RESUMO
In the Americas, L. infantum (syn. chagasi) is the main cause of human visceral leishmaniasis. The role of neutrophils as part of the innate response to Leishmania spp. infection is dubious and varies according to the species causing the infection. Global expression of coding RNAs, microRNAs and long non-coding RNAs changes as part of the immune response against pathogens. Changes in mRNA and non-coding RNA expression resulting from infection by Leishmania spp. are widely studied in macrophages, but scarce in neutrophils, the first cell to encounter the trypanosomatid, especially following infection by L. infantum. Herein, we aimed to understand the expression patterns of coding and non-coding transcripts during acute in vitro infection of human neutrophils by L. infantum. We isolated neutrophils from whole blood of healthy male donors (n = 5) and split into groups: 1) infected with L. infantum (MOI = 5:1), and 2) uninfected controls. After 3 hours of exposure of infected group to promastigotes of L. infantum, followed by 17 hours of incubation, total RNA was extracted and total RNA-Seq and miRNA microarray were performed. A total of 212 genes were differentially expressed in neutrophils following RNA-Seq analysis (log2(FC)±0.58, FDR≤0.05). In vitro infection with L. infantum upregulated the expression of 197 and reduced the expression of 92 miRNAs in human neutrophils (FC±2, FDR≤0.01). Lastly, 5 downregulated genes were classified as lncRNA, and of the 10 upregulated genes, there was only 1 lncRNA. Further bioinformatic analysis indicated that changes in the transcriptome and microtranscriptome of neutrophils, following in vitro infection with L. infantum, may impair phagocytosis, apoptosis and decrease nitric oxide production. Our work sheds light on several mechanisms used by L. infantum to control neutrophil-mediated immune response and identifies several targets for future functional studies, aiming at the development of preventive or curative treatments for this prevalent zoonosis.
Assuntos
Leishmania infantum , MicroRNAs , Neutrófilos , RNA Longo não Codificante , RNA Mensageiro , Humanos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Leishmania infantum/genética , Leishmania infantum/imunologia , RNA Longo não Codificante/genética , MicroRNAs/genética , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/genética , Adulto , Perfilação da Expressão GênicaRESUMO
The spleen plays a pivotal role in the pathogenesis of visceral leishmaniasis. In severe forms of the disease, the spleen undergoes changes that can compromise its function in surveilling blood-circulating pathogens. In this study, we present an integrated analysis of the structural and gene expression alterations in the spleens of three patients with relapsing visceral leishmaniasis, two of whom were coinfected with HIV. Our findings reveal that the IL6 signaling pathway plays a significant role in the disorganization of the white pulp, while BCL10 and ICOSLG are associated with spleen organization. Patients coinfected with HIV and visceral leishmaniasis exhibited lower splenic CD4+ cell density and reduced expression of genes such as IL15. These effects may contribute to a compromised immune response against L. infantum in coinfected individuals, further impacting the structural organization of the spleen.
Assuntos
Coinfecção , Infecções por HIV , Leishmaniose Visceral , Baço , Humanos , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/genética , Baço/patologia , Infecções por HIV/complicações , Coinfecção/virologia , Masculino , Adulto , Feminino , Linfócitos T CD4-Positivos/imunologia , Leishmania infantum/genética , Expressão GênicaRESUMO
Berardinelli-Seip congenital lipodystrophy (CGL), a rare autosomal recessive disorder, is characterized by a lack of adipose tissue. Infections are one of the major causes of CGL individuals' premature death. The mechanisms that predispose to infections are poorly understood. We used Leishmania infantum as an in vitro model of intracellular infection to explore mechanisms underlying the CGL infection processes, and to understand the impact of host mutations on Leishmania survival, since this pathogen enters macrophages through specialized membrane lipid domains. The transcriptomic profiles of both uninfected and infected monocyte-derived macrophages (MDMs) from CGL (types 1 and 2) and controls were studied. MDMs infected with L. infantum showed significantly downregulated expression of genes associated with infection-response pathways (MHC-I, TCR-CD3, and granzymes). There was a transcriptomic signature in CGL cells associated with impaired membrane trafficking and signaling in response to infection, with concomitant changes in the expression of membrane-associated genes in parasites (e.g. δ-amastins). We identified pathways suggesting the lipid storage dysfunction led to changes in phospholipids expression and impaired responses to infection, including immune synapse (antigen presentation, IFN-γ signaling, JAK/STAT); endocytosis; NF-kappaB signaling; and phosphatidylinositol biosynthesis. In summary, lipid metabolism of the host plays an important role in determining antigen presentation pathways.
