD25+ cells. Quantitative real-time PCR analyses were implemented to determine the level of expression of FOXP3 and IL-2Rα (CD25) genes in the peripheral blood mononucleated cells. RESULTS: We found a significant higher proportion of CD4+ T cells (p value < 0.001), along with a significantly reduced proportion of CD4+CD25+ Treg cells (p value = 0.001, p value = 0.02), in the peripheral blood of patient's with ALS. CONCLUSION: The results of our study are in line with the hypothesis that in the early phase of ALS, neuroprotective helper T cells infiltrate in the affected areas in the lumbar spinal cord. This was reflected in higher peripheral percentage of CD4+ helper T cells and higher expression of FOXP3 and IL-2Rα. The observed demise in the number of active CD4+CD25+ regulatory T cells might indicate early signs of progression to later stages of ALS in our study group. Interestingly, disease duration was the sole independent significant determining factor that predicted CD4+CD25+ regulatory T cell counts in the peripheral blood of patients at various stages of ALS, according to a logistic regression model.

MicroRNA-181 Variants Regulate T Cell Phenotype in the Context of Autoimmune Neuroinflammation.

BACKGROUND: Recent studies have revealed that multiple sclerosis (MS) lesions have distinct microRNA (miRNA) expression profiles. miR-181 family members show altered expression in MS tissues although their participation in MS pathogenesis remains uncertain. Herein, we investigated the involvement of miR-181a and miR-181b in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis (EAE). METHODS: miR-181a and -b levels were measured in the central nervous system (CNS) of patients with MS and mice with EAE as well as relevant leukocyte cultures by real-time RT-PCR. To examine the role of the miRNAs in leukocyte differentiation and function, miR-181a and -b mimic sequences were transfected into cultured primary macrophages and purified CD4+ T cells which were then analyzed by RT-PCR and flow cytometry. Luciferase reporter assays were performed to investigate the interaction of miR-181a and -b with the 3'-UTR of potential target transcripts, and the expression of target genes was measured in the CNS of EAE mice, activated lymphocytes, and macrophages. RESULTS: Expression analyses revealed a significant decrease in miR-181a and -b levels in brain white matter from MS patients as well as in spinal cords of EAE mice during the acute and chronic phases of disease. Suppression of miR-181a was observed following antigen-specific or polyclonal activation of lymphocytes as well as in macrophages following LPS treatment. Overexpression of miR-181a and -b mimic sequences reduced proinflammatory gene expression in macrophages and polarization toward M1 phenotype. miR-181a and -b mimic sequences inhibited Th1 generation in CD4+ T cells and miR-181a mimic sequences also promoted Treg differentiation. Luciferase assays revealed Suppressor of mothers against decapentaplegic 7 (Smad7), as a direct target of miR-181a and -b. CONCLUSION: Our data highlight the anti-inflammatory actions of miR-181a and -b in the context of autoimmune neuroinflammation. miR-181a and -b influence differentiation of T helper cell and activation of macrophages, providing potential therapeutic options for controlling inflammation in MS.

Cow's Milk Desensitization in Anaphylactic Patients: A New Personalized-dose Method.

Cow's milk allergy (CMA) is the most frequent food allergy in children and oral immunotherapy (OIT) is a promising approach for treatment of patients. The most challenging cases are anaphylactic with coexisting asthma and proposing safe protocols is crucial especially in high risk groups. Considering that CMA varies among patients, an individualized OIT protocol would be beneficial to achieve a safer and more efficient method of desensitization. 18 children more than 3 years of age with IgE-mediated CMA were enrolled. CMA was confirmed by positive skin prick test (SPT) and positive oral food challenge (OFC) and 60% of individuals had a convincing history of persistent asthma. SPT with milk extracts, whole fresh milk and serially diluted milk concentrations were performed.  The dilution of milk that induced 3-5 mm of wheal in each individual was selected as the starting dilution for OIT. Desensitization began by 1 drop of the defined dilution and continued increasingly. Overall, 16 out of 18 children (88.8%) achieved the daily intake of 120 mL of milk. Four out of these 16 children accomplished the protocol without any adverse allergic reactions. 12 patients experienced mild to severe reactions. Wheal and erythema in SPT (p≤0.001), and sIgE (p≤0.003) to most milk allergens were significantly decreased following desensitization. We successfully desensitized 16 of 18 children with IgE-mediated CMA by individualized desensitization protocol. Individualizing the OIT protocol would be helpful to save time and perhaps to relieve the allergic symptoms after ingesting cow's milk intake.

