Role of the sympathetic nervous system in cancer-associated cachexia and tumor progression in tumor-bearing BALB/c mice.

BACKGROUND: Adipose and muscle tissue wasting outlines the cachectic process during tumor progression. The sympathetic nervous system (SNS) is known to promote tumor progression and research suggests that it might also contribute to cancer-associated cachexia (CAC) energetic expenditure through fat wasting. METHODS: We sympathectomized L5178Y-R tumor-bearing male BALB/c mice by intraperitoneally administering 6-hydroxydopamine to evaluate morphometric, inflammatory, and molecular indicators of CAC and tumor progression. RESULTS: Tumor burden was associated with cachexia indicators, including a 10.5% body mass index (BMI) decrease, 40.19% interscapular, 54% inguinal, and 37.17% visceral adipose tissue loss, a 12% food intake decrease, and significant (p = 0.038 and p = 0.0037) increases in the plasmatic inflammatory cytokines IL-6 and IFN-γ respectively. Sympathectomy of tumor-bearing mice was associated with attenuated BMI and visceral adipose tissue loss, decreased interscapular Ucp-1 gene expression to basal levels, and 2.6-fold reduction in Mmp-9 relative gene expression, as compared with the unsympathectomized mice control group. CONCLUSION: The SNS contributes to CAC-associated morphometric and adipose tissue alterations and promotes tumor progression in a murine model.

Increased CD8+ tissue resident memory T cells, regulatory T cells and activated natural killer cells in systemic sclerosis lungs.

OBJECTIVE: Multiple observations indicate a role for lymphocytes in driving autoimmunity in SSc. While T and NK cells have been studied in SSc whole blood and bronchoalveolar lavage fluid, their role remains unclear, partly because no studies have analysed these cell types in SSc-interstitial lung disease (ILD) lung tissue. This research aimed to identify and analyse the lymphoid subpopulations in SSc-ILD lung explants. METHODS: Lymphoid populations from 13 SSc-ILD and 6 healthy control (HC) lung explants were analysed using Seurat following single-cell RNA sequencing. Lymphoid clusters were identified by their differential gene expression. Absolute cell numbers and cell proportions in each cluster were compared between cohorts. Additional analyses were performed using pathway analysis, pseudotime and cell ligand-receptor interactions. RESULTS: Activated CD16+ NK cells, CD8+ tissue resident memory T cells and Treg cells were proportionately higher in SSc-ILD compared with HC lungs. Activated CD16+ NK cells in SSc-ILD showed upregulated granzyme B, IFN-γ and CD226. Amphiregulin, highly upregulated by NK cells, was predicted to interact with epidermal growth factor receptor on several bronchial epithelial cell populations. Shifts in CD8+ T cell populations indicated a transition from resting to effector to tissue resident phenotypes in SSc-ILD. CONCLUSIONS: SSc-ILD lungs show activated lymphoid populations. Activated cytotoxic NK cells suggest they may kill alveolar epithelial cells, while their expression of amphiregulin suggests they may also induce bronchial epithelial cell hyperplasia. CD8+ T cells in SSc-ILD appear to transition from resting to the tissue resident memory phenotype.

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.

Synergistic Enhancement of Chemotherapy-Induced Cell Death and Antitumor Efficacy against Tumoral T-Cell Lymphoblasts by IMMUNEPOTENT CRP.

T-cell malignancies, including T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL), present significant challenges to treatment due to their aggressive nature and chemoresistance. Chemotherapies remain a mainstay for their management, but the aggressiveness of these cancers and their associated toxicities pose limitations. Immunepotent CRP (ICRP), a bovine dialyzable leukocyte extract, has shown promise in inducing cytotoxicity against various cancer types, including hematological cancers. In this study, we investigated the combined effect of ICRP with a panel of chemotherapies on cell line models of T-ALL and T-LBL (CEM and L5178Y-R cells, respectively) and its impact on immune system cells (peripheral blood mononuclear cells, splenic and bone marrow cells). Our findings demonstrate that combining ICRP with chemotherapies enhances cytotoxicity against tumoral T-cell lymphoblasts. ICRP + Cyclophosphamide (CTX) cytotoxicity is induced through a caspase-, reactive oxygen species (ROS)-, and calcium-dependent mechanism involving the loss of mitochondrial membrane potential, an increase in ROS production, and caspase activation. Low doses of ICRP in combination with CTX spare non-tumoral immune cells, overcome the bone marrow-induced resistance to CTX cell death, and improves the CTX antitumor effect in vivo in syngeneic Balb/c mice challenged with L5178Y-R. This led to a reduction in tumor volume and a decrease in Ki-67 proliferation marker expression and the granulocyte/lymphocyte ratio. These results set the basis for further research into the clinical application of ICRP in combination with chemotherapeutic regimens for improving outcomes in T-cell malignancies.

shRNA-Targeting Caspase-3 Inhibits Cell Detachment Induced by Pemphigus Vulgaris Autoantibodies in HaCaT Cells.

