RESUMEN
Visceral leishmaniasis (VL) is a chronic systemic disease. In Brazil this infection is caused by Leishmania (Leishmania) infantum. Extracellular vesicles (EVs) released by Leishmania species have different functions like the modulation of host immune systems and inflammatory responses, among others. This study evaluated the participation of EVs from L. (L.) infantum (Leish-EVs) in recognition of the humoral and cellular immune response of hosts with VL. Promastigotes were cultivated in 199 medium and, in the log phase of growth, they were centrifuged, washed, resus-pended in RPMI medium, and incubated for 2 to 24 h, at 25 °C or 37 °C to release Leish-EVs. This dynamic was evaluated using transmission (TEM) and scanning (SEM) electron microscopies, as well as nanoparticle tracking analysis (NTA). The results suggested that parasite penetration in mammal macrophages requires more Leish-EVs than those living in insect vectors, since promastigotes incubated at 37 °C released more Leish-EVs than those incubated at 25 °C. Infected THP-1 cells produced high EV concentration (THP-1 cells-EVs) when compared with those from the control group. The same results were obtained when THP-1 cells were treated with Leish-EVs or a crude Leishmania antigen. These data indicated that host-EV concentrations could be used to distinguish infected from uninfected hosts. THP-1 cells treated with Leish-EVs expressed more IL-12 than control THP-1 cells, but were unable to express IFN-γ. These same cells highly expressed IL-10, which inhibited TNF-α and IL-6. Equally, THP-1 cells treated with Leish-EVs up-expressed miR-21-5p and miR-146a-5p. In conclusion, THP-1 cells treated with Leish-EVs highly expressed miR-21-5p and miR-146a-5p and caused the dysregulation of IL-10. Indirectly, these results suggest that high expression of these miRNAs species is caused by Leish-EVs. Consequently, this molecular via can contribute to immunosuppression causing enhanced immunopathology in infected hosts.
RESUMEN
This retrospective cohort study analysed extracellular vesicles (EVs) and microRNAs (miRNAs) excreted in canine sera from dogs with canine visceral leishmaniasis (CanVL). A total of 56 canine sera were divided into Group I (28, from healthy dogs) and Group II (28, from the same dogs, but already with CanVL). CanVL was determined by clinical and laboratory diagnoses. Canine sera were ultra-centrifuged to recover EVs (Can-EVs). Analyses by transmission electron microscopy, nanoparticle tracking analysis (NTA), sodium dodecyl sulfate-poli-acrylammide gel eletroforesis (SDS-PAGE) and, Immunoblot confirmed the presence of (i) microvesicles/exosomes and (ii) the tetraspanins CD63 and CD9. EVs secreted by Leishmania (Leishmania) infantum-EVs were reactive against sera from dogs with CanVL (performed by ELISA and Immunoblot). NTA analyses exhibited that concentrations of Can-EVs from dogs with CanVL (7.78 × 1010 Can-EVs/mL) were higher (p < .0001) than the non-infected dogs (mean: 1.47 × 1010 Can-EVs/mL). These results suggested that concentrations of Can-EVs were able to distinguish dogs with CanVL from healthy dogs. The relative expressions of 11 miRNAs species (miR-21-5p, miR-146a-5p, miR-125b-5p, miR-144-3p, miR-194-5p, miR-346, miR-29c-3p, miR-155-5p, miR-24-3p, miR-181a-5p, and miR-9-5p) were estimated in purified miRNAs of 30 canine sera. Dogs with CanVL up-expressed miR-21-5p and miR-146a-5p when compared with healthy dogs. The other miRNA species were poorly or not expressed in canine sera. In conclusion, this study suggests that CanVL induces changes in size and concentration of Can-EVs, as well as, the up-expression of miR-21-5p and miR-146a-5p in infected dogs.
