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The present study compares the ability of distinct immunological assays (chemiluminescence immunoassay-CLIA, western blot-WB and flow cytometry-FC-Simplex and Duplex) to detect anti-HTLV (human T-lymphotropic virus) antibodies in candidates for blood donations at the Amazonas State Blood Center (Brazil) between January 2018 and December 2022. Overall, 257,942 samples from candidates for blood donations were screened using CLIA, which led to 0.15% seropositivity for HTLV (409 samples). A total of 151 candidates for blood donations were enrolled for retesting with CLIA followed by additional testing using WB and FC-Simplex and Duplex analysis. Our results demonstrated that 62% (93/151), 20% (30/151) and 17% (26/151) of the samples presented positive results with retesting using CLIA, WB and FC-Simplex analysis, respectively. Additional analysis of the CLIA, WB and FC-Simplex results revealed an overall agreement of 56% for CLIA and WB (22 co-negative; 30 co-positive samples), 48% for CLIA and FC-Simplex (21 co-negative; 24 co-positive samples) and 80% for WB and FC-Simplex (51 co-negative; 23 co-positive samples). Considering the WB as the reference standard for the diagnosis of infection with HTLV-1/2, we observed that the CLIA results of ≤3.0 RLU and >10.0 RLU in the retest can be used define a negative or positive result, respectively, and could be used as new specific cut-off values. The overall agreement between WB and FC-Duplex for accomplishing the differential diagnosis was evaluated and demonstrated 100% correspondence for the diagnosis of HTLV-1 (15/15) and HTLV-2 (7/7). Our findings demonstrate that gaps in the diagnosis of infection with HTLV-1/2 could be overcome by the simultaneous use of distinct immunological assays during retesting of candidates for blood donations.
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Donantes de Sangre , Infecciones por HTLV-I , Infecciones por HTLV-II , Virus Linfotrópico T Tipo 1 Humano , Virus Linfotrópico T Tipo 2 Humano , Humanos , Brasil , Infecciones por HTLV-I/diagnóstico , Infecciones por HTLV-I/sangre , Infecciones por HTLV-I/inmunología , Infecciones por HTLV-II/diagnóstico , Infecciones por HTLV-II/sangre , Virus Linfotrópico T Tipo 1 Humano/inmunología , Virus Linfotrópico T Tipo 1 Humano/aislamiento & purificación , Virus Linfotrópico T Tipo 2 Humano/inmunología , Masculino , Femenino , Adulto , Diagnóstico Diferencial , Persona de Mediana Edad , Western Blotting , Citometría de Flujo/métodos , Donación de SangreRESUMEN
The present study aimed at evaluating the YF-specific neutralizing antibody profile besides a multiparametric analysis of phenotypic/functional features of cell-mediated response elicited by the 1/5 fractional dose of 17DD-YF vaccine, administered as a single subcutaneous injection. The immunological parameters of each volunteer was monitored at two time points, referred as: before (Day 0) [Non-Vaccinated, NV(D0)] and after vaccination (Day 30-45) [Primary Vaccinees, PV(D30-45)]. Data demonstrated high levels of neutralizing antibodies for PV(D30-45) leading to a seropositivity rate of 93%. A broad increase of systemic soluble mediators with a mixed profile was also observed for PV(D30-45), with IFN-γ and TNF-α presenting the highest baseline fold changes. Integrative network mapping of soluble mediators showed increased correlation numbers in PV(D30-45) as compared to NV(D0) (532vs398). Moreover, PV(D30-45) exhibited increased levels of Terminal Effector (CD45RA+CCR7-) CD4+ and CD8+ T-cells and Non-Classical memory B-cells (IgD+CD27+). Dimensionality reduction of Mass Cytometry data further support these findings. A polyfunctional cytokine profile (TNF-α/IFN-γ/IL-10/IL-17/IL-2) of T and B-cells was observed upon in vitro antigen recall. Mapping and kinetics timeline of soluble mediator signatures for PV(D30-45) further confirmed the polyfunctional profile upon long-term in vitro culture, mediated by increased levels of IFN-γ and TNF-α along with decreased production of IL-10. These findings suggest novel insights of correlates of protection elicited by the 1/5 fractional dose of 17DD-YF vaccine.