Pemphigus is an autoimmune disease that affects the skin and mucous membranes, induced by the deposition of pemphigus IgG, which mainly targets desmogleins 1 and 3 (Dsg1 and 3). This autoantibody causes steric interference between Dsg1 and 3 and the loss of cell adhesion, producing acantholysis. This molecule and its cellular effects are clinically reflected as intraepidermal blistering. Pemphigus vulgaris-IgG (PV-IgG) binding involves p38MAPK-signaling-dependent caspase-3 activation. The present work assessed the in vitro effect of PV-IgG on the adherence of HaCaT cells dependent on caspase-3. PV-IgG induced cell detachment and apoptotic changes, as demonstrated by annexin fluorescent assays. The effect of caspase-3 induced by PV-IgG was suppressed in cells pre-treated with caspase-3-shRNA, and normal IgG (N-IgG) as a control had no relevant effects on the aforementioned parameters. The results demonstrated that shRNA reduces caspase-3 expression, as measured via qRT-PCR and via Western blot and immunofluorescence, and increases cell adhesion. In conclusion, shRNA prevented in vitro cell detachment and the late effects of apoptosis induced by PV-IgG on HaCaT cells, furthering our understanding of the molecular role of caspase-3 cell adhesion dependence in pemphigus disease.

A Quantitative Assessment of Staphylococcus aureus Community Carriage in Yuma, Arizona.

BACKGROUND: Disease control relies on pathogen identification and understanding reservoirs. Staphylococcus aureus infection prevention is based upon decades of research on colonization and infection, but diminishing returns from mitigation efforts suggest significant knowledge gaps. Existing knowledge and mitigation protocols are founded upon culture-based detection, with almost no information about pathogen quantities. METHODS: We used culture and a quantitative polymerase chain reaction assay on samples from 3 body sites to characterize colonization more comprehensively than previous studies by describing both prevalence and pathogen quantity. RESULTS: We show a much higher overall prevalence (65.9%) than previously documented, with higher quantities and prevalence associated with the nares, non-Hispanic males (86.9%), and correlating with colonization in other body sites. These results suggest that research and clinical practices likely misclassify over half of colonized persons, limiting mitigation measures and their impact. CONCLUSIONS: This work begins the process of rebuilding foundational knowledge of S aureus carriage with more accurate and wholistic approaches.

Extracellular Vesicles from Immune Cells: A Biomedical Perspective.

Research on the role of extracellular vesicles (sEV) in physiology has demonstrated their undoubted importance in processes such as the transportation of molecules with significance for cell metabolism, cell communication, and the regulation of mechanisms such as cell differentiation, inflammation, and immunity. Although the role of EVs in the immune response is actively investigated, there is little literature revising, in a comprehensive manner, the role of small EVs produced by immune cells. Here, we present a review of studies reporting the release of sEV by different types of leukocytes and the implications of such observations on cellular homeostasis. We also discuss the function of immune cell-derived sEV and their relationship with pathological states, highlighting their potential application in the biomedical field.

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.

Understanding the Molecular Conformation and Viscoelasticity of Low Sol-Gel Transition Temperature Gelatin Methacryloyl Suspensions.