Asunto(s)
Exosomas , Vesículas Extracelulares , Leishmaniasis Visceral , MicroARNs , Perros , Animales , Leishmaniasis Visceral/veterinaria , Leishmaniasis Visceral/metabolismo , Estudios Retrospectivos , MicroARNs/genéticaRESUMEN
Brazilian porcupine poxvirus (BPoPV) is a new poxvirus recently described in porcupines (Coendou prehensilis) from Brazil. Herein, we described a free-ranging adult male Coendou (Sphiggurus) spinosus rescued after being found lethargic on the ground in a rural area. The animal presented crusty, edematous, and suppurative skin lesions on the face, tail, and perineum, and yellowish ocular secretion. The diagnosis was performed by histopathology, transmission electron microscopy (TEM), PCR, and sequencing. Microscopically, proliferative and necrotizing dermatitis, subacute, multifocal with ballooning degeneration, and eosinophilic intracytoplasmic viral inclusion bodies were observed. TEM confirmed large brick-shaped virions inside the keratinocyte cytoplasm, measuring about 200-280 × 120-180 nm. Partial fragment of intracellular mature virion membrane protein gene and putative metalloproteinase gene was successfully amplified and sequenced, and the strain herein denoted IAL/21 V-102 was classified as BPoPV, showing 99.4% of nucleotide identity to the reference strain UFU/USP001. Enrofloxacin 10% (10 mg/kg) was administered every 24 h through intramuscular injection for 10 days, dipyrone/metamizole (25 mg/kg) every 24 h orally (PO) for 3 days, 0.5 ml (mL) of thymomodulin every 24 h PO for 30 days, and each 48 h for another 15 days. The lesions were cleaned and debrided every 15 days. Seventy-five days after the beginning of the treatment, the cutaneous lesions regressed, the animal gained weight, and was clinically stable. After treatment, the skin biopsy showed only mild epidermal acanthosis, intra-cellular edema, and mild lymphoplasmacytic perivascular dermatitis. No viral particles were observed by TEM and no poxviral DNA was amplified by PCR. This study documents the first case of confirmed and treated BPoPV infection in a hairy dwarf porcupine. The implemented therapeutic plan eliminated the infection and improved the general state of the animal.
Asunto(s)
Dermatitis , Puercoespines , Infecciones por Poxviridae , Animales , Masculino , Piel , Microscopía Electrónica de TransmisiónRESUMEN
This study characterized extracellular vesicles (EVs) of sera from mice infected with Toxoplasma gondii or immunized with EVs derived T gondii. EVs were purified of sera from four groups (5 A/Sn mice/group). EV-IM: Mice immunized with T gondii-released EVs; ACT: mice in acute infection; CHR: mice in chronic infection; and NI: normal mice. EVs were purified by ultracentrifugation. Concentration of serum-derived EVs from NI group was smaller than EV-IM, ACT and CHR groups. Most of the EVs from ACT and CHR groups were microvesicles, and they were bigger than the NI group. The same results were shown by Transmission Electron Microscopy. The presence of exosomes was shown in immunoblotting by tetraspanin (CD63 and CD9) evidence. Splenocytes of EV-IM, CHR and NI groups were stimulated with T. gondii derived EVs. EV-IM and CHR groups up-expressed IFN-γ; TNF-α and IL-17, when compared with the NI group. IL-10 was up-expressed only in the EV-IM group. EV-IM, ACT and CHR groups expressed more miR-155-5p, miR-29c-3p and miR-125b-5p than the NI group. Host-T gondii interaction can occur, also, via EVs. miRNAs participate in the modulation of cellular immune response against T gondii. These data give subsidies to propose the differentiation between infect or noninfect hosts by concentration of EVs.
