RESUMEN

The immune response to SARS-CoV-2 has been extensively studied following the pandemic outbreak in 2020; however, the presence of specific T cells against SARS-CoV-2 before vaccination has not been evaluated in Mexico. In this study, we estimated the frequency of T CD4+ and T CD8+ cells that exhibit a specific response to S (spike) and N (nucleocapsid) proteins in a Mexican population. We collected 78 peripheral blood samples from unvaccinated subjects, and the presence of antibodies against spike (RBD) and N protein was determined. Peripheral blood mononuclear cells were isolated and stimulated with a pool of S or N protein peptides (Wuhan-Hu-1 strain). IL-1ß, IL-4, IL-6, IL-10, IL-2, IL-8, TNF-α, IFN-γ, and GM-CSF levels were quantified in the supernatant of the activated cells, and the cells were stained to assess the activation and memory phenotypes. Differential activation frequency dependent on serological status was observed in CD4+ cells but not in CD8+ cells. The predominantly activated population was the central memory T CD4+ cells. Only 10% of the population exhibited the same phenotype with respect to the response to nucleocapsid peptides. The cytokine profile differed between the S and N responses. S peptides induced a more proinflammatory response compared with the N peptides. In conclusion, in a Mexican cohort before vaccination, there was a significant response to the S and N SARS-CoV-2 proteins resulting from previous infections with seasonal coronaviruses or previous undetected exposure to SARS-CoV-2.

Asunto(s)

Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Vacunación , Humanos , México/epidemiología , SARS-CoV-2/inmunología , COVID-19/inmunología , COVID-19/epidemiología , COVID-19/prevención & control , Femenino , Masculino , Adulto , Linfocitos T CD8-positivos/inmunología , Persona de Mediana Edad , Linfocitos T CD4-Positivos/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Citocinas/metabolismo , Vacunas contra la COVID-19/inmunología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Adulto Joven , Fosfoproteínas/inmunología , Anciano , Activación de Linfocitos/inmunología

RESUMEN

The nematodeHaemonchus placeiis a pathogenic parasite, the most seriously affecting ruminant's health and being responsible for enormous economic losses all over the world. The present protocol describes different in vitro techniques to select potential candidate antigens with immune-protective activity from excretory and secretory products (ESP) fromH. placeitransitory infective larvae (xL3). ESP from xL3were obtained from the in vitro infective larvae (L3) maintained in Hank's medium at 37 °C with 5% CO2for 48 h. Then, the presence of ESP proteins was confirmed by SDS-PAGE to be used in an in vitro proliferation assay with bovine peripheral blood mononuclear cells (PBMCs). The ESP were exposed to the PBMCs during two different periods (24 and 48 h). Genes associated with immune response against the nematode were analyzed using relative gene expression and bioinformatic tools. These are simple, economic, and helpful tools to identify potential immune-protective molecules under in vitro conditions for confirming the efficacy of future in vivo assays. Graphical overview.

RESUMEN

The human akna gene encodes an AT-hook transcription factor, the expression of which is involved in various cellular processes. The goal of this study was to identify potential AKNA binding sites in genes that participate in T-cell activation and validate selected genes. Here we analyzed ChIP-seq and microarray assays to determine AKNA-binding motifs and the cellular process altered by AKNA in T-cell lymphocytes. In addition, we performed a validation analysis by RT-qPCR to assess AKNA's role in promoting IL-2 and CD80 expression. We found five AT-rich motifs that are potential candidates as AKNA response elements. We identified these AT-rich motifs in promoter regions of more than a thousand genes in activated T-cells, and demonstrated that AKNA induces the expression of genes involved in helper T-cell activation, such as IL-2. The genomic enrichment and prediction of AT-rich motif analyses demonstrated that AKNA is a transcription factor that can potentially modulate gene expression by recognizing AT-rich motifs in a plethora of genes that are involved in different molecular pathways and processes. Among the cellular processes activated by AT-rich genes, we found inflammatory pathways potentially regulated by AKNA, suggesting AKNA is acting as a master regulator during T-cell activation.