Assuntos
Leishmania infantum , Lipodistrofia Generalizada Congênita , Macrófagos , Transdução de Sinais , Humanos , Macrófagos/metabolismo , Macrófagos/parasitologia , Macrófagos/imunologia , Lipodistrofia Generalizada Congênita/genética , Lipodistrofia Generalizada Congênita/metabolismo , Leishmania infantum/genética , Transcriptoma , Masculino , Feminino , Perfilação da Expressão Gênica , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/genética , Leishmaniose Visceral/metabolismoRESUMO
Introduction: Single nucleotide variations (SNVs) are specific genetic variations that commonly occur in a population and often do not manifest phenotypically. However, depending on their location and the type of nucleotide exchanged, an SNV can alter or inhibit the function of the gene in which it occurs. Immunoglobulin G (IgG) receptor genes have exhibited several polymorphisms, including rs1801274, which is found in the FcgRIIa gene. The replacement of A with T results in a Histidine (H) to Arginine (R) substitution, altering the affinity of the IgG receptor for IgG subtypes and C-reactive protein (CRP). In this study, we analyzed rs1801274 and its functional implications concerning L. Infantum uptake and cytokine production. Methods: We genotyped 201 individuals from an endemic area for visceral leishmaniasis to assess the presence of rs1801274 using Taqman probes for a candidate gene study. Additionally, we included seventy individuals from a non-endemic area for a functional study. Subsequently, we isolated and cultivated one-week adherent mononuclear cells (AMCs) derived from the peripheral blood of participants residing in the non-endemic region in the presence of L. infantum promastigotes, with and without antigen-specific IgG and/or CRP. We analyzed the rate of phagocytosis and the production of nitric oxide (NO), tumor necrosis factor (TNF)-a, interleukin (IL)-10, IL-12 p70, IL-1b, IL- 6, and IL-8 in the culture supernatants. Results and discussion: In participants from the endemic region, the A/G (H/R isoform) heterozygous genotype was significantly associated with susceptibility to the disease. Furthermore, SNVs induced a change in the phagocytosis rate in an opsonin-dependent manner. Opsonization with IgG increased the production of IL-10, TNF-a, and IL-6 in AMCs with the H/R isoform, followed by a decrease in NO production. The results presented here suggest that the rs1801274 polymorphism is linked to a higher susceptibility to visceral leishmaniasis.
Assuntos
Leishmania infantum , Leishmaniose Visceral , Humanos , Leishmaniose Visceral/genética , Leishmania infantum/genética , Receptores de IgG/genética , Interleucina-12 , Fator de Necrose Tumoral alfa , Nucleotídeos , Isoformas de Proteínas , Variação Genética , Imunoglobulina GRESUMO
Domestic dogs are the primary urban reservoirs of Leishmania infantum, the causative agent of visceral leishmaniasis. In Canine Leishmaniasis (CanL), modulation of the host's immune response may be associated with the expression of small non-coding RNAs called microRNA (miR). miR-194 expression increases in peripheral blood mononuclear cells (PBMCs) of dogs with leishmaniasis with a positive correlation with the parasite load and in silico analysis demonstrated that the TRAF6 gene is the target of miR-194 in PBMCs from diseased dogs. Here, we isolated PBMCs from 5 healthy dogs and 28 dogs with leishmaniasis, naturally infected with L. infantum. To confirm changes in miR-194 and TRAF6 expression, basal expression of miR-194 and gene expression of TRAF6 was measured using qPCR. PBMCs from healthy dogs and dogs with leishmaniasis were transfected with miR-194 scramble, mimic, and inhibitor and cultured at 37° C, 5% CO2 for 48 hours. The expression of possible targets was measured: iNOS, NO, T-bet, GATA3, and FoxP3 were measured using flow cytometry; the production of cytokines IL-1ß, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and TGF-ß in cell culture supernatants was measured using capture enzyme-linked immunosorbent assays (ELISA). Parasite load was measured using cytometry and qPCR. Functional assays followed by miR-194 inhibitor and IL-1ß blockade and assessment of NO production were also performed. Basal miR-194 expression was increased in PBMC from dogs with Leishmaniasis and was negatively correlated with TRAF6 expression. The mimic of miR-194 promoted an increase in parasite load. There were no significant changes in T-bet, GATA3, or FoxP3 expression with miR-194 enhancement or inhibition. Inhibition of miR-194 increased IL-1ß and NO in PBMCs from diseased dogs, and blockade of IL-1ß following miR-194 inhibition decreased NO levels. These findings suggest that miR-194 is upregulated in PBMCs from dogs with leishmaniasis and increases parasite load, possibly decreasing NO production via IL-1ß. These results increase our understanding of the mechanisms of evasion of the immune response by the parasite and the identification of possible therapeutic targets.
Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Leishmaniose , MicroRNAs , Animais , Cães , Citocinas/genética , Doenças do Cão/parasitologia , Fatores de Transcrição Forkhead , Leishmania infantum/fisiologia , Leishmaniose Visceral/genética , Leishmaniose Visceral/veterinária , Leucócitos Mononucleares , MicroRNAs/genética , Óxido Nítrico/metabolismo , Carga Parasitária , Fator 6 Associado a Receptor de TNF , Leishmaniose/veterinária , Interleucina-1beta/metabolismoRESUMO
Leishmaniases, a group of vector-borne diseases, are caused by the protozoan intracellular parasite Leishmania (L.) and are transmitted by the phlebotomine sandflies. A wide range of clinical manifestations in L- infection is observed. The clinical outcome ranges from asymptomatic, cutaneous leishmaniasis (CL) to severe mucosal leishmaniasis (ML) or visceral leishmaniasis (VL), depending on the L. species. Interestingly, only a fraction of L.-infected individuals progress to disease development, suggesting a key role of host genetics in the clinical outcome. NOD2 plays a critical role in the control of host defense and inflammation. The NOD2-RIK2 pathway is involved in developing a Th1- type response in patients with VL and C57BL/6 mice infected with L. infantum. We investigated whether variants in the NOD2 gene (R702W rs2066844, G908R rs2066845, and L1007fsinsC rs2066847) are associated with susceptibility to CL caused by L. guyanensis (Lg) in 837 patients with Lg-Cl and 797 healthy controls (HC) with no history of leishmaniasis. Both patients and HC are from the same endemic area of the Amazonas state of Brazil. The variants R702W and G908R were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and L1007fsinsC was by direct nucleotide sequencing. The minor allele frequency (MAF) of L1007fsinsC was 0.5% among the patients with Lg-CL and 0.6% in the healthy controls group. R702W genotypes frequencies were similar in both groups. Only 1% and 1.6% were heterozygous for G908R among the patients with Lg-CL and HC, respectively. None of the variants revealed any association with susceptibility to the development of Lg-CL. Correlations of genotypes with the level of plasma cytokines revealed that individuals with the mutant alleles of R702W tend to have low levels of IFN-γ. G908R heterozygotes also tend to have low IFN-γ, TNF-α, IL-17, and IL-8. Variants of NOD2 are not involved in the pathogenesis of Lg-CL.
Assuntos
Leishmaniose Cutânea , Leishmaniose Visceral , Proteína Adaptadora de Sinalização NOD2 , Animais , Camundongos , Citocinas/genética , Genótipo , Leishmania guyanensis , Leishmaniose Cutânea/genética , Leishmaniose Visceral/genética , Camundongos Endogâmicos C57BL , Humanos , Proteína Adaptadora de Sinalização NOD2/genéticaRESUMO
Gain-of-function mutations in the STAT1 gene have been initially associated with chronic mucocutaneous candidiasis. However, further research has shown that STAT1 GOF variants may increase susceptibility to infection by other intracellular pathogens. This report describes the first case of disseminated leishmaniasis associated with a STAT1 GOF mutation in a pediatric patient who did not have chronic mucocutaneous candidiasis. The patient was a four-year-old boy presenting with fever, severe asthenia, hepatosplenomegaly, pancytopenia, and liver failure. Bone marrow aspirate revealed hemophagocytosis and Leishmania parasites. Treatment consisted primarily of liposomal amphotericin B, as per the Hemophagocytic Lymphohistiocytosis 2004 protocol. After eight weeks of treatment, the patient did not improve and was submitted to diagnostic splenectomy. Activated macrophages and nodular spleen necrosis secondary to the visceral leishmaniasis were detected. Unfortunately, the patient died in the second week after splenectomy due to overwhelming systemic infection. DNA sequencing revealed a pathogenic (p. R274Q) GOF mutation in STAT1.
Assuntos
Candidíase Mucocutânea Crônica , Leishmaniose Visceral , Candidíase Mucocutânea Crônica/complicações , Candidíase Mucocutânea Crônica/genética , Criança , Pré-Escolar , Mutação com Ganho de Função , Humanos , Leishmaniose Visceral/complicações , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/genética , Masculino , Mutação , Fator de Transcrição STAT1/genéticaRESUMO
Visceral leishmaniasis (VL) is a vector-borne infectious disease that can be potentially fatal if left untreated. In Brazil, it is caused by Leishmania infantum parasites. Blood transcriptomics allows us to assess the molecular mechanisms involved in the immunopathological processes of several clinical conditions, namely, parasitic diseases. Here, we performed mRNA sequencing of peripheral blood from patients with visceral leishmaniasis during the active phase of the disease and six months after successful treatment, when the patients were considered clinically cured. To strengthen the study, the RNA-seq data analysis included two other non-diseased groups composed of healthy uninfected volunteers and asymptomatic individuals. We identified thousands of differentially expressed genes between VL patients and non-diseased groups. Overall, pathway analysis corroborated the importance of signaling involving interferons, chemokines, Toll-like receptors and the neutrophil response. Cellular deconvolution of gene expression profiles was able to discriminate cellular subtypes, highlighting the contribution of plasma cells and NK cells in the course of the disease. Beyond the biological processes involved in the immunopathology of VL revealed by the expression of protein coding genes (PCGs), we observed a significant participation of long noncoding RNAs (lncRNAs) in our blood transcriptome dataset. Genome-wide analysis of lncRNAs expression in VL has never been performed. lncRNAs have been considered key regulators of disease progression, mainly in cancers; however, their pattern regulation may also help to understand the complexity and heterogeneity of host immune responses elicited by L. infantum infections in humans. Among our findings, we identified lncRNAs such as IL21-AS1, MIR4435-2HG and LINC01501 and coexpressed lncRNA/mRNA pairs such as CA3-AS1/CA1, GASAL1/IFNG and LINC01127/IL1R1-IL1R2. Thus, for the first time, we present an integrated analysis of PCGs and lncRNAs by exploring the lncRNA-mRNA coexpression profile of VL to provide insights into the regulatory gene network involved in the development of this inflammatory and infectious disease.
