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1.
Apoptosis ; 24(3-4): 245-255, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30929105

RESUMO

Calreticulin (CRT) is a pleiotropic and highly conserved molecule that is mainly localized in the endoplasmic reticulum. Recently, CRT has gained special interest for its functions outside the endoplasmic reticulum where it has immunomodulatory properties. CRT translocation to the cell membrane serves as an "eat me" signal and promotes efferocytosis of apoptotic cells and cancer cell removal with completely opposite outcomes. Efferocytosis results in a silenced immune response and homeostasis, while removal of dying cancer cells brought about by anthracycline treatment, ionizing-irradiation or photodynamic therapy results in immunogenic cell death with activation of the innate and adaptive immune responses. In addition, CRT impacts phagocyte activation and cytokine production. The effects of CRT on cytokine production depend on its conformation, species specificity, degree of oligomerization and/or glycosylation, as well as its cellular localization and the molecular partners involved. The controversial roles of CRT in cancer progression and the possible role of the CALR gene mutations in myeloproliferative neoplasms are also addressed. The release of CRT and its influence on the different cells involved during efferocytosis and immunogenic cell death points to additional roles of CRT besides merely acting as an "eat me" signal during apoptosis. Understanding the contribution of CRT in physiological and pathological processes could give us some insight into the potential of CRT as a therapeutic target.


Assuntos
Calreticulina/imunologia , Imunidade/imunologia , Neoplasias/imunologia , Fagocitose/imunologia , Animais , Membrana Celular/imunologia , Retículo Endoplasmático/imunologia , Humanos
2.
Mem Inst Oswaldo Cruz ; 113(4): e170332, 2018 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-29513875

RESUMO

BACKGROUND: Trypanosoma cruzi is a protozoan parasite and an etiological agent of Chagas disease. There is a wide variability in the clinical outcome of its infection, ranging from asymptomatic individuals to those with chronic fatal mega syndromes. Both parasite and host factors, as well as their interplay, are thought to be involved in the process. OBJECTIVES: To evaluate the resistance to complement-mediated killing in two T. cruzi TcI strains with differential virulence and the subsequent effect on their infectivity in mammalian cells. METHODS: Tissue-culture derived trypomastigotes of both strains were incubated in guinea pig serum and subjected to flow cytometry in order to determine their viability and complement activations. Trypomastigotes were also incubated on host cells monolayers in the presence of serum, and infectivity was evaluated under different conditions of complement pathway inhibition. Relative expression of the main parasite-specific complement receptors between the two strains was assessed by quantitative real-time polymerase chain reaction. FINDINGS: In this work, we showed that two TcI strains, one with lower virulence (Ninoa) compared to the other (Qro), differ in their resistance to the lytic activity of complement system, hence causing a compromised ability of Ninoa strain to invade mammalian cells. These results correlate with the three-fold lower messenger RNA (mRNA) levels of complement regulatory protein (CRP), trypomastigote-decay acceleration factor (T-DAF), and complement C2 receptor inhibitor trispanning (CRIT) in Ninoa compared to those in Qro. On the other hand, calreticulin (CRT) mRNA and surface protein levels were higher in Ninoa strain and promoted its infectivity when the lectin pathway of the complement system was inhibited. MAIN CONCLUSIONS: This work suggests the complex interplay of CRP, T-DAF, CRIT, and CRT, and the diagnostic value of mRNA levels in the assessment of virulence potential of T. cruzi strains, particularly when dealing with isolates with similar genetic background.


Assuntos
Proteínas do Sistema Complemento/fisiologia , Trypanosoma cruzi/imunologia , Animais , Antígenos de Protozoários/análise , Antígenos de Protozoários/imunologia , Western Blotting , Calreticulina/análise , Sobrevivência Celular , Doença de Chagas/parasitologia , Chlorocebus aethiops , Citometria de Fluxo , Cobaias , Lectinas/metabolismo , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/imunologia , Proteínas de Protozoários/análise , Proteínas de Protozoários/imunologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/patogenicidade , Células Vero , Virulência
3.
BMC Vet Res ; 13(1): 289, 2017 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-28934965