Asunto(s)
Exosomas , Vesículas Extracelulares , MicroARNs , Toxoplasma , Toxoplasmosis , Animales , RatonesRESUMEN
This study investigated the participation extracellular vesicles (EVs) in Toxoplasma gondii-host interaction. EVs of three T. gondii strains (RH, ME-49 and VEG) were purified by chromatography and ELISA. Results of "nanoparticle tracking analysis" and scanning electron microscopy showed that RH strain released more EVs than other strains. Images of transmission electron microscopy showed that in beginning of incubation (culture medium), EVs were inside of tachyzoites preparing to be released. After 24 hours, they were largely produced inside tachyzoites and were released through plasma membrane. The parasite burden of mice infected with RH strain plus EVs was increased and with early death of 1-2 days compared of those that received only parasites. EV proteins of ME-49 and VEG strains were poorly reactive to sera of infected patients in imunoblot. However, those from RH strain were reactive against sera of patients with cerebral toxoplasmosis. EVs stimulated murine splenocytes caused similar production of IFN-γ and IL-10 levels. RH strain derived EVs stimulated more TNF-α than those stimulated by other strains. T. gondii and infected hosts can express the same miRNAs (miR-155-5p, miR-125b-5p, miR-423-3p). In conclusion, T. gondii derived EVs promote host-parasite interactions, modulate host immune responses, carry virulent factors and cause an imbalance in cellular immune response.
Asunto(s)
Vesículas Extracelulares/metabolismo , Toxoplasma/citología , Animales , Humanos , Inmunidad Celular , Interleucina-10/sangre , Ratones , MicroARNs/sangre , Toxoplasma/inmunología , Toxoplasmosis Animal/inmunología , Factor de Necrosis Tumoral alfa/sangreRESUMEN
The study aim was to analyze whether microvesicles and exosomes, named extracellular vesicles (EVs), purified from Toxoplasma gondii are able to stimulate the protective immunity of experimental mice when administered, as challenge, a highly virulent strain. EVs excreted from T. gondii tachyzoites (RH strain) were purified by chromatography and used for immunization assays in inbred mouse groups (EV-IM). Chronic infected (CHR) and naive (NI) mice were used as control groups, since the immune response is well known. After immunizations, experimental groups were challenged with 100 tachyzoites. Next, parasitemias were determined by real-time PCR (qPCR), and survival levels were evaluated daily. The humoral response was analyzed by detection of IgM, IgG, IgG1 and IgG2a, and opsonization experiments. The cellular response was evaluated in situ by immunohistochemistry on IFN-γ, IL-10, TNF-α and IL-17 expression in cells of five organs (brain, heart, liver, spleen and skeletal muscles). EV immunization reduced parasitemia and increased the survival index in two mouse lineages (A/Sn and BALB/c) infected with a lethal T. gondii strain. EV-IM mice had higher IgG1 levels than IgM or IgG2a. IgGs purified from sera of EV-IM mice were able to opsonize tachyzoites (RH strain), and mice that received these parasites had lower parasitemias, and mortality was delayed 48 h, compared with the same results from those receiving parasites opsonized with IgG purified from NI mice. Brain and spleen cells from EV-IM mice more highly expressed IFN-γ, IL-10 and TNF-α. In conclusion, EV-immunization was capable of inducing immune protection, eliciting high production of IgG1, IFN-γ, IL-10 and TNF-α.
