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1.
PLoS One ; 15(7): e0236375, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726331

RESUMO

BACKGROUND: Malaria in pregnancy causes maternal, fetal and neonatal morbidity and mortality, and maternal innate immune responses are implicated in pathogenesis of these complications. The effects of malaria exposure and obstetric and demographic factors on the early maternal immune response are poorly understood. METHODS: Peripheral blood mononuclear cell responses to Plasmodium falciparum-infected erythrocytes and phytohemagglutinin were compared between pregnant women from Papua New Guinea (malaria-exposed) with and without current malaria infection and from Australia (unexposed). Elicited levels of inflammatory cytokines at 48 h and 24 h (interferon γ, IFN-γ only) and the cellular sources of IFN-γ were analysed. RESULTS: Among Papua New Guinean women, microscopic malaria at enrolment did not alter peripheral blood mononuclear cell responses. Compared to samples from Australia, cells from Papua New Guinean women secreted more inflammatory cytokines tumor necrosis factor-α, interleukin 1ß, interleukin 6 and IFN-γ; p<0.001 for all assays, and more natural killer cells produced IFN-γ in response to infected erythrocytes and phytohemagglutinin. In both populations, cytokine responses were not affected by gravidity, except that in the Papua New Guinean cohort multigravid women had higher IFN-γ secretion at 24 h (p = 0.029) and an increased proportion of IFN-γ+ Vδ2 γδ T cells (p = 0.003). Cytokine levels elicited by a pregnancy malaria-specific CSA binding parasite line, CS2, were broadly similar to those elicited by CD36-binding line P6A1. CONCLUSIONS: Geographic location and, to some extent, gravidity influence maternal innate immunity to malaria.


Assuntos
Imunidade Inata/genética , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Complicações Parasitárias na Gravidez/imunologia , Adolescente , Adulto , Austrália/epidemiologia , Antígenos CD36/genética , Eritrócitos/imunologia , Eritrócitos/parasitologia , Eritrócitos/patologia , Feminino , Número de Gestações/imunologia , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-6/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/parasitologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/parasitologia , Leucócitos Mononucleares/patologia , Malária Falciparum/epidemiologia , Malária Falciparum/parasitologia , Pessoa de Meia-Idade , Papua Nova Guiné/epidemiologia , Plasmodium falciparum/patogenicidade , Gravidez , Complicações Parasitárias na Gravidez/parasitologia , Complicações Parasitárias na Gravidez/patologia , Linfócitos T/imunologia , Linfócitos T/parasitologia , Adulto Jovem
2.
PLoS Negl Trop Dis ; 14(1): e0008021, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961868

RESUMO

Domestic dogs are the main reservoir of Leishmania infantum, a causative agent of visceral leishmaniasis (VL). The number of human disease cases is associated with the rate of canine infection. Currently available drugs are not efficient at treating canine leishmaniasis (CanL) and months after the treatment most dogs show disease relapse, therefore the development of new drugs or new therapeutic strategies should be sought. In CanL, dogs lack the ability to mount a specific cellular immune response suitable for combating the parasite and manipulation of cytokine signaling pathway has the potential to form part of effective immunotherapeutic methods. In this study, recombinant canine cytokines (rcaIL-12, rcaIL-2, rcaIL-15 and rcaIL-7) and soluble receptor IL-10R1 (rcasIL-10R1), with antagonistic activity, were evaluated for the first time in combination (rcaIL-12/rcaIL-2, rcaIL-12/rcaIL-15, rcaIL-12/rcasIL-10R1, rcaIL-15/rcaIL-7) or alone (rcasIL-10R1) to evaluate their immunomodulatory capacity in peripheral blood mononuclear cells (PBMCs) from dogs with leishmaniasis. All the combinations of recombinant proteins tested were shown to improve lymphoproliferative response. Further, the combinations rcaIL-12/rcaIL-2 and rcaIL-12/rcaIL-15 promoted a decrease in programmed cell death protein 1 (PD-1) expression in lymphocytes. These same combinations of cytokines and rcaIL-12/rcasIL-10R1 induced IFN-γ and TNF-α production in PBMCs. Furthermore, the combination IL-12/IL-15 led to an increased in T-bet expression in lymphocytes. These findings are encouraging and indicate the use of rcaIL-12 and rcaIL-15 in future in vivo studies aimed at achieving polarization of cellular immune responses in dogs with leishmaniasis, which may contribute to the development of an effective treatment against CanL.


Assuntos
Doenças do Cão/tratamento farmacológico , Doenças do Cão/imunologia , Interleucina-12/administração & dosagem , Interleucina-15/administração & dosagem , Leishmaniose Visceral/imunologia , Animais , Doenças do Cão/genética , Doenças do Cão/parasitologia , Cães , Imunidade Celular , Interferon gama/genética , Interferon gama/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-15/genética , Interleucina-15/imunologia , Leishmania infantum/fisiologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/parasitologia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Receptores de Citocinas/genética , Receptores de Citocinas/imunologia
3.
Parasite Immunol ; 42(5): e12697, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31958344