RESUMO

BACKGROUND: Hepatitis E virus (HEV) infection is one of the most common causes of acute liver diseases in humans worldwide. In developing countries, HEV is commonly associated with waterborne outbreaks. Conversely, in industrialized countries, HEV infection is often associated with travel to endemic regions or ingestion of contaminated animal products. Limited information on both, human and animal HEV infection in Mexico is available. As a consequence, the distribution of the virus in the country is largely unknown. Here, we assessed the seroprevalence of HEV among swine in different geographical regions in Mexico. METHODS: Seroprevalence of anti-HEV antibodies in swine herds in Mexico was evaluated in a representative sample including 945 pig serum specimens from different regions of the country using a commercial enzyme-linked immunosorbent assay (ELISA). RESULTS: The overall prevalence of anti-HEV antibodies in swine was 59.4%. The northern region of Mexico exhibited the highest seroprevalence in the country (86.6%), while the central and southern regions in Mexico showed lower seroprevalence, 42.7% and 51.5%, respectively. CONCLUSIONS: In Mexico, HEV seroprevalence in swine is high. Importantly, northern Mexico showed the highest seroprevalence in the country. Thus, further studies are required to identify the risk factors contributing to HEV transmission among pigs in the country. Assessment of HEV human infection in the context of viral transmission in swine is required to better understand the epidemiology of hepatitis E in Mexico.


Assuntos
Anticorpos Antivirais/sangue , Hepatite E/veterinária , Doenças dos Suínos/virologia , Animais , Hepatite E/sangue , Hepatite E/epidemiologia , Hepatite E/imunologia , México/epidemiologia , Razão de Chances , Estudos Soroepidemiológicos , Suínos , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/imunologia
4.
Front Microbiol ; 15: 1402589, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39296294

RESUMO

Introduction: Human respiratory syncytial virus (hRSV) is a main cause of bronchiolitis in infants and its persistence has been described in immunocompromised subjects. However, limited evidence has been reported on the gene expression triggered by the hRSV and the effect of recombinant Taenia solium-derived calreticulin (rTsCRT). Methods: Using a comprehensive microarray approach, we analyzed the transcriptome profile of a macrophage cell line that has supported hRSV persistence for over 150 passages. We compared the gene expression of persistently infected and non-infected macrophages. We also evaluated the effect of rTsCRT on hRSV-infected macrophage gene transcription, as well as on cytokine production and number of copies of the persistent hRSV genome. Results: Our analysis showed that hRSV long-term virus infection significantly alters mRNA expression of antiviral, inflammatory, as well as arginine and lipid metabolism-associated genes, revealing a transcriptional signature that suggests a mixed M1/M2 phenotype. The resulting host-virus equilibrium allows for the regulation of viral replication, while evading the antiviral and proinflammatory responses. Interestingly, rTsCRT stimulus upregulated Tnfα, Il6 and Nos2 mRNA. We found increased levels of both proinflammatory cytokines and nitrite levels in the conditioned media of persistent macrophages treated with rTsCRT. This increase was associated with a significant reduction in viral genome copies. Discussion: hRSV persistently infected macrophages retain responsiveness to external stimuli and demonstrate that the profound changes induced by viral persistence are potentially reversible. Our observations contribute to the understanding of the mechanisms related to hRSV persistence in macrophages and have implications for the development of targeted therapies to eliminate persistent infections or reduce the negative effects related with chronic inflammatory diseases associated with hRSV infection.

5.
J Clin Microbiol ; 51(2): 629-32, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23224093

RESUMO

Here, we analyze the viral divergence among hepatitis C virus (HCV) chronic cases infected with genotype 1. The intrahost viral evolution was assessed by deep sequencing using the 454 Genome Sequencer platform. The results showed a rapid nucleotide sequence divergence. This notorious short-term viral evolution is of the utmost importance for the study of HCV transmission, because direct links between related samples were virtually lost. Thus, rapid divergence of HCV significantly affects genetic relatedness studies and outbreak investigations.


Assuntos
Variação Genética , Hepacivirus/genética , Hepatite C Crônica/virologia , Adulto , Idoso , Evolução Molecular , Feminino , Genoma Viral , Genótipo , Hepacivirus/classificação , Hepatite C Crônica/genética , Humanos , Interferons , Interleucinas/genética , Masculino , Pessoa de Meia-Idade , Filogenia , Fatores de Risco , Análise de Sequência de DNA
7.
Viral Immunol ; 36(8): 550-561, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37603294

