Immunological Response against Breast Lineage Cells Transfected with Human Papillomavirus (HPV).

Breast cancer is the most common neoplasm worldwide. Viral infections are involved with carcinogenesis, especially those caused by oncogenic Human Papillomavirus (HPV) genotypes. Despite the detection of HPV in breast carcinomas, the virus's activity against this type of cancer remains controversial. HPV infection promotes remodeling of the host's immune response, resulting in an immunosuppressive profile. This study assessed the individual role of HPV oncogenes in the cell line MDA-MB-231 transfected with the E5, E6, and E7 oncogenes and co-cultured with peripheral blood mononuclear cells. Immunophenotyping was conducted to evaluate immune system modulation. There was an increase in CD4+ T cell numbers when compared with non-transfected and transfected MDA-MB-231, especially in the Treg profile. Pro-inflammatory intracellular cytokines, such as IFN-γ, TNF-α, and IL-17, were impaired by transfected cells, and a decrease in the cytolytic activity of the CD8+ and CD56+ lymphocytes was observed in the presence of HPV oncogenes, mainly with E6 and E7. The E6 and E7 oncogenes decrease monocyte expression, activating the expected M1 profile. In the monocytes found, a pro-inflammatory role was observed according to the cytokines released in the supernatant. In conclusion, the MDA-MB-231 cell lineage transfected with HPV oncogenes can downregulate the number and function of lymphocytes and monocytes.

Whole Yeast Vaccine Displaying ZIKV B and T Cell Epitopes Induces Cellular Immune Responses in the Murine Model.

Improving antigen presentation is crucial for the success of immunization strategies. Yeasts are classically used as biofactories to produce recombinant proteins and are efficient vehicles for antigen delivery, in addition to their adjuvant properties. Despite the absence of epidemic outbreaks, several vaccine approaches continue to be developed for Zika virus infection. The development of these prophylactic strategies is fundamental given the severity of clinical manifestations, mainly due to viral neurotropism. The present study aimed to evaluate in vivo the immune response induced by P. pastoris recombinant strains displaying epitopes of the envelope (ENV) and NS1 ZIKV proteins. Intramuscular immunization with heat-attenuated yeast enhanced the secretion of IL-6, TNF-α, and IFN-γ, in addition to the activation of CD4+ and CD8+ T cells, in BALB/c mice. P. pastoris displaying ENV epitopes induced a more robust immune response, increasing immunoglobulin production, especially IgG isotypes. Both proposed vaccines showed the potential to induce immune responses without adverse effects, confirming the safety of administering P. pastoris as a vaccine vehicle. Here, we demonstrated, for the first time, the evaluation of a vaccine against ZIKV based on a multiepitope construct using yeast as a delivery system and reinforcing the applicability of P. pastoris as a whole-cell vaccine.

Pectin-like polysaccharide extracted from leaves Crataeva tapia promotes antioxidant, immunomodulatory and emulsifiers applied in therapeutic formulations.

The objective of this work was to isolate a polysaccharide similar to pectin from Crataeva tapia leaves, not yet reported in the literature, and to evaluate its antioxidant, cytotoxic and immunomodulatory profile. Pectin was extracted from the leaves in three stages, organic solvent followed by acidified water and ethanol precipitation. With the pectin obtained, the physicochemical characterization of the molecule was carried out using high-performance liquid chromatography, Fourier-transform infrared spectroscopy, nuclear magnetic resonance (13C and 1H) and different thermal and elemental analysis. Furthermore, the antioxidant activities were evaluated in vitro, and using human peripheral blood mononuclear cell culture, cytotoxicity and immunostimulatory actions were investigated. Physical and chemical analyses showed characteristic signs of pectin. Antioxidant activity tests showed that pectin had moderate to low antioxidant activity. Furthermore, pectin did not affect the viability of erythrocytes and PBMC and induced an immunostimulatory state when it promoted the production of cytokines IL-6, IL-10 and TNF-α and increased the activation of CD8 + T lymphocytes. This study showed that pectin from Crataeva tapia is not cytotoxic and promoted a pro-inflammatory profile in peripheral blood mononuclear cell with application as an immunostimulating and emulsifying compound.

