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In this work, the cell line SW620-GFP has been used in a complete magnetic hyperthermia assay, from the preparation of the ferrofluid with folate-coated iron oxide nanoparticles to in vivo experiments. The physical and chemical characterization of the nanoparticles evidenced their superparamagnetic behaviour, an average diameter of 12 ± 4 nm, a 2 nm coat thickness, and a high-power loss density. The main innovation of the work is the exclusive capability of viable SW620-GFP cells to emit fluorescence, enabling fast analysis of both, cell viability in vitro with an epifluorescence microscope and tumour size and shape in vivo in a non-invasive manner using the iBox technology. Moreover, with this imaging technique, it was possible to demonstrate the successful tumour size reduction in mice applying magnetic hyperthermia three times a week over 3 weeks.
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During the COVID-19 pandemic, the Ad5-nCoV vaccine was applied to the Mexican population before the WHO approved it. In a transversal study, we compare the CanSino vaccine efficacy and a natural SARS-CoV-2 infection in eliciting neutralizing antibodies against the SARS-CoV-2 Delta variant in Guadalajara, Mexico. Participants between 30-60 years were included in the study and classified into three groups: 1) Natural immunity (unvaccinated), 2) Vaccine-induced immunity (vaccinated individuals without a COVID-19 history), and 3) Natural immunity + vaccine-induced immunity. These groups were matched by age and gender. We assessed the ability of individuals' serum to neutralize the Delta variant and compared the results of the different groups using a neutralization test followed by plaque-forming units. Results showed that 39% of individuals' serum with a history of COVID-19 (natural immunity, Group 1) could not neutralize the Delta variant, compared to 33% in vaccinated individuals without COVID-19 (vaccine immunity, Group 2). In contrast, only 7% of vaccinated individuals with a history of COVID-19 (natural + vaccine immunities) could not neutralize the Delta variant. We concluded that the effectiveness of the Ad5-nCoV vaccine to induce neutralizing antibodies against the Delta variant is comparable to that of natural infection (61% vs. 67%). However, in individuals with both forms of immunity (Group 3), it increased to 93%. Based on these results, despite the Ad5-nCoV vaccine originally being designed as a single-dose regimen, it could be recommended that even those who have recovered from COVID-19 should consider vaccination to boost their immunity against this variant.
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COVID-19 , SARS-CoV-2 , Humanos , COVID-19/prevenção & controle , Anticorpos Neutralizantes , México/epidemiologia , Pandemias , Vacinas contra COVID-19 , Vacinação , Anticorpos AntiviraisRESUMO
Cervical cancer (CC) is a serious global health issue, and it is well-known that HPV infection is the main etiological factor that triggers carcinogenesis. In cancer, chemokine ligands and receptors are involved in tumor cell growth, metastasis, leukocyte infiltration, and angiogenesis; however, information on the role played by E6/E7 of HPV16/18 in the modulation of chemokines is very limited. Therefore, this study aimed to determine whether chemokines are differentially expressed in CC-derived cell lines; if E6/E7 oncoproteins from HPV16 and 18 are capable of mediating chemokine expression, what is the expression profile of chemokines in tissues derived from CC and what is their impact on the overall survival of patients with this pathology? For this purpose, RNA sequencing and real-time PCR were performed on SiHa, HeLa, and C33A tumorigenic cell lines, on the non-tumorigenic HaCaT cells, and the E6/E7 HPV-transduced HaCaT cell models. Furthermore, chemokine expression and survival analysis were executed on 304 CC and 22 normal tissue samples from The Cancer Genome Atlas (TCGA) repository. The results demonstrate that CXCL1, CXCL2, CXCL3, and CXCL8 are regulated by E6/E7 of HPV16 and 18, are overexpressed in CC biopsies, and that their higher expression is related to a worse prognostic survival.
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Epidermal growth factor receptor 2 (HER2) is the second target molecule most commonly used in breast cancer treatment. Both recurrence and metastasis are still deadly for HER2+ breast cancer patients. Hydrogels can be an option for developing three-dimensional (3D) cell culture systems that resemble tumor features better than monolayer cultures and could be used for preclinical screening for new biotherapeutics. Biopolymers (gelatin and alginate) were used to develop a hydrogel capable of encapsulating living HER2+ breast cancer cells BT-474/GFP. The hydrogel was physicochemically characterized, and the viability of embedded cells was evaluated. The hydrogel developed had suitable physical properties, with swelling of 38% of its original mass at 20 h capacity and pore sizes between 20 and 125 µm that allowed cells to maintain their morphology in a 3D environment, in addition to being biocompatible and preserving 90% of cell viability at 10 days. Furthermore, encapsulated BT-474/GFP cells maintained HER2 expression that could be detected by the Trastuzumab-fluorescent antibody, so this hydrogel could be used to evaluate new HER2-targeted therapies.
