Antibacterial efficacy of novel bismuth-silver nanoparticles synthesis on Staphylococcus aureus and Escherichia coli infection models.

Introduction: The emergence of multi-drug-resistant bacteria is one of the main concerns in the health sector worldwide. The conventional strategies for treatment and prophylaxis against microbial infections include the use of antibiotics. However, these drugs are failing due to the increasing antimicrobial resistance. The unavailability of effective antibiotics highlights the need to discover effective alternatives to combat bacterial infections. One option is the use of metallic nanoparticles, which are toxic to some microorganisms due to their nanometric size. Methods: In this study we (1) synthesize and characterize bismuth and silver nanoparticles, (2) evaluate the antibacterial activity of NPs against Staphylococcus aureus and Escherichia coli in several infection models (in vivo models: infected wound and sepsis and in vitro model: mastitis), and we (3) determine the cytotoxic effect on several cell lines representative of the skin tissue. Results and discussion: We obtained bimetallic nanoparticles of bismuth and silver in a stable aqueous solution from a single reaction by chemical synthesis. These nanoparticles show antibacterial activity on S. aureus and E. coli in vitro without cytotoxic effects on fibroblast, endothelial vascular, and mammary epithelium cell lines. In an infected-wound mice model, antibacterial effect was observed, without effect on in vitro mastitis and sepsis models.

Antitumoral and Immunogenic Capacity of ß-D-Glucose-Reduced Silver Nanoparticles in Breast Cancer.

Immunogenic cell death (ICD) is a type of cell death capable of stimulating immunity against cancer through danger signals that lead to an adaptive immune response. Silver nanoparticles (AgNPs) have been shown to have a cytotoxic effect on cancer cells; however, their mechanism of action is not fully understood. The present study synthesized, characterized, and evaluated the cytotoxic effect of beta-D-glucose-reduced AgNPs (AgNPs-G) against breast cancer (BC) cells in vitro; and assess the immunogenicity of cell death in vitro and in vivo. The results showed that AgNPs-G induce cell death in a dose-dependent manner on BC cell lines. In addition, AgNPs show antiproliferative effects by interfering with the cell cycle. Regarding the detection of damage-associated molecular patterns (DAMPs), it was found that treatment with AgNPs-G induces calreticulin exposure and the release of HSP70, HSP90, HMGB1, and ATP. In vivo, prophylactic vaccination did not prevent tumor establishment; however, tumor weight was significantly lower in AgNPs-G vaccinated mice, while the survival rate increased. In conclusion, we have developed a new method for the synthesis of AgNPs-G, with in vitro antitumor cytotoxic activity on BC cells, accompanied by the release of DAMPs. In vivo, immunization with AgNPs-G failed to induce a complete immune response in mice. Consequently, additional studies are needed to elucidate the mechanism of cell death that leads to the design of strategies and combinations with clinical efficacy.

Putative Wound Healing Induction Functions of Exosomes Isolated from IMMUNEPOTENT CRP.

Chronic wounds in diabetic patients can take months or years to heal, representing a great cost for the healthcare sector and impacts on patients' lifestyles. Therefore, new effective treatment alternatives are needed to accelerate the healing process. Exosomes are nanovesicles involved in the modulation of signaling pathways that can be produced by any cell and can exert functions similar to the cell of origin. For this reason, IMMUNEPOTENT CRP, which is a bovine spleen leukocyte extract, was analyzed to identify the proteins present and is proposed as a source of exosomes. The exosomes were isolated through ultracentrifugation and shape-size, characterized by atomic force microscopy. The protein content in IMMUNEPOTENT CRP was characterized by EV-trap coupled to liquid chromatography. The in silico analyses for biological pathways, tissue specificity, and transcription factor inducement were performed in GOrilla ontology, Panther ontology, Metascape, and Reactome. It was observed that IMMUNEPOTENT CRP contains diverse peptides. The peptide-containing exosomes had an average size of 60 nm, and exomeres of 30 nm. They had biological activity capable of modulating the wound healing process, through inflammation modulation and the activation of signaling pathways such as PIP3-AKT, as well as other pathways activated by FOXE genes related to specificity in the skin tissue.

Design, Synthesis, and Evaluation of Peptides Derived from L1 Protein Against Bovine Papillomavirus-1/2 Identified Along Mexico's Cattle Export Route.