Asunto(s)

Proteínas de Unión al ADN , Factores de Transcripción , Humanos , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/metabolismo , Interleucina-2/metabolismo , Proteínas Nucleares/genética , Linfocitos T/metabolismo , Biología Computacional

RESUMEN

Objectives: Infection with high-risk human papillomavirus is required to develop cervical cancer. Some viruses modulate the Fas/FasL signaling to evade the immune response; the role of these molecules in cervical cancer is not clear. In this study, we measured the expression levels of Fas and FasL mRNA, soluble proteins, and cell surface proteins in peripheral blood mononuclear cells from patients with low- and high-grade squamous intraepithelial lesions and cervical cancer in relation to healthy women, to gain new insights into the role of Fas/FasL in cervical cancer development. Materials and Methods: Fas/FasL mRNA expression was measured in cervical tissues and peripheral blood mononuclear cells from patients and healthy subjects; serum soluble proteins Fas/FasL were measured by ELISA, and cell-surface protein expression was detected by flow cytometry. Results: Varying expression levels were found for both molecules. Cervical Fas and FasL mRNA expression was decreased in low- and high-grade lesions, but it was increased in cervical cancer cases. While, systemic Fas mRNA expression increased as malignity progressed; systemic FasL mRNA expression was increased in low- and high-grade lesions, but it was decreased in cancer patients. Soluble FasL levels decreased as lesions progressed, while soluble Fas levels increased. Finally, overexpression of Fas/FasL on the surface of peripheral blood mononuclear cells was found in patients with low-grade lesion with respect to healthy donors. Conclusion: Fas and FasL act as negative modulators of the immune response, probably by removing specific cytotoxic T lymphocytes against papillomavirus -infected cells and tumor cells.

RESUMEN

Mushrooms have health benefits, including anti-tumoral properties. We evaluated the cytotoxic and cell death induction effects of water-soluble extracts of Pleurotus ostreatus and Pleurotus eryngii mycelium in the cervical cancer cell lines HeLa (HVP18+) and SiHa (HVP16+) as well as the non-tumoral cell line HaCaT. Both Pleurotus extracts presented similar protein patterns from 190 to 10 kDa and displayed protease activity on a gelatine substrate. The mycelium extracts of both Pleurotus strains induced a dose-dependent cytotoxic effect on HPV+ cells IC50 65 µg), whereas HaCaT cells were less susceptible (IC50 90 µg). The cytotoxic effect at the IC50 concentration was not associated with apoptosis, the activation of Caspases-3/7 was not significantive; only P. eryngii induced a moderate (1.2-fold) increase in SiHa cells. Pleurotus extracts induced autophagy, mainly in SiHa cells (4.3-fold). Neither extracts induced changes in p53 protein expression, suggesting that the cytotoxic effect could be due to p53-independent pathways.

Asunto(s)

Antineoplásicos , Pleurotus , Neoplasias del Cuello Uterino , Femenino , Humanos , Pleurotus/química , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias del Cuello Uterino/tratamiento farmacológico , Micelio/química , Células HeLa , Antineoplásicos/farmacología , Antineoplásicos/análisis , Apoptosis