Assuntos
Leishmania infantum , Leishmaniose Visceral , Leishmaniose , RNA Longo não Codificante , Humanos , Leishmania infantum/genética , Leishmaniose Visceral/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , TranscriptomaRESUMO
Visceral leishmaniasis in humans is a chronic and fatal disease if left untreated. Canine leishmaniasis (CanL) is a severe public health problem because infected animals are powerful transmitters of the parasite to humans via phlebotomine vectors. Therefore, dogs are an essential target for control measures. Progression of canine infection is accompanied by failure of cellular immunity with reduction of circulating lymphocytes and increased cytokines that suppress macrophage function. Studies showed that the regulation of the effector function of macrophages and T cells appears to depend on miRNAs; miRNA-21 (miR-21) shows increased expression in splenic leukocytes of dogs with CanL and targets genes related to the immune response. Mimics and inhibitors of miR-21 were used in vitro to transfect splenic leukocytes from dogs with CanL. After transfection, expression levels of the proteins FAS, FASL, CD69, CCR7, TNF-α, IL-17, IFN-γ, and IL-10 were measured. FAS, FASL, CD69, and CCR7 expression levels decreased in splenic leukocytes from dogs with CanL. The miR-21 mimic decreased CD69 expression in splenic leukocytes from CanL and healthy groups. The miR-21 inhibitor decreased IL-10 levels in culture supernatants from splenic leukocytes in the CanL group. These findings suggest that miR-21 alters the immune response in CanL; therefore, miR-21 could be used as a possible therapeutic target for CanL.
Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Leishmaniose , MicroRNAs , Animais , Doenças do Cão/parasitologia , Cães , Interleucina-10/genética , Interleucina-10/uso terapêutico , Leishmaniose/veterinária , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/genética , Leishmaniose Visceral/veterinária , MicroRNAs/genética , MicroRNAs/uso terapêutico , Receptores CCR7RESUMO
Visceral Leishmaniasis and HIV-AIDS coinfection (VL/HIV) is considered a life-threatening pathology when undiagnosed and untreated, due to the immunosuppression caused by both diseases. Serological tests largely used for the VL diagnosis include the direct agglutination test (DAT), ELISA and immunochromatographic (ICT) assays. For VL diagnosis in HIV infections, different studies have shown that the use of the DAT assay facilitates the VL diagnosis in co-infected patients, since the performance of the most widely used ELISA and ICT tests, based on the recombinant protein rK39, are much less efficient in HIV co-infections. In this scenario, alternative recombinant antigens may help the development of new serological diagnostic methods which may improve the VL diagnosis for the co-infection cases. This work aimed to evaluate the use of the recombinant Lci2 antigen, related to, but antigenically more diverse than rK39, for VL diagnosis in co-infected sera through ELISA assays. A direct comparison between recombinant Lci2 and rK39 was thus carried out. The two proteins were first tested using indirect ELISA with sera from VL afflicted individuals and healthy controls, with similar performances. They were then tested with two different sets of VL/HIV co-infected cases and a significant drop in performance, for one of these groups, was observed for rK39 (32% sensitivity), but not for Lci2 (98% sensitivity). In fact, an almost perfect agreement (Kappa: 0.93) between the Lci2 ELISA and DAT was observed for the coinfected VL/HIV patients. Lci2 then has the potential to be used as a new tool for the VL diagnosis of VL/HIV co-infections.
