ß-D-Glucose-Reduced Silver Nanoparticles Remodel the Tumor Microenvironment in a Murine Model of Triple-Negative Breast Cancer.

Breast cancer is the most diagnosed type of cancer worldwide and the second cause of death in women. Triple-negative breast cancer (TNBC) is the most aggressive, and due to the lack of specific targets, it is considered the most challenging subtype to treat and the subtype with the worst prognosis. The present study aims to determine the antitumor effect of beta-D-glucose-reduced silver nanoparticles (AgNPs-G) in a murine model of TNBC, as well as to study its effect on the tumor microenvironment. In an airbag model with 4T1 tumor cell implantation, the administration of AgNPs-G or doxorubicin showed antitumoral activity. Using immunohistochemistry it was demonstrated that treatment with AgNPs-G decreased the expression of PCNA, IDO, and GAL-3 and increased the expression of Caspase-3. In the tumor microenvironment, the treatment increased the percentage of memory T cells and innate effector cells and decreased CD4+ cells and regulatory T cells. There was also an increase in the levels of TNF-α, IFN-γ, and IL-6, while TNF-α was increased in serum. In conclusion, we suggest that AgNPs-G treatment has an antitumor effect that is demonstrated by its ability to remodel the tumor microenvironment in mice with TNBC.

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.

Aluminosilicates as a Double-Edged Sword: Adsorption of Aflatoxin B1 and Sequestration of Essential Trace Minerals in an In Vitro Gastrointestinal Poultry Model.

Aflatoxins can cause intoxication and poisoning in animals and humans. Among these molecules, aflatoxin B1 (AFB1) is the most dangerous because of its carcinogenic and mutagenic properties. To mitigate these effects, clay adsorbents are commonly included in the diet of animals to adsorb the carcinogens and prevent their absorption in the gastrointestinal tract. In this study, four clays, three smectites (C-1, C-2, and C-3), and one zeolite (C-4), were compared as adsorbents of AFB1 and trace inorganic nutrients using an in vitro gastrointestinal model for poultry. Characterization of the clays using Fourier transform infrared spectroscopy revealed characteristic bands of smectites in C-1, C-2, and C-3 (stretching vibrations of Si-O, Al-O-Si, and Si-O-Si). The C-4 presented bands related to the bending vibration of structural units (Si-O-Si and Al-O-Si). X-ray diffraction analysis showed that C-1 is a montmorillonite, C-2 is a beidellite, C-3 is a beidellite-Ca-montmorillonite, and C-4 is a clinoptilolite. The elemental compositions of the clays showed alumina, silica, iron, calcium, and sodium contents. The cation exchange capacity was higher in C-3 clay (60.2 cmol(+)/kg) in contrast with the other clays. The AFB1 adsorption of C-1 was the highest (98%; p ˂ 0.001), followed by C-2 (94%). However, all the clays also sequestered trace inorganic nutrients (Fe, Mn, Zn, and Se). Both smectites, montmorillonite and beidellite, were the most suitable for use as adsorbents of AFB1.

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.

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.

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.

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.

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.

The Inflammatory Process Modulates the Expression and Localization of WT1 in Podocytes Leading to Kidney Damage.

BACKGROUND/AIM: Wilms' tumor 1 (WT1) is involved in the development of the urogenital system and is expressed in podocytes throughout life. Inflammation of renal glomeruli causes renal damage-induced nephrotic syndrome and steroid-resistant nephrotic syndrome have mutations in the WT1 gene. The aim of this work was to determine if the inflammatory process modulates the expression and localization of WT1 in podocytes that cause kidney damage using lipopolysaccharide (LPS)-treated mice as a sepsis model. MATERIALS AND METHODS: In investigation of renal damage, proteinuria and histology were analyzed. WT1 modulation was analyzed by indirect immunofluorescence, immunohistochemistry and western blot assays, and proinflammatory cytokines were analyzed by quantitative polymerase chain reaction assay. RESULTS: WT1 expression decreased most at 24 and 36 h after the induction of inflammation and phosphorylated WT1 was mainly localized in the cytoplasm, reduced nephrin mRNA expression and increased mRNA expression of tumor necrosis factor α and interleukin 1ß. CONCLUSION: These results indicate that the immune system plays an important role in the modulation of WT1, leading to kidney damage.

