8 research outputs found
Transcriptional Activation of a Pro-Inflammatory Response (NF-κB, AP-1, IL-1β) by the <i>Vibrio cholerae</i> Cytotoxin (VCC) Monomer through the MAPK Signaling Pathway in the THP-1 Human Macrophage Cell Line
This study describes, to some extent, the VCC contribution as an early stimulation of the macrophage lineage. Regarding the onset of the innate immune response caused by infection, the β form of IL-1 is the most important interleukin involved in the onset of the inflammatory innate response. Activated macrophages treated in vitro with VCC induced the activation of the MAPK signaling pathway in a one-hour period, with the activation of transcriptional regulators for a surviving and pro-inflammatory response, suggesting an explanation inspired and supported by the inflammasome physiology. The mechanism of IL-1β production induced by VCC has been gracefully outlined in murine models, using bacterial knockdown mutants and purified molecules; nevertheless, the knowledge of this mechanism in the human immune system is still under study. This work shows the soluble form of 65 kDa of the Vibrio cholerae cytotoxin (also known as hemolysin), as it is secreted by the bacteria, inducing the production of IL-1β in the human macrophage cell line THP-1. The mechanism involves triggering the early activation of the signaling pathway MAPKs pERK and p38, with the subsequent activation of (p50) NF-κB and AP-1 (cJun and cFos), determined by real-time quantitation. The evidence shown here supports that the monomeric soluble form of the VCC in the macrophage acts as a modulator of the innate immune response, which is consistent with the assembly of the NLRP3 inflammasome actively releasing IL-1β
Culture supernatants from V. cholerae O1 ElTor strains isolated from different geographic areas induce cell vacuolation and cytotoxicity Cepas de V. cholerae O1 biotipo ElTor aisladas de diferente origen geográfico inducen vacuolización celular y citotoxicidad
OBJECTIVE: To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. MATERIAL AND METHODS: Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. RESULTS: All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+) and a non-toxigenic Mexican strain (CM 91-3, ctxAB-). Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. CONCLUSION: The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.<br>OBJETIVO: Analizar el efecto vacuolizante de cepas de V. cholerae O1 ElTor aisladas de diferente origen geográfico, incluyendo México. MATERIAL Y MÉTODOS: Se realizaron pruebas de hemolisis, vacuolización y citotoxicidad en células Vero, así como PCR, análisis por RFLP y clonación molecular. RESULTADOS: Todas las cepas indujeron el efecto vacuolizante. Las cepas del ribotipo 2, aisladas de las costas del Golfo en Estados Unidos, presentaron títulos altos de vacuolización. El gen hlyA fue amplificado en las nueve cepas mediante PCR, aunque sólo ocho fueron hemolíticas. Se clonó el gen hlyA de una cepa toxigénica (2514-88, ctxAB+) y de una cepa no toxigénica aislada en México (CM 91-3, ctxAB-). El sobrenadante de las clonas recombinantes indujo hemólisis, efecto vacuolizante y citotoxicidad. El RFLP mostró alta similitud del gen hlyA de las cepas estudiadas. CONCLUSIÓN: El efecto vacuolizante es un fenotipo ampliamente distribuido en cepas epidémicas de V. cholerae O1 biotipo ElTor
Perfil de polifenoles, capacidad antioxidante y efecto citotóxico in vitro en líneas celulares humanas de un extracto hidroalcohólico de pétalos de Calendula officinalis L.