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Vacuna contra la Fiebre Amarilla , Fiebre Amarilla , Humanos , Adulto , Anticuerpos Neutralizantes , Interleucina-10 , Anticuerpos Antivirales , Factor de Necrosis Tumoral alfa , Linfocitos T CD8-positivos , VacunaciónRESUMEN
Chagas disease, caused by Trypanosoma cruzi, remains a serious public health problem worldwide. The parasite was subdivided into six distinct genetic groups, called "discrete typing units" (DTUs), from TcI to TcVI. Several studies have indicated that the heterogeneity of T. cruzi species directly affects the diversity of clinical manifestations of Chagas disease, control, diagnosis performance, and susceptibility to treatment. Thus, this review aims to describe how T. cruzi genetic diversity influences the biology of the parasite and/or clinical parameters in humans. Regarding the geographic dispersion of T. cruzi, evident differences were observed in the distribution of DTUs in distinct areas. For example, TcII is the main DTU detected in Brazilian patients from the central and southeastern regions, where there are also registers of TcVI as a secondary T. cruzi DTU. An important aspect observed in previous studies is that the genetic variability of T. cruzi can impact parasite infectivity, reproduction, and differentiation in the vectors. It has been proposed that T. cruzi DTU influences the host immune response and affects disease progression. Genetic aspects of the parasite play an important role in determining which host tissues will be infected, thus heavily influencing Chagas disease's pathogenesis. Several teams have investigated the correlation between T. cruzi DTU and the reactivation of Chagas disease. In agreement with these data, it is reasonable to suppose that the immunological condition of the patient, whether or not associated with the reactivation of the T. cruzi infection and the parasite strain, may have an important role in the pathogenesis of Chagas disease. In this context, understanding the genetics of T. cruzi and its biological and clinical implications will provide new knowledge that may contribute to additional strategies in the diagnosis and clinical outcome follow-up of patients with Chagas disease, in addition to the reactivation of immunocompromised patients infected with T. cruzi.
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Enfermedad de Chagas , Variación Genética , Trypanosoma cruzi , Trypanosoma cruzi/genética , Humanos , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/parasitología , Animales , Interacciones Huésped-Parásitos/genética , Interacciones Huésped-Parásitos/inmunologíaRESUMEN
The re-emergence of yellow fever (YF) urged new mass vaccination campaigns and, in 2017, the World Health Organization approved the use of the fractional dose (FD) of the YF vaccine due to stock shortage. In an observational cross-sectional investigation, we have assessed viremia, antibodies, soluble mediators and effector and memory T and B-cells induced by primary vaccination of volunteers with FD and standard dose (SD). Similar viremia and levels of antibodies and soluble markers were induced early after immunization. However, a faster decrease in the latter was observed after SD. The FD led to a sustained expansion of helper T-cells and an increased expression of activation markers on T-cells early after vaccination. Although with different kinetics, expansion of plasma cells was induced upon SD and FD immunization. Integrative analysis reveals that FD induces a more complex network involving follicular helper T cells and B-cells than SD. Our findings substantiate that FD can replace SD inducing robust correlates of protective immune response against YF.
