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1.
Sci Rep ; 14(1): 15112, 2024 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956203

RESUMO

Cancer mice models are critical for immune-oncology research; they provide conditions to explore tumor immunoenviroment aiming to advance knowledge and treatment development. Often, research groups breed their own mice colonies. To assess the effect of C57BL/6 mice breeding nuclei in prostate cancer development and intratumoral macrophage populations, an isotransplantation experiment was performed. C57BL/6J mice from two breeding nuclei (nA and nB) were employed for prostate adenocarcinoma TRAMP-C1 cell implantation; tumor growth period and intratumoral macrophage profile were measured. BL/6nB mice (54%) showed tumor implantation after 69-day growth period while BL/6nA implantation reached 100% across tumor growth period (28 days). No difference in total macrophage populations was observed between groups within several tumoral regions; significantly higher M2 macrophage profile was observed in tumor microenvironments from both mice groups. Nevertheless, BL/6nB tumors showed around twice the population of M1 profile (11-27%) than BL6nA (4-15%) and less non-polarized macrophages. The M1:M2 average ratio was 1:8 for group A and 1:4 for B. Our results demonstrate different tumor progression and intratumoral macrophage populations among mice from the same substrain. Data obtained in this study shows the relevance of animal source renewal for better control of murine cancer model variables.


Assuntos
Modelos Animais de Doenças , Progressão da Doença , Macrófagos , Camundongos Endogâmicos C57BL , Neoplasias da Próstata , Microambiente Tumoral , Animais , Neoplasias da Próstata/patologia , Masculino , Camundongos , Macrófagos/imunologia , Linhagem Celular Tumoral
2.
BMC Infect Dis ; 21(1): 1121, 2021 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-34717584

RESUMO

BACKGROUND: More than 3 million infections were attributed to Chikungunya virus (CHIKV) in the 2014-2016 outbreak in Mexico, Central and South America, with over 500 deaths directly or indirectly related to this viral disease. CHIKV outbreaks are recurrent and no vaccine nor approved therapeutics exist to prevent or treat CHIKV infection. Reliable and robust diagnostic methods are thus critical to control future CHIKV outbreaks. Direct CHIKV detection in serum samples via highly specific and high affinity anti-CHIKV antibodies has shown to be an early and effective clinical diagnosis. METHODS: To isolate highly specific and high affinity anti-CHIKV, Chikungunya virions were isolated from serum of a patient in Veracruz, México. After purification and characterization via electron microscopy, SDS-PAGE and binding to well-characterized anti-CHIKV antibodies, UV-inactivated particles were utilized as selector in a solid-phase panning in combination with ALTHEA Gold Libraries™, as source of antibodies. The screening was based on ELISA and Next-Generation Sequencing. RESULTS: The CHIKV isolate showed the typical morphology of the virus. Protein bands in the SDS-PAGE were consistent with the size of CHIKV capsid proteins. UV-inactivated CHIKV particles bound tightly the control antibodies. The lead antibodies here obtained, on the other hand, showed high expression yield, > 95% monomeric content after a single-step Protein A purification, and importantly, had a thermal stability above 75 °C. Most of the antibodies recognized linear epitopes on E2, including the highest affinity antibody called C7. A sandwich ELISA implemented with C7 and a potent neutralizing antibody isolated elsewhere, also specific for E2 but recognizing a discontinuous epitope, showed a dynamic range of 0.2-40.0 mg/mL of UV-inactivated CHIKV purified preparation. The number of CHIKV particles estimated based on the concentration of E2 in the extract suggested that the assay could detect clinically meaningful amounts of CHIKV in serum. CONCLUSIONS: The newly discovered antibodies offer valuable tools for characterization of CHIKV isolates. Therefore, the strategy here followed using whole viral particles and ALTHEA Gold Libraries™ could expedite the discovery and development of antibodies for detection and control of emergent and quickly spreading viral outbreaks.