The hydro-alcoholic extract from Calendula officinalis petals, was evaluated showing beneficial effects against oxidative stress and therefore on diseases associated to inflammatory processes. The content of polyphenols of the extract was determined by TLC and HPLC-UV-DAD and the antioxidant efficiency estimated from Fe3+/Fe2+ reducing power, Cu2+ and Fe2+ chelation, •OH radical scavenging, ABTS [2, 2´-azino bis (3 ethylbenzothiazoline-6-sulphonic acid)] and DPPH (2,2´diphenyl-1-picrylhydrazyl) assays. The in vitro growth inhibition (IC50) in human cancer cell breast (MDA-MB-231), uterine cervix (HeLa), lung (A-549), colon (HT-29) and (Caco-2) was determined by MTT method. The extract showed that the polyphenols content in the hydro-alcoholic extract was 37.01±0.015 mg GAE g–1, being rutin the most abundant one. ABTS TEACand DPPHTEAC tests showed a high antioxidant capacity. The antioxidant activity profile displayed high reducing capacity and ability to chelate Cu2+ and Fe2+ as well as good •OH radical scavenging capacity. The extract showed a cytotoxic effect on human cancer cell lines of colon (HT-29)and (Caco-2),breast (MDA-MB-231), uterine cervix (HeLa),and lung (A-549)(IC50 = 10.84, 11.73, 11.26, 11.53, and 11.96 µg mL–1, respectively). The correlation analysis suggests that the Calendula officinalis polyphenols are directly related to the antioxidant efficiency of the extract and inversely to the cell viability.Se evaluó el extracto hidroalcohólico de los pétalos de Calendula officinalis, mostrando efectos benéficos contra el estrés oxidativo y enfermedades asociadas a procesos inflamatorios. El contenido de polifenoles del extracto fue determinado por TLC y HPLC-UV-DAD y la capacidad antioxidante se estimó del poder reductor de Fe3+/Fe2+, quelación de Cu2+ y Fe2+, eliminación de radicales •OH, y ensayos ABTS [2, 2´-azino-bis (3 etilbenzotiazolin-6-sulfónico) y DPPH (2,2´difenil-1-picrilhidracilo). La inhibición del crecimiento in vitro (IC50) en células cancerosas humanas de mama (MDA-MB-231), de cuello uterino (HeLa), pulmón (A-549), ycolon (HT-29 y Caco-2),fue determinado por el método de MTT.El extracto mostró que el contenido total de polifenoles fue de 37.01±0.015 mg GAe g-1, siendo la rutina la más abundante. Las pruebas ABTS TEAC y DPPH TEAC mostraron una alta capacidad antioxidante. El perfil de actividad antioxidante mostró buena capacidad reductora y capacidad para quelar Cu2+ y Fe2+, así como una buena capacidad de eliminación de radicales •OH. El extracto también mostró efecto citotóxico en células de colon (HT-29 y Caco-2), de mama (MDA-MB-231), de cuello uterino (HeLa) y pulmón (A-549) (IC50 = 10.84, 11.73, 11.26, 11.53 y 11.96 μg mL-1, respectivamente). El análisis de correlación sugiere que los polifenoles de Calendula officinalis están directamente relacionados con la capacidad antioxidante del extracto y se asocian inversamente con la viabilidad celular
Overexpression of MMP-3 and uPA with Diminished PAI-1 Related to Metastasis in Ductal Breast Cancer Patients Attending a Public Hospital in Mexico City
Extracellular matrix metalloproteases and the fibrinolytic system are important protease systems interacting with each other in charge of remodeling and recycling of tissues. Their role in tumor invasion and metastasis is often discussed. In this study several metalloproteases such as MMP-1, MMP-3, MMP-9, and TIMP-1 together with molecules from the fibrinolytic system like uPA, its receptor uPAR, and its inhibitor, PAI-1, were studied by immune-histochemistry to establish a comparison with and without metastasis. From the (118) primary tumors of Mexican patients with ductal breast cancer studied, 56% were grade II and 69% were size T2; the group with metastatic ganglia included 64 samples (54.3%). In patients with metastasis the estimated expression of MMP-3 and uPA (resp., 28% and 45%) was higher than that from no metastatic tumors; it means there is higher expression of both markers in metastatic tumors (p<0.05). At the same time, metastatic tumors showed statistically significant lower signal of PAI-1 (24%) than tumors without metastasis (p<0.05). We concluded that overexpression of MMP-3 and uPA, altogether with diminished expression of PAI-1 from metastatic tumors, might be a crucial step towards metastasis in ductal breast cancer. Nevertheless, additional studies in different populations are necessary to establish a pattern
ADIPOQ and ADIPOR2 gene polymorphisms: association with overweight/obesity in Mexican children
Background: ENSANUT 2012 showed a combined prevalence of overweight and obesity of 34.4% in Mexican children. Single nucleotide polymorphisms (SNPs) of the ADIPOQ and ADIPOR2 genes have been reported in many populations, but their association with obesity has not been confirmed in other studies. Our aim was to determine the association of SNPs from ADIPOQ and ADIPOR2 genes with obesity in Mexican children.
