Royal Jelly Nanoparticle Alleviates Experimental Model of Breast Cancer Through Suppressing Regulatory T Cells and Upregulating TH1 Cells.

BACKGROUND: Royal jelly, a natural product from bees' hypopharyngeal glands, is commonly used in biomedicine due to its antioxidant and anti-tumor activities. The aim of this study was to compare royal jelly in free form and loaded in layered double hydroxide (LDH) nanoparticle for the treatment of breast cancer with a focus on Th1 and T regulatory parameters in an animal model. METHODS: Nanoparticles were produced using the coprecipitation method and characterized using DLS, FTIR, and SEM techniques. Forty female BALB/c mice were inoculated with 7.5 x 105 4T1 cells and treated with royal jelly in free and nanoparticle form. Clinical signs and tumor volume were assessed weekly. The effect of royal jelly products on the serum level of IFN-γ and TGF-ß was measured by ELISA. In addition, the mRNA expression of these cytokines and Th1 and regulatory T cells' transcription factors (T-bet and FoxP3) was assessed by real-time PCR in the splenocytes of tumor-bearing mice. RESULTS: The physicochemical analysis of nanoparticles confirmed the synthesis of LDH nanoparticles and loading of royal jelly into the LDH structures (RJ-LDH). Animal studies showed that royal jelly and RJ-LDH significantly reduced the size of tumor in BALB/c mice. Additionally, treatment with RJ-LDH significantly inhibited TGF-ß and increased IFN-γ production. The data also revealed that RJ-LDH inhibited the differentiation of regulatory T cells, while promoting Th1 cell differentiation via regulating their master transcription factors. CONCLUSIONS: These results indicated that royal jelly and RJ-LDH could inhibit breast cancer progression by in-hibiting regulatory T cells and expansion of Th1 cell. Furthermore, the current study demonstrated the therapeutic efficacy of royal jelly is enhanced by LDH nanoparticles; hence, RJ-LDH is significantly more efficient than Free-RJ in the treatment of breast cancer.

Increased expression of PD-1 in CD8 + CD3 + T cells correlates with EBV viral load in MS patients.

Multiple sclerosis (MS) is one of the common autoimmune diseases. The exact etiology of MS is still unclear, but recent studies have shown the possibility of infectious agent involvement such as Epstein-Barr virus (EBV) in MS pathophysiology. In this study, CD3 + CD8 + T cells of 25 new case MS patients were compared with healthy donors for expression of exhaustion marker, PD-1, using flow cytometry. Also, the expression of the EBV gene, BRCF-1, in PBMCs was analyzed using real-time PCR. Results revealed a lower frequency of CD3 + CD8 + T cells in MS patients. Also, increased expression of PD-1 was observed on CTLs which correlated with higher viral loads. Therefore, a lower frequency of CD8 + T cells but a higher exhaustion marker in MS patients reveals a new mechanism of EBV pathogenesis in MS development. The results suggest that inefficient immune control of EBV in patients with MS may cause exacerbation of the disease. Future studies on the mechanism of T cell exhaustion and chronic infections may aid in a better understanding of the disease and the design of effective therapies.

Catechin-Induced changes in PODXL, DNMTs, and miRNA expression in Nalm6 cells: an integrated in silico and in vitro approach.

BACKGROUND: This study explored the impact of predicted miRNAs on DNA methyltransferases (DNMTs) and the PODXL gene in Nalm6 cells, revealing the significance of these miRNAs in acute lymphocytic leukemia (ALL). METHODS: A comprehensive approach was adopted, integrating bioinformatic analyses encompassing protein structure prediction, molecular docking, dynamics, and ADMET profiling, in conjunction with evaluations of gene and miRNA expression patterns. This methodology was employed to elucidate the therapeutic potential of catechin compounds in modulating the activity of DNA methyltransferases (DNMTs) and the PODXL gene. RESULTS: The findings from our investigation indicate that catechins possess the capability to inhibit DNMT enzymes. This inhibitory effect is associated with the upregulation of microRNAs miR-200c and miR-548 and a concurrent downregulation of PODXL gene expression. These molecular interactions culminate in an augmented apoptotic response within ALL (Nalm6) cells. CONCLUSION: The study posits that catechins may represent a viable therapeutic avenue for inducing apoptosis in ALL cells. This is achieved through the modulation of epigenetic mechanisms and alterations in gene expression profiles, highlighting the potential of catechins as agents for cancer therapy.