Introduction: Bovine papillomatosis affects animal health and represents one of the greatest economic losses in the livestock sector. New control and prevention methods to protect the livestock industry from this disease are necessary. The aim of this study was to evaluate a candidate peptide for antibody production against bovine papillomavirus (BPV). Material and Methods: A total of 64 cattle underwent wart excision among 5,485 cattle distributed over 2 to 4 farms per state and 12 farms in total in the four Mexican states of Tabasco, Chiapas, Veracruz, and Nuevo León. The prevalence of bovine papillomatosis per farm was calculated by wart visualisation. The warts were genotyped by PCR and sequenced, then a phylogenetic tree was built using MEGA X software. A synthetic peptide was designed in the ABCpred, Bepipred 2.0, Bepipred IDBT, Bepitope, LBtope, and MHC II predictor online server software's based on the C-terminal region of the L1 protein. Mice antibody production was induced by subcutaneous immunisation with 50 µg of synthetic peptide and evaluated by indirect ELISA. Results: The prevalence of BPV was higher in Tabasco, Chiapas, and Veracruz. Bovine papillomaviruses 1 and 2 were found in all representative samples. A phylogenetic tree showed that Mexican sequences were located in exclusive clades yet were highly related to international ones. The peptide immunisation induced antibody titres of 1 : 10,000/1 : 1,000,000 against synthetic peptide and whole wart lysate (WWL), respectively. Conclusion: Co-infections of BPV-1 and -2 were found in all four states. Immunisation of BALB/C mice with BPV-1/2-derived synthetic peptide based on the C-terminal region of the major viral capsid protein L1 induced the production of specific antibodies able to recognise BPV-1/2 viral particles from bovine WWL.

In vitro chemosensitivity of a canine tumor venereal transmissible cancer cell line.

The canine transmissible venereal tumor (CTVT) is the most common malignity in dogs. Because there are reports that this tumor is resistant to vincristine sulfate, the chemotherapeutic options are scarce, and the development of new therapeutic approaches is necessary. In this study, we evaluated the cytotoxic activity of vincristine, doxorubicin, temozolomide, panobinostat, toceranib, gemcitabine, cisplatin, fluorouracil, cyclophosphamide, and methotrexate on a CTVT cell line, determining that all drugs decreased the viability in a dose-dependent manner. Furthermore, they inhibit cellular migration in a time- and drug-dependent manner, as evaluated by the wound healing assay. On the other hand, vincristine, panobinostat, gemcitabine, toceranib, cyclophosphamide, and methotrexate increased the percentage of cells in the subG1 phase, and doxorubicin, temozolomide, gemcitabine, toceranib, and methotrexate decreased the percentage of cells in the synthesis phase. To efficientize the use of vincristine, only toceranib increased the cytotoxic effect of vincristine in a synergistic manner. Our results confirm the use of vincristine as the gold standard for CTVT treatment as monotherapy and suggest the use of a combinatorial and sequential treatment with toceranib.

Orthotopic and heterotopic triple negative breast cancer preclinical murine models: A tumor microenvironment comparative.

Breast cancer is the most frequent type of cancer worldwide and triple negative breast cancer is a particularly aggressive subtype. Novel therapies for the treatment of cancer patients focus on the remodeling of the tumor microenvironment (TME). Orthotopic and heterotopic syngeneic mice are the most common model used to study the TME in preclinical research. Despite this, there are no published studies that address the differences between orthotopic and heterotopic murine breast cancer models at the TME level. In this report we compared proliferation, immune cell infiltrates, extracellular matrix, vascular density, and response to chemotherapy between the mammary fat pad orthotopic model, and the air pouch heterotopic model. Our study shows that the orthotopic tumors form more metastasis, however, the heterotopic tumors grow larger, have a higher FOXP3 cell infiltrate, and resemble more accurately the breast cancer TME. Our findings show that both models are very similar, there are however some differences that should be considered in the experimental design of preclinical studies.

Evaluation of the cytotoxic and immunogenic potential of temozolamide, panobinostat, and Lophophora williamsii extract against C6 glioma cells.

Glioblastoma multiforme is a malignant neoplasm of the brain with poor prognosis. The first-line drug against glioblastoma is the alkylating agent temozolamide (TMZ); unfortunately, treatment resistance and tumor re-incidence are common. In some cases, immunogenic cell death (ICD) inducers can decrease treatment resistance and tumor recurrence by stimulating an antitumor specific immune response. Not all ICD inducers, however, are suitable for glioma patients because of the low permeability of the blood-brain barrier (BBB). Panobinostat (PAN), a histone deacetylase inhibitor and Lophophora williamsii (LW) extract can pass through the BBB and have antitumor properties. The aim of this study is to evaluate the cytotoxic potential of TMZ, PAN and LW extract against the glioma C6 cell line, and its role in the release of damage-associated molecular patterns (DAMPs), which is a hallmark of ICD. Our results indicate that all treatments induce cellular death in a time- and concentration-dependent manner, and that PAN and LW extract induce apoptosis, whereas TMZ induces apoptosis and necrosis. Also, that some of the treatments and their sequential administration induce the release of DAMPs. Furthermore, in a rat glioma model, we observed that all treatments decreased tumor volume, but the in vivo cell death mechanism was not ICD. Our findings indicate that TMZ, PAN, and LW combination have a cytotoxic effect against glioma cells but do not induce ICD.