RESUMO

Mechanisms of immune activation in effector cells during Haemonchus contortus infection in sheep are currently unknown. Microarray experiments have been performed on tissues of H contortus infected sheep of varying parasite resistance during early and late points of infection, but not in immune effector cells. The purpose of this study was to compare early gene activation in peripheral blood mononuclear cells (PBMC) from primed parasite susceptible (Suffolk) and resistant (St. Croix) sheep in response to H contortus larval antigen (HcLA). Peripheral blood mononuclear cells were cultured for 6 hours with HcLA, and RNA-sequencing was performed. St. Croix PBMC upregulated 499 unique genes in response to HcLA while Suffolk PBMC upregulated 130 unique genes and 25 genes were shared between the two breeds. St. Croix PBMC had increased expression of genes associated with immune function, signal transduction, response to stress and others. In addition, while mechanisms of innate recognition of H contortus are unknown, multiple pattern recognition receptors were found to be upregulated in St. Croix PBMC cultured with HcLA and none were found to be upregulated in Suffolk PBMC. These patterns of immune gene activation may contribute to St. Croix's rapid response and ability to resist H contortus infection.


Assuntos
Hemoncose/veterinária , Haemonchus/imunologia , Leucócitos Mononucleares/imunologia , RNA de Helmintos/genética , Doenças dos Ovinos/parasitologia , Animais , Cruzamento , Hemoncose/genética , Hemoncose/imunologia , Haemonchus/fisiologia , Larva/imunologia , Larva/fisiologia , Leucócitos Mononucleares/parasitologia , RNA de Helmintos/metabolismo , RNA-Seq , Ovinos , Doenças dos Ovinos/genética , Doenças dos Ovinos/imunologia , Carneiro Doméstico
4.
Biochim Biophys Acta Mol Basis Dis ; 1866(3): 165629, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31816438

RESUMO

One of the greatest challenges in Chagas disease research is the search for tools that will enable the assessment of pharmacological treatment efficacy. A recently described set of serological biomarkers composed of four parasite antigens and established criteria of treatment efficacy allowed the evaluation of the impact of benznidazole treatment a short/medium time after the treatment. In addition, cellular immunological parameters have also been described as potential indicators of the treatment response. The cytotoxic CD8+ T cells specific to five epitopes in the PFR2, PFR3, TcCA-2 and KMP11 antigens have been analysed, and these epitopes have been shown to be recognized, processed and presented in the context of a natural T. cruzi infection. In the present manuscript, we characterized these antigen-specific CD8+ T cells in indeterminate chronic Chagas disease patients both before and after (from 11 to 28 months) benznidazole treatment. The results indicate that there is a differential memory CD8+ T cell profile depending on the antigenic epitope and that the benznidazole treatment modulates the memory, differentiation and senescence phenotypes of the epitope-specific CD8+ T cells. Moreover, in these patients, the reactivity of sera against the referred set of biomarkers was evaluated. The data obtained show that the patients who met the established therapeutic efficacy criteria presented a differential phenotypic profile of the antigen-specific CD8+ T cells even prior to treatment compared to the patients who did not meet the therapeutic efficacy criteria, and this behaviour is associated with a better functionality of these CD8+ T cells.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Doença de Chagas/tratamento farmacológico , Doença de Chagas/imunologia , Epitopos/imunologia , Nitroimidazóis/uso terapêutico , Adulto , Biomarcadores/sangue , Linfócitos T CD8-Positivos/parasitologia , Doença de Chagas/parasitologia , Citocinas/imunologia , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/parasitologia , Masculino , Fenótipo , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/imunologia
5.
Front Immunol ; 10: 2081, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31555289

RESUMO

The parasitic helminth Fasciola hepatica (liver fluke) causes economic loss to the livestock industry globally and also causes zoonotic disease. New control strategies such as vaccines are urgently needed, due to the rise of drug resistance in parasite populations. Vaccine development requires a comprehensive understanding of the immunological events during infection. Previous in vivo studies by our group have investigated global differentially expressed genes (DEGs) in ovine peripheral blood mononuclear cells (PBMC) in response to both acute and chronic F. hepatica infection. This work demonstrated that pathways involved in the pathogenesis of ovine fasciolosis included fibrosis, inhibition of macrophage nitric oxide production, and antibody isotype switching, among others. Transcriptomic changes in PBMC populations following F. hepatica infection in cattle, in which the disease phenotype is quite different, have not yet been examined. Using RNA sequencing we investigated gene expression changes in PBMC isolated from 9 non-infected and 11 F. hepatica-experimentally-infected calves immediately before infection, at 1 and at 14 weeks post-infection. Longitudinal time-course comparisons between groups revealed 21 and 1,624 DEGs driven exclusively by F. hepatica infection in cattle at acute and chronic stages, respectively. These results show that fewer DEGs at the acute stage of infection can be identified in cattle, as compared with sheep. In addition, the log2 fold-changes of these DEGs were relatively low (-1 to 3) reflecting the different clinical presentation of F. hepatica infection in cattle. Gene pathways for hepatic fibrosis and hepatic cholestasis along with apoptosis of antigen-presenting cells were enriched at chronic stages. Our results reflect the major differences in the disease phenotype between cattle and sheep and may indicate pathways to target in vaccine development.