Neutralizing tumor-related inflammation and reprogramming of cancer-associated fibroblasts by Curcumin in breast cancer therapy.

Tumor-associated inflammation plays a vital role in cancer progression. Among the various stromal cells, cancer-associated fibroblasts are promising targets for cancer therapy. Several reports have indicated potent anti-inflammatory effects attributed to Curcumin. This study aimed to investigate whether inhibiting the inflammatory function of cancer-associated fibroblasts (CAFs) with Curcumin can restore anticancer immune responses. CAFs were isolated from breast cancer tissues, treated with Curcumin, and co-cultured with patients' PBMCs to evaluate gene expression and cytokine production alterations. Blood and breast tumor tissue samples were obtained from 12 breast cancer patients with stage II/III invasive ductal carcinoma. Fibroblast Activation Protein (FAP) + CAFs were extracted from tumor tissue, treated with 10 µM Curcumin, and co-cultured with corresponding PBMCs. The expression of smooth muscle actin-alpha (α-SMA), Cyclooxygenase-2(COX-2), production of PGE2, and immune cell cytokines were evaluated using Real-Time PCR and ELISA, respectively. Analyzes showed that treatment with Curcumin decreased the expression of genes α-SMA and COX-2 and the production of PGE2 in CAFs. In PBMCs co-cultured with Curcumin-treated CAFs, the expression of FoxP3 decreased along with the production of TGF-ß, IL-10, and IL-4. An increase in IFN-γ production was observed that followed by increased T-bet expression. According to our results, Curcumin could reprogram the pro-tumor phenotype of CAFs and increase the anti-tumor phenotype in PBMCs. Thus, CAFs, as a component of the tumor microenvironment, are a suitable target for combination immunotherapies of breast cancer.

Evaluation of apoptosis-related gene Fas (CD95) and FasL (CD178) polymorphisms in Iranian rheumatoid arthritis patients.

Apoptosis signals are essential for establishing homeostasis and adequate immune response. Dysregulation of apoptosis-related genes in the immune system, which could be due to gene polymorphisms, conduct to autoimmune diseases including rheumatoid arthritis. In the current study, the apoptosis-related gene Fas_-670A>G, FasL_844C>T, and FasLIVS2nt_124A>G polymorphisms were genotyped in 120 Iranian patients with rheumatoid arthritis (RA) and 112 unrelated healthy controls using PCR-RFLP method. Among the 120 RA patients being heterozygous in the promoter region of Fas_-670A/G (OR 1.42,CI 0.92-1.52, P = 0.18) and FasL_-844C/T (OR 1.42, CI 0.92-1.52, P = 0.18) and homozygous in the minor allele for FasLIVS2nt_124G/G (OR 1.43, CI 0.76-1.81, P = 0.7), the frequency of these polymorphisms is higher in the cases than in controls and the elevated risk of RA were observed when the patient compared with controls, although this is not statistically significant.

Enterococci as Intestinal Microbiota: Investigation of Characteristics and Probiotic Potential in Isolates from Adults and Breast-Fed Infants.