Fatal Case of Rabies in a Captive White-Tailed Deer: A Case Report from Chiapas, Mexico.

Rabies is a fatal viral infection that causes enc ephalitis in warm-blooded animals, including humans. Dog-transmitted rabies is considered eradicated in Mexico; however, rabies is not being tested in livestock with neurological symptoms (one of the main manifestations of rabies disease). In this case report, we describe a rabies case in a white-tailed deer in the Santo Domingo ranch, in Catazajá, Chiapas, Mexico, where white-tailed deer are kept under captivity, and are meant for human consumption. This is the first report of a rabies case in white-tailed deer in Mexico. We also describe the challenges to obtain a rabies diagnosis and the lack of public health policies to ensure containment of the disease, as well as the lack of awareness among farmers in the area. One single confirmed case of rabies indicates that more animals are affected by the disease. The risk for human health and economical losses will remain unknown until rabies tests are routinely performed in animals that present neurological symptoms.

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.

Triggering of protease-activated receptors (PARs) induces alternative M2 macrophage polarization with impaired plasticity.

Protease-activated receptors (PARs) have been described in a wide diversity of vertebrate cells, including human immune cells. Macrophages are pivotal cells in the host-pathogen interaction and their polarization in M1 or M2 cells has been described as a new central paradigm in the immune response to pathogens. In this context, we explored the involvement of PAR activation by serine proteases on M1/M2 macrophage differentiation and their impact on the Th1/Th2 cytokine profile in response to Mycobacterium tuberculosis antigen. Our results demonstrate that the serine proteases, thrombin and trypsin, induce interleukin (IL)-4 release from human monocytes, together with upregulation of the macrophage mannose receptor (CD206) in the same way that alternative M2a differentiated cells with M-CSF/IL-4. Protease stimulation of monocytes in the presence of PAR-1 (SCH-79797) or PAR-2 (FSLLRY-NH2) antagonists abolished IL-4 release from monocytes, whereas the use of the peptide agonist for PAR-1 (SFLLRNPNDKYEPF-NH2) or PAR-2 (SLIGKV-NH2) induced the secretion of IL-4 at a level comparable to thrombin or trypsin. When these protease-induced M2 macrophages from healthy human PPD + donors were co-cultured with autologous lymphocyte population in the presence of Mycobacterium tuberculosis antigen, we found a consistent inhibition of IFN-γ/IL-12 release together with persistent IL-4 expression, in contrast to the expected Th1 profile obtained with M2a macrophages. To our knowledge, this is the first observation that proteolytic activation of PAR1/2 receptors in monocytes induces M2-like macrophages with impaired plasticity and their implication in the driving of the Th1/Th2 cytokine profile.

Inhibitory Effect of Cuphea aequipetala Extracts on Murine B16F10 Melanoma In Vitro and In Vivo.