Assuntos
Febre de Chikungunya , Vírus Chikungunya , Anticorpos Neutralizantes , Anticorpos Antivirais , Febre de Chikungunya/diagnóstico , Humanos , Proteínas do Envelope Viral
3.
Mycopathologia ; 184(6): 747-757, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31637573

RESUMO

Candida albicans is a commensal fungus of the skin and mucous membranes in humans, but it is also responsible for mucocutaneous and systemic infections in immunocompromised patients like low birth weight neonates and premature newborns. The epicutaneous application of C. albicans is widely used to study the immune response against this pathogen in adult mice models. However, the immune response of newborns against infections caused by the genus Candida is poorly understood. In order to mimic premature human infection, we developed a model of C. albicans epicutaneous infection in newborn mice. We found that yeasts were able to colonize while the pseudohyphae invaded the epidermis. Recruitment of polymorphonuclear and mononuclear cells at the infection zone was observed. Fungal invasion, fungal burden and cellular infiltration displayed a time- and dose-dependent response. Interestingly, newborn mice were able to control C. albicans primary infection. Finally, we showed that the epicutaneous infection of C. albicans in newborn mice at birth results in the induction of cell-mediated immunity as evinced by delayed-type hypersensitivity assays.


Assuntos
Animais Recém-Nascidos/microbiologia , Candida albicans/imunologia , Candidíase/imunologia , Imunidade Celular , Animais , Candida albicans/crescimento & desenvolvimento , Candidíase/microbiologia , Epiderme/microbiologia , Camundongos , Modelos Animais , Pele/microbiologia
4.
Cell Immunol ; 320: 46-55, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28950987

RESUMO

BACKGROUND: The expression of CD73 in tumor cells plays a significant role in the production of adenosine (Ado) that suppresses antitumor effector cells. METHODS: In this study we analyzed the capability of HPV-positive (HPV+) cervical cancer (CeCa) cell lines CaSki, SiHa, HeLa, and RoVa; and HPV-negative (HPV-) cell lines C33A and ViBo to produce Ado and inhibit effector functions of CD8+ T cells. RESULTS: HPV+ CeCa cells expressed significantly higher levels of CD73 in the membrane (p<0.01) than HPV- CeCa cells and this expression was associated with the production of larger amounts of Ado (>400µM) compared to HPV-CeCa cells (<200µM) in the presence of AMP, as well asa stronger inhibition of (>50%) proliferation, activation, and cytotoxic activity of CD8+ T cells via interaction with A2A adenosine receptor. We also provide evidence that silenced E6/E7 expression in CeCa cells, strongly reduced its CD73 expression level and its capability to generate Ado. CONCLUSION: This results suggest that HPV infection, which is associated with more than 99% of CeCa cases, may present an increased constitutive expression of CD73 in cervical neoplasia to contribute to the suppression of the immune response mediated by the production of large amounts of Ado.


Assuntos
5'-Nucleotidase/metabolismo , Adenosina/metabolismo , Papillomaviridae/imunologia , Infecções por Papillomavirus/imunologia , Receptor A2A de Adenosina/metabolismo , Linfócitos T Citotóxicos/imunologia , Neoplasias do Colo do Útero/metabolismo , Monofosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Proteínas de Ligação a DNA/genética , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Terapia de Imunossupressão , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , RNA Interferente Pequeno/genética , Proteínas Repressoras/genética , Evasão Tumoral , Neoplasias do Colo do Útero/imunologia
5.
Int J Tuberc Lung Dis ; 20(12): 1681-1688, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28000588

RESUMO

SETTING: Mexico City, Mexico. OBJECTIVE: To identify proteins synthetised by Mycobacterium tuberculosis in hypoxic culture, which resemble more closely a granuloma environment than aerobic culture, and to determine if they are recognised by antibodies from patients with active pulmonary tuberculosis (PTB). DESIGN: Soluble extracts from M. tuberculosis H37Rv cultured under aerobic or hypoxic conditions were analysed using two-dimensional polyacrylamide gel electrophoresis, and proteins over-expressed under hypoxia were identified by mass spectrometry. The presence of immunoglobulin (Ig) G, IgA and IgM antibodies against these proteins was determined in the serum of 42 patients with active PTB and 42 healthy controls. RESULTS: We selected three M. tuberculosis H37Rv proteins (alpha-crystallin protein [Acr, Rv2031c], universal stress protein Rv2623 and isocitrate lyase [ICL, RV0467]) that were over-expressed under hypoxia. Titres of anti-Acr and anti-ICL IgA antibodies were higher in patients than in healthy controls, with an area under the receiver operating characteristic curve of 0.71 for anti-ICL IgA antibodies. CONCLUSION: ICL could be used in combination with other M. tuberculosis antigens to improve the sensitivity and specificity of current serological TB diagnostic methods.