RESUMEN

The excretory/secretory (E/S) products released by infective transitory larvae (xL3) of Haemonchus placei have an important biological function in stimulating immune mechanisms during the invasive process. Our objective was to analyse the modulatory activity of 15 and 70 kDa E/S products from H. placei xL3. Both E/S products were collected from xL3in vitro cultures at 24 and 72 h. Proteins were confirmed by SDS-PAGE, and the corresponding spots were elicited by gel isoelectrofocusing (IEF) and characterised by mass spectrometry. Additionally, flow cytometry of CD4+/γδ+ T cells and immune gene expression were performed by proliferation assays using each E/S product to stimulate lymphocyte and peripheral blood mononuclear cells (PBMCs) from non-infected calves. The IEF results displayed two spots of 7.0 and 5.7 pI for the 15 and 70 kDa products, respectively. Additionally, 29 and 17 peptides from the 15 and 70 kDa E/S products, respectively, were identified with the hypothetical neurotransmitter and enzymatic functions necessary for larval development. The relative expression displayed upregulation of IL4, 5, 6, 8, 10, 13, IFNγ, and FCεR1A genes (from 2.0- to 17.6-fold, p < 0.05) stimulated by the 15 and 70 kDa proteins, indicating specific genes against haemonchosis. Although the percentage of median florescence intensity (MFI%) of CD4+/γδ+ T cells did not change for both E/S products compared to the negative control and concanavalin-A stimulated cells as the positive control (p > 0.05), the 15-kDa protein reduced the levels of both T cells, and the 70-kDa proteins increased the γδ+ cells slightly. Additionally, there was increased PBMCs proliferation by the 70 kDa proteins (p < 0.05), denoting the biological role of other immune cells. The 15 and 70 kDa protein E/S products from H. placei xL3 showed modulation of the immune response, and although more studies are required, they indicate important functions in the host/parasite interaction.

Asunto(s)

Antígenos Helmínticos , Haemonchus , Larva , Leucocitos Mononucleares , Linfocitos , Animales , Antígenos Helmínticos/farmacología , Bovinos , Haemonchus/química , Interacciones Huésped-Parásitos/inmunología , Factores Inmunológicos/farmacología , Larva/química , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Linfocitos/efectos de los fármacos , Linfocitos/inmunología

RESUMEN

Secundiflorol G (SG) is an isoflavan isolated from the root bark of Aeschynomene fascicularis, a Mayan medicinal plant used to treat cancer-like symptoms. SG has been shown to have cytotoxic effects on cervical cancer cells (HeLa). Assays were done to identify the mechanisms of SG's cytotoxic effect.HeLa cells treated with SG exhibited early and late apoptosis, and caspase-9, -8 and -3 activities. It also induces generation of reactive oxygen species and disrupted mitochondrial membrane potential.SG isolated from A. fascicularis induces apoptosis through extrinsic and intrinsic pathways on HeLa cells. SG could be a candidate for in vivo studies and a promising natural compound in cervical cancer treatment.

Asunto(s)

Apoptosis/efectos de los fármacos , Benzopiranos/aislamiento & purificación , Benzopiranos/farmacología , Fabaceae/química , Isoflavonas/aislamiento & purificación , Isoflavonas/farmacología , Plantas Medicinales/química , Neoplasias del Cuello Uterino/patología , Antineoplásicos/farmacología , Benzopiranos/química , Caspasas/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Activación Enzimática/efectos de los fármacos , Femenino , Células HeLa , Humanos , Isoflavonas/química , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno/metabolismo , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/enzimología , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

The aim of the present study was to assess the expression of cytokines and FCεR1A receptor stimulated by Haemonchus placei larval excretory and secretory (ES) products associated with the pathogenesis in calves. Bovine peripheral blood mononuclear cells (PBMC) were stimulated in in vitro assays with H. placei L4 ES product at 8, 12, 16 and 24 h. ES products were collected in in vitro assays at 48 h with molecular weight of 72/60 kDa and isoelectric point of 7.2 pI. Specific IgG for infected and control calves, positive and negative, were employed to recognise H. placei larval ES products by indirect ELISA, showing a mean of 1.8, 0.83 and 0.28 OD, respectively, (p ≤ 0.001). The quantification of relative gene expression was performed using a set of cytokines (IL-2, IFNγ, TGFß, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-13), FCεR1A receptor and housekeeping (GAPDH, ß-actin and ß-2-microglobulin) by RT-qPCR. An early increased expression, 2.2- to 3.4-fold change, of IL-2 (p ≤ 0.001), IL-5 and TGFß (p ≥ 0.05) was determined, followed by TGFß (30.7 and 14.14), IL-8 (102.8 and 1504.4) and IL-10 (60.4 and 1.7) (p ≤ 0.05) after 12 and 16 h, respectively, and reducing the expression level at 24 h. In addition, IL-6, IL-13 and FCεR1A receptor also displayed mild expression level, 2.1 - to 7.60-fold change, at 24 h (p ≥ 0.05). We conclude that ES products of 72/60 kDa collected in vitro from H. placei larvae are recognised by infected hosts and have the ability to induce diverse immune factors to modulate the nematode damage.