RESUMO

Current evidence shows higher production of cytokines and antibodies against severe acute respiratory coronavirus 2 (SARS-CoV-2) in severe and critical cases of Coronavirus Disease 2019 (COVID-19) in comparison with patients with moderate or mild disease. A recent hypothesis proposes an important role of genotoxicity and cytotoxicity in the induction of the cytokine storm observed in some patients at later stages of the disease. Interestingly, in this study, we report significantly higher levels of interleukin (IL)-1ß, IL-6, MCP-1, and IL-4 cytokines in mild COVID-19 patients versus severe cases, as well as a high frequency of karyorrhexis (median [Me] = 364 vs. 20 cells) and karyolysis (Me = 266 vs. 52 cells) in the mucosal epithelial cells of both groups of patients compared with uninfected individuals. Although we observed higher levels of anti-SARS-CoV-2 IgM and IgG antibodies in COVID-19 patients, IgM antibodies were significantly higher only in mild cases, for the N and the S viral antigens. High levels of IgG antibodies were observed in both mild and severe cases. Our results showed elevated concentrations of proinflammatory and anti-inflammatory cytokines in mild cases, which may reflect an active innate immune response and could be related to the higher IgM and IgG antibody levels found in those patients. In addition, we found that SARS-CoV-2 infection induces cytotoxic damage in the oral mucosa, highlighting the importance of studying the genotoxic and cytotoxic events induced by infection and its role in the pathophysiology of COVID-19.


Assuntos
COVID-19 , Humanos , Citocinas , SARS-CoV-2 , Anticorpos Antivirais , Imunoglobulina G , Imunoglobulina M
8.
J Clin Microbiol ; 50(2): 281-7, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22116161

RESUMO

The use of telaprevir and boceprevir, both protease inhibitors (PI), as part of the specifically targeted antiviral therapy for hepatitis C (STAT-C) has significantly improved sustained virologic response (SVR) rates. However, different clinical studies have also identified several mutations associated with viral resistance to both PIs. In the absence of selective pressure, drug-resistant hepatitis C virus (HCV) mutants are generally present at low frequency, making mutation detection challenging. Here, we describe a mismatch amplification mutation assay (MAMA) PCR method for the specific detection of naturally occurring drug-resistant HCV mutants. MAMA PCR successfully identified the corresponding HCV variants, while conventional methods such as direct sequencing, endpoint limiting dilution (EPLD), and bacterial cloning were not sensitive enough to detect circulating drug-resistant mutants in clinical specimens. Ultradeep pyrosequencing was used to confirm the presence of the corresponding HCV mutants. In treatment-naïve patients, the frequency of all resistant variants was below 1%. Deep amplicon sequencing allowed a detailed analysis of the structure of the viral population among these patients, showing that the evolution of the NS3 is limited to a rather small sequence space. Monitoring of HCV drug resistance before and during treatment is likely to provide important information for management of patients undergoing anti-HCV therapy.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , Hepacivirus/efeitos dos fármacos , Hepatite C/virologia , Mutação , Oligopeptídeos/farmacologia , Prolina/análogos & derivados , Adulto , Biota , Feminino , Hepacivirus/classificação , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Prolina/farmacologia , Virologia/métodos
9.
J Clin Microbiol ; 50(4): 1461-3, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22301026

RESUMO

Here, we describe a transmission event of hepatitis C virus (HCV) among injection drug users. Next-generation sequencing (NGS) was used to assess the intrahost viral genetic variation. Deep amplicon sequencing of HCV hypervariable region 1 allowed for a detailed analysis of the structure of the viral population. Establishment of the genetic relatedness between cases was accomplished by phylogenetic analysis. NGS is a powerful tool with applications in molecular epidemiology studies and outbreak investigations.


Assuntos
Hepacivirus/genética , Hepatite C/transmissão , Sequenciamento de Nucleotídeos em Larga Escala , Abuso de Substâncias por Via Intravenosa/complicações , Adulto , Usuários de Drogas , Hepacivirus/classificação , Hepatite C/etiologia , Hepatite C/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Tipagem Molecular , Filogenia , Análise de Sequência de DNA , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética
10.
Exp Parasitol ; 132(3): 334-40, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22921496