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A ferrofluid with 1,2-Benzenediol-coated iron oxide nanoparticles was synthesized and physicochemically analyzed. This colloidal system was prepared following the typical co-precipitation method, and superparamagnetic nanoparticles of 13.5 nm average diameter, 34 emu/g of magnetic saturation, and 285 K of blocking temperature were obtained. Additionally, the zeta potential showed a suitable colloidal stability for cancer therapy assays and the magneto-calorimetric trails determined a high power absorption density. In addition, the oxidative capability of the ferrofluid was corroborated by performing the Fenton reaction with methylene blue (MB) dissolved in water, where the ferrofluid was suitable for producing reactive oxygen species (ROS), and surprisingly a strong degradation of MB was also observed when it was combined with H2O2. The intracellular ROS production was qualitatively corroborated using the HT-29 human cell line, by detecting the fluorescent rise induced in 2,7-dichlorofluorescein diacetate. In other experiments, cell metabolic activity was measured, and no toxicity was observed, even with concentrations of up to 4 mg/mL of magnetic nanoparticles (MNPs). When the cells were treated with magnetic hyperthermia, 80% of cells were dead at 43 °C using 3 mg/mL of MNPs and applying a magnetic field of 530 kHz with 20 kA/m amplitude.
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Coloides/química , Coloides/farmacologia , Hipertermia Induzida/métodos , Nanopartículas Magnéticas de Óxido de Ferro/química , Espécies Reativas de Oxigênio/metabolismo , Catecóis/química , Linhagem Celular , Coloides/síntese química , Citotoxinas/síntese química , Citotoxinas/química , Citotoxinas/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Magnetismo , Microscopia Eletrônica de Transmissão , Oxidantes/síntese química , Oxidantes/química , Oxidantes/farmacologia , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Difração de Raios XRESUMO
Background: Several studies have shown that patients with cancer have antibodies in serum that react with cellular autoantigens, known as Tumor-Associated Antigens (TAA). The present work aimed to determine whether a mini-array comprising four recombinant TAA increases the detection of specific serum antibodies for the diagnosis of early-stage breast cancer. Methods: The mini-array included Alpha 1-AntiTrypsin (A1AT), TriosePhosphate Isomerase 1 (TPI1), Peptidyl-Prolyl cis-trans Isomerase A (PPIA), and PeroxiReDoXin 2 (PRDX2) full-length recombinant proteins. The proteins were produced after gene cloning, expression, and purification, and were verified by Western blot assays. Then, Dot-Blot was performed to find antibodies against the four TAA in 12 sera from women with early-stage breast cancer (stage II) and 12 sera from healthy women. Results: Antibody detection against individual TAA in early-stage breast cancer sera ranged from 58.3% to 83.3%. However, evaluation of the four TAA showed that there was a positive antibody reaction reaching a sensitivity of 100% and a specificity of 85% in early-stage breast cancer, suggesting that this mini-array must be evaluated as a clinical diagnostic tool for early-stage breast cancer in a larger sample size. Conclusion: Our results suggest that TAA mini-arrays may provide a promising and powerful method for improving the detection of breast cancer in Mexican women.
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Biomarcadores Tumorais/análise , Técnicas Biossensoriais , Neoplasias da Mama/diagnóstico , Soro/química , Adulto , Antígenos de Neoplasias , Neoplasias da Mama/sangue , Feminino , Testes Hematológicos , Humanos , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Breast cancer is a multifactorial and heterogeneous disease with distinct molecular features and histopathologic subtypes involving different therapeutic responses and clinical outcomes. Classification of breast cancer in molecular subtypes has made possible an approach to develop therapeutic strategies in order to have a better understanding of the breast cancer development. Due to the heterogeneity of the disease, there are still features to be elucidated in the behavior, etiology and clinical outcomes of each molecular subtype in breast cancer. METHODS: Variables measured in 1,695 cases of invasive breast carcinoma were age, histopathological diagnosis, histopathological grade, expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER2), cell proliferation marker (Ki67) and basal cytokeratins (CK 5/6). P values were obtained using Chi square test and hazard ratios were calculated with 95% confidence intervals. RESULTS: An increase of aggressive molecular subtypes of breast cancer was observed. The mean age of incidence of breast cancer patients is decreasing, and breast cancer Patients younger than 40-years-old showed higher risk to exhibit Triple negative and Basal-like tumors. CONCLUSIONS: The mean age for this pathology is decreasing in our population and there is predominance in the differential occurrence of etiologically distinct entities of breast cancer affecting to the young women.