Assuntos
Anticorpos Antiprotozoários/isolamento & purificação , Infecções por HIV/genética , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Proteínas Recombinantes/isolamento & purificação , Testes de Aglutinação , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Coinfecção/diagnóstico , Coinfecção/genética , Coinfecção/parasitologia , Ensaio de Imunoadsorção Enzimática , HIV/patogenicidade , Infecções por HIV/complicações , Infecções por HIV/parasitologia , Infecções por HIV/virologia , Humanos , Leishmania infantum/genética , Leishmania infantum/patogenicidade , Leishmaniose Visceral/genética , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/virologia , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/genéticaRESUMO
Visceral Leishmaniasis (VL) is a neglected tropical disease, caused by L. infantum in the New World, where dogs are the main reservoir. These parasites can regulate host immune response through miRNA differential expression in the early stages of infection; however such early response has not yet been investigated in the canine model. PBMC from healthy dogs were exposed to L. infantum in vitro and microarray analysis showed an upregulation of miR-206, miR-302d, miR-433, miR-214, miR-493, miR-514, miR-1835, miR-210, miR-539, miR-432, miR-188, miR-345 and downregulation of miR-489 and miR-503 in comparison to non-exposed control cells, at 24 h post-exposure. In silico target prediction showed that the upregulated miRNAs target 1541 genes, which can modulate important pathways involved in the early immune responses, like the "MAPK signaling pathway", one of the most relevant pathways to Leishmania survival inside host cells. These findings shed light on parasite modulation of host immunity following Leishmania infection, which in turn can be explored for drug development.
Assuntos
Doenças do Cão/parasitologia , Leishmania infantum , Leishmaniose Visceral/veterinária , Leucócitos Mononucleares/metabolismo , MicroRNAs/metabolismo , Animais , Células Cultivadas , Doenças do Cão/sangue , Doenças do Cão/genética , Doenças do Cão/imunologia , Cães , Regulação para Baixo , Leishmaniose Visceral/sangue , Leishmaniose Visceral/genética , Leishmaniose Visceral/imunologia , Sistema de Sinalização das MAP QuinasesRESUMO
This study aimed to determine the seroprevalence, factors associated with seropositivity to Leishmania infection in dogs and spatial analysis in six municipalities in the semiarid region of Pernambuco, Brazil. Blood samples were collected from 462 dogs, 77 in each municipality, and used for serological analysis [dual path platform (DPP®) and enzyme-linked immunosorbent assay (ELISA)]. Clinical signs of dogs were evaluated and associated factors for Leishmania infection were analyzed using robust Poisson regression model. A seroprevalence of 42.8% (198/462, IC: 95% = 38.6%-47.6%) was detected in dogs that tested positive in both tests, ranging from 29.8% to 55.8%, with higher prevalence in the municipality of Cabrobó (55.8%; P = 0.006). About 67% (132/198) of the seropositive dogs showed one or more clinical signs suggestive of canine leishmaniasis (CanL), such as lymphadenomegaly, skin lesions and conjunctivitis, which were associated with seropositivity. High seroprevalence levels were identified in urban and rural areas in all the municipalities, and the buffer for sand flies around cases covered almost these entire areas. Spatial analysis revealed a significant cluster, showing a relative risk of 1.88 in the urban area of Cabrobó. The higher density of seropositive dogs in urban areas indicates the need effective control measures against CanL to prevent the emergence of canine and human diseases.(AU)
Este estudo objetivou determinar a soroprevalência, fatores associados com a soropositividade da infecção por Leishmania em cães e análise espacial em seis municípios do semiárido de Pernambuco. Amostras de sangue foram coletadas de 462 cães, 77 em cada município, e utilizadas para análise sorológica [teste rápido imunocromatográfico (DPP®) e ensaio de imunoabsorção enzimática (ELISA)]. Os sinais clínicos dos cães foram avaliados, e os fatores associados à infecção por Leishmania foram analisados, usando-se modelo robusto de regressão de Poisson. Foi detectada uma soroprevalência de 42,8% (198/462, IC: 95% = 38,6%-47,6%) em cães com resultados positivos em ambos os testes, variando de 29,8% a 55,8%, com maior prevalência no município de Cabrobó (55,8%; P = 0,006). Cerca de 67% (132/198) dos cães apresentaram um ou mais sinais clínicos sugestivos de CanL, como linfadenomegalia, lesões de pele e conjuntivite, os quais foram associados à soropositividade. Altos níveis de soroprevalência foram identificados nas áreas urbanas e rurais de todos os municípios, e os buffers da área de vôo do flebotomíneo em torno dos casos cobriam quase todas as áreas dos municípios. A análise espacial revelou um cluster significativo, mostrando um risco relativo de 1,88 na área urbana de Cabrobó. A maior densidade de casos em áreas urbanas indica a necessidade de medidas efetivas de controle contra o CanL, para evitar o surgimento da doença canina e humana.(AU)
Assuntos
Animais , Cães , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/genética , SorologiaRESUMO
Visceral leishmaniasis (VL) is considered a major public health concern in Brazil and several regions of the world. A recent advance in the diagnosis of infectious diseases was the development of loop-mediated isothermal amplification (LAMP). The aim of this study was to develop and evaluate a new LAMP assay for detection of K26 antigen-coding gene of L. donovani complex. A total of 219 blood samples of immunocompetent patients, including 114 VL cases and 105 non-VL cases, were analyzed for the diagnosis of VL in the present study. Diagnostic accuracy was calculated against a combination of parasitological and/or serological tests as a reference standard. The results were compared to those of kDNA Leishmania-PCR. The detection limit for the K26-Lamp assay was 1fg L. infantum purified DNA and 100 parasites/mL within 60 min of amplification time with visual detection for turbidity. The assay was specific for L. donovani complex. Sensitivity, specificity, and accuracy were 98.2%, 98.1%, and 98.2%, respectively, for K26-LAMP and 100%, 100%, and 100%, respectively, for kDNA Leishmania-PCR. Excellent agreement was observed between K26-LAMP and kDNA Leishmania-PCR assays (K = 0.96). A highly sensitive and specific LAMP assay targeting K26 antigen-coding gene of L. donovani complex was developed for diagnosis in peripheral blood samples of VL patients.