MicroRNA-142 regulates inflammation and T cell differentiation in an animal model of multiple sclerosis.

BACKGROUND: MicroRNAs have emerged as an important class of modulators of gene expression. These molecules influence protein synthesis through translational repression or degradation of mRNA transcripts. Herein, we investigated the potential role of miR-142a isoforms, miR-142a-3p and miR-142a-5p, in the context of autoimmune neuroinflammation. METHODS: The expression levels of two mature isoforms of miR-142 were measured in the brains of patients with multiple sclerosis (MS) and the CNS tissues from mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Expression analyses were also performed in mitogen and antigen-stimulated splenocytes, as well as macrophages and astrocytes using real-time RT-PCR. The role of the mature miRNAs was then investigated in T cell differentiation by transfection of CD4+ T cells, followed by flow cytometric analysis of intracellular cytokines. Luciferase assays using vectors containing the 3'UTR of predicted targets were performed to confirm the interaction of miRNA sequences with transcripts. Expression of targets were then analyzed in activated splenocytes and MS/EAE tissues. RESULTS: Expression of miR-142-5p was significantly increased in the frontal white matter from MS patients compared with white matter from non-MS controls. Likewise, expression levels of miR-142a-5p and miR-142a-3p showed significant upregulation in the spinal cords of EAE mice at days 15 and 25 post disease induction. Splenocytes stimulated with myelin oligodendrocyte glycoprotein (MOG) peptide or anti-CD3/anti-CD28 antibodies showed upregulation of miR-142a-5p and miR-142a-3p isoforms, whereas stimulated bone marrow-derived macrophages and primary astrocytes did not show any significant changes in miRNA expression levels. miR-142a-5p overexpression in activated lymphocytes shifted the pattern of T cell differentiation towards Th1 cells. Luciferase assays revealed SOCS1 and TGFBR1 as direct targets of miR-142a-5p and miR-142a-3p, respectively, and overexpression of miRNA mimic sequences suppressed the expression of these target transcripts in lymphocytes. SOCS1 levels were also diminished in MS white matter and EAE spinal cords. CONCLUSIONS: Our findings suggest that increased expression of miR-142 isoforms might be involved in the pathogenesis of autoimmune neuroinflammation by influencing T cell differentiation, and this effect could be mediated by interaction of miR-142 isoforms with SOCS1 and TGFBR-1 transcripts.

CD73 specific siRNA loaded chitosan lactate nanoparticles potentiate the antitumor effect of a dendritic cell vaccine in 4T1 breast cancer bearing mice.

The efficacy of conventional anti-tumor immunotherapeutic approaches is markedly affected by the immunosuppressive microenvironment of tumor. Since adenosine is one of the main orchestra leaders in immunosuppression symphony of tumor, targeting its producing molecules such as CD73 can help to achieve a better clinical outcome following conventional cancer immunotherapeutic approaches. In the present study, we evaluated the efficacy of CD73-specific siRNA-loaded chitosan-lactate nanoparticles (ChLa NPs) in combination with tumor lysate pulsed dendritic cells (DCs) vaccine in treatment of 4T1 (murine derived) breast cancer bearing mice. Our results showed that intravenous administration of CD73-specific siRNA-loaded NPs led to reduced expression of CD73 in tumor cells which was associated with decreased tumor growth and metastasis, and improved mice survival. Furthermore, we found that the mechanism by which combination therapy inhibits tumor growth is in part related to downregulation of regulatory T (Treg), myeloid derived suppressor cells (MDSCs), and tumor associated macrophages, an augmented CTL effector function, improved proliferation status of T cells, increased production of inflammatory cytokines interferon (IFN)-γ and interleukin (IL)-17 and reduced levels of IL-10. Moreover, this treatment protocol attenuated the expression and activities of matrix metalloproteinases (MMPs) 2 and 9 which could be associated to the prevention of lung metastasis. In conclusion, our findings indicate that the use of CD73-specific siRNA-loaded NPs provides an immune potentiating function, thereby improves the efficacy of DC based cancer immunotherapy.