Assuntos
Anticorpos Antibacterianos/sangue , Imunoglobulina A/sangue , Isocitrato Liase/imunologia , Tuberculose Pulmonar/diagnóstico , alfa-Cristalinas/imunologia , Adulto , Idoso , Antígenos de Bactérias/sangue , Estudos de Casos e Controles , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , México , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Sensibilidade e Especificidade , Tuberculose Pulmonar/sangue , Adulto Jovem
6.
Immunol Lett ; 158(1-2): 183-8, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24447863

RESUMO

Uncontrolled and intricate production of inflammatory factors is the characteristic feature of dengue infection. The triggering receptor expressed in myeloid cells-1 (TREM-1), expressed on the surface of monocytes and neutrophils, is capable of enhancing and regulating the inflammatory response via the production of different mediators in bacterial and viral infections. Here, both the expression of TREM-1 on human monocytes and neutrophils from peripheral blood of dengue infected individuals, as well as the levels of the soluble form of TREM-1 (sTREM-1) in the sera of these patients were compared against healthy controls. A significant reduction of TREM-1 expression was observed in neutrophils during the first days of infection, followed by a gradual recovery throughout the course of infection. Also, sera from DENV-infected patients exhibited significantly higher sTREM-1 levels than healthy individuals. The difference was more pronounced during the first 5 days after the onset of symptoms. These findings highlight the dynamic process of TREM-1 expression during DENV infection. We hypothesized that increment of free sTREM-1 could be a compensatory mechanism aiming to counteract the inflammatory process elicited during DENV infection.


Assuntos
Vírus da Dengue/imunologia , Dengue/imunologia , Glicoproteínas de Membrana/biossíntese , Monócitos/imunologia , Neutrófilos/imunologia , Receptores Imunológicos/biossíntese , Adolescente , Adulto , Células Cultivadas , Criança , Progressão da Doença , Feminino , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Imunomodulação , Masculino , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Monócitos/virologia , Neutrófilos/virologia , Receptores Imunológicos/sangue , Receptores Imunológicos/genética , Receptor Gatilho 1 Expresso em Células Mieloides , Adulto Jovem
7.
Int J Oncol ; 39(6): 1491-9, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21805029

RESUMO

Natural killer cells play a role in the immune antitumor response by recognizing and eliminating tumor cells through the engagement of NKG2D receptors with their ligands on target cells. This work aimed to investigate whether epigenetic drugs are able to increase MICA and MICB expression as well as NK cell cytotoxicity. Prostate, colon, breast and cervical cancer cell lines were analyzed for the expression of MICA and MICB at the mRNA and protein levels by RT-PCR, Western blot, flow cytometry and ELISA. The activating mark H3K4m2 at the MICA and MICB promoters was investigated by ChIP assays. Cytotoxicity of NK cells against the target epithelial cancer cells was investigated with the CD107 cytotoxicity assay. The results show that hydralazine and valproic acid not only increase the expression of MICA and MICB ligands of target cells, but also reduce their shedding to the supernatant. This upregulation occurs at the transcriptional level as revealed by increase of the H3K4 activating mark at the promoter of MICA and MICB genes. These effects are paralleled by increased cytotoxicity of NK cells, which was attenuated at different degrees by using blocking antibodies against the NKG2D receptor and ligands. In conclusion, our results demonstrate the ability of hydralazine and valproate to increase the NK activity against epithelial cancer cell lines and suggest that these drugs could reduce the levels of soluble MICA and MICB helping in avoiding tumor-induced suppression of NK cytotoxicity against the tumor.


Assuntos
Antineoplásicos/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Hidralazina/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Regulação para Cima/efeitos dos fármacos , Ácido Valproico/farmacologia , Linhagem Celular Tumoral , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Ligantes , Neoplasias/genética , Neoplasias/imunologia , Ligação Proteica/imunologia
8.
Clin Exp Immunol ; 140(3): 443-9, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15932505