Assuntos
Doenças dos Bovinos/genética , Doenças dos Bovinos/imunologia , Fasciola hepatica/imunologia , Fasciolíase/genética , Fasciolíase/imunologia , Leucócitos Mononucleares/imunologia , Transcriptoma/imunologia , Animais , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Apoptose/genética , Apoptose/imunologia , Bovinos , Doenças dos Bovinos/parasitologia , Colestase/genética , Colestase/imunologia , Colestase/parasitologia , Fasciolíase/parasitologia , Expressão Gênica/genética , Expressão Gênica/imunologia , Leucócitos Mononucleares/parasitologia , Fígado/imunologia , Fígado/parasitologia , Masculino
6.
EBioMedicine ; 45: 278-289, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31257148

RESUMO

BACKGROUND: Leukocyte-associated immunoglobulin like receptor-1 (LAIR1) is a transmembrane inhibitory receptor that influences susceptibility to a myriad of inflammatory diseases. Our recent investigations of severe malarial anaemia (SMA) pathogenesis in Kenyan children discovered that novel LAIR1 genetic variants which were associated with decreased LAIR1 transcripts enhanced the longitudinal risk of SMA and all-cause mortality. METHODS: To characterize the molecular mechanism(s) responsible for altered LAIR1 signalling in severe malaria, we determined LAIR1 transcripts and protein, sLAIR1, sLAIR2, and complement component 1q (C1q) in children with malarial anaemia, followed by a series of in vitro experiments investigating the LAIR1 signalling cascade. FINDINGS: Kenyan children with SMA had elevated circulating levels of soluble LAIR1 (sLAIR1) relative to non-SMA (1.69-fold P < .0001). The LAIR1 antagonist, sLAIR2, was also elevated in the circulation of children with SMA (1.59 fold-change, P < .0001). There was a positive correlation between sLAIR1 and sLAIR2 (ρ = 0.741, P < .0001). Conversely, circulating levels of complement component 1q (C1q), a LAIR1 natural ligand, were lower in SMA (-1.21-fold P = .048). These in vivo findings suggest that reduced membrane-bound LAIR1 expression in SMA is associated with elevated production of sLAIR1, sLAIR2 (antagonist), and limited C1q (agonist) availability. Since reduced LAIR1 transcripts in SMA were associated with increased acquisition of haemozoin (PfHz) by monocytes (P = .028), we explored the relationship between acquisition of intraleukocytic PfHz, LAIR1 expression, and subsequent impacts on leukocyte signalling in cultured PBMCs from malaria-naïve donors stimulated with physiological concentrations of PfHz (10 µg/mL). Phagocytosis of PfHz reduced LAIR1 transcript and protein expression in a time-dependent manner (P < .050), and inhibited LAIR1 signalling through decreased phosphorylation of LAIR1 (P < .0001) and SH2-domain containing phosphatase-1 (SHP-1) (P < .001). This process was associated with NF-κB activation (P < .0001) and enhanced production of IL-6, IL-1ß, and TNF-α (all P < .0001). INTERPRETATION: Collectively, these findings demonstrate that SMA is characterized by reduced LAIR1 transmembrane expression, reduced C1q, and enhanced production of sLAIR1 and sLAIR2, molecular events which can promote enhanced production of cytokines that contribute to the pathogenesis of SMA. These investigations are important for discovering immune checkpoints that could be future targets of immunotherapy to improve disease outcomes.


Assuntos
Anemia/sangue , Malária Falciparum/sangue , Receptores Imunológicos/genética , Anemia/genética , Anemia/parasitologia , Pré-Escolar , Complemento C1q/metabolismo , Feminino , Hemeproteínas/administração & dosagem , Humanos , Lactente , Interleucina-1beta/sangue , Interleucina-6/sangue , Leucócitos Mononucleares/parasitologia , Malária Falciparum/genética , Malária Falciparum/parasitologia , Masculino , Fagocitose , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/genética
7.
EBioMedicine ; 45: 290-302, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31278068

RESUMO

BACKGROUND: Severe malarial anaemia (SMA) is a leading cause of childhood mortality in holoendemic Plasmodium falciparum regions. METHODS: To gain an improved understanding of SMA pathogenesis, whole genome and transcriptome profiling was performed in Kenyan children (n=144, 3-36months) with discrete non-SMA and SMA phenotypes. Leukocyte associated immunoglobulin like receptor 1 (LAIR1) emerged as a predictor of susceptibility to SMA (P<1×10-2, OR: 0.44-1.37), and was suppressed in severe disease (-1.69-fold, P=0.004). To extend these findings, the relationship between LAIR1 polymorphisms [rs6509867 (16231C>A); rs2287827 (18835G>A)] and clinical outcomes were investigated in individuals (n=1512, <5years) at enrolment and during a 36-month longitudinal follow-up. FINDINGS: Inheritance of the 16,231 recessive genotype (AA) increased susceptibility to SMA at enrolment (OR=1.903, 95%CI: 1.252-2.891, P=0.003), and longitudinally (RR=1.527, 95%CI: 1.119-2.083, P=0.008). Carriage of the 18,835 GA genotype protected against SMA cross-sectionally (OR=0.672, 95%CI: 0.480-0.9439, P=0.020). Haplotype carriage (C16231A/G18835A) also altered cross-sectional susceptibility to SMA: CG (OR=0.717, 95%CI: 0.527-0.9675, P=0.034), CA (OR=0.745, 95%CI: 0.536-1.036, P=0.080), and AG (OR=1.641, 95%CI: 1.160-2.321, P=0.005). Longitudinally, CA carriage was protective against SMA (RR=0.715, 95%CI: 0.554-0.923, P=0.010), while AG carriage had an additive effect on enhanced SMA risk (RR=1.283, 95%CI: 1.057-1.557, P=0.011). Variants that protected against SMA had elevated LAIR1 transcripts, while those with enhanced risk had lower expression (P<0.05). Inheritance of 18,835 GA reduced all-cause mortality by 44.8% (HR=0.552, 95%CI: 0.329-0.925, P=0.024), while AG haplotype carriage increased susceptibility by 68% (HR=1.680, 95%CI: 1.020-2.770, P=0.040). INTERPRETATION: These findings suggest LAIR1 is important for modulating susceptibility to SMA and all-cause childhood mortality.