In vitro cytotoxicity of four calcium silicate-based endodontic cements on human monocytes, a colorimetric MTT assay.

OBJECTIVES: This study was performed to evaluate the cytotoxicity of four calcium silicate-based endodontic cements at different storage times after mixing. MATERIALS AND METHODS: Capillary tubes were filled with Biodentine (Septodont), Calcium Enriched Mixture (CEM cement, BioniqueDent), Tech Biosealer Endo (Tech Biosealer) and ProRoot MTA (Dentsply Tulsa Dental). Empty tubes and tubes containing Dycal were used as negative and positive control groups respectively. Filled capillary tubes were kept in 0.2 mL microtubes and incubated at 37℃. Each material was divided into 3 groups for testing at intervals of 24 hr, 7 day and 28 day after mixing. Human monocytes were isolated from peripheral blood mononuclear cells and cocultered with 24 hr, 7 day and 28 day samples of different materials for 24 and 48 hr. Cell viability was evaluated using an MTT assay. RESULTS: In all groups, the viability of monocytes significantly improved with increasing storage time regardless of the incubation time (p < 0.001). After 24 hr of incubation, there was no significant difference between the materials regarding monocyte viability. However, at 48 hr of incubation, ProRoot MTA and Biodentine were less cytotoxic than CEM cement and Biosealer (p < 0.01). CONCLUSIONS: Biodentine and ProRoot MTA had similar biocompatibility. Mixing ProRoot MTA with PBS in place of distilled water had no effect on its biocompatibility. Biosealer and CEM cement after 48 hr of incubation were significantly more cytotoxic to on monocyte cells compared to ProRoot MTA and Biodentine.

In vitro induction of potent tumor-specific cytotoxic T lymphocytes using TLR agonist-activated AML-DC.

Dendritic cells (DCs) are recognized as key regulators of the immune system. Active DC immunization protocols are quickly obtaining interest as an alternative therapeutic approach in acute myeloid leukemia patients. Despite apparent progress in DC-based immunotherapy, some discrepancies were reported in generating potent DCs and their source. In addition to monocytes, DCs can be differentiated from leukemic blasts of acute myeloid leukemia (AML) patients (AML-DC) possessing the ability of stimulating anti-leukemic immune response. In this study, we differentiated peripheral blood blasts of 16 out of 20 AML patients in vitro in the presence of GM-CSF and IL-4 into immature AML-DC. Then, DCs matured using different combinations of Toll-like receptor (TLR) ligands to obtain functional DCs as demonstrated by cell morphology, immunophenotype, and functional activity. Autologous cytotoxic T cell induction of matured DCs was evaluated in four patients and compared with immature counterparts. Our results showed that although the TLR3 agonist (Poly I:C) has a synergistic effect on the TLR4 agonist (lipopolysaccharide, LPS), the addition of the TLR7/8 agonist (R848) is necessary to reinforce the effect of LPS or LPS + POLY(I:C) to produce efficient DCs with the higher level of IL-12 (30 to 90 times). Such DCs activate allogeneic T cells and effectively prime autologous cytotoxic T cells in vitro. In contrast, FSL-1 as a TLR2/6 agonist has a negative effect on LPS + Poly(I:C) and LPS + R848 to produce IL-12. Thus, DCs prepared using a maturation mixture including a TLR7/8 agonist may be used as a potential tool for DC-based immunotherapy purposes in leukemic patients.