Assuntos
DNA de Protozoário , Leishmania donovani/genética , Leishmaniose Visceral , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Brasil , DNA de Protozoário/sangue , DNA de Protozoário/genética , Feminino , Humanos , Leishmania donovani/metabolismo , Leishmaniose Visceral/sangue , Leishmaniose Visceral/genética , MasculinoRESUMO
Detrimental effects of malnutrition on immune responses to pathogens have long been recognized and it is considered a main risk factor for various infectious diseases, including visceral leishmaniasis (VL). Thymus is a target of both malnutrition and infection, but its role in the immune response to Leishmania infantum in malnourished individuals is barely studied. Because we previously observed thymic atrophy and significant reduction in cellularity and chemokine levels in malnourished mice infected with L. infantum, we postulated that the thymic microenvironment is severely compromised in those animals. To test this, we analyzed the microarchitecture of the organ and measured the protein abundance in its interstitial space in malnourished BALB/c mice infected or not with L. infantum. Malnourished-infected animals exhibited a significant reduction of the thymic cortex:medulla ratio and altered abundance of proteins secreted in the thymic interstitial fluid. Eighty-one percent of identified proteins are secreted by exosomes and malnourished-infected mice showed significant decrease in exosomal proteins, suggesting that exosomal carrier system, and therefore intrathymic communication, is dysregulated in those animals. Malnourished-infected mice also exhibited a significant increase in the abundance of proteins involved in lipid metabolism and tricarboxylic acid cycle, suggestive of a non-proliferative microenvironment. Accordingly, flow cytometry analysis revealed decreased proliferation of single positive and double positive T cells in those animals. Together, the reduced cortical area, decreased proliferation, and altered protein abundance suggest a dysfunctional thymic microenvironment where T cell migration, proliferation, and maturation are compromised, contributing for the thymic atrophy observed in malnourished animals. All these alterations could affect the control of the local and systemic infection, resulting in an impaired response to L. infantum infection.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Leishmania infantum/imunologia , Leishmaniose Visceral/imunologia , Desnutrição/imunologia , Linfócitos T/imunologia , Timo/imunologia , Animais , Transporte Biológico , Movimento Celular , Proliferação de Células , Ciclo do Ácido Cítrico/genética , Ciclo do Ácido Cítrico/imunologia , Exossomos/imunologia , Exossomos/metabolismo , Exossomos/parasitologia , Líquido Extracelular/imunologia , Líquido Extracelular/metabolismo , Líquido Extracelular/parasitologia , Galectina 1/genética , Galectina 1/imunologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Imunidade Inata , Leishmania infantum/crescimento & desenvolvimento , Leishmaniose Visceral/genética , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/parasitologia , Metabolismo dos Lipídeos , Masculino , Desnutrição/genética , Desnutrição/metabolismo , Desnutrição/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Plasminogênio/genética , Plasminogênio/imunologia , Proteoma/genética , Proteoma/imunologia , Linfócitos T/parasitologia , Timo/metabolismo , Timo/parasitologiaRESUMO
There is no safe and efficacious vaccine against human leishmaniasis available and live attenuated vaccines have been used as a prophylactic alternative against the disease. In order to obtain an attenuated Leishmania parasite for vaccine purposes, we generated L. infantum KHARON1 (KH1) null mutants (ΔLikh1). This gene was previously associated with growth defects in L. mexicana. ΔLikh1 was obtained and confirmed by PCR, qPCR and Southern blot. We also generate a KH1 complemented line with the introduction of episomal copies of KH1. Although ΔLikh1 promastigote forms exhibited a growth pattern similar to the wild-type line, they differ in morphology without affecting parasite viability. L. infantum KH1-deficient amastigotes were unable to sustain experimental infection in macrophages, forming multinucleate cells which was confirmed by in vivo attenuation phenotype. The cell cycle analysis of ΔLikh1 amastigotes showed arrested cells at G2/M phase. ΔLikh1-immunized mice presented reduced parasite burden upon challenging with virulent L. infantum, when compared to naïve mice. An effect associated with increased Li SLA-specific IgG serum levels and IL-17 production. Thus, ΔLikh1 parasites present an infective-attenuated phenotype due to a cytokinesis defect, whereas it induces immunity against visceral leishmaniasis in mouse model, being a candidate for antileishmanial vaccine purposes.
