Plant extracts modulate cellular stress to inhibit replication of mouse Coronavirus MHV-A59.

The Covid-19 infection outbreak led to a global epidemic, and although several vaccines have been developed, the appearance of mutations has allowed the virus to evade the immune response. Added to this is the existing risk of the appearance of new emerging viruses. Therefore, it is necessary to explore novel antiviral therapies. Here, we investigate the potential in vitro of plant extracts to modulate cellular stress and inhibit murine hepatitis virus (MHV)-A59 replication. L929 cells were treated with P2Et (Caesalpinia spinosa) and Anamu SC (Petiveria alliacea) plant extracts during infection and virus production, ROS (reactive oxygen species), UPR (unfolded protein response), and autophagy were assessed. P2Et inhibited virus replication and attenuated both ROS production and UPR activation induced during infection. In contrast, the sustained presence of Anamu SC during viral adsorption and replication was required to inhibit viral infection, tending to induce pro-oxidant effects, and increasing UPR gene expression. Notably, the loss of the PERK protein resulted in a slight decrease in virus yield, suggesting a potential involvement of this UPR pathway during replication. Intriguingly, both extracts either maintained or increased the calreticulin surface exposure induced during infection. In conclusion, our findings highlight the development of antiviral natural plant extracts that differentially modulate cellular stress.

Modulation of Myeloid-Derived Suppressor Cells in the Tumor Microenvironment by Natural Products.

During carcinogenesis, the microenvironment plays a fundamental role in tumor progression and resistance. This tumor microenvironment (TME) is characterized by being highly immunosuppressive in most cases, which makes it an important target for the development of new therapies. One of the most important groups of cells that orchestrate immunosuppression in TME is myeloid-derived suppressor cells (MDSCs), which have multiple mechanisms to suppress the immune response mediated by T lymphocytes and thus protect the tumor. In this review, we will discuss the importance of modulating MDSCs as a therapeutic target and how the use of natural products, due to their multiple mechanisms of action, can be a key alternative for modulating these cells and thus improve response to therapy in cancer patients.

Plant extract from Caesalpinia spinosa inhibits cancer-associated fibroblast-like cells generation and function in a tumor microenvironment model.

Interactions in the tumor microenvironment (TME) between tumor cells and stromal cells such as cancer-associated fibroblasts (CAF) favor increased survival, progression, and transformation of cancer cells by activating mechanisms of invasion and metastasis. The design of new therapies to modulate or eliminate the CAF phenotype or functionality has been the subject of recent research including natural product-based therapies. We have previously described the generation of a standardized extract rich in polyphenols obtained from the Caesalpinia spinosa plant (P2Et), which present antitumor activities in breast cancer and melanoma models through activities that modulate the metabolism of tumor cells or induce the development of the immune response. In this work, a model of CAF generation was initially developed from the exposure of 3T3 fibroblasts to the cytokine TGFß1. CAF-like cells generated in this way exhibited changes in the expression of Caveolin-1 and α-SMA, and alterations in glucose metabolism and redox status, typical of CAFs isolated from tumor tissues. Then, P2Et was shown to counteract in vitro-induced CAF-like cell generation, preventing caveolin-1 loss and attenuating changes in glucose uptake and redox profile. This protective effect of P2Et translates into a decrease in the functional ability of CAFs to support colony formation and migration of 4T1 murine breast cancer tumor cells. In addition to the functional interference, the P2Et extract also decreased the expression of genes associated with the epithelial-mesenchymal transition (EMT) and functional activities related to the modulation of the cancer stem cells (CSC) population. This work is an in vitro approach to evaluate natural extracts' effect on the interaction between CAF and tumor cells in the tumor microenvironment; thus, these results open the chance to design a more profound and mechanistic analysis to explore the molecular mechanisms of P2Et multimolecular activity and extent this analysis to an in vivo perspective. In summary, we present here a standardized polymolecular natural extract that has the potential to act in the TME by interfering with CAF generation and functionality.

Regulatory Role of the RUNX2 Transcription Factor in Lung Cancer Apoptosis.