Assuntos
Anemia/sangue , Malária Falciparum/sangue , Receptores Imunológicos/genética , Anemia/genética , Anemia/parasitologia , Pré-Escolar , Feminino , Regulação da Expressão Gênica , Genótipo , Haplótipos/genética , Humanos , Lactente , Quênia/epidemiologia , Leucócitos Mononucleares/parasitologia , Malária Falciparum/genética , Malária Falciparum/parasitologia , Masculino , Fagocitose , Transdução de Sinais/genética
8.
Vet Immunol Immunopathol ; 211: 6-9, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31084896

RESUMO

Helminth parasites are highly prevalent in swine production, causing chronic infections and considerable morbidity due to growth retardation. Moreover, helminths actively modulate host immune responses to other pathogens and/or vaccines. Here, we investigated the modulatory effects of Ascaris suum adult body fluid (ABF) and Trichuris suis Soluble Products (TsSP) on the cytokine response in porcine peripheral blood mononuclear cells (PBMCs) and the intestinal epithelial cell line IPEC-J2. In PBMCs, TsSP induced the secretion of IL-6, IL-10 and IL-1ß, but not TNF-α. Moreover, TsSP significantly enhanced the production of bacterial lipopolysaccharide (LPS)-induced IL-6 and IL-10 but suppressed the production of LPS-induced TNF-α. ABF did not induce cytokine secretion from PBMC, but suppressed LPS-induced secretion of TNF-α and IL-6. ABF did not have any effect on cytokine production in IPEC-J2 cells. In contrast, TsSP selectively induced the secretion of IL-6, and enhanced the IL-6 response induced by LPS. The IL-6 response appeared to be a conserved response to T. suis products, as significant secretion was also observed in alveolar macrophages. Thus, T. suis products have diverse modulatory effects on cytokine secretion in vitro, with IL-6 production a consistent feature of the innate host response.


Assuntos
Antígenos de Helmintos/imunologia , Ascaris suum/imunologia , Citocinas/metabolismo , Células Epiteliais/imunologia , Leucócitos Mononucleares/imunologia , Doenças dos Suínos/parasitologia , Trichuris/imunologia , Animais , Ascaríase/imunologia , Ascaríase/parasitologia , Ascaríase/veterinária , Citocinas/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/parasitologia , Feminino , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/parasitologia , Masculino , Suínos/imunologia , Suínos/parasitologia , Doenças dos Suínos/imunologia , Tricuríase/imunologia , Tricuríase/parasitologia , Tricuríase/veterinária
9.
Parasit Vectors ; 12(1): 182, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31023355

RESUMO

BACKGROUND: Pattern recognition receptors (PRRs) can recognize pathogen-associated molecular patterns and activate downstream signalling pathways, resulting in modulation of host immunity against pathogens. Here, we investigated whether PRR-mediated recognition is involved in host immune responses to the blood-feeding nematode Haemonchus contortus. METHODS: During blood-feeding, H. contortus secretes immune-modulating antigens into host blood. Therefore, we stimulated sheep peripheral blood mononuclear cells (PBMCs) with H. contortus soluble extract (HcAg) and performed transcriptional profiling. RESULTS: HcAg upregulated two genetically linked CLRs (CLEC2L and KLRG2), two NLRs attenuating inflammation (NLRP12 and NLRC3) and one G protein-coupled receptor with potent anti-inflammatory effects (HCAR2). Furthermore, several Th2-related transcription factors (ATF3, IRF4, BCL3 and NFATC) were also upregulated, which may confer anti-inflammatory type 2 immune responses to HcAg. CONCLUSIONS: Together, our preliminary studies provide new insights into how the host innate immune system controls type 2 immunity to H. contortus. Further work will be needed to identify H. contortus products recognized by the host innate immune system and determine the Th2 polarization ability of these putative PRR ligands.