Methods: A total of 2,634 children from 6 to 12 years old were enrolled in the study from four IMSS Units in Mexico City. We selected 1,469 unrelated children (745 normal weight and 724 overweight/obese). Phenotype characterization included anthropometric measurements, blood pressure, biochemical parameters, insulin concentrations and presence of acanthosis nigricans (AN). Analysis of the SNPs rs182052, rs266729, rs2241766, rs822393 of ADIPOQ and rs11061971 of ADIPOR2 was carried out in the DNA samples.
Results: The study showed significant differences (p <0.05) between groups in waist circumference, blood pressure, presence of AN, insulin concentrations, HOMA-IR, fasting glucose and lipid parameters, being higher in obese children. No associations in ADIPOQ variants with the presence of overweight/obesity were found. The presence of the variant rs11061971 of ADIPOR2 in children had a significant association with protection of overweight/obesity (OR 0.79, 95% CI 0.68-0.93, p = 0.003). Also, the log-additive model confirmed the association by codominant and dominant models (p <0.05).
Conclusions: The presence of rs11061971 of ADIPOR2 variant confers protection against obesity and could be used as a marker in Mexican children
Development of a multiplex PCR assay to detect gastroenteric pathogens in the feces of mexican children
Abstract Acute gastroenteritis (AGE) is a major cause of
childhood morbidity and mortality worldwide; the etiology
ofAGEincludes viruses, bacteria, and parasites.Amultiplex
PCR assay to simultaneously identify human Astrovirus
(HAstV), Calicivirus (HuCVs), Entamoeba histolytica
(E. histolytica), and enteroinvasive Escherichia coli (EIEC)
in stool samples is described. A total of 103 samples were
individually analyzed by ELISA (enzyme-linked immunosorbent
assays) and RT-PCR/PCR. HAstV and HuCVs were
detected in four out of 103 samples (3.8 %) by RT-PCR, but
ELISAs found only one sample as positive for HuCVs
(2.5 %). E. histolytica was identified in two out of 19 samples
(10.5 %) and EIEC in 13 out of 20 samples (70 %) by PCR,
and all PCR products were sequenced to verify their identities.
Our multiplex PCR results demonstrate the simultaneous
amplification of different pathogens such as HAstV,
EIEC, and E. histolytica in the same reaction, though the
HuCVs signal was weak in every replicate. Regardless, this
multiplex PCR protocol represents a novel tool for the identification of distinct pathogens and may provide support for the diagnosis of AGE in children
<i>Brachybacterium conglomeratum</i> Is Associated with Cervicovaginal Infections and Human Papilloma Virus in Cervical Disease of Mexican Female Patients
Brachybacterium conglomeratum, traditionally considered an environmental bacterium, has recently garnered attention for its potential involvement in human health. While prior research hinted at its pathogenic role in humans, our study aims to determine its prevalence and associations in diverse clinical contexts. We examined vaginal swabs from three distinct patient groups: patients with low-grade squamous intraepithelial lesions (LSIL), patients with cervicovaginal infections, and patients with a history of precancerous lesions undergoing follow-up. B. conglomeratum was present in all three patient groups, with the highest prevalence observed in the LSIL group. Statistically significant associations were primarily identified in the LSIL group, where B. conglomeratum was present in 60% of cases. Notably, the LSIL group exhibited coinfections with multiple high-risk oncogenotypes of human papillomavirus (HPV), suggesting potential synergistic effects, and understanding these microbial relationships and their influence on viral persistence, particularly with HPV, holds promise for mitigating HPV-related carcinogenesis. Furthermore, Gardnerella vaginalis and Atopobium vaginae were frequently detected in this group, along with Ureaplasma parvum as the predominant sexually transmitted bacterium. In all cases, B. conglomeratum was found in association with these microorganisms rather than as a sole pathogen. This coexistence underscores the intricate microbial interactions within cervicovaginal infections and precancerous lesions. This study marks the first report of B. conglomeratum prevalence in women with these clinical conditions