RESUMO

Oral immunization with functional recombinant Taenia solium calreticulin (rTsCRT) induces 37% reduction in tapeworm burden in the experimental model of intestinal taeniosis in hamsters. Furthermore, tapeworms recovered from vaccinated animals exhibit diminished length, being frequently found in more posterior parts of the small intestine. The aim of this study was to analyze the immunological mechanisms involved in protection in response to rTsCRT oral immunization. Hamsters were orally immunized with rTsCRT using cholera toxin (CT) as adjuvant, weekly for 4 weeks. Fifteen days after the last boost animals were challenged with four T. solium cysticerci. Reduction in the adult worm recovery and increased transcription of mRNA for IL-4 and IFN-γ in the mucosa of rTsCRT+CT immunized animals were observed. Immunization also induced goblet cell hyperplasia in the mucosa surrounding the implantation site of the parasite. Specific IgG and IgA antibodies in serum and fecal supernatants were detected after the second immunization, being more pronounced after challenge. Our data suggest that oral vaccination with rTsCRT+CT regulates a local expression of IL-4 and IFN-γ, stimulating secretion of IgA that, together with the increase of goblet cells and mucin production, could result in an unfavorable environment for T. solium promoting an impaired tapeworm development.


Assuntos
Calreticulina/imunologia , Taenia solium/imunologia , Teníase/prevenção & controle , Vacinação/métodos , Administração Oral , Animais , Anticorpos Anti-Helmínticos/análise , Anticorpos Anti-Helmínticos/sangue , Calreticulina/administração & dosagem , Cricetinae , Fezes/química , Feminino , Imunização , Imunoglobulina A/análise , Imunoglobulina G/sangue , Mesocricetus , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Suínos , Taenia solium/química , Teníase/imunologia
11.
Infect Genet Evol ; 101: 105288, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35489699

RESUMO

Drug resistant tuberculosis (DR-TB) is an important public health issue in different parts of the world. Mycobacterium tuberculosis complex variants (MTBC vars) preferentially infect certain hosts, limiting their distribution to different ecosystems. However, MTBC vars can infect other hosts beyond their preferred target potentially contributing to persistence of drug resistance (DR) in other niches. Here, we performed a comprehensive intra-host genetic analysis for the identification of DR-related mutations among all MTBC minor vars whole genome sequences (8,095 strains) publicly available worldwide. High confidence drug-resistance mutations in katG (isoniazid), rpsL (streptomycin), pncA (pyrazinamide), rpoB (rifampicin) and gyrA (fluoroquinolones) genes were identified among intrahost minor sub-populations in 197 different strains (2.43%) belonging to vars africanum, bovis, caprae, microti, orygis and pinnipedii. In addition, a three-dimensional structure modeling analysis to assess the role of novel mutations was also performed. Our findings highlight the importance of detecting discrete intra-host populations carrying DR mutations.


Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Resistência a Medicamentos , Ecossistema , Humanos , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia
12.
J Clin Microbiol ; 49(9): 3370-4, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21775538

RESUMO

Dengue virus (DENV) is the most important arthropod-borne viral infection in humans. Here, the genetic relatedness among autochthonous DENV Mexican isolates was assessed. Phylogenetic and median-joining network analyses showed that viral strains recovered from different geographic locations are genetically related and relatively homogeneous, exhibiting limited nucleotide diversity.


Assuntos
Vírus da Dengue/classificação , Vírus da Dengue/isolamento & purificação , Dengue/epidemiologia , Dengue/virologia , Análise por Conglomerados , Vírus da Dengue/genética , Variação Genética , Genótipo , Humanos , México/epidemiologia , Epidemiologia Molecular , Filogeografia , RNA Viral/genética
13.
J Clin Microbiol ; 49(7): 2706-10, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21613433

RESUMO

Several studies have identified associations between single nucleotide polymorphisms (SNPs) occurring near the interleukin-28B (IL-28B) gene and response to antiviral treatment among hepatitis C virus (HCV) patients. Here, we describe a reliable melt-mismatch amplification mutation assay (melt-MAMA) PCR-based genotyping method for IL-28B which can be used in the management of HCV patients, helping to better define the course of therapy.