INTRODUCCIÓN: El cáncer de mama es una enfermedad heterogénea y multifactorial. Presenta distintas características clínicas, moleculares e histopatológicas, las cuales se asocian con la respuesta a los esquemas terapéuticos, así como al resultado clínico. La clasificación en subtipos moleculares (luminales, HER2, triple negativo y basales) ha permitido el desarrollo y aplicación de estrategias terapéuticas particulares. Sin embargo, dada la gran heterogeneidad de la enfermedad, existen aún características por elucidar en el comportamiento, etiología y resultados clínicos de cada subtipo molecular de cáncer de mama. MÉTODOS: Se obtuvieron datos de 1695 casos de cáncer de mama invasor. Se realizaron correlaciones entre las siguientes variables: edad, diagnóstico histopatológico, grado histológico, expresión del receptor de estrógenos (ER), receptor de progesterona (PR), receptor 2 del factor de crecimiento epidérmico humano (HER2), marcador de proliferación celular (Ki67) y citoqueratinas basales (CK 5/6). Los valores de p fueron calculados utilizando Chi cuadrada y el cociente de riesgo fue calculado con un intervalo de confianza de 95%. RESULTADOS: Se observó un incremento en la frecuencia de los subtipos moleculares más agresivos, así como una disminución en el valor de la media de la edad en las pacientes diagnosticadas con cáncer de mama. El análisis de la información indica que en pacientes menores de 40 años existe mayor riesgo a la presencia de tumores triple negativo o basales. CONCLUSIONES: En población mexicana, la media de edad para el diagnóstico primario de cáncer de está disminuyendo y hay mayor frecuencia de subtipos moleculares más agresivos en pacientes jóvenes.
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B7H6, an endogenous ligand expressed on tumor cell surfaces, triggers NKp30-mediated activation of human NK cells. In contrast, the release of soluble B7H6 has been proposed as a novel mechanism by which tumors might evade NK cell-mediated recognition. Since NK cells are critical for the maintenance of early pregnancy, it is not illogical that soluble B7H6 might also be an important factor in directing NK cell activity during normal pregnancy. Thus, this study was focused on the characterization of soluble B7H6 during the development of normal pregnancy. Serum samples were obtained from healthy pregnant women who were experiencing their second pregnancies (n=36). Additionally, 17 of these pregnant participants were longitudinally studied for the presence of B7H6 during their second and third trimesters. Age-matched healthy non-pregnant women served as controls (n=30). The presence of soluble B7H6 was revealed by Western blotting. A further characterization was performed using an immunoproteomic approach based on 2DE-Western blotting combined with MALDI-MS. The results show that sera from all pregnant women were characterized by the presence of two novel isoforms of B7H6, both with lower MW than the reported of 51kDa. These isoforms were either a heavy (â¼37kDa) or a light isoform (â¼30kDa) and were mutually exclusive. N-glycosylation did not completely explain the different molecular weights exhibited by the two isoforms, as was demonstrated by enzymatic deglycosylation with PNGase F. The confirmation of the identity and molecular mass of each isoform indicates that B7H6, while maintaining the C- and N-termini, is most likely released during pregnancy by a mechanism distinct from proteolytic cleavage. We found that both isoforms, but mainly the heavier B7H6, were released via exosomes; and that the lighter isoform was also released in an exosome-free manner that was not observed in the heavy isoform samples. In conclusion, we find that soluble B7H6 is constitutively expressed during pregnancy and that, moreover, the soluble B7H6 is present in two new isoforms, which are released by exosomal and exosome-free mechanisms.