Association of Rhizophora mangle and ascorbic acid in hydrogels: Evaluation of cytotoxic and immunomodulatory effects

Abstract Polyphenolics from Rhizophora mangle (R. mangle) have shown wound healing and anti- inflammatory effects that may be potentiated by being associated with ascorbic acid, an important substance for collagen and elastin synthesis that plays a role in tissue repair. In our study, we aimed to formulate an association of R. mangle and ascorbic acid in hydrogels and evaluate the association's cytotoxic and immunomodulatory effects. In a pre-formulation study, three gelling polymers (i.e.xanthan gum, poloxamer and hydroxyethyl cellulose) were tested. The selected polymer (i.e. xanthan gum) was used to evaluate cytotoxic and immunomodulatory effects using flow cytometry. Xanthan gum (1.5%) had a homogeneous appearance, an orange colour, a smooth surface, intense brightness and the typical odour, as well as non-Newtonian pseudoplastic behaviour. With a pH of 5.0-5.3 and a non-cytotoxic profile, xanthan gum induced the proliferation and activation of CD4 +, CD8+ and NK T lymphocytes and the production of IL- 2, IL-4, IL-10, IL-17 and TNF-α cytokines in stimulated splenocytes. The results suggest that the association of R. mangle and ascorbic acid in 1.5% xanthan gum hydrogel may be promising in preparations for wound-healing processes.

Third-Generation Vaccines: Features of Nucleic Acid Vaccines and Strategies to Improve Their Efficiency.

Gene immunization comprises mRNA and DNA vaccines, which stand out due to their simple design, maintenance, and high efficacy. Several studies indicate promising results in preclinical and clinical trials regarding immunization against ebola, human immunodeficiency virus (HIV), influenza, and human papillomavirus (HPV). The efficiency of nucleic acid vaccines has been highlighted in the fight against COVID-19 with unprecedented approval of their use in humans. However, their low intrinsic immunogenicity points to the need to use strategies capable of overcoming this characteristic and increasing the efficiency of vaccine campaigns. These strategies include the improvement of the epitopes' presentation to the system via MHC, the evaluation of immunodominant epitopes with high coverage against emerging viral subtypes, the use of adjuvants that enhance immunogenicity, and the increase in the efficiency of vaccine transfection. In this review, we provide updates regarding some characteristics, construction, and improvement of such vaccines, especially about the production of synthetic multi-epitope genes, widely employed in the current gene-based vaccines.

Biocompatibility and immunostimulatory properties of fish collagen and shrimp chitosan towards peripheral blood mononuclear cells (PBMCs).

Several naturally occurring biopolymers are commercially produced from livestock and farmed animals processing wastes, including aquatic organisms. These wastes are considered valuable coproducts of fishery processing industry, from which biopolymers may be recovered and exploited for their bioactive potential. The aim of this work was to prepare polymeric films from collagen and chitosan solutions, extracted from fishery discards, and investigate the cytotoxicity and immunomodulatory activity towards human peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from healthy donors and treated with Chitosan, Collagen, Chitosan+Collagen solutions and Chitosan+Collagen film in order to measure the changes in cell viability, cytosolic calcium concentration ([Ca2+]cyt), mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS) levels, differentiation and activation of T CD8+ and CD4+ lymphocytes, and cytokine production. Results showed that collagen and chitosan preparations did not show cytotoxic effect, while cellular IL-6, IL-10, and TNF-α release was observed. Chitosan and collagen were able to promote non-cytotoxic PBMCs activation through cytosolic and mitochondrial ROS production. There was a noteworthy phenotyping of lymphocytes T CD8+ and CD4+ counting and an increase of [Ca2+] cyt and ΔΨm levels. These results suggest that chitosan/collagen-based biomaterials produce immunostimulatory effects on PBMC with potential to biomedical approaches.