Assuntos
Hemoncose/veterinária , Haemonchus/química , Proteínas de Helminto/farmacologia , Imunidade Inata , Leucócitos Mononucleares/imunologia , Extratos de Tecidos/farmacologia , Animais , Antígenos de Helmintos/imunologia , Perfilação da Expressão Gênica , Hemoncose/sangue , Proteínas de Helminto/imunologia , Interações Hospedeiro-Patógeno , Leucócitos Mononucleares/parasitologia , Proteínas NLR/genética , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/imunologia , Ovinos , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/parasitologia , Fatores de Transcrição/genética
10.
Methods Mol Biol ; 1955: 315-337, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30868538

RESUMO

Human CD8+ and CD4+ T cell lines and clones are valuable tools to explore the role of these cells in the context of diseases, especially in cases in which the main underlying actor is the immune response, like Chagas disease. These cell lines and clones provide a good experimental system to address the phenotypic and functional features of specific T cell subpopulations and furthermore settle the framework necessary for analyzing their antigen/peptide specificity.This chapter details a culture method for the establishment of T. cruzi-specific memory T cell lines from mononuclear cells isolated from Chagas disease patients' peripheral blood. The presented protocol comprises (1) enrichment of memory CD4+ T cells, (2) stimulation with parasite lysate, (3) evaluation of specificity, and (4) expansion and maintenance of specific T cell lines.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Técnicas de Cultura de Células/métodos , Doença de Chagas/imunologia , Leucócitos Mononucleares/imunologia , Trypanosoma cruzi/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/parasitologia , Linhagem Celular , Proliferação de Células , Células Cultivadas , Doença de Chagas/parasitologia , Citocinas/imunologia , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/parasitologia
11.
Methods Mol Biol ; 1955: 339-348, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30868539

RESUMO

Chagas disease is the highest impact parasitic disease in Latin America. In recent years, the use of immune-related biomarkers to predict diagnostic and treatment efficacy or to monitor diseases has been considered a promising tool. Our group has worked for the past 20 years on the characterization of different immunological aspects of the T-cell responses to T. cruzi antigens. We have shown that monitoring of appropriate immunological responses can provide earlier and robust measures of treatment.The Enzyme-Linked ImmunoSPOT (ELISPOT) assays are powerful tools to evaluate antigen-specific immune responses at the single-cell level. Herein, we describe uses of the ELISPOT assay to determine the T. cruzi-specific T-cell populations in PBMCs from chronic chagasic subjects.


Assuntos
Doença de Chagas/diagnóstico , Técnicas Imunoenzimáticas/métodos , Linfócitos T/imunologia , Trypanosoma cruzi/imunologia , Células Cultivadas , Doença de Chagas/tratamento farmacológico , Doença de Chagas/imunologia , Doença de Chagas/parasitologia , Doença Crônica , Humanos , Imunidade Celular , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/parasitologia , Nitroimidazóis/farmacologia , Nitroimidazóis/uso terapêutico , Prognóstico , Linfócitos T/parasitologia , Resultado do Tratamento , Tripanossomicidas/farmacologia , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/efeitos dos fármacos
12.
Methods Mol Biol ; 1955: 363-380, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30868541

RESUMO

The thiol moieties of cysteinyl residues in proteins undergo a number of modifications including nitrosylation, oxidation, persulfidation, sulfenylation, and others. These protein modifications may influence gain as well as loss of function in biological and disease conditions. Herein, we describe a quantitative approach that combines accurate, sensitive fluorescence modification of cysteinyl-S-nitrosyl (SNOFlo) groups that leaves electrophoretic mobility unaffected and offers the measurement of changes in S-nitrosylation (SNO) status relative to protein abundance. This approach has been useful in evaluating the global protein abundance and SNO profile of Chagas seropositive individuals that were categorized in clinically asymptomatic (C/A) and clinically symptomatic (C/S) subgroups and compared to normal healthy (N/H) controls. Through analyzing the proteome datasets with different bioinformatics and statistics tools, potential pathologic mechanisms in disease progression are identified. We also propose a panel of protein biomarkers that have a potential to identify the infected individuals at risk of developing clinical Chagas disease.


Assuntos
Doença de Chagas/sangue , Leucócitos Mononucleares/parasitologia , Proteínas/análise , Proteômica/métodos , Biomarcadores/análise , Doença de Chagas/patologia , Doença Crônica , Cisteína/análise , Eletroforese em Gel Bidimensional/métodos , Fluorescência , Humanos , Espectrometria de Massas/métodos , Óxido Nítrico/análise
13.
Parasit Vectors ; 12(1): 105, 2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30871600

RESUMO

BACKGROUND: Hepatocellular carcinoma-associated antigen 59 (HCA59), which is one of the most important excretory/secretory products of Haemonchus contortus (HcESPs), is known to have antigenic functions. However, its immunomodulatory effects on host cells are poorly understood. METHODS: Here, we cloned the HCA59 gene and expressed the recombinant protein of HCA59 (rHCA59). Binding activities of rHCA59 to goat peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) were checked by immunofluorescence assay (IFA) and the immunoregulatory effects of rHCA59 on cytokine secretions, cell migration, cell proliferation, nitric oxide production, and changes in expression of genes in related pathways were observed by co-incubation of rHCA59 with goat PBMCs and DCs. Monocyte phagocytosis and characterization of goat blood DC subsets were detected by flow cytometry. RESULTS: The IFA results revealed that rHCA59 could bind to PBMCs and DCs. Treatment of PBMCs with rHCA59 significantly increased cellular proliferation and NO production in a dose-dependent manner, while cell migration was vigorously blocked. Treatment with rHCA59 significantly suppressed monocytes phagocytosis. The quantity of surface marker CD80 on DCs increased significantly after rHCA59 treatment. In addition, the expression of genes included in the WNT pathway was related to the differentiation and maturation of DCs, and the production of IL-10 and IL-17 produced by PBMCs was altered. CONCLUSIONS: Our findings illustrated that rHCA59 could enhance host immune responses by regulating the functions of goat PBMCs and DCs, which would benefit our understanding of HCA59 from parasitic nematodes contributing to the mechanism of parasitic immune evasion.


Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Carcinoma Hepatocelular/veterinária , Hemoncose/veterinária , Haemonchus/imunologia , Proteínas de Helminto/imunologia , Neoplasias Hepáticas/imunologia , Animais , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/parasitologia , Diferenciação Celular , Movimento Celular , Proliferação de Células , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Feminino , Cabras , Hemoncose/parasitologia , Proteínas de Helminto/genética , Imunomodulação , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/parasitologia , Neoplasias Hepáticas/parasitologia , Masculino , Monócitos/imunologia , Monócitos/parasitologia , Óxido Nítrico/metabolismo , Ratos , Proteínas Recombinantes
14.
Artigo em Inglês | MEDLINE | ID: mdl-30800644

RESUMO

In biology, models are experimental systems meant to recreate aspects of diseases or human tissue with the goal of generating inferences and approximations that can contribute to the resolution of specific biological problems. Although there are many models for studying intracellular parasites, their data have produced critical contradictions, especially in immunological assays. Peripheral blood mononuclear cells (PBMCs) represent an attractive tissue source in pharmacogenomics and in molecular and immunologic studies, as these cells are easily collected from patients and can serve as sentinel tissue for monitoring physiological perturbations due to disease. However, these cells are a very sensitive model due to variables such as temperature, type of stimulus and time of collection as part of posterior processes. PBMCs have been used to study Toxoplasma gondii and other apicomplexan parasites. For instance, this model is frequently used in new therapies or vaccines that use peptides or recombinant proteins derived from the parasite. The immune response to T. gondii is highly variable, so it may be necessary to refine this cellular model. This mini review highlights the major approaches in which PBMCs are used as a model of study for T. gondii and other apicomplexan parasites. The variables related to this model have significant implications for data interpretation and conclusions related to host-parasite interaction.


Assuntos
Apicomplexa/crescimento & desenvolvimento , Apicomplexa/imunologia , Interações Hospedeiro-Patógeno , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/parasitologia , Modelos Teóricos , Infecções por Protozoários/fisiopatologia , Animais , Pesquisa Biomédica/tendências , Humanos , Infecções por Protozoários/imunologia , Infecções por Protozoários/parasitologia
15.
PLoS One ; 13(12): e0206876, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30517108

RESUMO

Visceral leishmaniasis (VL) in humans is a chronic and often fatal disease if left untreated. Dogs appear to be the main reservoir host for L. infantum infection, however, in many regions other canids such as jackals, foxes, wolves and other mammals, such as hares or black rats, have been implicated as wild reservoirs. Most dogs cannot form an effective immune response against this infection, and this could be modulated by small non-coding RNAs, called microRNAs, responsible for post-transcriptional control of gene expression. Here, we evaluated the expression of miRNAs in peripheral blood mononuclear cells (PBMC) of symptomatic dogs naturally infected with Leishmania (L.) infantum (n = 10) and compared to those of healthy dogs (n = 5). Microarray analysis revealed that miR-21, miR-424, miR-194 and miR-451 had a 3-fold increase in expression, miR-192, miR-503, and miR-371 had a 2-fold increase in expression, whereas a 2-fold reduction in expression was observed for miR-150 and miR-574. Real-time PCR validated the differential expression of miR-21, miR-150, miR-451, miR-192, miR-194, and miR-371. Parasite load of PBMC was measured by real-time PCR and correlated to the differentially expressed miRNAs, showing a strong positive correlation with expression of miR-194, a regular positive correlation with miR-371 expression, and a moderate negative correlation with miR-150 expression in PBMC. These findings suggest that Leishmania infection interferes with miRNAs expression in PBMC, and their correlation with parasite load may help in the identification of therapeutic targets in Canine Visceral Leishmaniasis (CVL).


Assuntos
Doenças do Cão , Regulação da Expressão Gênica , Leishmania infantum , Leishmaniose Visceral , Leucócitos Mononucleares/metabolismo , MicroRNAs/biossíntese , Animais , Doenças do Cão/sangue , Doenças do Cão/parasitologia , Cães , Leishmaniose Visceral/sangue , Leishmaniose Visceral/veterinária , Leucócitos Mononucleares/parasitologia
16.
Artigo em Inglês | MEDLINE | ID: mdl-30406037