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Antígenos B7/sangue , Exossomos/metabolismo , Células Matadoras Naturais/imunologia , Receptor 3 Desencadeador da Citotoxicidade Natural/agonistas , Isoformas de Proteínas/genética , Antígenos B7/genética , Feminino , Regulação da Expressão Gênica , Glicosilação , Humanos , Ativação Linfocitária , Gravidez , Terceiro Trimestre da GravidezRESUMO
Bovine colostrum contains compounds, which provide passive immune protection from mother to newborn calves. Little is known about cytokine levels and their role in bovine colostrum. Moreover, the capacity of bovine colostrum cells to mount specific immune responses after natural exposure to bovine tuberculosis (bTB) antigens in dairy herds has not been studied, thus far. The purpose of this study was to identify biomarkers for bTB infection measurable in bovine colostrum. The present study reveals that isolated-immune colostrum cells can mount a specific immune response against bTB antigens, by measuring the novo IFN-γ release in cell culture. We found that IFN-γ levels in the responders (Bov+) to bTB antigen were higher than in non-responders (Bov-). On the other hand, proinflammatory cytokines contained in colostrum's whey were tested in Tuberculin Skin Test (TST) reactor (TST+) and non-reactor (TST-) animals to assess their potential role as biomarker. We observed that IFN-γ levels were lower or undetectable, as opposed to IL4 levels were measurable, the TNF-α level was higher in TST- than TST+, while IL-6 levels showed the opposite reaction and with no statistical significance. Moreover, IL-1α mRNA expression levels were higher in colostrum mononuclear cells (CMC) in Bov+ cattle. Collectively, these data suggest that the differential expression of pro and anti-inflammatory cytokines could have relevant value to diagnose bTB in cattle.
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Biomarcadores , Colostro/metabolismo , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Mycobacterium bovis/imunologia , Tuberculose Bovina/imunologia , Tuberculose Bovina/metabolismo , Animais , Antígenos de Bactérias/imunologia , Bovinos , Citocinas/genética , Feminino , Expressão Gênica , Testes de Liberação de Interferon-gama , Tuberculose Bovina/genéticaRESUMO
BACKGROUND: Assembly of recombinant capsid proteins into virus-like particles (VLPs) still represents an interesting challenge in virus-based nanotechnologies. The structure of VLPs has gained importance for the development and design of new adjuvants and antigen carriers. The potential of Tobacco etch virus capsid protein (TEV CP) as adjuvant has not been evaluated to date. FINDINGS: Two constructs for TEV CP expression in Escherichia coli were generated: a wild-type version (TEV-CP) and a C-terminal hexahistidine (His)-tagged version (His-TEV-CP). Although both versions were expressed in the soluble fraction of E. coli lysates, only His-TEV-CP self-assembled into micrometric flexuous filamentous VLPs. In addition, the His-tag enabled high yields and facilitated purification of TEV VLPs. These TEV VLPs elicited broader IgG2-specific antibody response against a novel porcine reproductive and respiratory syndrome virus (PRRSV) protein when compared to the potent IgG1 response induced by the protein alone. CONCLUSIONS: His-TEV CP was purified by immobilized metal affinity chromatography and assembled into VLPs, some of them reaching 2-µm length. TEV VLPs administered along with PRRSV chimeric protein changed the IgG2/IgG1 ratio against the chimeric protein, suggesting that TEV CP can modulate the immune response against a soluble antigen.
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Adjuvantes Imunológicos/administração & dosagem , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/administração & dosagem , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Potyvirus/imunologia , Vacinas Virais/imunologia , Virossomos/administração & dosagem , Citoesqueleto de Actina/metabolismo , Adjuvantes Imunológicos/metabolismo , Proteínas do Capsídeo/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Imunoglobulina G/sangue , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Virossomos/metabolismoRESUMO
Acaricidal activity of essential oils extracted from cumin seeds (Cuminum cyminum), allspice berries (Pimenta dioica) and basil leaves (Ocimum basilicum) were tested on 10-day-old Rhipicephalus (Boophilus) microplus tick larvae using the LPT. Two-fold dilutions of the three essential oils were tested from a starting dilution of 20% down to 1.25%. Results showed a high toxicological effect for cumin, producing 100% mortality in all tested concentrations on R. microplus larvae. Similarly, allspice essential oil produced 100% mortality at all concentrations with the exception of a dramatic decrease at 1.25% concentration. Conversely, basil essential oil was not shown to be toxic against R. microplus larvae. The most common compounds detected by gas chromatography-mass spectrometry were as follows: cumin: cuminaldehyde (22.03%), γ-terpinene (15.69%) and 2-caren-10-al (12.89%); allspice: methyl eugenol (62.7%) and eugenol (8.3%); basil: linalool (30.61%) and estragole (20.04%). Results clearly indicate that C. cyminum and P. dioica essential oils can be used as an effective alternative for R. microplus tick control, and there is a high probability they can be used for other ticks affecting cattle in Mexico and throughout the world, thereby reducing the necessity for traditional and unfriendly synthetic acaricides.
