Exosomes isolated from IMMUNEPOTENT CRP, a hemoderivative, to accelerate diabetic wound healing.

The increasing risk of amputation due to diabetic foot ulcer calls for new therapeutic options; for that, we determined the role of IMMUNEPOTENT CRP (ICRP) and its parts in the wound healing process of superficial wounds in diabetic BALB/c mice. A potency test was performed to confirm the batch of ICRP, and then its parts were separated into pellets, supernatants, and exosomes, and another group of exosomes loaded with insulin was added. Viability and scratch healing were assessed in NIH-3T3, HUVEC, and HACAT cell lines. Diabetes was induced with streptozotocin, and wounds were made by dissecting the back skin. Treatments were topically applied, and closure was monitored; inflammatory cytokines in sera were also evaluated by flow cytometry, and histological analysis was performed by Masson's staining and immunohistochemistry for p-AKT, p-FOXO, p-P21, and p-TSC2. ICRP pellets and exosomes increased cellular viability, and exosomes and exosome-insulin accelerated scratch healing in vitro. Exosome-insulin releases insulin constantly over time in vitro. In vivo, treatments accelerated wound closure, and better performance was observed in pellet, exosome, and exosome-insulin treatments. Best collagen expression was induced by ICRP. P-AKT and p-FOXO were overexpressed in healing tissues. Inflammatory cytokines were downregulated by all treatments. In conclusion, IMMUNEPOTENT CRP components, especially exosomes, and the process of encapsulation of exosome-insulin accelerate diabetic wound healing and enhance cellular proliferation, collagen production, and inflammation modulation through the phosphorylation of components of the AKT pathway.

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.

Development of a new generation of miniemulsion based on cottonseed oil with α-tocopherol and ZnO and evaluation of its adjuvant activity.

Background: Emulsions have been widely used as immunological adjuvants. But the use of materials derived from plants such as cottonseed oil, alpha-tocopherol, or minerals such as zinc, as well as their use at the nanometric scale has been little explored. In this study, we develop a new miniemulsion and evaluated its antioxidant and phagocytic capacity, as well as parameters related to immune response stimulation by cytokine expression and antibodies production in a mice model. Methods: Formulated CN (cottonseed oil miniemulsion) and CNZ (cottonseed oil miniemulsion whit zinc oxide nanoparticles) miniemulsions were characterized by scanning electronic microscopy SEM, DLS and FT-IR. In murine macrophages, splenocytes and thymocytes primary cultures safety and cytotoxicity were determined by MTT. In macrophages the antioxidant and phagocytic capacity was evaluated. In BALB/c mice, the stimulation of the immune system was determined by the expression of cytokines and the production of antibodies. Results: The CN and CNZ presented stability for 90 days. Immediately after preparation, the CN presented a higher particle size (543.1 nm) than CNZ (320 nm). FT-IR demonstrated the correct nanoparticle synthesis by the absence of sulfate groups. CN and CNZ (1.25 to 10 µL/mL) had no toxic effect on macrophages (p = 0.108), splenocytes (p = 0.413), and thymocytes (p = 0.923). All CN and CNZ doses tested induced nitric oxide and antioxidants production in dose dependent manner when compared with control. CN-ovalbumin and CNZ-ovalbumin treatments in femoral subcutaneous tissue area showed inflammation with higher leukocyte infiltration compared with FCA. The intraperitoneal administration with CN, CNZ, and FCA showed a higher total intraperitoneal cells recruitment (CD14+) after 24 h of inoculation than control (p = 0.0001). CN and CNZ increased the phagocyte capacity with respect to untreated macrophages in the Candida albicans-phagocytosis assay. The evaluation of residual CFU indicated that only CN significantly decreased (p = 0.004) this value at 3 h. By other side, only CN increased (p = 0.002) the nitric oxide production. CNZ stimulated a major INFγ secretion compared with FCA at day 7. A major IL-2 secretion was observed at days 7 and 14, stimulated with CN and CNZ. Both miniemulsions did not affect the antibody isotypes production (IgG1, IgG2a, IgG3, IgA and IgM) at days 7, 14, 28, and 42. CN induced a significant IgG production against OVA, but lesser than FCA. Conclusions: The two new miniemulsions with adjuvant and antioxidant capacity, were capable of generating leukocyte infiltration and increased cytokines and antibodies production.

Antitumor efficacy of silver nanoparticles reduced with ß-D-glucose as neoadjuvant therapy to prevent tumor relapse in a mouse model of breast cancer.

Introduction: Neoadjuvant therapy constitutes a valuable modality for diminishing tumor volume prior to surgical resection. Nonetheless, its application encounters limitations in the context of recurrent tumors, which manifest resistance to conventional treatments. Silver nanoparticles (AgNPs) have emerged as a promising alternative for cancer treatment owing to their cytotoxic effects. Methods: Cellular viability was assessed by Alamar blue assay in 4T1 breast cancer cell line. Silver biodistribution was detected by an inductively coupled plasma optical emission spectrometer in an in vivo mice model. For neoadjuvant evaluation, mice were randomized and treated intratumoral with AgNPs-G or intraperitoneally with doxorubicin (DOX) as a control. Recurrence was determined after 170 days by counting lung metastatic nodules (dyed with Bouin solution) with histological confirmation by H&E. Masson's stain, Ki67 immunohistochemistry, and a TUNEL assay were performed in lungs from treated mice. Results: AgNPs-G reduced 4T1 cell viability and in an ex vivo assay the AgNPs-G decreased the tumor cell viability. After intravenous administration of AgNPs-G were detected in different organs. After intratumor administration, AgNPs-G are retained. The AgNPs-G treatment significantly reduced tumor volume before its surgical resection. AgNPs-G reduced the development of lung metastatic nodules and the expression of Ki67. TUNEL assay indicated that AgNPs-G didn't induce apoptosis. Conclusions: We concluded that intratumor administration of AgNPs-G reduced tumor volume before surgical resection, alongside a reduction in lung metastatic nodules, and Ki67 expression. These findings provide valuable insights into the AgNPs-G potential for intratumor and neoadjuvant cancer therapies. However, further research is needed to explore their full potential and optimize their use in clinical settings.

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.

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.