For biomedical applications, gelatin is usually modified with methacryloyl groups to obtain gelatin methacryloyl (GelMA), which can be crosslinked by a radical reaction induced by low wavelength light to form mechanically stable hydrogels. The potential of GelMA hydrogels for tissue engineering has been well established, however, one of the main disadvantages of mammalian-origin gelatins is that their sol-gel transitions are close to room temperature, resulting in significant variations in viscosity that can be a problem for biofabrication applications. For these applications, cold-water fish-derived gelatins, such as salmon gelatin, are a good alternative due to their lower viscosity, viscoelastic and mechanical properties, as well as lower sol-gel transition temperatures, when compared with mammalian gelatins. However, information regarding GelMA (with special focus on salmon GelMA as a model for cold-water species) molecular conformation and the effect of pH prior to crosslinking, which is key for fabrication purposes since it will determine final hydrogel's structure, remains scarce. The aim of this work is to characterize salmon gelatin (SGel) and salmon methacryloyl gelatin (SGelMA) molecular configuration at two different acidic pHs (3.6 and 4.8) and to compare them to commercial porcine gelatin (PGel) and methacryloyl porcine gelatin (PGelMA), usually used for biomedical applications. Specifically, we evaluated gelatin and GelMA samples' molecular weight, isoelectric point (IEP), their molecular configuration by circular dichroism (CD), and determined their rheological and thermophysical properties. Results showed that functionalization affected gelatin molecular weight and IEP. Additionally, functionalization and pH affected gelatin molecular structure and rheological and thermal properties. Interestingly, the SGel and SGelMA molecular structure was more sensitive to pH changes, showing differences in gelation temperatures and triple helix formation than PGelMA. This work suggests that SGelMA presents high tunability as a biomaterial for biofabrication, highlighting the importance of a proper GelMA molecular configuration characterization prior to hydrogel fabrication.

CAR-T Cell Therapy: From the Shop to Cancer Therapy.

Cancer is a worldwide health problem. Nevertheless, new technologies in the immunotherapy field have emerged. Chimeric antigen receptor (CAR) technology is a novel biological form to treat cancer; CAR-T cell genetic engineering has positively revolutionized cancer immunotherapy. In this paper, we review the latest developments in CAR-T in cancer treatment. We present the structure of the different generations and variants of CAR-T cells including TRUCK (T cells redirected for universal cytokine killing. We explain the approaches of the CAR-T cells manufactured ex vivo and in vivo. Moreover, we describe the limitations and areas of opportunity for this immunotherapy and the current challenges of treating hematological and solid cancer using CAR-T technology as well as its constraints and engineering approaches. We summarize other immune cells that have been using CAR technology, such as natural killer (NK), macrophages (M), and dendritic cells (DC). We conclude that CAR-T cells have the potential to treat not only cancer but other chronic diseases.

Changes in the natural killer cell repertoire and function induced by the cancer immune adjuvant candidate IMMUNEPOTENT-CRP.

Natural killer (NK) cells are CD3-, CD16+, CD56+ that play a crucial role in immune response by recognizing and eliminating a variety of virus-infected, malignant, and antibody-coated target cells. We examined activation; repertoire changes and effector functions of human NK cells normal donors treated with IMMUNEPOTENT-CRP (I-CRP), a bovine dialyzable leukocyte extract (DLE) containing a mixture of low molecular weight molecules. I-CRP induces human NK cells activation and increase CD56Dim CD16- subset, without inducing proliferation. Human NK cells showed an increase on NKp30, NKp44, NKp46, NKG2D, NKG2C and KIR receptors, whereas no significant differences on CD160, CD85j and CD226 where observed. I-CRP-treated human NK cells exhibited an increased degranulation activity against K562 target cells, as shown by CD107a assay, and this correlates with cytotoxicity against K562 cells observed in calcein release assay. These results indicate that I-CRP can modify human NK cells receptor repertoire leading to an increased cytotoxic activity, supporting evidence for its use to stimulate NK cells.

The bovine dialysable leukocyte extract IMMUNEPOTENT CRP induces immunogenic cell death in breast cancer cells leading to long-term antitumour memory.

BACKGROUND: Cancer recurrence is a serious problem in breast cancer (BC) patients, and immunogenic cell death (ICD) has been proposed as a strategy to overcome this recurrence. IMMUNEPOTENT CRP (ICRP) acts as an immunomodulator and can be cytotoxic to cancer cells. Thus, we evaluated if ICRP induces ICD in BC cells. METHODS: Immunogenicity of ICRP-induced cell death was evaluated in vitro, analysing the principal biochemical characteristics of ICD in MCF-7, MDA-MB-231 and 4T1 cells. Ex vivo, we assessed the ability of killed cancer cells (KCC) obtained from ICRP-treated 4T1 cells (ICRP-KCC) to induce DC maturation, T-cell priming and T-cell-mediated cancer cytotoxicity. In vivo, we evaluated tumour establishment and antitumour immune memory after prophylactic ICRP-KCC vaccination in BALB/c mice. RESULTS: ICRP induced caspase-independent, ROS-dependent cell death, autophagosome formation, P-eIF2α, chaperone protein exposure, CD47 loss, ATP and HMBG1 release in BC cells. Additionally, ICRP-KCC promoted DC maturation, which triggered T-cell priming and cancer cytotoxicity. Prophylactic vaccination with ICRP-KCC prevented tumour establishment and induced long-term antitumour memory in BALB/c mice, involving DC maturation in lymph nodes, CD8+ T-cell augmentation in lymph nodes, peripheral blood and tumour site and ex vivo tumour-specific cytotoxicity by splenocytes. CONCLUSIONS: ICRP induces ICD in BC cells, leading to long-term antitumour memory.