Pectin-like Polysaccharide Extracted from the Leaves Caesalpinia pulcherrima is a Promising Antioxidant and Immunomodulator Agent

Abstract Studies have shown that Caesalpinia pulcherrima extracts promote antioxidant, healing, immunomodulating and antiparasitic activities and its polysaccharides can be used as functional food. In this sense, this work had as objective the isolation and characterization of a polysaccharide-like pectin, extracted from the C. pulcherrima leaves and its possible applications as an antioxidant and immunomodulator agent. The molecule was characterized by high performance liquid chromatography, fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. Its antioxidant potential was evaluated through the methods of phosphomolybdenum, ABTS radical scavenging [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid], DPPH (1,1-diphenyl-2-picrylhydrazyl) and nitric oxide radical. The immunostimulating effects of pectin were tested in splenocytes to evaluate its toxic, proliferative and cell activator and immunomodulatory potential. The polysaccharide obtained has structural characteristics similar to pectins. Pectin showed high in vitro antioxidant activity for ABTS radical scavenging, moderate activity for phosphomolybdenum and low activity for DPPH and nitric oxide. In vitro immunomodulation assays showed that pectin obtained did not promote a cytotoxic effect (viability > 90%). The increase in cytosolic ROS levels indicates a possible mechanism of cell activation without causing damage. Immunophenotyping showed that pectin increased a subpopulation of CD8+ T lymphocytes and monocytes. In addition, it promoted a mostly pro-inflammatory response confirmed by the production of cytokines IL-2, -4, -6, IFN-γ and TNF-α. These results reinforce the ethnopharmacological use of C. pulcherrima leaves and expand the use of this plant for future applications as herbal medicines.

Development of synthetic antigen vaccines for COVID-19.

The current pandemic called COVID-19 caused by the SARS-CoV-2 virus brought the need for the search for fast alternatives to both control and fight the SARS-CoV-2 infection. Therefore, a race for a vaccine against COVID-19 took place, and some vaccines have been approved for emergency use in several countries in a record time. Ongoing prophylactic research has sought faster, safer, and precise alternatives by redirecting knowledge of other vaccines, and/or the development of new strategies using available tools, mainly in the areas of genomics and bioinformatics. The current review highlights the development of synthetic antigen vaccines, focusing on the usage of bioinformatics tools for the selection and construction of antigens on the different vaccine constructions under development, as well as strategies to optimize vaccines for COVID-19.

Characterization of a lignin from Crataeva tapia leaves and potential applications in medicinal and cosmetic formulations.

Lignins are phenolic macromolecules that have several applications. In this work, we examine some biological activities of a lignin-like macromolecule isolated from the Crataeva tapia leaves, not yet studied to evaluate its potential applications in medicinal and cosmetic formulations. Lignin was obtained by alkaline delignification and its physical-chemical characterization was made by means of FT-IR, UV-Vis, NMR spectroscopy, elementary analysis, molecular mass determination and thermal analysis. Lignin is of the GSH type, with levels of hydrogen (5.10%), oxygen (27.18%), carbon (67.60%), nitrogen (0.12%) and phenolic content of 189.6 ± 9.6 mg GAE/g. In addition, it is a thermally stable macromolecule with low antioxidant activity. Cytotoxicity and cytokine production were assessed by flow cytometry. The photoprotective activity was evaluated by adding different concentrations of lignin to a commercial cream. Lignin was not cytotoxic, it stimulated the production of TNF-α, IL-6 and IL-10 and did not promote a significant change in nitric oxide levels. In addition, this macromolecule was able to promote increased absorption of ultraviolet light from a commercial cream. These results reinforce the ethnopharmacological use of C. tapia leaves and suggest the need for further studies to determine the potential medicinal and cosmetic applications (sunscreen) of lignin from C. tapia leaves.

Evaluation of the In Vivo Acute Toxicity and In Vitro Hemolytic and Immunomodulatory Activities of the Moringa oleifera Flower Trypsin Inhibitor (MoFTI).