Asunto(s)
Vesículas Extracelulares , Toxoplasma , Animales , Inmunización , Ratones , Ratones Endogámicos BALB C , VacunaciónRESUMEN
AIM: This study analyzed microvesicles and exosomes, called as extracellular vesicles (EVs) excreted in serum and cerebrospinal fluid (CSF) from patients with cerebral or gestational toxoplasmosis. METHODS: Clinical samples from 83 individuals were divided into four groups. Group I, 20 sera from healthy individuals and pregnant women (seronegative for toxoplasmosis); group II, 21 sera from seropositive patients for toxoplasmosis (cerebral or gestational forms); group III, 26 CSF samples from patients with cerebral toxoplasmosis/HIV co-infection (CT/HIV) (seropositive for toxoplasmosis); and group IV, 16 CSF samples from seronegative patients for toxoplasmosis, but with HIV infection and other opportunistic infections (OI/HIV). Serum and CSF samples were ultracentrifuged to recover EVs. Next, vesicle size and concentration were characterized by Nanoparticle Tracking Analysis (NTA). RESULTS: Concentrations of serum-derived EVs from toxoplasmosis patients (mean: 2.4 x 1010 EVs/mL) were statically higher than of non-infected individuals (mean: 5.9 x 109 EVs/mL). Concentrations of CSF-derived EVs were almost similar in both groups. CT/HIV (mean: 2.9 x 109 EVs/mL) and OI/HIV (mean: 4.8 x 109 EVs/mL). Analyses by NTA confirmed that CSF-derived EVs and serum-derived EVs had size and shape similar to microvesicles and exosomes. The mean size of EVs was similar in serum and CSF. Thus, the concentration, and not size was able distinguish patients with toxoplasmosis than healthy individuals. Presence of exosomes was also confirmed by transmission electron microscopy and evidence of tetraspanins CD63 and CD9 in immunoblotting. Relative expressions of miR-146a-5p, miR-155-5p, miR-21-5p, miR-29c-3p and miR-125b-5p were estimated in exosomal miRNA extracted of EVs. Serum-derived EVs from group II (cerebral and gestational toxoplasmosis) up-expressed miR-125b-5p and miR-146a-5p. CSF-derived EVs from CT/HIV patients) up-expressed miR-155-5p and miR-21-5p and were unable to express miR-29c-3p. CONCLUSION: These data suggest the participation of EVs and exosomal miRNAs in unbalance of immune response as elevation of TNF-α, IL-6; and downregulation of IFN-γ in cerebral and gestational forms of toxoplasmosis.
Asunto(s)
Complicaciones Parasitarias del Embarazo/sangre , Complicaciones Parasitarias del Embarazo/líquido cefalorraquídeo , Toxoplasmosis Cerebral/sangre , Toxoplasmosis Cerebral/líquido cefalorraquídeo , Toxoplasmosis/complicaciones , Micropartículas Derivadas de Células/genética , Micropartículas Derivadas de Células/patología , Exosomas/genética , Exosomas/patología , Vesículas Extracelulares/genética , Vesículas Extracelulares/patología , Femenino , Expresión Génica , Infecciones por VIH/sangre , Infecciones por VIH/líquido cefalorraquídeo , Infecciones por VIH/complicaciones , Voluntarios Sanos , Humanos , MicroARNs/sangre , MicroARNs/líquido cefalorraquídeo , MicroARNs/genética , Microscopía Electrónica de Transmisión , Embarazo , Complicaciones Parasitarias del Embarazo/genética , Toxoplasmosis/sangre , Toxoplasmosis/líquido cefalorraquídeo , Toxoplasmosis Cerebral/genéticaRESUMEN
This study established a protocol to purify Toxoplasma gondii tachyzoite microvesicles and exosomes, called as extracellular vesicles (EVs). In addition, the investigations were conducted to determine the kinetic of EV release by tachyzoites and whether EV proteins are able to modulate the host immune response. The particle size and concentration released by tachyzoites in culture medium at different incubation-period were characterized by nanoparticle tracking analysis. Tachyzoites (1 × 106 ) released around 4.37 ± 0.81 × 108 EVs/mL/h, with size varying between 138.2 and 171.9 nm. EVs released into the medium were purified by gel-exclusion chromatography and screened by ELISA, using a pool of human positive sera for toxoplasmosis. EV-fractions contained high concentration of proteins, and EVs were analyzed by scanning and transmission electron microscopies. Tachyzoites released EVs into the culture medium throughout all membrane surface, and these vesicles contain small RNAs/miRNA. Pooled sera from chronically infected human or mice (infected with 2 different T. gondii strains) recognized distinct EV electrophoretic patterns in immunoblotting. T. gondii EVs significantly induced IL-10, TNF-α and iNOS in murine macrophages. In conclusion, this study shows that T. gondii secrete/excrete EVs (microvesicles and exosomes) contain miRNA and they were immunologically recognized by host immune response.