Enterococci act as symbionts in human gastrointestinal tract. The present study aimed to evaluate the characteristics of fecal enterococci isolated from infants and adults, and to compare them to the known probiotic bacteria, including lactobacilli species and E. faecalis Symbioflor 1. In total, sporadic distribution of virulence genes was detected among the studied enterococci. Furthermore, the frequency of genes encoding for sex pheromones (ccf and cob), collagen adhesion (ace), cell wall adhesion (efaAfs), and gelatinase (gelE) was observed to be significantly higher in those isolates obtained from infants compared to those obtained from adults. Although the ability of biofilm formation was found in all isolates, the strong biofilm formation was observed in enterococci from infants and strong correlation was observed between the capacities to form biofilm and attachment to Caco-2 cells. Cell-free culture supernatant showed some inhibitory effects on indicator strains, which were related to the production of organic acids (against P. aeruginosa and enteropathogenic E. coli) or both organic acids and proteinaceous antimicrobial agents (against L. monocytogenes and E. faecalis). Approximately, 79% and 71% of the isolates showed strong inhibitory effects on P. aeruginosa and L. monocytogenes, respectively. Unlike lactobacilli, enterococcal cell-free supernatants had no toxicity on intestinal cells. In conclusion, this study shows that some enterococcal isolates obtained from fecal microbiota have characteristics, which are comparable with the known probiotic bacteria. Therefore, these isolates should be considered to find probiotic candidate. The proteinaceous identity of antimicrobial substances derived from these isolates highlighted the probable contribution of bacteriocins into this issue.

Effect of shark cartilage derived protein on the NK cells activity.

CONTEXT: Shark cartilage has been used for its beneficial effects on various diseases. There are evidences, that shark cartilage stimulates cellular and humoral immune responses, which makes it an anti-tumor and immunomodulator candidate. OBJECTIVE: The immunostimulatory effect of shark cartilage derived proteins on the cytotoxic activity of natural killer (NK) cells from healthy human peripheral blood mononuclear cells was studied. MATERIAL AND METHODS: The shark cartilage was extracted and its bioactive proteins were purified using ion-exchange chromatography (DE-52) and sequential fractionation on Amicon ultrafiltration membranes. The effect of each protein fraction on the modulation of cytotoxic activity of NK cells, as effectors, against K562, as target cells, was assayed by enzymatic lactate dehydrogenase test. RESULTS: The most immunostimulatory effect on the cytotoxic activity of NK cells was observed for AR10 fraction, containing proteins with molecular weight of about 14.5 kDa on the reducible discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis. DISCUSSION: Among the examined shark cartilage derived proteins, the most immunostimulatory effects on the NK cells cytotoxicity was found for AR10 fraction with molecular weight of about 14 kDa. We propose-the direct interactions of shark cartilage derived proteins with NK cells surface receptors may lead to the enhancing in the cytotoxic activity of NK cells. CONCLUSION: Thus AR10 fraction, proteins of about 14.5 kDa, has a novel immunostimulatory effect on the NK cells activity in vitro and if confirmed by in vivo trials, it may lead to its future clinical applications as, immunotherapy of cancer, HIV, and augmentation of host immune system related immunodeficiency disorders.

Chemotherapeutic drugs: Cell death- and resistance-related signaling pathways. Are they really as smart as the tumor cells?

Chemotherapeutic drugs kill cancer cells or control their progression all over the patient's body, while radiation- and surgery-based treatments perform in a particular site. Based on their mechanisms of action, they are classified into different groups, including alkylating substrates, antimetabolite agents, anti-tumor antibiotics, inhibitors of topoisomerase I and II, mitotic inhibitors, and finally, corticosteroids. Although chemotherapeutic drugs have brought about more life expectancy, two major and severe complications during chemotherapy are chemoresistance and tumor relapse. Therefore, we aimed to review the underlying intracellular signaling pathways involved in cell death and resistance in different chemotherapeutic drug families to clarify the shortcomings in the conventional single chemotherapy applications. Moreover, we have summarized the current combination chemotherapy applications, including numerous combined-, and encapsulated-combined-chemotherapeutic drugs. We further discussed the possibilities and applications of precision medicine, machine learning, next-generation sequencing (NGS), and whole-exome sequencing (WES) in promoting cancer immunotherapies. Finally, some of the recent clinical trials concerning the application of immunotherapies and combination chemotherapies were included as well, in order to provide a practical perspective toward the future of therapies in cancer cases.

A Three-Dimensional Scaffold-Based System for Modeling the Bone Marrow Tissue.