IMMUNEPOTENT CRP plus doxorubicin/cyclophosphamide chemotherapy remodel the tumor microenvironment in an air pouch triple-negative breast cancer murine model.

In 1889, Steven Paget postulated the theory that cancer cells require a permissive environment to grow. This permissive environment is known as the tumor microenvironment (TME) and nowadays it is evident that the TME is involved in the progression and response to therapy of solid cancer tumors. Triple-negative breast cancer is one of the most lethal types of cancer for women worldwide and chemotherapy remains the standard treatment for these patients. IMMUNEPOTENT CRP is a bovine dialyzable leukocyte extract with immunomodulatory and antitumor properties. The combination of chemotherapy and IMMUNEPOTENT CRP improves clinical parameters of breast cancer patients. In the current study, we aimed to evaluate the antitumor effect of doxorubicin/cyclophosphamide chemotherapy plus IMMUNEPOTENT CRP and its impact over the tumor microenvironment in a triple-negative breast cancer murine model. We evaluated CD8+, CD4+, T regulatory cells, memory T cells, myeloid-derived suppressor cells, CD71+, innate effector cells and molecules such as α-SMA, VEGF, CTLA-4, PD-L1, Gal-3, IDO, IL-2, IFN-γ, IL-12, IL-6, MCP-1, and IL-10 as part of the components of the TME. Doxorubicin/cyclophosphamide + IMMUNEPOTENT CRP decreased tumor volume, prolonged survival, increased infiltrating and systemic CD8+ T cells and decreased tumor suppressor molecules (such as PD-L1, Gal-3, and IL-10 among others). In conclusion, we suggest that IMMUNEPOTENT CRP act as a modifier of the TME and the immune response, potentiating or prolonging anti-tumor effects of doxorubicin/cyclophosphamide in a triple-negative breast cancer murine model.

A Novel Gold Calreticulin Nanocomposite Based on Chitosan for Wound Healing in a Diabetic Mice Model.

The development of new nanomaterials to promote wound healing is rising, because of their topical administration and easy functionalization with molecules that can improve and accelerate the process of healing. A nanocomposite of gold nanoparticles (AuNPs) functionalized with calreticulin was synthetized and evaluated. The ability of the nanocomposite to promote proliferation and migration was determined in vitro, and in vivo wound healing was evaluated using a mice model of diabetes established with streptozotocin (STZ). In vitro, the nanocomposite not affect the cell viability and the expression of proliferating cell nuclear antigen (PCNA). Moreover, the nanocomposite promotes the clonogenicity of keratinocytes, endothelial cells, and fibroblasts, and accelerates fibroblast migration. In vivo, mice treated with the nanocomposite presented significantly faster wound healing. The histological evaluation showed re-epithelization and the formation of granular tissue, as well as an increase of collagen deposition. Therefore, these results confirm the utility of AuNPs⁻calreticulin nanocomposites as potential treatment for wound healing of diabetic ulcers.

Immunotherapy for the treatment of canine transmissible venereal tumor based in dendritic cells pulsed with tumoral exosomes.

Context: Exosomes secreted by tumor cells are a good source of cellular components that stimulate the immune response, such as alarmins (mRNA, tetraspanins (CD9, CD63, CD81), heat-shock proteins, major histocompatibility complex class I molecules) and tumor-associated antigens. These properties permit to pulsed dendritic cells in the immunotherapy for many cancers types. The aim of this study was to demonstrate the use of exosomes derived from canine transmissible venereal tumor (CTVT) as an antigen to pulsed dendritic cells and its administration in dogs with CTVT as treatment against this disease. Material and methods: From primary culture of CTVT cells the exosomes were isolated and characterized by scanning electron microscopy assay, dot blot and protein quantification. The monocytes of each patient were differentiated to dendritic cells (DC) and pulsed with CTVT exosomes (CTVTE). Phagocytosis, tumor size, populations of lymphocytes and IFN-c levels were evaluated. Results: The CTVTE showed a size around 90 nm. CD81, CD63, CD9 and Hsp70 were expressed. Monocytes showed an expression of 85.71% for CD14+, 12.3% for CD80+, 0.1% for CD83+ and 0.8% for DLA-II. In DC 5.1% for CD14+, 86.7% for CD80+, 90.1% for CD83+ and 92.6% for DLA-II and a phagocytosis of 63% was obtained by FITC Dextran test. No side effects were observed in the experimental groups with our therapy. Tumor regression was of 100% at the seventh week, as well as an increase in the level of IFN-γ (142 pg/ml), and CD4+ (28%) and CD8+ (34%) cell percentage. Discusion and conclusion: These results have shown that DC pulsed with tumor exosomes induce regression of the TVT in dogs.