Asunto(s)
Vesículas Extracelulares/inmunología , Toxoplasma/inmunología , Toxoplasmosis/parasitología , Animales , Ensayo de Inmunoadsorción Enzimática , Exosomas/inmunología , Exosomas/parasitología , Vesículas Extracelulares/parasitología , Humanos , Immunoblotting , Interleucina-10/genética , Interleucina-10/inmunología , Macrófagos/inmunología , Macrófagos/parasitología , Ratones , Toxoplasma/genética , Toxoplasmosis/genética , Toxoplasmosis/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
A great variety of viruses which cause exanthema share other clinical manifestations, making the etiologic identification a very difficult task, relying exclusively on the clinical examination. Rubella virus (RV) infection during the early stages of pregnancy can lead to serious birth defects, known as congenital rubella syndrome (CRS). In the present report, we described the presence of Zika virus (ZIKV) particles in urine samples and also ZIKV isolation in SIRC cells from the urine of a patient in acute phase of suspected rubella disease. The 50-year-old unvaccinated woman living in Sao Paulo, Brazil, was admitted to the emergency room with fever, headache, rash, arthralgia and prostration. Urine samples were collected for virus isolation and RT-qPCR. SIRC and Vero cells were inoculated with urine samples during 7 days. RT-qPCR was performed using measles virus (MV) and RV primers and both were found to be negative. After this result, RT-qPCR was performed for parvovirus B19, herpes virus 6 and ZIKV. The urine sample and the isolate were positive by Real Time PCR for ZIKV and negative for all other viruses tested. The sequences isolated are from the Asiatic lineage.
Asunto(s)
Rubéola (Sarampión Alemán)/diagnóstico , Infección por el Virus Zika/orina , Virus Zika/aislamiento & purificación , Brasil , Células Cultivadas , Medios de Cultivo , Diagnóstico Diferencial , Femenino , Genotipo , Humanos , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Infección por el Virus Zika/diagnóstico , Infección por el Virus Zika/virologíaRESUMEN
The present study described a group A rotavirus (RVA) outbreak in an age-care facility in Brazil, using epidemiologic and molecular diagnostic methods. A descriptive clinical, epidemiological and environmental investigation was conducted. Stool samples were collected and screened for RVA, Norovirus (NoV), Enteric Adenovirus 40/41 (AdV 40/41) and Astrovirus (AstV) using ELISA, RT-PCR, qRT-PCR, electron microscopy and sequencing methods. Outbreak occurred during 26th-29th October, 2015; 28 individuals affected (22 residents; 6 staff). The attack rate was 25.9% and 8.5% among residents (median-age: 85.5 years) and staff (median-age: 28 years), respectively. Female staff was identified as the index case. RVA G2P[4] genotype was detected in 87.5% (7/8). Genetic analysis demonstrated that the outbreak involved one single strain, suggesting a common-source infection. RVA should be considered during outbreaks investigations in residential facilities, and raise the question if the current licensed RVA vaccines for children could also be helpful for the elderly.