Cuphea aequipetala (C. aequipetala) has been used in Mexican traditional medicine since prehispanic times to treat tumors. In this paper, we evaluated the antiproliferative and apoptotic effect of the methanolic and aqueous extracts of C. aequipetala on several cancer cell lines including the B16F10 cell line of murine melanoma and carried a murine model assay. In vitro assay analyzed the effect in the cellular cycle and several indicators of apoptosis, such as the caspase-3 activity, DNA fragmentation, phosphatidylserine exposure (Annexin-V), and induction of cell membrane permeabilization (propidium iodide) in the B16F10 cells. In vivo, groups of C57BL/6 female mice were subcutaneously injected with 5x105 B16F10 cells and treated with 25 mg/mL of C. aequipetala extracts via oral. Aqueous and methanolic extracts showed a cytotoxic effect in MCF-7, HepG2, and B16F10 cell lines. The methanolic extract showed more antiproliferative effect with less concentration, and for this reason, the in vitro experiments were only continued with it. This extract was able to induce accumulation of cells on G1 phase of the cell cycle; moreover, it was able to induce DNA fragmentation and increase the activity of caspase-3 in B16F10 cells. On the other hand, in the murine model of melanoma, the aqueous extract showed a greater reduction of tumor size in comparison with the methanolic extract, showing an 80% reduction versus one of around 31%, both compared with the untreated control, indicating a better antitumor effect of C. aequipetala aqueous extract via oral administration. In conclusion, the in vitro data showed that both C. aequipetala extracts were able to induce cytotoxicity through the apoptosis pathway in B16F10 cells, and in vivo, the oral administration of aqueous extract reduces the melanoma tumoral mass, suggesting an important antitumoral effect and the perspective to search for effector molecules involved in it.

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.

Immunogenic potential of three transmissible venereal tumor cell lysates to prime canine-dendritic cells for cancer immunotherapy.

Whole tumor cell lysates consist of a mixture of tumor antigens and danger associated molecular patterns (DAMPs) that can be used for dendritic cell maturation and consequently for the activation of a polyclonal T cell-specific tumor response. We evaluated the in vitro efficacy of three different preparations of canine transmissible venereal tumor (TVT) cell lysates: hypochlorous acid-whole tumor cell lysates (HOCl-L), heat shock-whole tumor cell lysates (HS-L), and freeze-thaw cycles-whole tumor cell lysates (FT-L) for the maturation of canine-derived dendritic cells. Our results showed calreticulin, HSP70, and HSP90 release in the three tumor lysates preparations (HOCl-L, HS-L, and FT-L); however, HMGB1 was detected only in HOCl-L and FT-L. Additionally, the uptake by HOCl-L pulsed dendritic cell (DC) increased compared to HS-L and FT-L pulsed DC; and dendritic cell maturation was confirmed by the appropriate cell surface markers (CD11c, CD80, CD83, and MHCII). Furthermore, dendritic cells pulsed with HOCl-L, HS-L or FT-L were cultured with canine lymphocytes. There was an increase of Th1-type cytokines (IL-12, TNF-α, and IFN-γ), in all the tumor cell lysates co-cultures, this correlates with T lymphocyte activation and cytotoxic response. Our data confirm that TVT cell lysates can induce functional canine-DC and that HOCl-L is the most effective one. This preparation of TVT cell lysates with HOCl is an attractive approach that allows the recognition of neoantigens as potential tumor targets and DC priming and therefore could be used for cancer immunotherapy against TVT.

IMMUNEPOTENT CRP induces cell cycle arrest and caspase-independent regulated cell death in HeLa cells through reactive oxygen species production.

BACKGROUND: Regulated cell death (RCD) is a mechanism by which the cell activates its own machinery to self-destruct. RCD is important for the maintenance of tissue homeostasis and its deregulation is involved in diseases such as cervical cancer. IMMUNEPOTENT CRP (I-CRP) is a dialyzable bovine leukocyte extract that contains transfer factors and acts as an immunomodulator, and can be cytotoxic to cancer cell lines and reduce tumor burden in vivo. Although I-CRP has shown to improve or modulate immune response in inflammation, infectious diseases and cancer, its widespread use has been limited by the absence of conclusive data on the molecular mechanism of its action. METHODS: In this study we analyzed the mechanism by which I-CRP induces cytotoxicity in HeLa cells. We assessed cell viability, cell death, cell cycle, nuclear morphology and DNA integrity, caspase dependence and activity, mitochondrial membrane potential, and reactive oxygen species production. RESULTS: I-CRP diminishes cell viability in HeLa cells through a RCD pathway and induces cell cycle arrest in the G2/M phase. We show that the I-CRP induces caspase activation but cell death induction is independent of caspases, as observed by the use of a pan-caspase inhibitor, which blocked caspase activity but not cell death. Moreover, we show that I-CRP induces DNA alterations, loss of mitochondrial membrane potential, and production of reactive-oxygen species. Finally, pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger, prevented both ROS generation and cell death induced by I-CRP. CONCLUSIONS: Our data indicate that I-CRP treatment induced cell cycle arrest in G2/M phase, mitochondrial damage, and ROS-mediated caspase-independent cell death in HeLa cells. This work opens the way to the elucidation of a more detailed cell death pathway that could potentially work in conjunction with caspase-dependent cell death induced by classical chemotherapies.