Assuntos
Antivirais/administração & dosagem , Hepatite C Crônica/tratamento farmacológico , Interleucinas/genética , Mutação , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , Temperatura de Transição , Idoso , Feminino , Genótipo , Humanos , Interferons , Masculino , Pessoa de Meia-Idade , Prognóstico , Resultado do Tratamento
14.
Virol J ; 8: 370, 2011 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-21794170

RESUMO

BACKGROUND: Varicella (chickenpox) exhibits a characteristic epidemiological pattern which is associated with climate. In general, primary infections in tropical regions are comparatively less frequent among children than in temperate regions. This peculiarity regarding varicella-zoster virus (VZV) infection among certain age groups in tropical regions results in increased susceptibility during adulthood in these regions. Moreover, this disease shows a cyclic behavior in which the number of cases increases significantly during winter and spring. This observation further supports the participation of environmental factors in global epidemiology of chickenpox. However, the underlying mechanisms responsible for this distinctive disease behavior are not understood completely. In a recent publication, Philip S. Rice has put forward an interesting hypothesis suggesting that ultra-violet (UV) radiation is the major environmental factor driving the molecular evolution of VZV. DISCUSSION: While we welcomed the attempt to explain the mechanisms controlling VZV transmission and distribution, we argue that Rice's hypothesis takes lightly the circulation of the so called "temperate VZV genotypes" in tropical regions and, to certain degree, overlooks the predominance of such lineages in certain non-temperate areas. Here, we further discuss and present new information about the overwhelming dominance of temperate VZV genotypes in Mexico regardless of geographical location and climate. SUMMARY: UV radiation does not satisfactorily explain the distribution of VZV genotypes in different tropical and temperate regions of Mexico. Additionally, the cyclic behavior of varicella does not shown significant differences between regions with different climates in the country. More studies should be conducted to identify the factors directly involved in viral spreading. A better understanding of the modes of transmissions exploited by VZV and their effect on viral fitness is likely to facilitate the implementation of preventive measures for disease control.


Assuntos
Varicela/epidemiologia , Varicela/virologia , Evolução Molecular , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/efeitos da radiação , Raios Ultravioleta , Criança , Pré-Escolar , Clima , Genótipo , Humanos , México/epidemiologia
15.
PLoS Negl Trop Dis ; 15(2): e0009145, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33591982

RESUMO

Identifying the Mycobacterium tuberculosis resistance mutation patterns is of the utmost importance to assure proper patient's management and devising of control programs aimed to limit spread of disease. Zoonotic Mycobacterium bovis infection still represents a threat to human health, particularly in dairy production regions. Routinary, molecular characterization of M. bovis is performed primarily by spoligotyping and mycobacterial interspersed repetitive units (MIRU) while next generation sequencing (NGS) approaches are often performed by reference laboratories. However, spoligotyping and MIRU methodologies lack the resolution required for the fine characterization of tuberculosis isolates, particularly in outbreak settings. In conjunction with sophisticated bioinformatic algorithms, whole genome sequencing (WGS) analysis is becoming the method of choice for advanced genetic characterization of tuberculosis isolates. WGS provides valuable information on drug resistance and compensatory mutations that other technologies cannot assess. Here, we performed an analysis of the most frequently identified mutations associated with tuberculosis drug resistance and their genetic relationship among 2,074 Mycobacterium bovis WGS recovered primarily from non-human hosts. Full-length gene sequences harboring drug resistant associated mutations and their phylogenetic relationships were analyzed. The results showed that M. bovis isolates harbor mutations conferring resistance to both first- and second-line antibiotics. Mutations conferring resistance for isoniazid, fluoroquinolones, streptomycin, and aminoglycosides were identified among animal strains. Our findings highlight the importance of molecular surveillance to monitor the emergence of mutations associated with multi and extensive drug resistance in livestock and other non-human mammals.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/genética , Tuberculose/veterinária , América/epidemiologia , Animais , Antituberculosos/farmacologia , Mutação , Filogenia , Tuberculose/microbiologia , Sequenciamento Completo do Genoma
16.
Methods ; 49(4): 346-50, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19651215

RESUMO

Neurocysticercosis in humans is caused by the tapeworm Taenia solium and generates substantial morbidity in Latin America, Africa and Asia.The life cycle of T. solium includes pigs as intermediate hosts and human beings as definitive hosts. Tapeworm carriers are the main risk factor for acquiring cysticercosis in the household, thus prevention and control programs are being developed. Infected people have no symptoms, therefore are difficult to identify and treat, thus vaccination against the adult tapeworm is an alternative control measure. Since the infection occurs naturally only in human beings, experimental models have been standardized. Hamsters are believed to be good models to study the infection but they have not been properly evaluated for vaccination. Since taeniosis is gained by ingesting pork meat with cysticerci, oral vaccination was evaluated, and given that intestinal immunity is enhanced with adjuvants, cholera toxin was used, because it is one of the most potent adjuvants, in view of the fact that it increases epithelium permeability enhancing entrance of the co-administered unrelated antigens. Recombinant functional T. solium calreticulin was employed for the standardization of the methodology and the evaluation of oral vaccination. Protection was associated with the type of cysticerci and the age of the hamsters used. When reddish bigger parasites were orally introduced in hamsters as challenge, protection was around 40%, while when yellowish small parasites were used, protection increased to 100%, suggesting that the characteristics of cysticerci are determinant. Protection was gained in 9month old hamsters, but not in 3month old animals.