Asunto(s)
Brotes de Enfermedades , Gastroenteritis , Salud Pública , Jubilación , Infecciones por Rotavirus , Rotavirus/clasificación , Rotavirus/genética , Adulto , Anciano de 80 o más Años , Brasil , Heces/virología , Femenino , Gastroenteritis/epidemiología , Gastroenteritis/virología , Genotipo , Humanos , Masculino , Norovirus/aislamiento & purificación , Factores de Riesgo , Rotavirus/aislamiento & purificación , Infecciones por Rotavirus/epidemiología , Infecciones por Rotavirus/virologíaRESUMEN
A expressão do oncogene E7 do HPV de alto risco é um dos responsáveis pela carcinogênese cervical, através da sua interferência sobre a via Cdk-Rb-E2F resultando na progressão do ciclo celular anormal e superexpressão das proteínas p16 e Ki-67. O objetivo deste estudo descritivo foi avaliar, em estudo de corte transversal, a positividade das expressões imunocitoquímicas das proteínas p16 e Ki-67 e sua distribuição celular, nas 199 amostras citológicas de base líquida (CBL) de casos com diagnóstico negativo e ASC-US com testesde HPV de alto risco positivo determinados em pelo menos um dos métodos de PCR e/ou HC2. Através da técnica imunocitoquímica, a expressão positiva da proteína p16 foi observada no citoplasma e núcleo enquanto que a proteína Ki- 67 foi apenas nuclear, quando comparada aos seus respectivos controles positivos. A proteína p16 foi positiva em 71/101 amostras de citologia negativa e em 12/16 amostras ASC-US, ambas associadas aos testes de HC2 e/ou PCR positivos para HPV de alto risco. A proteína Ki-67 foi positiva em 76/101 amostras de citologia negativa e em 12/16 amostras ASC-US, ambas também associadas aos testes de HC2 e/ou PCR positivos para HPV de alto risco. A proporção de positividade de Ki-67 aumentou em paralelo com o aumento do grau da positividade de p16 (p 0,001). A expressão da proteína p16 e proteína Ki-67 em amostras citológicas cérvico-vaginais colhidas em meio líquido, com HC2 e/ou PCR positivos para HPV de alto risco em casos com diagnóstico citológico negativo ou ASCUS detectou precocemente a presença de DNA-HPV através da imuno-expressão destas proteínas. Concluímos que estes resultados poderão ser utilizados em estratégias de programas de prevenção, e monitorar com eficiência e efetividade pacientes que poderão desenvolver lesões mais graves, através da detecção precoce das alterações do mecanismo de controle do ciclo celular.
Asunto(s)
Técnicas Citológicas , Frotis VaginalRESUMEN
A expressão do oncogene E7 do HPV de alto risco é um dos responsáveis pela carcinogênese cervical, através da sua interferência sobre a via Cdk-Rb-E2F resultando na progressão do ciclo celular anormal e superexpressão das proteínas p16 e Ki-67. O objetivo deste estudo descritivo foi avaliar, em estudo de corte transversal, a positividade das expressões imunocitoquímicas das proteínas p16 e Ki-67 e sua distribuição celular, nas 199 amostras citológicas de base líquida (CBL) de casos com diagnóstico negativo e ASC-US com testesde HPV de alto risco positivo determinados em pelo menos um dos métodos de PCR e/ou HC2. Através da técnica imunocitoquímica, a expressão positiva da proteína p16 foi observada no citoplasma e núcleo enquanto que a proteína Ki- 67 foi apenas nuclear, quando comparada aos seus respectivos controles positivos. A proteína p16 foi positiva em 71/101 amostras de citologia negativa e em 12/16 amostras ASC-US, ambas associadas aos testes de HC2 e/ou PCR positivos para HPV de alto risco. A proteína Ki-67 foi positiva em 76/101 amostras de citologia negativa e em 12/16 amostras ASC-US, ambas também associadas aos testes de HC2 e/ou PCR positivos para HPV de alto risco. A proporção de positividade de Ki-67 aumentou em paralelo com o aumento do grau da positividade de p16 (p 0,001). A expressão da proteína p16 e proteína Ki-67 em amostras citológicas cérvico-vaginais colhidas em meio líquido, com HC2 e/ou PCR positivos para HPV de alto risco em casos com diagnóstico citológico negativo ou ASCUS detectou precocemente a presença de DNA-HPV através da imuno-expressão destas proteínas. Concluímos que estes resultados poderão ser utilizados em estratégias de programas de prevenção, e monitorar com eficiência e efetividade pacientes que poderão desenvolver lesões mais graves, através da detecção precoce das alterações do mecanismo de controle do ciclo celular.