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BACKGROUND: Leprosy is a highly neglected disease that is considered a serious public health problem in many countries. This illness is characterised by a variety of clinical and histopathological manifestations that are related to the patient immune response. OBJECTIVES: This work aimed evaluate the profile of circulating immune mediators in the plasma from patients classified clinically as paucibacillary (PB), multibacillary (MB), households contacts (HHC), type1 leprosy reaction (T1R), type2 leprosy reaction (T2R) and control individuals without medical history of leprosy (CTL). METHODS: To assessment of the plasma immune mediators was used multiplex microbeads immunoassay "Luminex". FINDINGS: The results showed that patients (PB) had a regulatory-biased profile, while MB revealed a pro-inflammatory trend of highly expressed biomarkers. HHC display conspicuously increased levels in the plasma of the chemokines (CCL2, CCL3, CCL4, CCL5 and CXCL8), pro-inflammatory cytokines (IFN-γ,TNF and IL-1ß), modulating cytokines (IL-9 and IL-1Ra) and growth factors (PDGF, G-CSF and IL-2). Interestingly, HHC displayed superior production of IFN-γ as compared to other leprosy groups, indicating a putative protective role for this cytokine during chronic Mycobacterium leprae exposure. MAIN CONCLUSION: Further investigations are currently underway to elucidate the potential of these mediators as biomarkers applicable to the diagnosis/prognosis of leprosy and also T1R and T2R leprosy reactions.
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Citocinas , Lepra , Humanos , Mycobacterium leprae , Quimiocinas , BiomarcadoresRESUMEN
BACKGROUND Leprosy is a highly neglected disease that is considered a serious public health problem in many countries. This illness is characterised by a variety of clinical and histopathological manifestations that are related to the patient immune response. OBJECTIVES This work aimed evaluate the profile of circulating immune mediators in the plasma from patients classified clinically as paucibacillary (PB), multibacillary (MB), households contacts (HHC), type1 leprosy reaction (T1R), type2 leprosy reaction (T2R) and control individuals without medical history of leprosy (CTL). METHODS To assessment of the plasma immune mediators was used multiplex microbeads immunoassay "Luminex". FINDINGS The results showed that patients (PB) had a regulatory-biased profile, while MB revealed a pro-inflammatory trend of highly expressed biomarkers. HHC display conspicuously increased levels in the plasma of the chemokines (CCL2, CCL3, CCL4, CCL5 and CXCL8), pro-inflammatory cytokines (IFN-γ,TNF and IL-1β), modulating cytokines (IL-9 and IL-1Ra) and growth factors (PDGF, G-CSF and IL-2). Interestingly, HHC displayed superior production of IFN-γ as compared to other leprosy groups, indicating a putative protective role for this cytokine during chronic Mycobacterium leprae exposure. MAIN CONCLUSION Further investigations are currently underway to elucidate the potential of these mediators as biomarkers applicable to the diagnosis/prognosis of leprosy and also T1R and T2R leprosy reactions.
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In the present study, the levels of serum and airway soluble chemokines, pro-inflammatory/regulatory cytokines, and growth factors were quantified in critically ill COVID-19 patients (total n=286) at distinct time points (D0, D2-6, D7, D8-13 and D>14-36) upon Intensive Care Unit (ICU) admission. Augmented levels of soluble mediators were observed in serum from COVID-19 patients who progress to death. An opposite profile was observed in tracheal aspirate samples, indicating that systemic and airway microenvironment diverge in their inflammatory milieu. While a bimodal distribution was observed in the serum samples, a unimodal peak around D7 was found for most soluble mediators in tracheal aspirate samples. Systems biology tools further demonstrated that COVID-19 display distinct eccentric soluble mediator networks as compared to controls, with opposite profiles in serum and tracheal aspirates. Regardless the systemic-compartmentalized microenvironment, networks from patients progressing to death were linked to a pro-inflammatory/growth factor-rich, highly integrated center. Conversely, patients evolving to discharge exhibited networks of weak central architecture, with lower number of neighborhood connections and clusters of pro-inflammatory and regulatory cytokines. All in all, this investigation with robust sample size landed a comprehensive snapshot of the systemic and local divergencies composed of distinct immune responses driven by SARS-CoV-2 early on severe COVID-19.