Total and Unbound Bictegravir Concentrations and Viral Suppression in Cerebrospinal Fluid of Human Immunodeficiency Virus-Infected Patients (Spanish HIV/AIDS Research Network, PreEC/RIS 56).

We determined total and unbound concentrations of bictegravir (BIC) in cerebrospinal fluid (CSF) in 15 asymptomatic, virologically suppressed patients. The median plasma and CSF total BIC concentrations were 1837.1 ng/mL (interquartile range [IQR], 1237.2-2586.7) and 6.9 (IQR, 4.8-10.9), respectively. Median unbound BIC concentration was 2.48 ng/mL (IQR, 1.6-3.7). Total and unbound BIC CSF concentrations were above the half-maximal effective concentration value in all patients, and all subjects had human immunodeficiency virus viral suppression in plasma and CSF. Bictegravir may contribute to inhibit viral replication in this compartment.

IMMUNEPOTENT CRP induces DAMPS release and ROS-dependent autophagosome formation in HeLa and MCF-7 cells.

BACKGROUND: IMMUNEPOTENT CRP (ICRP) can be cytotoxic to cancer cell lines. However, its widespread use in cancer patients has been limited by the absence of conclusive data on the molecular mechanism of its action. Here, we evaluated the mechanism of cell death induced by ICRP in HeLa and MCF-7 cells. METHODS: Cell death, cell cycle, mitochondrial membrane potential and ROS production were evaluated in HeLa and MCF-7 cell lines after ICRP treatment. Caspase-dependence and ROS-dependence were evaluated using QVD.oph and NAC pre-treatment in cell death analysis. DAMPs release, ER stress (eIF2-α phosphorylation) and autophagosome formation were analyzed as well. Additionally, the role of autophagosomes in cell death induced by ICRP was evaluated using SP-1 pre-treatment in cell death in HeLa and MCF-7 cells. RESULTS: ICRP induces cell death, reaching CC50 at 1.25 U/mL and 1.5 U/mL in HeLa and MCF-7 cells, respectively. Loss of mitochondrial membrane potential, ROS production and cell cycle arrest were observed after ICRP CC50 treatment in both cell lines, inducing the same mechanism, a type of cell death independent of caspases, relying on ROS production. Additionally, ICRP-induced cell death involves features of immunogenic cell death such as P-eIF2α and CRT exposure, as well as, ATP and HMGB1 release. Furthermore, ICRP induces ROS-dependent autophagosome formation that acts as a pro-survival mechanism. CONCLUSIONS: ICRP induces a non-apoptotic cell death that requires an oxidative stress to take place, involving mitochondrial damage, ROS-dependent autophagosome formation, ER stress and DAMPs' release. These data indicate that ICRP could work together with classic apoptotic inductors to attack cancer cells from different mechanisms, and that ICRP-induced cell death might activate an immune response against cancer cells.

CD47 agonist peptide PKHB1 induces immunogenic cell death in T-cell acute lymphoblastic leukemia cells.