Assuntos
Citocinese , Leishmania infantum , Leishmaniose Visceral , Mutação , Animais , Citocinese/genética , Citocinese/imunologia , Modelos Animais de Doenças , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/imunologia , Humanos , Leishmania infantum/genética , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/imunologia , Leishmaniose Visceral/genética , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/prevenção & controle , Pontos de Checagem da Fase M do Ciclo Celular/genética , Pontos de Checagem da Fase M do Ciclo Celular/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Plasmídeos/genética , Plasmídeos/imunologia , Plasmídeos/metabolismo , Células THP-1RESUMO
Visceral leishmaniasis (VL) is a major public health issue reported as the second illness in mortality among all tropical diseases. Clinical trials have shown that protection against VL is associated with robust T cell responses, especially those producing IFN-γ. The Leishmania amastigote 2 (A2) protein has been repeatedly described as immunogenic and protective against VL in different animal models; it is recognized by human T cells, and it is also commercially available in a vaccine formulation containing saponin against canine VL. Moving toward a more appropriate formulation for human vaccination, here, we tested a new optimized version of the recombinant protein (rA2), designed for Escherichia coli expression, in combination with adjuvants that have been approved for human use. Moreover, aiming at improving the cellular immune response triggered by rA2, we generated a recombinant live vaccine vector using Trypanosoma cruzi CL-14 non-virulent strain, named CL-14 A2. Mice immunized with respective rA2, adsorbed in Alum/CpG B297, a TLR9 agonist recognized by mice and human homologs, or with the recombinant CL-14 A2 parasites through homologous prime-boost protocol, were evaluated for antigen-specific immune responses and protection against Leishmania infantum promastigote challenge. Immunization with the new rA2/Alum/CpG formulations and CL-14 A2 transgenic vectors elicited stronger cellular immune responses than control groups, as shown by increased levels of IFN-γ, conferring protection against L. infantum challenge. Interestingly, the use of the wild-type CL-14 alone was enough to boost immunity and confer protection, confirming the previously reported immunogenic potential of this strain. Together, these results support the success of both the newly designed rA2 antigen and the ability of T. cruzi CL-14 to induce strong T cell-mediated immune responses against VL in animal models when used as a live vaccine vector. In conclusion, the vaccination strategies explored here reveal promising alternatives for the development of new rA2 vaccine formulations to be translated human clinical trials.
Assuntos
Antígenos de Protozoários/imunologia , Imunidade Celular , Leishmania infantum/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/prevenção & controle , Linfócitos T/imunologia , Trypanosoma cruzi/imunologia , Animais , Antígenos de Protozoários/genética , Feminino , Leishmania infantum/genética , Vacinas contra Leishmaniose/genética , Leishmaniose Visceral/genética , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/patologia , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/patologiaRESUMO
Background: Little is known about the genetic diversity of Leishmania infantum isolates in the state of Minas Gerais, Brazil. Methods: Molecular characterization of hsp70, mpi and ITS1 was carried out for 29 isolates of L. infantum from the bone marrow of naturally infected dogs from the cities of Divinópolis, Pará de Minas and Brumadinho, located in the central-west, central and central regions of the state of Minas Gerais, Brazil, respectively. Results: Analysis of the parasite nucleotide sequences demonstrated very high homogeneity of the studied samples. Conclusions: In the endemic regions studied, parasites are genotypically indistinguishable.