Asunto(s)

Citocinas/metabolismo , Haemonchus/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Receptores de IgE/metabolismo , Animales , Bovinos , Proliferación Celular/efectos de los fármacos , Citocinas/genética , Haemonchus/genética , Haemonchus/inmunología , Inmunoglobulina G/metabolismo , Larva/genética , Larva/inmunología , Larva/metabolismo , Leucocitos Mononucleares/metabolismo , Masculino , ARN Mensajero/metabolismo , Regulación hacia Arriba

RESUMEN

Persistent infection with high-risk Human Papillomavirus (HR-HPV) is the main requisite for cervical cancer development. Normally, HPV is limited to the site of infection and regulates a plethora of cellular elements to avoid the immune surveillance by inducing an anti-inflammatory state, allowing the progress through the viral cycle and the carcinogenic process. Recent findings suggest that the AT-hook transcriptional factor AKNA could play a role in the development of cervical cancer. AKNA is strongly related to the expression of co-stimulatory molecules such CD40/CD40L to achieve an anti-tumoral immune response. To date, there is no evidence demonstrating the effect of the HPV E6 oncoprotein on the AT-hook factor AKNA. In this work, minimal expression of AKNA in cervical carcinoma compared to normal tissue was found. We show the ability of E6 from high-risk HPVs 16 and 18 to interact with and down-regulate AKNA as well as its co-stimulatory molecule CD40 in a proteasome dependent manner. We also found that p53 interacts with AKNA and promotes AKNA expression. Our results indicate that the de-regulation of CD40 and AKNA is induced by the HPV E6 oncoprotein, and this event involves the action of p53 suggesting that the axis E6/p53A/AKNA might play an important role in the de-regulation of the immune system during the carcinogenic process induced by HR-HPV.

RESUMEN

The E6 oncoprotein can interfere with the ability of infected cells to undergo programmed cell death through the proteolytic degradation of proapoptotic proteins such as p53, employing the proteasome pathway. Therefore, inactivation of the proteasome through MG132 should restore the activity of several proapoptotic proteins. We investigated whether in HPV16 E6-expressing keratinocytes (KE6 cells), the restoration of p53 levels mediated by MG132 and/or activation of the CD95 pathway through apoptosis antigen-1 (APO-1) antibody are responsible for the induction of apoptosis. We found that KE6 cells underwent apoptosis mainly after incubation for 24 h with MG132 alone or APO-1 plus MG132. Both treatments activated the extrinsic and intrinsic apoptosis pathways. Autophagy was also activated, principally by APO-1 plus MG132. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in the elevation of p53 protein levels and its phosphorylation in Ser46 and Ser20; the p53 protein was localized mainly at nucleus after treatment with MG132 or APO-1 plus MG132. In addition, induction of its transcriptional target genes such as p21, Bax and TP53INP was observed 3 and 6 h after treatment. Also, LC3 mRNA was induced after 3 and 6 h, which correlates with lipidation of LC3B protein and induction of autophagy. Finally, using pifithrin alpha we observed a decrease in apoptosis induced by MG132, and by APO-1 plus MG132, suggesting that restoration of APO-1 sensitivity occurs in part through an increase in both the levels and the activity of p53. The use of small molecules to inhibit the proteasome pathway might permit the activation of cell death, providing new opportunities for CC treatment.