Lung cancer is the leading cause of cancer death globally. Numerous factors intervene in the onset and progression of lung tumors, among which the participation of lineage-specific transcription factors stands out. Several transcription factors important in embryonic development are abnormally expressed in adult tissues and thus participate in the activation of signaling pathways related to the acquisition of the tumor phenotype. RUNX2 is the transcription factor responsible for osteogenic differentiation in mammals. Current studies have confirmed that RUNX2 is closely related to the proliferation, invasion, and bone metastasis of multiple cancer types, such as osteosarcoma, breast cancer (BC), prostate cancer, gastric cancer, colorectal cancer, and lung cancer. Thus, the present study is aimed at evaluating the role of the RUNX2 transcription factor in inhibiting the apoptosis process. Loss-of-function assays using sh-RNA from lentiviral particles and coupled with Annexin/propidium iodide (PI) assays (flow cytometry), immunofluorescence, and quantitative PCR analysis of genes related to cell apoptosis (BAD, BAX, BCL2, BCL-XL, and MCL1) were performed. Silencing assays and Annexin/PI assays demonstrated that when RUNX2 was absent, the percentage of dead cells increased, and the expression levels of the BCL2, BCL-XL, and MCL1 genes were downregulated. Furthermore, to confirm whether the regulatory role of RUNX2 in the expression of these genes is related to its binding to the promoter region, we performed chromatin immunoprecipitation (ChIP) assays. Here, we report that overexpression of the RUNX2 gene in lung cancer may be related to the inhibition of the intrinsic apoptosis pathway, specifically, through direct transcriptional regulation of the antiapoptotic gene BCL2 and indirect regulation of BCL-XL and MCL1.

Randomized double-blind clinical study in patients with COVID-19 to evaluate the safety and efficacy of a phytomedicine (P2Et).

Background: It has been proposed that polyphenols can be used in the development of new therapies against COVID-19, given their ability to interfere with the adsorption and entrance processes of the virus, thus disrupting viral replication. Seeds from Caesalpinia spinosa, have been traditionally used for the treatment of inflammatory pathologies and respiratory diseases. Our team has obtained an extract called P2Et, rich in polyphenols derived from gallic acid with significant antioxidant activity, and the ability to induce complete autophagy in tumor cells and reduce the systemic inflammatory response in animal models. Methods: In this work, a phase II multicenter randomized double-blind clinical trial on COVID-19 patients was designed to evaluate the impact of the P2Et treatment on the clinical outcome and the immunological parameters related to the evolution of the disease. The Trial was registered with the number No. NCT04410510*. A complementary study in an animal model of lung fibrosis was carried out to evaluate in situ lung changes after P2Et in vivo administration. The ability of P2Et to inhibit the viral load of murine and human coronaviruses in cellular models was also evaluated. Results: Patients treated with P2Et were discharged on average after 7.4 days of admission vs. 9.6 days in the placebo group. Although a decrease in proinflammatory cytokines such as G-CSF, IL-15, IL-12, IL-6, IP10, MCP-1, MCP-2 and IL-18 was observed in both groups, P2Et decreased to a greater extent G-CSF, IL-6 and IL-18 among others, which are related to lower recovery of patients in the long term. The frequency of T lymphocytes (LT) CD3+, LT double negative (CD3+CD4-CD8-), NK cells increased in the P2Et group where the population of eosinophils was also significantly reduced. In the murine bleomycin model, P2Et also reduced lung inflammation and fibrosis. P2Et was able to reduce the viral replication of murine and human coronaviruses in vitro, showing its dual antiviral and anti-inflammatory role, key in disease control. Conclusions: Taken together these results suggest that P2Et could be consider as a good co-adjuvant in the treatment of COVID-19. Clinical trail registration: https://clinicaltrials.gov/ct2/show/NCT04410510, identifier: NCT04410510.

The delay in cell death caused by the induction of autophagy by P2Et extract is essential for the generation of immunogenic signals in melanoma cells.

P2Et extract obtained from the Caesalpinia spinosa plant is abundant in phenolic compounds such as gallic acid and ethyl gallate and can generate signals to activate the immune response by inducing a mechanism known as immunogenic cell death in murine models of breast cancer and melanoma. Immunogenic cell death involves mechanisms such as autophagy, which can be modulated by various natural compounds, including phenolic compounds with a structure similar to those found in P2Et extract. Here, we determine the role of autophagy in apoptosis and the generation of immunogenic signals using murine wild-type B16-F10 melanoma cells and cells with beclin-1 gene knockout. We show that P2Et extract and ethyl gallate induced autophagy, partially protecting tumor cells from death and promoting calreticulin exposure and the release of ATP. Although ethyl gallate showed a mechanism similar to that of P2Et, the induction of apoptosis and immunogenic signals was significantly weaker. In contrast, gallic acid-induced autophagy acted by blocking autophagic flux, which was associated with increased cell death. However, this compound did not induce any of the immunogenic death signals evaluated. Therefore, the complex extract has greater antitumor potential than isolated compounds. Here, we show that inducing autophagic flux with P2Et protects cancer cells from cell death and that this delay in cell death is required for the generation of immunogenic signals.