BACKGROUND: Protease inhibitors have been isolated from plants and present several biological activities, including immunomodulatory action. OBJECTIVE: This work aimed to evaluate a Moringa oleifera flower trypsin inhibitor (MoFTI) for acute toxicity in mice, hemolytic activity on mice erythrocytes and immunomodulatory effects on mice splenocytes. METHODS: The acute toxicity was evaluated using Swiss female mice that received a single dose of the vehicle control or MoFTI (300 mg/kg, i.p.). Behavioral alterations were observed 15-240 min after administration, and survival, weight gain, and water and food consumption were analyzed daily. Organ weights and hematological parameters were analyzed after 14 days. Hemolytic activity of MoFTI was tested using Swiss female mice erythrocytes. Splenocytes obtained from BALB/c mice were cultured in the absence or presence of MoFTI for the evaluation of cell viability and proliferation. Mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) levels were also determined. Furthermore, the culture supernatants were analyzed for the presence of cytokines and nitric oxide (NO). RESULTS: MoFTI did not cause death or any adverse effects on the mice except for abdominal contortions at 15-30 min after administration. MoFTI did not exhibit a significant hemolytic effect. In addition, MoFTI did not induce apoptosis or necrosis in splenocytes and had no effect on cell proliferation. Increases in cytosolic and mitochondrial ROS release, as well as Δψm reduction, were observed in MoFTI-treated cells. MoFTI was observed to induce TNF-α, IFN-γ, IL-6, IL-10, and NO release. CONCLUSION: These results contribute to the ongoing evaluation of the antitumor potential of MoFTI and its effects on other immunological targets.

Lignin isolated from Caesalpinia pulcherrima leaves has antioxidant, antifungal and immunostimulatory activities.

Caesalpinia pulcherrima is a shrub with worldwide distribution used as an ornamental plant. In this study, we extracted a lignin from the C. pulcherrima leaves and investigated its biological functions. The lignin was characterized by FT-IR, UV-Vis, GPC, TGA and nuclear magnetic resonance (1H and 13C). The antioxidant activity was evaluated using phosphomolybdenum complexation methods (TAA), sequestration of DPPH and ABTS radicals, reducing power, formation of nitrite radical and iron chelating activity (Fe2 +). Antifungal activity was made using Candida spp., Aspergillus spp. and Cryptococcus neoformans strains. Cytotoxicity, oxidative stress, and cytokine production were performed using mouse splenocytes. The lignin showed maximal UV-Vis at ~280 nm, 22.27 L/g·cm of absorptivity and, 2,503 kDa of molecular weight. Phenolic compounds (41.33 ± 0.65 mg GAE/g) and indications of a guaiacyl-syringyl-hydroxyphenyl (GSH)-type composition were found. Antioxidant activities of lignin to TAA (40±1.2%) and to DPPH (16.9±0.2%) was high and showed antifungal potential, especially against Candida spp. (IC50 = 31.3 µg/mL) and C. neoformans (15.6 µg/mL). In mouse splenocytes, the lignin was not cytotoxic and stimulated the cell proliferation and cytokine release. These results indicate that C. pulcherrima lignin has the potential to be used as antifungal and immunostimulant compound.

Schinus terebinthifolia leaf lectin (SteLL) is an immunomodulatory agent by altering cytokine release by mice splenocytes.

Schinus terebinthifolia leaf lectin (SteLL) was reported to be an antimicrobial and antitumor agent. In this work, we evaluated the immunomodulatory activity of SteLL on mice splenocytes and also determined its native molecular mass and putative sequence similarities with plant proteins. The effects of SteLL (12.5 µg/mL) on viability, cytosolic Ca2+ concentration ([Ca2+]cyt), cytosolic and mitochondrial levels of reactive oxygen species (ROS), and mitochondrial transmembrane potential (ΔΨm) of mice splenocytes were determined. In addition, the culture supernatants were collected for quantification of interleukins (IL), tumor necrosis factor (TNF), interferon-gamma (IFN-γ) and nitric oxide (NO). SteLL showed a native molecular mass of 12.4 kDa and tandem mass spectrometry (MS/MS) ions search revealed similarities with adenosine triphosphate (ATP) synthase and F1-ATPase from plants (4% and 6% coverage, respectively). SteLL was not toxic to splenocytes, did not alter the [Ca2+]cyt and ROS levels, and slightly reduced ΔΨm. The presence of SteLL stimulated the cells to release pro-inflammatory cytokines (IL-17A, TNF-α, IFN-γ and IL-2) and also of IL-4, an anti-inflammatory cytokine that can prevent exacerbated inflammation. SteLL induced decrease in the secretion of NO. In conclusion, SteLL has biotechnological potential as an immunomodulator agent for use in studies employing cultures of immune cells. In addition, the anti-infectious and antitumor properties of the leaves may involve the immunomodulation property of SteLL.