Hematopoietic stem and progenitor cells (HPC) niche, consisting of HPC and their surrounding stromal components, is the fundamental unit for bone marrow (BM) tissue engineering. Previously, mouse BM-derived cell complexes with HPC niche unit properties called "niche-like units" were isolated and characterized. This study was aimed to evaluate the possibility of bioengineering marrow tissue in heterotypic sites using niche-like units in combination with three-dimensional scaffolds. BM niche-like units were isolated from GFP-transgenic C57BL/6 mice and seeded on electrospun poly (L-lactide) nanofiber scaffolds, which were then roll-folded and aseptically implanted into the peritoneal cavity of irradiated wild-type mice. One month after implantation, donor-derived cells were detected in peripheral blood of the recipients and contributed to restoration of all blood lineages. The transplanted bioengineered tissue histologically resembled native BM structure and was connected to the mouse systemic circulation. Long-term self-renewal was confirmed by serial transplantation into tertiary recipients. In conclusion, this study establishes a novel system for BM tissue engineering, which can be used to improve the HPC transplantation outcomes especially in cases where HPC niche is damaged and also as an in vivo model to test the effects of different factors on hematopoiesis.

BCc1, the novel antineoplastic nanocomplex, showed potent anticancer effects in vitro and in vivo.

PURPOSE: In spite of all the efforts and researches on anticancer therapeutics, an absolute treatment is still a myth. Therefore, it is necessary to utilize novel technologies in order to synthesize smart multifunctional structures. In this study, for the first time, we have evaluated the anticancer effects of BCc1 nanocomplex by vitro and in vivo studies, which is designed based on the novel nanochelating technology. METHODS: Human breast adenocarcinoma cell line (MCF-7) and mouse embryonic fibroblasts were used for the in vitro study. Antioxidant potential, cell toxicity, apoptosis induction, and CD44 and CD24 protein expression were evaluated after treatment of cells with different concentrations of BCc1 nanocomplex. For the in vivo study, mammary tumor-bearing female Balb/c mice were treated with different doses of BCc1 and their effects on tumor growth rate and survival were evaluated. RESULTS: BCc1 decreased CD44 protein expression and increased CD24 protein expression. It induced MCF-7 cell apoptosis but at the same concentrations did not have negative effects on mouse embryonic fibroblasts viability and protected them against oxidative stress. Treatment with nanocomplex increased survival and reduced the tumor size growth in breast cancer-bearing balb/c mice. CONCLUSION: These results demonstrate that BCc1 has the capacity to be assessed as a new anticancer agent in complementary studies.

Tumor Associated Mesenchymal Stromal Cells Show Higher Immunosuppressive and Angiogenic Properties Compared to Adipose Derived MSCs.

BACKGROUND: Differentiation, migratory properties and availability of Mesenchymal Stromal Cells (MSC) have become an important part of biomedical research. However, the functional heterogeneity of cells derived from different tissues has hampered providing definitive phenotypic markers for these cells. OBJECTIVE: To characterize and compare the phenotype and cytokines of adipose derived MSCs (AD-MSCs) and tumoral-MSCs (T-MSCs) isolated from mammary tumors of BALB/c mice. METHODS: Immunophenotyping and in vitro differentiation tests were used for MSC characterization. Cytokine and enzyme profiles were assessed using ELISA and Real-time PCR, respectively. RESULTS: T-MSCs expressed significantly higher levels of HLA-DR (p=0.04). Higher levels of PGE2 and COX-2 enzyme were also observed in T-MSCs (p=0.07 and p=0.00, respectively). Additionally, T-MSCs expressed higher levels of iNOS and MMP9 (p=0.01 and p=0.01, respectively). T-MSCs were also able to induce higher levels of proliferation and migration of HUVEC endothelial cells in wound scratch assay compared to AD-MSCs (p=0.015). CONCLUSION: Functional differences showed by the surface markers of MSCs, cytokine and enzyme production indicate the effect of different microenvironments on MSCs phenotype and function.

Long acting propranolol and HSP-70 rich tumor lysate reduce tumor growth and enhance immune response against fibrosarcoma in Balb/c mice.