Assuntos
Modelos Animais de Doenças , Taenia solium , Teníase/prevenção & controle , Vacinação/métodos , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Administração Oral , Animais , Cricetinae , Feminino , Humanos , Masculino , Mesocricetus , Suínos , Taenia solium/efeitos dos fármacos , Taenia solium/imunologia , Teníase/imunologia , Teníase/parasitologia
17.
Arch Med Res ; 51(1): 65-75, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32097797

RESUMO

BACKGROUND AND AIMS: Calreticulin is a chaperone and master regulator of intracellular calcium homeostasis. Several additional functions have been discovered. Human and parasite calreticulin have been shown to suppress mammary tumor growth in vivo. Here, we explored the capacity of recombinant Taenia solium calreticulin (rTsCRT) to modulate cancer cell growth in vitro. METHODS: We used different concentrations of rTsCRT to treat cancer cell lines and analyzed viability and colony formation capacity. We also tested the combination of the IC20 or IC50 doses of rTsCRT and of the chemotherapeutic drug 5-fluorouracil on MCF7 and SKOV3 cell lines. As a control, the non-tumorigenic cell line MCF10-A was employed. The effect of the drug combinations was also assessed in cancer stem-like cells. Additionally, scavenger receptor ligands were employed to identify the role of this receptor in the rTsCRT anti-tumoral effect. RESULTS: rTsCRT has a dose-dependent in vitro anti-tumoral effect, being SKOV3 the most sensitive cell line followed by MCF7. When rTsCRT/5-fluorouracil were used, MCF7 and SKOV3 showed a 60% reduction in cell viability; colony formation capacity was also diminished. Treatment of cancer stem-like cells from MCF7 showed a higher reduction in cell viability, while those from SKOV3 were more sensitive to colony disaggregation. Finally, pharmacological inhibition of the scavenger receptor, abrogated the reduction in viability induced by rTsCRT in both the parental and stem-like cells. CONCLUSION: Our data suggest that rTsCRT alone or in combination with 5-fluorouracil inhibits the growth of breast and ovarian cancer cell lines through its interaction with scavenger receptors.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Calreticulina/uso terapêutico , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Proteínas Recombinantes/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/patologia , Calreticulina/genética , Calreticulina/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Sinergismo Farmacológico , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/farmacologia , Células HeLa , Humanos , Células MCF-7 , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Taenia solium/genética
18.
Eur J Gastroenterol Hepatol ; 32(1): 10-16, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31651650

RESUMO

OBJECTIVE: Ulcerative colitis and Crohn's disease are the two clinical forms of inflammatory bowel disease (IBD). Diverse studies have shown the association of single nucleotide polymorphism (SNP) in molecules of the immune system and the occurrence of IBD. Here, several SNPs of the immune system with controversial results for their association with UC and CD were evaluated in a Mexican population. METHODS: SNPs rs1800896, rs3024505 (IL-10); rs11209026 (IL23R); rs2066844, rs2066845 (NOD-2), and rs2241880 (ATG16L1) were assessed in 93 patients with IBD and 200 healthy controls by hybridization probes and quantitative PCR. RESULTS: The AG genotype for rs1800896 was associated with an increased risk for both UC and CD (P = 0.005 and P = 0.026, respectively); whereas the AA genotype presents a negative association (P = 0.011 for UC, and 0.0038 for CD). For this SNP, G allele was associated with risk of UC (P = 0-043) but not for CD. For the rs3024505 in IL-10, T allele was associated with UC (P = 0.011). Moreover, this allele was associated with early onset of UC (P = 0.033) and with the use of steroid treatment (P = 0.019). No significant differences for NOD2 (rs2066844T and rs2066845C), IL23R (rs11209026), and ATG16L1 (rs22411880) were found between cases and controls and the homozygous TT genotype for rs2066844 and CC for rs2066845 were not observed. CONCLUSION: Our results show both genotypic and phenotypic associations of IL-10 SNPs with IBD but not with the other immune-related SNPs studied in this Mexican cohort.