A proposed eye ex vivo permeation approach to evaluate pesticides: Case dimethoate.

This study aims to propose different ex vivo methodologies for pesticide evaluation when in contact with ocular mucosa. The first ex vivo study was performed using vertical Franz cells with fresh and refrigerated excised bovine corneas. The second evaluated the permeation through the cornea by using fresh, refrigerated and damaged whole bovine eyes. Both experiments were evaluated by applying 50 µl (of 5 mg/ml solution) of an emulsifiable pesticide formulation (Dimetoato 500 EC®). In the first study, dimethoate profiles and permeation fluxes showed no statistical differences (p < .05) between fresh and refrigerated excised corneas, probably due to the excision procedure generating damage to the cornea by altering stromal structure, irrespective of the refrigeration procedure. In relation to the results obtained for developed ex vivo permeation method in whole eyes, there was a statistical difference (p < .05) between experiments performed with fresh eyes and those performed with refrigerated/damaged eyes. Damage generated by both the refrigeration process and exposure to irritant products led to a five-fold reduction in the amount of corneal-permeated pesticide as well as a two-fold increase in retention. Thus, the ex vivo permeation approach with whole eyes seems to be a simple and reproducible method to evaluate pesticides. It is evident that further evaluations need to be performed using other pesticides with distinct physicochemical characteristics.

Immunostimulatory and antioxidant activities of a lignin isolated from Conocarpus erectus leaves.

In this work, we investigated the antioxidant and immunomodulatory activities of a lignin isolated from Conocarpus erectus leaves. The lignin was characterized by FTIR, 1H NMR, 13C NMR and gel permeation chromatography analysis as well as ultraviolet/visible absorption spectra. The lignin was evaluated for total antioxidant activity (TAA), DPPH and ABTS+ scavenging abilities, and by a lipid peroxidation inhibition assay. Immunomodulatory activity of the lignin (10 µg/mL) on human peripheral blood mononuclear cells (PBMCs) was determined. The C. erectus lignin was found to be of the guaiacyl-syringyl-p-hydroxyphenyl (G-S-H) type, with an average molecular weight of 2709 Da (polydispersity index: 2.1). It showed low TAA (17.92%) and moderate antioxidant activity against DPPH and ABTS+ (IC50: 231.16 and 356.03 µg/mL, respectively). It also inhibited lipid peroxidation by 42.14%. The lignin promoted an increase in mitochondrial ROS levels as well as cytosolic Ca2+ in PBMCs. In addition, it promoted the differentiation and activation of CD8+ T lymphocytes, differentiation of CD14+ monocytes, and stimulated the release of nitric oxide and cytokines, mainly those linked to a Th1 response. The results showed that the C. erectus lignin may be used in future studies in which the modulation of the immune response is a key factor.