BACKGROUND: Noradrenaline (NA), the principal neurotransmitter released from sympathetic nerve terminals, influences T-cell maturation, not only directly in developing T cells, but also indirectly, by acting on the thymic nonlymphoid cells. In vitro and in vivo studies have demonstrated the anti-proliferative, anti-migratory, anti-angiogenic and cytotoxic properties of propranolol, ß-AR blocker, against various cancers. OBJECTIVES: To evaluate the effect of propranolol on efficacy of HSP-70 rich lysate vaccine in immunotherapy of fibrosarcoma. METHODS: Mouse fibrosarcoma WEHI-164 cells were used to immunize tumor-bearing mice with or without propranolol and HSP-70. Splenocytes proliferation, cytotoxicity activity of the splenocytes, naturally occurring CD4+ CD25high T-reg cells and IFN-γ and IL-4 secretion as well as tumor size, were assessed to describe the anti-tumor immune response. RESULTS: A significant increase in the level of IFN-γ in the mice vaccinated with WEHI-164 cells enriched with HSP-70 and co-treated with propranolol was observed compared to controls. However, HSP enrichment or propranolol treatment alone did not enhance the immune response as measured by the level of IFN-γ. Likewise, a decrease in tumor growth in the test group (p<0.01) and a significant increase in CTL activity (p<0.05) was observed. CONCLUSION: HSP enriched vaccine shows anti-tumor activity, probably due to the modulation of immune responses.

Co-administration of GP96 and Her2/neu DNA vaccine in a Her2 breast cancer model.

Heat-shock proteins have biochemical and immunological roles in chaperoning/signaling and activation of innate and adaptive immune responses, respectively. Their effect on the immune response is due to a phenomenon known as cross-priming of antigen, in which exogenous antigens are presented via MHC class I by antigen presenting cells. GP96 exerts adjuvant activity with some viral and bacterial antigens when applied in the form of a DNA vaccine. In this study, animals with Her2-expressing tumors were vaccinated by co-administration of GP96+ Her2/neu DNA vaccines. Analyses of the immune response, 2 weeks after the last immunization revealed decreased CD4+ CD25+ Foxp3+ naturally occurring regulatory T cells (Tregs) at the tumor site and increased IFN-γ/IL-4 level. Nevertheless, the graph of tumor size demonstrated a bi-phasic pattern in which partial control of tumor progression initially occurred, but finally its effectiveness was inversely affected by tumor size.

A comparison of low-dose cyclophosphamide treatment with artemisinin treatment in reducing the number of regulatory T cells in murine breast cancer model.

BACKGROUND: Artemisinin (ART) is a sesquiterpene lactone. Possessing an endoperoxide bridge is unique among antimalarial drugs, and now much attention is focused on the anti-cancer properties of ART. In this study we aimed at the immunomodulatory effects of artemisinin in the treatment of breast cancer in comparison to the conventional anti-cancer drug, cyclophosphamide (CTX). METHODS: We examined delayed-type hypersensitivity, antibody and IL-4 and IFN gamma production, tumour volume, tumour infiltrated regulatory T cells (Treg) and spleen lymphocyte proliferation assay. Briefly three groups of five 4-6 week old female Balb/c tumour-bearing mice (mouse mammary tumour) were treated with 2.8 mg/kg ART and 20mg/kg CTX intraperitoneally for 20 consecutive days. Tumour volume was measured using a digital vernier calliper (with accuracy of 0.01). Mice were sacrificed and percentage of tumour infiltrating Tregs were obtained using flow cytometry (BD, USA). Proliferation of splenocytes was obtained using BrdU proliferation assay (Roche). RESULTS: Our results showed that ART can reduce the number of Tregs in tumour stroma (P-value or=0.05) and control. Furthermore ART increased IFN gamma/IL-4 ratio produced in splenocyte culture (P-value or=0.05). DISCUSSION: Cancer is a multi-factorial disease which needs a multi-approach treatment. Early accumulation of Treg cells in the tumour tissue correlates with tumour progression and is an indication of bad prognosis. According to the obtained results, ART can reduce the number of Tregs. We suggest using artemisinin, with its dual action mechanism. It can effectively kill cancer cells along with reducing the suppressive microenvironment.