Assuntos
Medula Óssea/parasitologia , Doenças do Cão/parasitologia , Leishmania infantum/genética , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/veterinária , Animais , Brasil/epidemiologia , Cidades , Reservatórios de Doenças/parasitologia , Reservatórios de Doenças/estatística & dados numéricos , Doenças do Cão/epidemiologia , Doenças do Cão/transmissão , Cães , Leishmania infantum/patogenicidade , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/genética , Leishmaniose Visceral/transmissão , Filogenia , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: The analysis of modular gene co-expression networks is a well-established method commonly used for discovering the systems-level functionality of genes. In addition, these studies provide a basis for the discovery of clinically relevant molecular pathways underlying different diseases and conditions. RESULTS: In this paper, we present a fast and easy-to-use Bioconductor package named CEMiTool that unifies the discovery and the analysis of co-expression modules. Using the same real datasets, we demonstrate that CEMiTool outperforms existing tools, and provides unique results in a user-friendly html report with high quality graphs. Among its features, our tool evaluates whether modules contain genes that are over-represented by specific pathways or that are altered in a specific sample group, as well as it integrates transcriptomic data with interactome information, identifying the potential hubs on each network. We successfully applied CEMiTool to over 1000 transcriptome datasets, and to a new RNA-seq dataset of patients infected with Leishmania, revealing novel insights of the disease's physiopathology. CONCLUSION: The CEMiTool R package provides users with an easy-to-use method to automatically implement gene co-expression network analyses, obtain key information about the discovered gene modules using additional downstream analyses and retrieve publication-ready results via a high-quality interactive report.
Assuntos
Regulação da Expressão Gênica , Redes Reguladoras de Genes , Software , Automação , Bases de Dados Genéticas , Dengue/genética , Perfilação da Expressão Gênica , Humanos , Leishmaniose Visceral/genética , Psoríase/genética , Análise de Sequência de RNA , Transcriptoma/genéticaRESUMO
Manifestations of Leishmania infantum infection range from asymptomatic to symptomatic visceral leishmaniasis (VL). People with symptomatic VL (sVL) have suppressed immune responses against Leishmania antigens that are reversed after clinical cure. The intradermal leishmanin skin test (LST) is negative during sVL, but it becomes positive after treatment. The aim of this study was to compare T cell responses in individuals with sVL, recovered VL (RecVL), and endemic controls. Endemic controls were household contacts of a VL case and they were grouped by their LST results, either positive (LST+) or negative (LST-). Mononuclear cells were studied ex vivo or after stimulation with soluble Leishmania antigens (SLA); cell surface markers and cytokines were determined. T cells, ex vivo, from individuals with sVL and from LST+ individuals presented a higher activation for CD4+ and CD8+ cells expressing CD69. However, lymphocytes from sVL stimulated with SLA had lower percentages of CD4+ and CD8+ cells expressing CD69 and CD8+ cells expressing CD25, with no release of interferon-γ or tumor necrosis factor. sVL subjects had lower percentage of memory cells (CD4+ CD45RO+), ex vivo, without SLA stimulation than RecVL, LST+, or LST- (P = 0.0022). However, individuals with sVL had fewer regulatory cells after SLA stimulation (CD4+ CD25HIGH, P = 0.04 and CD4+ FOXP3+, P = 0.02) than RecVL. The decrease in specific memory and activated CD4+ and CD8+ cells, as in response to Leishmania antigens, could explain, in part, the immune impairment during sVL. Finally, protective T cell responses are long lasting because both RecVL or LST+ individuals maintain a specific protective response to Leishmania years after the primary infection.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunidade Celular , Memória Imunológica , Leishmania infantum/imunologia , Leishmaniose Visceral/imunologia , Adolescente , Adulto , Idoso , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Protozoários/farmacologia , Doenças Assintomáticas , Brasil , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/parasitologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/parasitologia , Estudos de Casos e Controles , Criança , Feminino , Expressão Gênica , Humanos , Interferon gama/genética , Interferon gama/imunologia , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Leishmania infantum/crescimento & desenvolvimento , Leishmaniose Visceral/genética , Leishmaniose Visceral/parasitologia , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/imunologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Testes Cutâneos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Different Leishmania proteins have been evaluated in order to find a potential vaccine candidate or diagnostic marker capable of providing long lasting protection against infection or helping to identify infected mammalian hosts, respectively. However, just few molecules have fulfilled all the requirements to be evaluated. In the current study, we evaluated the prophylactic and diagnostic value against visceral leishmaniasis (VL) of a small glutamine-rich tetratricopeptide repeat-containing (SGT) protein from Leishmania infantum species. In a first step, the immune response elicited by the immunization using the recombinant protein (rSGT) plus saponin was evaluated in BALB/c mice. Immunized animals had a low parasitism in all evaluated organs. They developed a specific Th1 immune response, which was based on protein-specific production of IFN-γ, IL-12 and GM-CSF, and a humoral response dominated by antibodies of the IgG2a isotype. Both CD4+ and CD8+ T cells contributed to the IFN-γ production, showing that both T cell subtypes contribute to the resistance against infection. Regarding its value as a diagnostic marker, rSGT showed maximum sensitivity and specificity to serologically identify L. infantum-infected dog and human sera. No cross-reactivity with sera from humans or dogs that had other diseases was found. Although further studies are necessary to validate these findings, data showed here suggest immunogenicity of rSGT and its protective effect against murine VL, as well as its potential for the serodiagnosis of human and canine VL.