Evaluation of antitumor activity and toxicity of Schinus terebinthifolia leaf extract and lectin (SteLL) in sarcoma 180-bearing mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Schinus terebinthifolia Raddi is a plant broadly used in folk medicine and the use of its leaf extract as an antitumor agent has been reported. AIM OF THE STUDY: To evaluate the antitumor potential and the toxicity of saline extract (SE) and lectin (SteLL) from S. terebinthifolia leaves in sarcoma 180-bearing mice. MATERIALS AND METHODS: Cytotoxicity to sarcoma 180 cells was tested in vitro, and antitumor assay was performed using Swiss female mice. The treatments (0.15 M NaCl, negative control; methotrexate 1.5 mg/kg, positive control; SE 100 mg/kg; SteLL 1 and 5 mg/kg) by intraperitoneal injections started on the 8th day after tumor inoculation and lasted 7 days. It was analyzed: tumor weight; number and gauge of tumor vessels; hematological and biochemical parameters; histopathological changes; and occurrence of micronuclei in bone marrow cells. RESULTS: SE and SteLL showed IC50 values (concentrations that reduced cell viability to 50%) of 301.65 and 8.30 µg/mL, respectively. The lectin was able to induce apoptosis. Treatments with the extract and lectin caused a 57.6-73.6% reduction in tumor weight, which was not significantly different from the reduction in the methotrexate group. Tumors of animals treated with SteLL at 5 mg/kg showed reduced number of secondary vessels while the gauge was lower in all treated groups. In the groups treated with SteLL, tumors showed reduced and slightly vascularized parenchyma, with necrosis in the center and at the periphery. No alterations in the blood levels of urea, creatine, and glucose were detected while serum AST level was moderately increased in the SE group. Histopathological analysis revealed vacuolization and steatosis in the liver of animals treated with the extract and lectin. In addition, the treatments with SE and SteLL resulted in the reduction of filtration space and alterations in tubular architecture in kidneys. In respect to hematological parameters, it was only detected increase in the number of monocytes in SE group. The extract and lectin did not induce the formation of micronuclei in the bone marrow cells. CONCLUSIONS: SE and SteLL had antitumor effect against sarcoma 180 without inducing hematological changes and genotoxic effects in mice; however, some degree of hepatic and renal toxicity was observed, suggesting the evaluation of drug delivery strategies in the future.

Lignins isolated from Prickly pear cladodes of the species Opuntia fícus-indica (Linnaeus) Miller and Opuntia cochenillifera (Linnaeus) Miller induces mice splenocytes activation, proliferation and cytokines production.

Opuntia fícus-indica and Opuntia cochenillifera are species of Cactaceae, found in the arid regions of the planet. They present water, cellulose, hemicellulose, pectins, extractives, ashes and lignins. Here we aimed to study the immunomodulatory action of lignins from these two species against mice splenocytes, since no study for this purpose has yet been reported. The antioxidant activities of these lignins were evaluated by the DPPH, ABTS, NO assays and total antioxidant activity. Cytotoxicity was evaluated through Annexin V-FITC and propidium iodide-PE probs and cell proliferation was determined by CFSE. Immunomodulation studies with Opuntia lignins obtained were performed through investigation of ROS levels, cytosolic calcium release, changes on mitochondrial membrane potential, cytokine production and NO release. Results showed that Opuntia cochenillifera lignin presented more phenolic amount and antioxidant activities than Opuntia ficius-indica. Both lignins showed high cell viability (>96%) and cell proliferation. Activation signal was observed for both lignins with increase of ROS and cytosolic calcium levels, and changes in mitochondrial membrane potential. In addition, lignins induced high TNF-α, IL-6 and IL-10 production and reduced NO release. Therefore, these lignins present great potential to be used as molecules with a proinflammatory profile, being shown as a promising therapeutic agent.

Synthesis, in vitro and in vivo biological evaluation, COX-1/2 inhibition and molecular docking study of indole-N-acylhydrazone derivatives.

The objective of this work was to obtain and evaluate anti-inflammatory in vitro, in vivo and in silico potential of novel indole-N-acylhydrazone derivatives. In total, 10 new compounds (3a-j) were synthesized in satisfactory yields, through a condensation reaction in a single synthesis step. In the lymphoproliferation assay, using mice splenocytes, 3a and 3b showed inhibition of lymphocyte proliferation of 62.7% (±3.5) and 50.7% (±2), respectively, while dexamethasone presented an inhibition of 74.6% (±2.4). Moreover, compound 3b induced higher Th2 cytokines production in mice splenocytes cultures. The results for COX inhibition assays showed that compound 3b is a selective COX-2 inhibitor, but with less potency when compared to celecoxib, and compound 3a not presented selectivity towards COX-2. The molecular docking results suggest compounds 3a and 3b interact with the active site of COX-2 in similar conformations, but not with the active site of COX-1, and this may be the main reason to the COX-2 selectivity of compound 3b. In vivo carrageenan-induced paw edema assays were adopted for the confirmation of the anti-inflammatory activity. Compound 3b showed better results in suppressing edema at all tested concentrations and was able to induce an edema inhibition of 100% after 5 h of carrageenan injection at the 30 mg kg-1 dosage, corroborating with the COX inhibition and lymphoproliferation results. I addition to our experimental results, in silico analysis suggest that compounds 3a and 3b present a well-balanced profile between pharmacodynamics and pharmacokinetics. Thus, our preliminary results revealed the potentiality of a new COX-2 selective derivative in the modulation of the inflammatory process.