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COVID-19 , Enfermedad Crítica , Citocinas/metabolismo , Humanos , Cinética , SARS-CoV-2RESUMEN
A panoramic analysis of chemokines, pro-inflammatory/regulatory cytokines, and growth factors was performed in serum samples from patients with acute DENV infection (n=317) by a high-throughput microbeads array. Most soluble mediators analyzed were increased in DENV patients regardless of the DENV serotype. The substantial increase (≥10-fold) of CXCL10, IL-6, and IFN-γ, and decreased levels of PDGF (<0.4-fold) was universally identified in all DENV serotypes. Of note, increased levels of CXCL8, CCL4, and IL-12 (≥3-9-fold) were selectively observed in DENV2 as compared to DENV1 and DENV4. Heatmap and biomarker signatures further illustrated the massive release of soluble mediators observed in DENV patients, confirming the marked increase of several soluble mediators in DENV2. Integrative correlation matrices and networks showed that DENV infection exhibited higher connectivity among soluble mediators. Of note, DENV2 displayed a more complex network, with higher connectivity involving a higher number of soluble mediators. The timeline kinetics (Day 0-1, D2, D3, D4-6) analysis additionally demonstrated differences among DENV serotypes. While DENV1 triggers a progressive increase of soluble mediators towards D3 and with a decline at D4-6, DENV2 and DENV4 develop with a progressive increase towards D4-6 with an early plateau observed in DENV4. Overall, our results provided a comprehensive overview of the immune response elicited by DENV infection, revealing that infection with distinct DENV serotypes causes distinct profiles, rhythms, and dynamic network connectivity of soluble mediators. Altogether, these findings may provide novel insights to understand the pathogenesis of acute infection with distinct DENV serotypes.
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Virus del Dengue , Dengue , Anticuerpos Antivirales , Humanos , Serogrupo , SueroRESUMEN
The aim of this study was to improve the quality of frozen-thawed equine sperm by the addition of caffeine to it. Semen from nine stallions was frozen and different concentrations of caffeine (3, 5 and 7.5 mM) were added to frozen-thawed semen. The sperm kinetic parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome reacted sperm were evaluated with a computer-assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite and hydroperoxide concentrations of frozen-thawed semen were measured using spectrophotometry. Sperm fertility was evaluated by artificial insemination (AI) of 16 mares with thawed ejaculates (control and 5 mM caffeine-treated groups). Compared to that in the control, the addition of 5 mM caffeine induced an increase in sperm motility (38.9 ± 2.8 versus 32.6 ± 3.4%), and a decrease in nitrite concentration (11.4 ± 2.1 versus 12.8 ± 2.9 µM/µg protein, p < .05). Moreover, the pregnancy rate from AI in the caffeine group was significantly higher (62.5%) than that in the control group (12.5%). These data suggest that caffeine reduced the nitrite concentration and enhanced sperm motility in thawed equine sperm, thus increasing the fertility rate in mares inseminated with caffeine-treated equine semen.
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Preservación de Semen , Motilidad Espermática , Animales , Cafeína/farmacología , Criopreservación , Femenino , Caballos , Humanos , Inseminación Artificial/veterinaria , Masculino , Nitritos , Embarazo , Preservación de Semen/veterinaria , EspermatozoidesRESUMEN
Papillomaviruses are among the most widespread animal viruses, with many hosts harbouring multiple virus types. The present study aimed to evaluate the haematological and immunophenotypic profile of cattle infected with bovine papillomavirus (BPV). Blood samples were collected from 10 animals with clinical cutaneous BPV and without clinical papillomatosis (control). Haematological analysis demonstrated a significant reduction in haemoglobin and haematocrit for BPV-infected animals. The results also showed an increase of natural killer cells and a decrease of γδ+ T-cells and the CD4+/CD8+ ratio for the BPV group when compared to the control group. The infection was also found to stimulate a pro-inflammatory profile with the participation of CD8+T cells producing elevated IFN-γ and IL-17. These findings, although preliminary, provide a better understanding of the immune response of cattle infected with BPV.