T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis derived from its genetic heterogeneity, which translates to a high chemoresistance. Recently, our workgroup designed thrombospondin-1-derived CD47 agonist peptides and demonstrated their ability to induce cell death in chronic lymphocytic leukemia. Encouraged by these promising results, we evaluated cell death induced by PKHB1 (the first-described serum-stable CD47-agonist peptide) on CEM and MOLT-4 human cell lines (T-ALL) and on one T-murine tumor lymphoblast cell-line (L5178Y-R), also assessing caspase and calcium dependency and mitochondrial membrane potential. Additionally, we evaluated selectivity for cancer cell lines by analyzing cell death and viability of human and murine non-tumor cells after CD47 activation. In vivo, we determined that PKHB1-treatment in mice bearing the L5178Y-R cell line increased leukocyte cell count in peripheral blood and lymphoid organs while recruiting leukocytes to the tumor site. To analyze whether CD47 activation induced immunogenic cell death (ICD), we evaluated damage-associated molecular patterns (DAMP) exposure (calreticulin, CRT) and release (ATP, heat shock proteins 70 and 90, high-mobility group box 1, CRT). Furthermore, we gave prophylactic antitumor vaccination, determining immunological memory. Our data indicate that PKHB1 induces caspase-independent and calcium-dependent cell death in leukemic cells while sparing non-tumor murine and human cells. Moreover, our results show that PKHB1 can induce ICD in leukemic cells as it induces CRT exposure and DAMP release in vitro, and prophylactic vaccinations inhibit tumor establishment in vivo. Together, our results improve the knowledge of CD47 agonist peptides potential as therapeutic tools to treat leukemia.

Advantages of adipose tissue stem cells over CD34+ mobilization to decrease hepatic fibrosis in Wistar rats.

INTRODUCTION AND OBJECTIVES: Chronic liver inflammation may lead to hepatic cirrhosis, limiting its regenerative capacity. The clinical standard of care is transplantation, although stem cell therapy may be an alternative option. The study aim was to induce endogenous hematopoietic stem cells (HSCs) with granulocyte colony stimulating factor (G-CSF) and/or intravenous administration of adipose tissue-derived mesenchymal stem cells (MSCs) to decrease hepatic fibrosis in an experimental model. MATERIAL AND METHODS: A liver fibrosis model was developed with female Wistar rats via multiple intraperitoneal doses of carbon tetrachloride. Three rats were selected to confirm cirrhosis, and the rest were set into experimental groups to evaluate single and combined therapies of G-CSF-stimulated HSC mobilization and intravenous MSC administration. RESULTS: Treatment with MSCs and G-CSF significantly improved alanine amino transferase levels, while treatment with G-CSF, MSCs, and G-CSF+MSCs decreased aspartate amino transferase levels. Hepatocyte growth factor (HGF) and interleukin 10 levels increased with MSC treatment. Transforming growth factor ß levels were lower with MSC treatment. Interleukin 1ß and tumor necrosis factor alpha levels decreased in all treated groups. Histopathology showed that MSCs and G-CSF reduced liver fibrosis from F4 to F2. CONCLUSIONS: MSC treatment improves liver function, decreases hepatic fibrosis, and plays an anti-inflammatory role; it promotes HGF levels and increased proliferating cell nuclear antigen when followed by MSC treatment mobilization using G-CSF. When these therapies were combined, however, fibrosis improvement was less evident.

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.

Immunization with a Synthetic Helicobacter pylori Peptide Induces Secretory IgA Antibodies and Protects Mice against Infection.

Helicobacter pylori is a spiral Gram-negative bacterium associated with inflammation of the gastric mucosa, peptic ulcer, and gastric adenocarcinoma, whose treatment has failed due to antibiotic resistance and side effects. Furthermore, because there are no vaccines effective against H. pylori, an appropriate vaccine design targeting conserved/essential genes must be identified. In the present study, a H. pylori 50-52 kDa immunogen-derived peptide antigen with the sequence Met-Val-Thr-Leu-Ile-Asn-Asn-Glu (MVTLINNE) was used to immunize against H. pylori infection. For this, mice received an intraperitoneal injection of 100 µg of H. pylori peptide on the first week, followed by two weekly subcutaneous reinforcements and further 109 bacteria administration in the drinking water for 3 weeks. Thymic cells proliferative responses to concanavalin A, serum levels of IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ, and TNF-α cytokines, and IgG1, IgG2a, IgG2b, IgG3 IgM, and IgA immunoglobulins were evaluated. Significant (p < 0.05) increases on lymphoproliferation and spleen weights after immunization were observed. In contrast, infection significantly (p < 0.05) decreased lymphoproliferation, which was recovered in immunized mice. In addition, levels of serum TH1 and TH2 cytokines were not altered after immunization, except for the significant increase in IL-6 production in immunized and/or infected animals. Moreover, immunization correlated with plasma secretory IgA and IgG, whereas infection alone only elicited IgM antibodies. Peptide immunization protected 100% of mice against virulent H. pylori. MVTLINNE peptide deserves further research as an approach to the prophylaxis of H. pylori infection.

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.