Calliandra surinamensis lectin (CasuL) does not impair the functionality of mice splenocytes, promoting cell signaling and cytokine production.

CasuL is a lectin (carbohydrate-binding protein) isolated from the leaf pinnulae of Calliandra surinamensis that is toxic against cancer cells. In this study, the effects of CasuL on the activation of immune cells were evaluated in BALB/c mice splenocytes. Assays measuring the changes in cytosolic calcium concentration ([Ca2+]cyt), mitochondrial membrane potential (ΔΨm), and reactive oxygen species (ROS) levels associated with cell viability, proliferation, and cytokine and nitric oxide production were performed. The lectin (3.12-100 µg/mL) did not induce apoptosis or necrosis of splenocytes, and treatment for 48 h at 12.5 µg/mL stimulated cell proliferation. High cytosolic ROS levels were found in cells incubated with CasuL (12.5 µg/mL), but it did not affect [Ca2+]cyt, mitochondrial ROS, and ΔΨm levels. Furthermore, CasuL promoted high IL-2 and TNF-α production but did not affect nitric oxide release. In conclusion, CasuL was able to promote oxidative stress in mouse immune cells without inducing cell damage, and stimulated proliferation and cytokine production. These findings suggest the potential use of CasuL in future antitumoral and immunological targets.

Activities of stromal and immune cells in HPV-related cancers.

The immune system is composed of immune as well as non-immune cells. As this system is a well-established component of human papillomavirus- (HPV)-related carcinogenesis, high risk human papillomavirus (hrHPV) prevents its routes and mechanisms in order to cause the persistence of infection. Among these mechanisms are those originated from stromal cells, which include the cancer-associated fibroblasts (CAFs), the myeloid-derived suppressor cells (MDSCs) and the host infected cells themselves, i.e. the keratinocytes. These types of cells play central role since they modulate immune cells activities to create a prosperous milieu for cancer development, and the knowledge how such interactions occur are essential for prognostic assessment and development of preventive and therapeutic approaches. Nevertheless, the precise mechanisms are not completely understood, and this lack of knowledge precluded the development of entirely efficient immunotherapeutic strategies for HPV-associated tumors. As a result, an intense work for attaining how host immune response works, and developing of effective therapies has been applied in the last decade. Based on this, this review aims to discuss the major mechanisms of immune and non-immune cells modulated by hrHPV and the potential and existing immunotherapies involving such mechanisms in HPV-related cancers. It is noticed that the combination of immunotherapies has been demonstrated to be essential for obtaining better results, especially because the possibility of increasing the modulating capacity of the HPV-tumor microenvironment has been shown to be central in strengthening the host immune system.

Viral Modulation of TLRs and Cytokines and the Related Immunotherapies for HPV-Associated Cancers.

The modulation of the host innate immune system is a well-established carcinogenesis feature of several tumors, including human papillomavirus- (HPV-) related cancers. This virus is able to interrupt the initial events of the immune response, including the expression of Toll-like receptors (TLRs), cytokines, and inflammation. Both TLRs and cytokines play a central role in HPV recognition, cell maturation and differentiation as well as immune signalling. Therefore, the imbalance of this sensitive control of the immune response is a key factor for developing immunotherapies, which strengthen the host immune system to accomplish an efficient defence against HPV and HPV-infected cells. Based on this, the review is aimed at exposing the HPV immune evasion mechanisms involving TLRs and cytokines and at discussing existing and potential immunotherapeutic TLR- and cytokine-related tools.