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Papillomavirus Bovino 1/aislamiento & purificación , Enfermedades de los Bovinos/inmunología , Infecciones por Papillomavirus/veterinaria , Animales , Papillomavirus Bovino 1/patogenicidad , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Estudios de Casos y Controles , Bovinos , Enfermedades de los Bovinos/sangre , Enfermedades de los Bovinos/virología , Femenino , Inmunofenotipificación/veterinaria , Células Asesinas Naturales , Masculino , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/virologíaRESUMEN
Macrophages are critical mediators of the inflammatory process, playing a relevant role in the pathogenesis of Salmonella Typhimurium. The protocols for isolation, culture, and differentiation of monocytes into macrophages and their interaction with Salmonella are well established in humans and murine models, but little information is available in swine. The aims of this study were to establish an efficient protocol for macrophage culture and to evaluate the interaction of the invA mutant strain and the wild type (WT) Salmonella Typhimurium with porcine macrophages. Peripheral blood monocyte-derived macrophages from pigs were obtained, separated by density-gradient centrifugation, and cultured in Teflon vials for 10 days. After the differentiation period, cultures consisted of 92.4% CD14+ cells. In addition, these cells showed phagocytic ability, demonstrated by the presence of the same amount of WT and invA mutant Salmonella Typhimurium 1 h after interaction with macrophages. The early cytotoxic effect was Salmonella pathogenicity island (SPI)-[1]dependent, in which log-phase WT strains were more efficient (p < 0.01) than the invA mutant strain at inducing the death of macrophages.
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Macrófagos/microbiología , Salmonelosis Animal/patología , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/patogenicidad , Animales , Proteínas Bacterianas/genética , Células Cultivadas , Femenino , Islas Genómicas/genética , Salmonella typhimurium/clasificación , Salmonella typhimurium/genética , Porcinos , VirulenciaRESUMEN
In this investigation, machine-enhanced techniques were applied to bring about scientific insights to identify a minimum set of phenotypic/functional memory-related biomarkers for post-vaccination follow-up upon yellow fever (YF) vaccination. For this purpose, memory status of circulating T-cells (Naïve/early-effector/Central-Memory/Effector-Memory) and B-cells (Naïve/non-Classical-Memory/Classical-Memory) along with the cytokine profile (IFN/TNF/IL-5/IL-10) were monitored before-NV(day0) and at distinct time-points after 17DD-YF primary vaccination-PV(day30-45); PV(year1-9) and PV(year10-11). A set of biomarkers (eEfCD4; EMCD4; CMCD19; EMCD8; IFNCD4; IL-5CD8; TNFCD4; IFNCD8; TNFCD8; IL-5CD19; IL-5CD4) were observed in PV(day30-45), but not in NV(day0), with most of them still observed in PV(year1-9). Deficiencies of phenotypic/functional biomarkers were observed in NV(day0), while total lack of memory-related attributes was observed in PV(year10-11), regardless of the age at primary vaccination. Venn-diagram analysis pre-selected 10 attributes (eEfCD4, EMCD4, CMCD19, EMCD8, IFNCD4, IL-5CD8, TNFCD4, IFNCD8, TNFCD8 and IL-5CD4), of which the overall mean presented moderate accuracy to discriminate PV(day30-45)&PV(year1-9) from NV(day0)&PV(year10-11). Multi-parameter approaches and decision-tree algorithms defined the EMCD8 and IL-5CD4 attributes as the top-two predictors with moderated performance. Together with the PRNT titers, the top-two biomarkers led to a resultant memory status observed in 80% and 51% of volunteers in PV(day30-45) and PV(year1-9), contrasting with 0% and 29% found in NV(day0) and PV(year10-11), respectively. The deficiency of memory-related attributes observed at PV(year10-11) underscores the conspicuous time-dependent decrease of resultant memory following17DD-YF primary vaccination that could be useful to monitor potential correlates of protection in areas under risk of YF transmission.