RESUMO

Calreticulin (CRT) is a highly conserved protein in the endoplasmic reticulum that plays important roles in the regulation of key cellular functions. Little is known about the participation of E. histolytica CRT (EhCRT) in the processes of pathogenicity or in the modulation of the host immune response. The aim of this study was to evaluate the role of CRT in the proliferation and the cytokine profile in peripheral blood mononuclear cells (PBMCs) from patients with amebic liver abscess (ALA) during the acute phase (AP-ALA) of the disease compared to patients during the resolution phase (R-ALA). The PBMCs from each participant were cocultured with EhCRT and tested by the colorimetric method to evaluate their proliferation index (PI). The supernatants were subjected to an enzyme-linked immunosorbent assay (ELISA) to evaluate the concentration of cytokines. The mean values of all groups were compared using the independent t-test. When the PIs of individuals without diagnosis of liver abscess (NEG) were compared, there were no statistically significant differences in the proliferation of PBMCs between patients with AP-ALA and R-ALA when stimulated with EhCRT or concanavalin A (ConA). However, the levels of interleukins [IL-6, IL-10, granulocyte colony stimulating factor (GCSF), and transforming growth factor ß1 (TGFß1)] were higher in patients with AP-ALA, whereas in patients with R-ALA, higher levels of interferon gamma (IFNγ) were detected. These results suggest that EhCRT acts as a mitogen very similar to the activity of ConA. In addition, EhCRT is an excellent immunogen for the specific activation of PBMCs, inducing the differential expression of ILs depending on the outcome of disease, determining the type of immune response: a Th2 cytokine profile during the acute phase and a Th1 profile during the resolution phase.


Assuntos
Calreticulina/metabolismo , Citocinas/biossíntese , Entamoeba histolytica/crescimento & desenvolvimento , Entamoeba histolytica/imunologia , Interações Hospedeiro-Patógeno , Leucócitos Mononucleares/parasitologia , Abscesso Hepático Amebiano/parasitologia , Calreticulina/imunologia , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura/química , Entamoeba histolytica/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Humanos , Leucócitos Mononucleares/imunologia , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo
17.
Parasit Vectors ; 11(1): 579, 2018 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-30400957

RESUMO

BACKGROUND: Fasciola gigantica-induced immunomodulation is a major hurdle faced by the host for controlling infection. Here, we elucidated the role of F. gigantica Ras-related protein Rab10 (FgRab10) in the modulation of key functions of peripheral blood mononuclear cells (PBMCs) of goats. METHODS: We cloned and expressed recombinant FgRab10 (rFgRab10) protein and examined its effects on several functions of goat PBMCs. Protein interactors of rFgRab10 were predicted in silico by querying the databases Intact, String, BioPlex and BioGrid. In addition, a total energy analysis of each of the identified interactions was also conducted. Gene Ontology (GO) enrichment analysis was carried out using FuncAssociate 3.0. RESULTS: The FgRab10 gene (618 bp), encodes 205-amino-acid residues with a molecular mass of ~23 kDa, had complete nucleotide sequence homology with F. hepatica Ras family protein gene (PIS87503.1). The rFgRab10 protein specifically cross-reacted with anti-Fasciola antibodies as shown by Western blot and immunofluorescence analysis. This protein exhibited multiple effects on goat PBMCs, including increased production of cytokines [interleukin-2 (IL-2), IL-4, IL-10, transforming growth factor beta (TGF-ß) and interferon gamma (IFN-γ)] and total nitric oxide (NO), enhancing apoptosis and migration of PBMCs, and promoting the phagocytic ability of monocytes. However, it significantly inhibited cell proliferation. Homology modelling revealed 63% identity between rFgRab10 and human Rab10 protein (Uniprot ID: P61026). Protein interaction network analysis revealed more stabilizing interactions between Rab proteins geranylgeranyltransferase component A 1 (CHM) and Rab proteins geranylgeranyltransferase component A 2 (CHML) and rFgRab10 protein. Gene Ontology analysis identified RabGTPase mediated signaling as the most represented pathway. CONCLUSIONS: rFgRab10 protein exerts profound influences on various functions of goat PBMCs. This finding may help explain why F. gigantica is capable of provoking recognition by host immune cells, less capable of destroying this successful parasite.


Assuntos
Fasciola/genética , Proteínas de Helminto/genética , Interações Hospedeiro-Parasita/imunologia , Leucócitos Mononucleares/parasitologia , Proteínas rab de Ligação ao GTP/genética , Animais , Western Blotting , Proliferação de Células , Simulação por Computador , Citocinas , Fasciola hepatica/genética , Fasciolíase/parasitologia , Ontologia Genética , Cabras/sangue , Imunomodulação , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Homologia de Sequência , Homologia Estrutural de Proteína , Proteínas rab de Ligação ao GTP/imunologia , Proteínas rab de Ligação ao GTP/farmacologia
18.
BMC Infect Dis ; 18(1): 500, 2018 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-30285653