Upregulation of CD200 is associated with Foxp3+ regulatory T cell expansion and disease progression in acute myeloid leukemia.

Immunosuppression in acute myeloid leukemia (AML) is an important mechanism of tumor escape. CD200, as an immunosuppressive molecule, is overexpressed in some hematological malignancies and it has also been shown to be an independent prognostic factor in AML. In the current study, simultaneous CD200 expression and Foxp3(+) regulatory T cell levels were investigated in Iranian patients with AML by flow cytometry. We also assessed the effect of CD200-CD200R blockade on Th1 and T-reg cytokine production and T cell proliferation in autologous AML- and monocyte-DC mixed lymphocyte reactions (MLRs). ELISA assay was performed to detect IL-2, IL-12, IFN-γ, IL-10, and TGF-ß production in MLR supernatants. Expression of Foxp3, IL-10, and TGF-ß mRNAs in MLRs were detected by real-time PCR. Our results demonstrated significant overexpression of CD200 (P = 0.001) in association with higher frequencies of Foxp3(+) T cells in AML patients (r = 0.8, P < 0.001). Blocking of CD200-CD200R interaction demonstrated a significant decrease in TGF-ß and IL-10 expression in AML-DC MLRs and a significant increase in IL-12 and IFN-γ expression in monocyte-DC MLRs. Elevated T cell levels with lower Foxp3 intensity was also shown in CD200-CD200R-blocked MLRs. Expression of IL-10 mRNA declined significantly only in AML-DC MLRs where CD200-CD200R interaction was blocked and the same result was observed for TGF-ß and Foxp3 mRNA in both AML- and monocyte-DC MLRs. These data present a significant role for CD200 in suppressing anti-tumor immune response through stimulation of regulatory mechanisms in AML patients and suggest that CD200 may have a prognostic value in this malignancy and its blockade may be used as a target for AML immunotherapy.

Synergistic effect of Toll-like receptor 4 and 7/8 agonists is necessary to generate potent blast-derived dendritic cells in Acute Myeloid Leukemia.

Leukemic cells from AML patients can be differentiated to dendritic cells (DCs). Such DCs have potential for immunotherapy of patients. Blasts from 15 AML patients were differentiated into DCs and matured by different TLR agonists. We could generate AML-DCs from 73% of patients mostly with M4 or M5 subtypes. The DC recoveries ranged from 28% to 50%. The results showed that concomitant use of TLR4 and TLR7/8 agonists induced proficient DCs. Therefore, a combination of TLR4 and 7/8 agonists can be considered as an appropriate maturation cocktail for AML-DC production in order to use in the immunotherapy of AML patients.

B-cell-T-cell activation and interaction in common variable immunodeficiency.

Common variable immunodeficiency (CVID) is a heterogeneous group of disorders, characterized by hypogammaglobulinemia and normal or low numbers of B cells, which predispose patients to recurrent infections. Peripheral blood mononuclear cells from 19 patients with CVID, and age- and sex-matched controls, were subjected to an in vitro assay of B-cell-T-cell activation and interaction, using anti-immunoglobulin (Ig)-D conjugated to dextran (alpha-delta-dex), as a polyclonal T independent type 2 antigen mimic, with and without anti-CD3/anti-CD28, as polyclonal T-cell stimuli. Stimulation of lymphocytes with either anti-CD3 or anti-CD3 plus anti-CD28 induced T-cell activation and proliferation in CVID patients who were similar to age- and sex-matched controls, but B cells of patients were significantly less activated when peripheral blood mononuclear cells were stimulated with polyclonal T-cell agonists alone. Comparison of CD86 expression in the patients with matched controls revealed that patients had low B-cell activation in response to T-cell stimuli (bystander T-cell help). In conclusion, this sample of CVID patients exhibits a defect of T-cell "help" to B cells, and/or B-cell response to T-cell help.