The novel immunomodulator IMMUNEPOTENT CRP combined with chemotherapy agent increased the rate of immunogenic cell death and prevented melanoma growth.

Immunogenic cell death is a cell death modality that stimulates the immune system to combat cancer cells. IMMUNEPOTENT CRP (ICRP) is a mixture of substances of low molecular weight obtained from bovine spleens that exhibits in vitro cytotoxic activity on different tumor cell lines and modulates the immune response in vivo. The aim of the present study was to determine whether the cytotoxic effect of ICRP and its combination with oxaliplatin (OXP) on murine melanoma B16F10 cells was due to immunogenic cell death. The cytotoxic assay was performed using flow cytometry to detect Annexin V and propidium iodide staining, and calreticulin (CRT) exposure. Adenosine triphosphate, heat shock protein (HSP) 70, HSP90 and high mobility group box 1 (HMGB1) release were identified using bioluminescence, western blot and ELISA assays, respectively. The present in vitro study demonstrated that treatments with ICRP or OXP induced cell death in a time-dependent manner, but treatment with the combination of ICRP + OXP increased the cytotoxic effect following 24 h of treatment. CRT exposure and release of adenosine triphosphate (ATP), HSP70, HSP90 and HMGB1 were induced by treatment with ICRP, and the combination of ICRP + OXP increased the exposure and release of damage-associated molecular patterns (DAMPs), while OXP treatment only induced CRT exposure, ATP and HMGB1 release. The in vivo experiments demonstrated that administration of tumor-derived DAMP-rich cell lysates derived from B16F10 cells treated with ICRP and the combination of ICRP + OXP prevented melanoma growth; however, OXP treatment did not. These results suggested that IMMUNEPOTENT CRP may be used as an agent to increase the ability of antitumor drugs to induce immunogenic cell death and prevent the growth of melanoma.

Cytoplasmic Localization of WT1 and Decrease of miRNA-16-1 in Nephrotic Syndrome.

Nephrotic syndrome (NS) is a glomerular disease that is defined by the leakage of protein into the urine and is associated with hypoalbuminemia, hyperlipidemia, and edema. Steroid-resistant NS (SRNS) patients do not respond to treatment with corticosteroids and show decreased Wilms tumor 1 (WT1) expression in podocytes. Downregulation of WT1 has been shown to be affected by certain microRNAs (miRNAs). Twenty-one patients with idiopathic NS (68.75% were SSNS and 31.25% SRNS) and 10 healthy controls were enrolled in the study. Podocyte number and WT1 location were determined by immunofluorescence, and the serum levels of miR-15a, miR-16-1, and miR-193a were quantified by RT-qPCR. Low expression and delocalization of WT1 protein from the nucleus to the cytoplasm were found in kidney biopsies of patients with SRNS and both nuclear and cytoplasmic localization were found in steroid-sensitive NS (SSNS) patients. In sera from NS patients, low expression levels of miR-15a and miR-16-1 were found compared with healthy controls, but only the miR-16-1 expression levels showed statistically significant decrease (p = 0.019). The miR-193a expression levels only slightly increased in NS patients. We concluded that low expression and delocalization from the WT1 protein in NS patients contribute to loss of podocytes while modulation from WT1 protein is not associated with the miRNAs analyzed in sera from the patients.