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Acaricidas/farmacologia , Cuminum/química , Ixodidae/efeitos dos fármacos , Ocimum basilicum/química , Óleos/farmacologia , Pimenta/química , Acaricidas/química , Animais , Bovinos , Doenças dos Bovinos/parasitologia , Cromatografia Gasosa-Espectrometria de Massas , Larva/efeitos dos fármacos , Óleos/química , Óleos Voláteis/química , Óleos Voláteis/farmacologia , Testes de Sensibilidade Parasitária , Óleos de Plantas/química , Óleos de Plantas/farmacologia , Infestações por Carrapato/parasitologia , Infestações por Carrapato/veterináriaRESUMO
This study combines two methodologies - vector expression of a genomic library and proteomics - to identify immunogenic proteins of Mycobacterium bovis. Immunization of BALB/c mice with a plasmid DNA pool from the library, containing approximately 8000 clones, induced a humoral response that facilitated the detection of 12 antigenic proteins by Western blotting. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry identified four proteins (Cpn60-1, HSP70, EF-Tu, and AdoHcyase). Such genomic immunization offers the possibility of in vivo screening of potential candidate M. bovis antigens.
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Antígenos de Bactérias/genética , DNA Bacteriano/genética , Biblioteca Genômica , Mycobacterium bovis/genética , Proteômica/métodos , Tuberculose Bovina/diagnóstico , Animais , Antígenos de Bactérias/imunologia , Western Blotting/veterinária , Bovinos , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida/veterinária , Espectrometria de Massas/veterinária , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/imunologia , Tuberculose Bovina/genética , Tuberculose Bovina/imunologiaRESUMO
Trichomonas vaginalis is a parasitic protist incapable of de novo purine and pyrimidine biosynthesis. The lack of these de novo syntheses of nucleotides is supplemented with purine and pyrimidine salvage pathways. Likewise, T. vaginalis is incapable of converting its ribonucleotides to deoxyribonucleotides. Therefore, the parasite must rely on the salvage of exogenous deoxyribonucleosides for DNA synthesis. It has been demonstrated that the parasite can incorporate external adenine and guanine in vitro, but no in vivo nucleotide source has been identified so far. Accordingly, we set out to determine if the parasite could incorporate 3H-thymidine from the nuclei of a cervical-derived cell line into its own DNA. By light and electron microscopy we found that the parasite was able to interact directly, both with mechanically isolated HeLa cell nuclei and with the nuclei released after the disruption of HeLa cell monolayers by the parasite. This study shows that T. vaginalis was capable of incorporating 3H-thymidine from labeled HeLa cells into its own DNA suggesting that the nuclei of this cervical cell line could be an in vivo source of nucleotides for T. vaginalis.
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Núcleo Celular/metabolismo , Células HeLa/metabolismo , Ribonucleosídeos/metabolismo , Timidina/metabolismo , Trichomonas vaginalis/metabolismo , Animais , Núcleo Celular/parasitologia , DNA/metabolismo , Células HeLa/parasitologia , Células HeLa/ultraestrutura , Interações Hospedeiro-Parasita/fisiologia , Humanos , Purinas/metabolismo , Trichomonas vaginalis/crescimento & desenvolvimento , Trichomonas vaginalis/ultraestruturaRESUMO
Trichomonosis, a chronic sexually transmitted disease, remains a public health problem affecting yearly over 170 million people worldwide. This disease is caused by Trichomonas vaginalis, a protozoan flagellate rich in cysteine proteinases (CPs). Although CPs are involved in trichomonal cytopathogenicity, only few of them have been defined as virulence factors. In this study, we characterize a T. vaginalis 39-kDa proteinase (CP39) found in vaginal secretions from patients with trichomonosis. The CP39 proteinase bound to HeLa epithelial cells, vaginal epithelial cells (VECs), and human prostatic cancer cells (DU-145). CP39 did not bind to a human colon cancer (CaCo) cell line, suggesting tissue-specific binding. CP39 was found in six fresh trichomonad isolates tested. In two-dimensional gels, CP39 appeared as a single spot with a pI 4.5. CP39 is inhibited by E-64, stable at 50 degrees C, and active in a wide pH range (3.6-9.0), with an optimum pH at 7.0. In addition, CP39 degraded collagens I, III, IV, and V, human fibronectin, human hemoglobin, and human immunoglobulins A and G. Indirect immunofluorescence detected CP39 on the parasite surface with specific polyclonal antibody to purified CP39. Finally, CP39 was found to be immunogenic, as evidenced by detection on immunoblots with serum of patients with trichomonosis, but not control individuals. These data suggest that CP39 may play a role during trichomonal infection.