Assuntos
Colite Ulcerativa , Doenças Inflamatórias Intestinais , Proteínas Relacionadas à Autofagia/genética , Estudos de Casos e Controles , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/genética , Predisposição Genética para Doença , Genótipo , Humanos , Doenças Inflamatórias Intestinais/genética , Interleucina-10/genética , Proteína Adaptadora de Sinalização NOD2/genética , Polimorfismo de Nucleotídeo Único , Receptores de Interleucina/genética
19.
J Biomol Tech ; 29(3): 61-70, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30034295

RESUMO

The dextran sodium sulfate (DSS) model of colitis is widely used as a result of its simplicity and reproducibility and because it mimics clinicopathological disease features. Its effectiveness depends on the mouse strain, DSS MW, and brand. Quantitative RT-PCR (qRT-PCR) is highly sensitive for analyzing cytokine mRNA expression. We analyzed an acute model of DSS treatment in Balb/c mice for the onset of colitis using qRT-PCR for the quantification of a mouse cytokine transcript. We compared differences among 1--and 2-step qRT-PCR for transcript quantification, the effect of multiple concentrations of DSS, and the use of 2 reference genes in 3 portions of the colon. A reliable and sensitive 1-step protocol for qRT-PCR was established with a modified double LiCl precipitation for RNA isolation. The variability of 2 reference genes, ß-actin and eukaryotic elongation factor 2, was compared, and expression of IL-6 was analyzed in 3 segments of the colon. The RNA cleaning protocol prevented inhibition of qRT-PCR by DSS, and RNA loss was minimized. No clinical differences among the different DSS concentrations were seen on d 7, but higher concentrations resulted in the appearance of earlier symptoms. Higher efficiency and sensitivity of the 1-step qRT-PCR reaction using eukaryotic elongation factor 2 were obtained and also less variability. Although expression levels of IL-6 were high in the middle and distal colon, the middle section had consistently less variability in values. Thus, this segment is recommended for future studies. These factors influence the statistical significance of data and need to be considered to get accurate and reliable results and to improve comparisons of the published colitis experiments.


Assuntos
Colite/genética , Perfilação da Expressão Gênica/métodos , RNA Mensageiro/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Precipitação Química , Colite/induzido quimicamente , Colite/patologia , Colo/patologia , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Relação Dose-Resposta a Droga , Feminino , Interleucina-6/genética , Camundongos Endogâmicos BALB C , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Taq Polimerase/antagonistas & inibidores
20.
PLoS One ; 12(10): e0186510, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29036211

RESUMO

Intestinal helminth antigens are inducers of type 2 responses and can elicit regulatory immune responses, resulting in dampened inflammation. Several platyhelminth proteins with anti-inflammatory activity have been reported. We have identified, cloned and expressed the Taenia solium calreticulin (rTsCRT) and shown that it predominantly induces a type 2 response characterized by IgG1, IL-4 and IL-5 production in mice. Here, we report the rTsCRT anti-inflammatory activity in a well-known experimental colitis murine model. Mice were orally immunized with purified rTsCRT and colitis was induced with trinitrobenzene sulfonic acid (TNBS). Clinical signs of disease, macroscopic and microscopic tissue inflammation, cytokine production and micronuclei formation, as a marker of genotoxicity, were measured in order to assess the effect of rTsCRT immunization on experimentally induced colitis. rTsCRT administration prior to TNBS instillation significantly reduced the inflammatory parameters, including the acute phase cytokines TNF-α, IL-1ß and IL-6. Dampened inflammation was associated with increased local expression of IL-13 and systemic IL-10 and TGF-ß production. Genotoxic damage produced by the inflammatory response was also precluded. Our results show that oral treatment with rTsCRT prevents excessive TNBS-induced inflammation in mice and suggest that rTsCRT has immunomodulatory properties associated with the expression of type 2 and regulatory cytokines commonly observed in other helminths.


Assuntos
Calreticulina/administração & dosagem , Calreticulina/farmacologia , Colite/imunologia , Colite/prevenção & controle , Intestinos/efeitos dos fármacos , Intestinos/imunologia , Taenia solium/química , Administração Oral , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacologia , Antígenos de Helmintos/imunologia , Colite/metabolismo , Citocinas/metabolismo , Dano ao DNA , Modelos Animais de Doenças , Imunomodulação/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Camundongos
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