RESUMO

BACKGROUND: Visceral leishmaniasis (VL), caused by Leishmania donovani complex parasites, is a neglected parasitic disease that is generally fatal if untreated. Despite decades of research to develop a sensitive VL diagnostic test, definitive diagnosis of VL still mainly relies on the visualization of the parasite in aspirates from the spleen, liver or bone marrow, an invasive and dangerous process with variable sensitivity. A sensitive assay that can detect Leishmania antigen from blood samples will help confirm cause, cure or recurrence of VL. METHODS: In this study, rabbit polyclonal antibodies were raised against eight recombinant Leishmania proteins that are highly abundant in Leishmania. The antibodies were purified and labeled with biotin for developing a prototype sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: The ELISA for the Leishmania 40S ribosomal protein S12 detected target antigen with the highest sensitivity and specificity and could detect 1 pg of purified protein or as few as 60 L. donovani parasites. The 40S ribosomal protein S12 sandwich ELISA could detect the target antigen from Peripheral Blood Mononuclear Cell (PBMC) samples in 68% of VL patients and post-kala-azar dermal leishmaniasis (PKDL) patients, providing an estimation of parasitemia ranging from 15 to 80 amastigotes per ml of blood. CONCLUSION: These results indicate that the 40S ribosomal protein S12 sandwich ELISA warrants further tests with more clinical samples of VL patients and other parasitic diseases. It is hopeful that this ELISA could become a useful tool for confirming VL diagnosis, monitoring treatment progress, disease recurrence and possibly detecting asymptomatic Leishmania infections with a high parasite load.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Leishmaniose Visceral/sangue , Proteínas Ribossômicas/imunologia , Adolescente , Adulto , Animais , Antígenos de Protozoários/imunologia , Infecções Assintomáticas , Estudos de Casos e Controles , Feminino , Humanos , Leishmania/imunologia , Leishmania/patogenicidade , Leishmaniose/sangue , Leishmaniose/diagnóstico , Leishmaniose/parasitologia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/parasitologia , Leucócitos Mononucleares/parasitologia , Masculino , Pessoa de Meia-Idade , Doenças Negligenciadas , Carga Parasitária , Parasitemia/sangue , Parasitemia/diagnóstico , Coelhos , Proteínas Ribossômicas/genética , Sensibilidade e Especificidade
19.
EBioMedicine ; 37: 442-452, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30337251

RESUMO

BACKGROUND: Plasmodium falciparum and Plasmodium vivax are two major parasites responsible for malaria which remains a threat to almost 50% of world's population despite decade-long eradication program. One possible reason behind this conundrum is that the bases of clinical variability in malaria caused by either species are complex and poorly understood. METHODS: Whole-genome transcriptome was analyzed to identify the active and predominant pathways in the PBMC of P. falciparum and P. vivax infected malaria patients. Deregulated genes were identified and annotated using R Bioconductor and DAVID/KEGG respectively. Genetic and functional regulation of CD14, a prioritized candidate, were established by quantitative RT-PCR, genotyping using RFLP and resequencing, mapping of transcription factor binding using CONSITE and TFBIND, dual luciferase assay, western blot analysis, RNAi- mediated gene knockdown and chromatin-immunoprecipation. FINDINGS: The study highlighted that deregulation of host immune and inflammatory genes particularly CD14 as a key event in P. falciparum malaria. An abundance of allele-C of rs5744454, located in CD14 promoter, in severe malaria motivated us to establish an allele-specific regulation of CD14 by SP1. An enhancement of SP1 and CD14 expression was observed in artemisinin treated human monocyte cell line. INTERPRETATION: Our data not only reinstates that CD14 of TLR pathway plays a predominant role in P. falciparum malaria, it establishes a functional basis for genetic association of rs5744454 with P. falciparum severe malaria by demonstrating a cis-regulatory role of this promoter polymorphism. Moreover, the study points towards a novel pharmacogenetic aspect of artemisinin-based anti-malarial therapy. FUND: DST-SERB, Govt. of India, SR/SO/HS-0056/2013.


Assuntos
Regulação da Expressão Gênica , Leucócitos Mononucleares/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Malária Falciparum/metabolismo , Plasmodium falciparum , Plasmodium vivax , Fator de Transcrição Sp1/metabolismo , Transcriptoma , Adulto , Feminino , Humanos , Leucócitos Mononucleares/parasitologia , Leucócitos Mononucleares/patologia , Malária Vivax , Masculino , Pessoa de Meia-Idade
20.
PLoS One ; 13(10): e0204177, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30300360

RESUMO

The lack of suitable animal models for the study of cytoadhesion of P. falciparum-infected erythrocytes (IEs) has necessitated in vitro studies employing a range of cell lines of either human tumour origin (e.g., BeWo and C32 cells) or non-human origin (e.g., CHO cells). Of the human cells available, many were isolated from adults, or derived from a pool of donors (e.g., HBEC-5i). Here we demonstrate, for the first time, the successful isolation of blood outgrowth endothelial cells (BOECs) from frozen stabilates of peripheral blood mononuclear cells obtained from small-volume peripheral blood samples from paediatric malaria patients. BOECs are a sub-population of human endothelial cells, found within the peripheral blood. We demonstrate that these cells express receptors such as Intercellular Adhesion Molecule 1 (ICAM-1/CD54), Endothelial Protein C Receptor (EPCR/CD201), platelet/endothelial cell adhesion molecule 1 (PECAM-1/CD31), Thrombomodulin (CD141), and support adhesion of P. falciparum IEs.


Assuntos
Técnicas de Cultura de Células/métodos , Eritrócitos/citologia , Leucócitos Mononucleares/citologia , Malária Falciparum/sangue , Plasmodium falciparum/fisiologia , Animais , Antígenos de Superfície/metabolismo , Células CHO , Adesão Celular , Linhagem Celular , Criança , Pré-Escolar , Cricetulus , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/parasitologia , Receptor de Proteína C Endotelial/metabolismo , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/parasitologia , Malária Falciparum/parasitologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo
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