Phyto-Immunotherapy, a Complementary Therapeutic Option to Decrease Metastasis and Attack Breast Cancer Stem Cells.

In this review, we report on the complexity of breast cancer stem cells as key cells in the emergence of a chemoresistant tumor phenotype, and as a result, the appearance of distant metastasis in breast cancer patients. The search for mechanisms that increase sensitivity to chemotherapy and also allow activation of the tumor-specific immune response is of high priority. As we observed throughout this review, natural products isolated or in standardized extracts, such as P2Et or others, could act synergistically, increasing tumor sensitivity to chemotherapy, recovering the tumor microenvironment, and participating in the induction of a specific immune response. This, in turn, would lead to the destruction of cancer stem cells and the decrease in metastasis. Source of Data: Relevant studies were found using the following keywords or medical subject headings (MeSH) in PubMed, and Google Scholar: "immune response" and "polyphenols" and "natural products" and "BCSC" and "therapy" and "metabolism" and "immunogenic cell death." The focus was primarily on the most recent scientific publication.

Immunodeficiency in a Patient with 22q11.2 Distal Deletion Syndrome and a p.Ala7dup Variant in the MAPK1 Gene.

The genetic basis for sporadic immunodeficiency in patients with 22q11.2 distal deletion syndrome is unknown. We report an adult with a type 1 (D-F) 22q11.2 distal deletion syndrome and recurrent severe infections due to herpes zoster virus, presenting mild T cell lymphopenia and diminished frequency of naive CD4+ T cells, but increased frequencies of central, effector, and terminally differentiated memory T cells. Antigen-specific CD4+ and CD8+ T cells to influenza, rotavirus, and SEB were conserved in the patient, but responses to tetanus toxoid were temporarily undetectable. Exomic sequencing identified the c.20_22dupCGG (NM_002745.4) variant in the remaining MAPK1 gene of the patient, which adds 1 alanine to the polyalanine amino-terminal tract of the protein (p.Ala7dup). The mother, unlike the father, was heterozygote for the variant. Western blot analysis with the patient's activated PBMCs showed a 91% reduction in the MAPK1 protein. Further studies will be necessary to determine whether or not the variant present in the remaining MAPK1 gene of the patient is pathogenic.

Polyphenol-rich extract induces apoptosis with immunogenic markers in melanoma cells through the ER stress-associated kinase PERK.

Polyphenols elicit antitumor activities, in part, through the induction of anti- or pro-oxidant effects in cancer cells which promote priming of protective anti-tumor immunity. We recently characterized a polyphenol-rich extract from Caesalpinia spinosa (P2Et) that stimulates in vivo antitumor responses against breast and melanoma tumor models via the promotion of immunogenic cancer cell death (ICD). However, the primary mediators whereby P2Et promotes ICD remained unknown. Here, we sought to elucidate the role that severe endoplasmic reticulum (ER) stress plays in mediating P2Et-induced apoptosis and ICD in murine melanoma cells. Our findings demonstrate a substantial selective induction of specific ER-stress mediators in B16-F10 melanoma cells treated with P2Et. While knockout of the ER stress-associated PKR-like ER kinase (PERK) prevented induction of apoptosis and expression of ICD markers in P2Et-treated cells, deletion of X-box binding protein 1 (Xbp1) did not. P2Et-driven activation of PERK in melanoma cells was found to promote ER-calcium release, disrupt mitochondrial membrane potential, and trigger upregulation of ICD drivers, surface calreticulin expression, and extracellular release of ATP and HMGB1. Notably, calcium release inhibition, but not targeting of PERK-driven integrated stress responses, prevented P2Et-induced apoptosis. Collectively, these results underline the central role of PERK-directed calcium release in mediating the antitumor and immunogenic actions of P2Et in melanoma cells.

[Homocysteine levels and polymorphisms of MTHFR and CBS genes in Colombian patients with superficial and deep venous thrombosis]. / Niveles de homocisteína y polimorfismos de los genes de la MTHFR y la CBS en pacientes colombianos con trombosis venosa superficial y profunda.