Asunto(s)

Apoptosis/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Neoplasias del Cuello Uterino/genética , Receptor fas/genética , Anticuerpos/farmacología , Autofagia/efectos de los fármacos , Benzotiazoles/farmacología , Proteínas Portadoras/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Proteínas de Choque Térmico/genética , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/virología , Leupeptinas/farmacología , Proteínas Oncogénicas Virales/genética , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Proteolisis/efectos de los fármacos , Proteínas Represoras/genética , Tolueno/análogos & derivados , Tolueno/farmacología , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/virología , Proteína X Asociada a bcl-2/genética , Receptor fas/metabolismo

RESUMEN

Toxoplasma gondii invades any nucleated cell, but different replication speed and effects on survival/apoptosis processes have been found depending on cell type. There are scarce and controversial results regarding the effect of this parasite on host cell apoptosis within the brain. The invasion and replication of T. gondii RH strain within newborn mouse astrocytes were evaluated in the present work. At 4 hpi>90% cells were infected and harbored one to three parasitophorous vacuoles with one tazchyzoite/vacuole. Cell culture massive destruction started after 24 h of exposure, when the parasite already replicated, with a duplication time of around 5 h. The effect of T. gondii infection on apoptosis was also evaluated by changes in some anti- and pro-apoptotic markers. At early infection times decreased Bcl-2, Survivin and PUMA and increased Noxa expression was found, although Survivin and Noxa mRNA levels reverted towards an anti-apoptotic phenotype after 6 h. Caspases 3/7 activity decreased three hours after infection, although it returned to normal levels thereafter. This enzymatic activity was strongly stimulated by Cisplatin (anti-neoplasic drug) but it was inhibited by previous T. gondii infection. Likewise, parasite invasion prevented PARP-1 fragmentation and cell apoptosis induced by the same drug. In conclusion, astrocytes seem to activate some apoptosis signals shortly after infection, but the parasite takes control of the cell and inhibits programmed death for up to 24 h, until it replicates, egresses and generates cellular destruction.

Asunto(s)

Proteínas Reguladoras de la Apoptosis/genética , Astrocitos/parasitología , Proteínas Protozoarias/genética , Toxoplasma/fisiología , Animales , Animales Recién Nacidos , Antineoplásicos/farmacología , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Astrocitos/citología , Astrocitos/efectos de los fármacos , Encéfalo/citología , Caspasas/metabolismo , Cisplatino/farmacología , Femenino , Regulación de la Expresión Génica , Genes bcl-2/genética , Proteína Ácida Fibrilar de la Glía , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Organismos Libres de Patógenos Específicos , Survivin , Toxoplasma/crecimiento & desarrollo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

Toxoplasma gondii is a cosmopolitan protozoan which infects all homoeothermic species, including humans. This parasite may cause severe neurological problems in congenitally infected newborns or immunocompromised individuals, but it also provokes psychiatric and neurological disorders as well as behavioural and sensory deficit. There is controversy regarding the effect of T. gondii upon astrocytes, which may serve as parasite proliferation recipients or protective immune response activators within the central nervous system. This apparent contradiction could partially be due to the infection degree obtained in the different experiments reported. Thus, we decided to systematically review the in vitro models used to study these phenomena. Fifteen articles from which direct invasion and replication data could be gathered were found. Very heterogeneous results emerged, mainly due to diversity of models in relation to parasite strain (virulence), host species, parasite dose and evaluation times after infection. Also, the results were measured in diverse ways, i.e. some reported percent infected cells, while others informed parasites pervacuole or cell, or parasitic vacuoles per cell. Very few conclusions could be drawn, among them that human astrocytoma cell lines and mouse astrocytes seem more susceptible to infection and less resistant to tachyzoite proliferation than human primary culture astrocytes. The present study supports the need to reanalyse T. gondii astrocyte invasion and replication processes, especially with the use of actual technology, which allows detailed mechanistic studies.

Asunto(s)

Astrocitos/parasitología , Toxoplasma/crecimiento & desarrollo , Toxoplasma/patogenicidad , Animales , Técnicas de Cultivo de Célula , Células Cultivadas , Humanos , Ratones , Toxoplasma/inmunología