A global metagenomic map of urban microbiomes and antimicrobial resistance
We present a global atlas of 4,728 metagenomic samples from mass-transit systems in 60 cities over 3 years, representing the first systematic, worldwide catalog of the urban microbial ecosystem. This atlas provides an annotated, geospatial profile of microbial strains, functional characteristics, antimicrobial resistance (AMR) markers, and genetic elements, including 10,928 viruses, 1,302 bacteria, 2 archaea, and 838,532 CRISPR arrays not found in reference databases. We identified 4,246 known species of urban microorganisms and a consistent set of 31 species found in 97% of samples that were distinct from human commensal organisms. Profiles of AMR genes varied widely in type and density across cities. Cities showed distinct microbial taxonomic signatures that were driven by climate and geographic differences. These results constitute a high-resolution global metagenomic atlas that enables discovery of organisms and genes, highlights potential public health and forensic applications, and provides a culture-independent view of AMR burden in cities.Funding: the Tri-I Program in Computational Biology and Medicine (CBM) funded by NIH grant 1T32GM083937; GitHub; Philip Blood and the Extreme Science and Engineering Discovery Environment (XSEDE), supported by NSF grant number ACI-1548562 and NSF award number ACI-1445606; NASA (NNX14AH50G, NNX17AB26G), the NIH (R01AI151059, R25EB020393, R21AI129851, R35GM138152, U01DA053941); STARR Foundation (I13- 0052); LLS (MCL7001-18, LLS 9238-16, LLS-MCL7001-18); the NSF (1840275); the Bill and Melinda Gates Foundation (OPP1151054); the Alfred P. Sloan Foundation (G-2015-13964); Swiss National Science Foundation grant number 407540_167331; NIH award number UL1TR000457; the US Department of Energy Joint Genome Institute under contract number DE-AC02-05CH11231; the National Energy Research Scientific Computing Center, supported by the Office of Science of the US Department of Energy; Stockholm Health Authority grant SLL 20160933; the Institut Pasteur Korea; an NRF Korea grant (NRF-2014K1A4A7A01074645, 2017M3A9G6068246); the CONICYT Fondecyt Iniciación grants 11140666 and 11160905; Keio University Funds for Individual Research; funds from the Yamagata prefectural government and the city of Tsuruoka; JSPS KAKENHI grant number 20K10436; the bilateral AT-UA collaboration fund (WTZ:UA 02/2019; Ministry of Education and Science of Ukraine, UA:M/84-2019, M/126-2020); Kyiv Academic Univeristy; Ministry of Education and Science of Ukraine project numbers 0118U100290 and 0120U101734; Centro de Excelencia Severo Ochoa 2013–2017; the CERCA Programme / Generalitat de Catalunya; the CRG-Novartis-Africa mobility program 2016; research funds from National Cheng Kung University and the Ministry of Science and Technology; Taiwan (MOST grant number 106-2321-B-006-016); we thank all the volunteers who made sampling NYC possible, Minciencias (project no. 639677758300), CNPq (EDN - 309973/2015-5), the Open Research Fund of Key Laboratory of Advanced Theory and Application in Statistics and Data Science – MOE, ECNU, the Research Grants Council of Hong Kong through project 11215017, National Key RD Project of China (2018YFE0201603), and Shanghai Municipal Science and Technology Major Project (2017SHZDZX01) (L.S.