INTRODUCTION: Thrombosis develops when the hemostatic system is incorrectly activated due to the unbalance between procoagulant, anticoagulant and fibrinolytic mechanisms allowing the formation of a clot within a blood vessel. The risk factors of this pathology can be acquired or can be genetic. OBJECTIVES: To analyze in a Colombian population with diagnosis of venous thrombosis, lipid profile, glucose and homocystein levels, to calculate the alleles and genotypic frequencies of polymorphisms c.699 C>T, c.1080 C>T, c.844ins68 of the cystathionine ß synthase and the c.677 C>T of the methylenetetrahydrofolate reductase (MTHFR) genes. MATERIALS AND METHODS: Thirty three patients and their controls were studied. The biochemical test was carried out by colorimetric methods and immunoassay. In this survey we used the restriction fragments longitude polymorphism (RLFP) technique to identify the polymorphisms mentioned. The association study was performed through the chi square test. RESULTS: We confirmed that gene alterations increase risk for pathology; we found statistically significant differences in the group with hypercholesterolemia in presence of the polymorphism c.699 C>T in the CBS gene, showing a protective effect in the individuals carrying this genetic variation. Likewise, we found a statistical trend for an eventual protective effect of the CBS c.844ins68 polymorphism to venous thrombotic disease. CONCLUSIONS: There were not any statistically significant differences in homocystein levels between cases and controls; nevertheless, the variability in the plasma concentrations was greater in the group of cases.

Niveles de homocisteína y polimorfismos de los genes de la MTHFR y la CBS en pacientes colombianos con trombosis venosa superficial y profunda / Homocysteine levels and polymorphisms of MTHFR and CBS genes in Colombian patients with superficial and deep venous thrombosis

Introducción. Se produce trombosis cuando en el sistema hemostático se desequilibran los mecanismos procoagulantes, anticoagulantes y fibrinolíticos, y se forman coágulos dentro de los vasos sanguíneos. Los factores de riesgo para el desarrollo de esta enfermedad pueden ser adquiridos o genéticos, polimorfismos o mutaciones en genes que conducen a hiperhomocisteinemia o que están comprometidos en las vías de coagulación. Objetivos. Analizar, en una población colombiana con diagnóstico de trombosis venosa el perfil lipídico, los niveles de glucosa y homocisteína, y calcular las frecuencias alélicas y genotípicas de los polimorfismos c.677C>T del gen de la metilen-tetra-hidrofolato reductasa (MTHFR) y c. 699C>T, c.1080 C>T, c.844ins68 del gen de la cistationina betasintasa (CBS).Materiales y métodos. Se estudiaron 33 pacientes con sus respectivos controles. Las pruebas bioquímicas se realizaron por métodos colorimétricos y de inmunoensayo. Se utilizó la técnica de fragmentos de longitud polimórfica para la identificación de los polimorfismos mencionados. El estudio de asociación se hizo mediante la prueba de de ji al cuadrado. Resultados. Se confirmó el papel de algunos factores de riesgo ya establecidos para el desarrollo de enfermedad trombótica venosa y se encontró un efecto protector del polimorfismo CBS c.699C>T para el riesgo de hipercolesterolemia con diferencia estadísticamente significativa en el grupo de los casos al compararlo con los controles. Por otra parte, se encontró una tendencia estadística que podría indicar un efecto protector del polimorfismo 844ins68 para el desarrollo de enfermedad trombótica venosa. Conclusiones. No se encontraron diferencias estadísticamente significativas en los niveles de homocisteína entre el grupo de casos y de controles. Sin embargo, la variabilidad en las concentraciones plasmáticas fue mayor en los casos.

Introduction. Thrombosis develops when the hemostatic system is incorrectly activated due to the unbalance between procoagulant, anticoagulant and fibrinolytic mechanisms allowing the formation of a clot within a blood vessel. The risk factors of this pathology can be acquired or can be genetic. Objectives. To analyze in a Colombian population with diagnosis of venous thrombosis, lipid profile, glucose and homocystein levels, to calculate the alleles and genotypic frequencies of polymorphisms c.699 C>T, c.1080 C>T, c.844ins68 of the cystathionine ß synthase and the c.677 C>T of the methylenetetrahydrofolate reductase (MTHFR) genes. Materials and methods. Thirty three patients and their controls were studied. The biochemical test was carried out by colorimetric methods and immunoassay. In this survey we used the restriction fragments longitude polymorphism (RLFP) technique to identify the polymorphisms mentioned. The association study was performed through the chi square test. Results. We confirmed that gene alterations increase risk for pathology; we found statistically significant differences in the group with hypercholesterolemia in presence of the polymorphism c.699 C>T in the CBS gene, showing a protective effect in the individuals carrying this genetic variation. Likewise, we found a statistical trend for an eventual protective effect of the CBS c.844ins68 polymorphism to venous thrombotic disease. Conclusions. There were not any statistically significant differences in homocystein levels between cases and controls; nevertheless, the variability in the plasma concentrations was greater in the group of cases.