RESUMO

It has been shown recently that different genotypes of Mycobacterium tuberculosis induce distinct immune responses in the host, as reflected by variations in cytokine and iNOS expression. Because these molecules are probably regulated by multiple factors in vivo this complex phenomenon was partially analysed by assessing cytokine and iNOS expression by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) in an in vitro model of bone marrow-derived macrophages infected with three different M. tuberculosis genotypes: Canetti, H37 Rv and Beijing. Although the three genotypes induced production of iNOS and the different cytokines tested at 24 h post-infection, macrophages infected with the Beijing isolate expressed the highest levels of mRNA for iNOS, interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, IL-12 cytokines and lower levels of IL-10 compared with cells infected with other genotypes. This expression pattern has been associated with infection control, but during infection in vivo with the Beijing genotype it is lost upon progression to chronic phase. The failure to control infection is likely to be influenced by cytokines produced by other cell types and bacterial molecules expressed during the course of disease. Results presented in this work show that each genotype has the ability to induce different levels of cytokine expression that could be related to its pathogenesis during infection.


Assuntos
Citocinas/imunologia , Macrófagos/imunologia , Tuberculose/imunologia , Animais , Células da Medula Óssea/imunologia , Células Cultivadas , Genótipo , Interleucina-1/imunologia , Interleucina-10/imunologia , Interleucinas/imunologia , Camundongos , Mycobacterium tuberculosis/genética , Óxido Nítrico Sintase/imunologia , Óxido Nítrico Sintase Tipo II , Fagocitose/imunologia , RNA Mensageiro/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fator de Crescimento Transformador beta/imunologia , Tuberculose/genética
9.
Scand J Immunol ; 60(4): 329-37, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15379857

RESUMO

Iron is known to play an important role in different bacterial infections and, in particular, in their development. One example is infection with Mycobacterium tuberculosis where iron contributes to growth and survival of the bacteria within the host cell. The majority of studies performed on tuberculosis have focused on the direct effect of iron on bacterial growth; however, little is known about how iron modifies the mycobacterial-host interaction. In order to address this, we have investigated the effect of iron on intracellular growth of M. tuberculosis in J774 macrophages and the molecular mechanisms that are affected during this interaction. We observed that iron modifies intracellular growth of the mycobacteria and that their growth kinetics was modified from that observed for the extracellular situation in the presence of iron. Similarly, when iron was present during the infection, there was a reduced release of tumour necrosis factor-alpha and it was related to a higher number of bacilli inside the host cell and low expression of interleukin-1 (IL-1) and IL-6 mRNA. Hence, this work demonstrates that iron, besides promoting mycobacterial growth, also regulates the relationship between macrophage and bacteria.


Assuntos
Citocinas/biossíntese , Ferro/farmacologia , Macrófagos/imunologia , Macrófagos/microbiologia , Mycobacterium tuberculosis/patogenicidade , Citocinas/genética , Mediadores da Inflamação/metabolismo , Interleucina-1/biossíntese , Interleucina-1/genética , Interleucina-6/biossíntese , Interleucina-6/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética
10.
Immunol Lett ; 67(3): 167-77, 1999 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-10369123

RESUMO

In this work we eluted peptides from purified class I MHC molecules, isolated from a novel human cervical carcinoma cell line (INBL), generated in our laboratory and positive for HPV-18 infection. A fraction of these peptides was capable of stimulating T lymphocytes obtained from a donor matched for HLA-Cw4 and who was also HPV-18+. Direct N-terminal Edman degradation of these peptides, revealed the sequence (XQFPIFLQF) that matched 85% with the sequence NVFPIFLQM localized in between the 54 and 62 residues of the HPV-18 L1 protein. After stimulation with the synthetic peptide NVFPIFLQM, T lymphocytes from the donor were capable to lyse INBL cells. Our results provide evidence of the existence of naturally occurring viral epitopes presented on cervical cancer cells by the HLA-Cw4 allele, that could be useful for immunotherapy on this type of patient.


Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Papillomaviridae/imunologia , Peptídeos/imunologia , Neoplasias do Colo do Útero/virologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Antígenos Virais/química , Antígenos Virais/imunologia , Citotoxicidade Imunológica , Epitopos/imunologia , Feminino , Antígenos de Histocompatibilidade Classe I/química , Humanos , Ativação Linfocitária , Espectrometria de Massas , Dados de Sequência Molecular , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Peptídeos/química , Peptídeos/isolamento & purificação , Linfócitos T/imunologia , Células Tumorais Cultivadas , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/virologia , Neoplasias do Colo do Útero/imunologia , Proteínas Virais/química , Proteínas Virais/isolamento & purificação
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