Análisis descriptivo de los niveles de homocisteína, polimorfismos C699T, C1080T y 844Ins68pb de la CBS, C677T de la MTHFR y otros factores de riesgo cardiovascular en una población colombiana con síndrome coronario agudo / Descriptive analysis of homocysteine levels, polymorphisms C699T, C1080T and 844Ins68pb of CBS, C677T from the MTHFR, and other cardiovascular risk factors in a Colombian population with acute coronary syndrome

La enfermedad cardiovascular es la primera causa de muerte en el mundo occidental. Por esta razón, es necesario describir los factores de riesgo conocidos, al igual que los factores genéticos, nutricionales y ambientales emergentes, como la hiperhomocisteinemia y la deficiencia de la vitamina B12 y de ácido fólico en la población colombiana, que permitan proponer estrategias comunitarias de control de la enfermedad. El objetivo de este estudio fue describir los factores de riesgo conocidos y los emergentes,principalmente la hiperhomocisteinemia y los polimorfismos relacionados con ella en pacientes con síndrome coronario agudo. El estudio incluyó 156 pacientes, a quienes se les cuantificó perfil lipídico, glucosa, creatinina, urea, homocisteína, vitamina B12 y ácido fólico, y se describieron las frecuencias de las variantes polimórficas c.677C/T, de la MTHFR (5,10-methylenetetrahydrofolate reductase y c.699C/T, c.1080 C/T y c.844ins68pb de CBS (Cystathionine â-Synthase). El 43,6% de los pacientes con síndrome coronario agudo correspondió a las mujeres y el 56,4% a los hombres. Los valores medios de colesterol total, cLDL, cHDL, glucosa, homocisteína, vitaminas B12 y ácido fólico, se encontraron en el rango normal. Sin embargo, se pudo observar que la homociste ína presentaba una tendencia al aumento con la edad, tanto en hombres como en mujeres. Los niveles de cHDL en el grupo de hombres y de mujeres, presentaron diferencia significativa (p=0,0379) e, igualmente, la diferencia fue significativa en los niveles de creatinina (p=0007), de vitamina B12 (p=0,0341) y en la diabetes mellitus (p=0,0436). Con este estudio se realizó una aproximación a la descripción de los niveles del perfil lipídico, glucemia, hiperhomocisteinemia y de polimorfismos en genes involucrados en la vía de la homocisteínametionina, en pacientes con enfermedad cardiovascular en la población colombiana...

Cardiovascular diseases are the main cause of death in the western world. Therefore, it is necessary to describe the associated genetic, nutritional and environmental risk factors, including hyperhomocysteinemia, vitamin B12 and folic acid deficiencies in the Colombian population. Through this survey we want to propose strategies to the community in order to control cardiovascular diseases. The goal of this study was to describe the known risk factors and also the emerging ones such as hyperhomocysteinemia and some polymorphisms, in a Colombian population Our study included 156 patients with acute coronary artery syndrome, whose lipid, glucose, creatinine, homocysteine, vitamin B12 and folic acid levels were measured and the identification of polymorphisms 677C/T, from the MTHFR and 699C/T, 1080C/T, 844ins68pb of CBS. Overall, 43.6% of patients with acute coronary artery syndrome corresponded to women, and 56.4% to men who participated in this study. The results of cholesterol CLDL, CHDL, glucose, homocysteine, vitamin B12 and folic acid levels were found in normal ranges. However, we were able to observe that the homocysteine presented a tendency to increase with age in men and women,the CHDL levels within the group of men and women showed a significant difference (p=0.0379)as well as in the levels of creatinine (p=0.0007) of vitamin B12 (p=0.0341) and diabetes mellitus (p=0.0436). In this study, we propose a rough description of the lipid, glycemia, and hyperhomocysteinemia levels and polymorphisms in genes involved the homocysteine-methionine metabolism in patients with cardiovascular disease in the population of Colombia...