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Anticuerpos Antivirales/sangre , Biomarcadores/sangre , Vacunación , Vacuna contra la Fiebre Amarilla/inmunología , Fiebre Amarilla/prevención & control , Virus de la Fiebre Amarilla/inmunología , Adolescente , Adulto , Anciano , Linfocitos B/inmunología , Linfocitos T CD8-positivos/inmunología , Citocinas/sangre , Humanos , Masculino , Persona de Mediana Edad , Factores de Tiempo , Fiebre Amarilla/inmunología , Fiebre Amarilla/virología , Adulto JovenRESUMEN
Vaccination against Anaplasma marginale has been considered an important control strategy for bovine anaplasmosis. Recently, mice immunized with rMSP1 a linked to carbon nanotubes (MWNT) showed significant immune responses, generating a new possibility for use of an inactivated vaccine. The objective of this study was to investigate the cellular and humoral responses in calves immunized with MWNT+rMSP1a , associated with inactivated vaccine of A. marginale produced in vitro, and evaluate the toxic effects of the MWNT on renal and hepatic function. rMSP1a was covalently linked to MWNT. Inactivated vaccine (AmUFMG2) was produced by cultivating A. marginale in IDE8 cells. Twenty-four Holstein calves were divided (four groups) and immunized subcutaneously with PBS and non-carboxylated MWNT (control, G1), AmUFMG2 (G2), MWNT+rMSP1a (G3), and AmUFMG2 with MWNT+rMSP1a (G4). Blood samples were collected for total leukocyte counts, biochemical profiling and evaluation of the cellular and humoral response. Immunization with MWNT+rMSP1a induced increase in the total number of leukocytes, NK cells, in the lymphocyte populations and higher levels of antibodies compared to calves immunized only with AmUFMG2. Furthermore, MWNT did not induce changes in the biochemical profile. These data indicate that MWNT+rMSP1a were able to induce the immune responses more efficiently than AmUFMG2 alone, without generating toxicity.(AU)
Vacinação contra Anaplasma marginale tem sido considerada uma importante estratégia de controle da anaplasmose bovina. Recentemente, camundongos imunizados com rMSP1a funcionalizada à nanotubos de carbono (MWNT) apresentaram resposta imune significante, gerando nova possibilidade para o uso da vacina inativada. O objetivo desse estudo foi investigar a resposta celular e humoral em bezerros imunizados com MWNT+rMSP1a, associado com a vacina inativada de A. marginale produzida in vitro, e avaliar os efeitos tóxicos dos MWNT nas funções hepática e renal. rMSP1 a foi ligada covalentemente aos MWNT. Vacina inativada (AmUFMG2) foi produzida através do cultivo de A. marginale em células IDE8. Vinte e quatro bezerros Holandeses foram divididos (quatro grupos) e imunizados subcutaneamente com: PBS e MWNT não-carboxilados (controle, G1), AmUFMG2 (G2), MWNT+rMSP1 a (G3), e AmUFMG2 com MWNT+rMSP1a (G4). Amostras de sangue foram coletadas para contagem de leucócitos, perfil bioquímico e avaliação da resposta celular e humoral. Imunização com MWNT+rMSP1a induziu aumento dos leucócitos totais, células NK, na população de linfócitos e altos níveis de anticorpos comparado com animais imunizados apenas com AmUFMG2. Além disso, MWNT não induziu alterações no perfil bioquímico. Esses dados indicam que MWNT+rMSP1a foram capazes de induzir eficientemente a resposta imune comparado com AmUFMG2 sozinho, sem gerar toxicidade.(AU)
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Animales , Bovinos , Anaplasma marginale , Anaplasmosis/inmunología , Proteína 1 de Superficie de Merozoito , Nanotubos de Carbono , Inmunidad Humoral , Vacunas de Productos InactivadosRESUMEN
Abstract Vaccination against Anaplasma marginale has been considered an important control strategy for bovine anaplasmosis. Recently, mice immunized with rMSP1 a linked to carbon nanotubes (MWNT) showed significant immune responses, generating a new possibility for use of an inactivated vaccine. The objective of this study was to investigate the cellular and humoral responses in calves immunized with MWNT+rMSP1a , associated with inactivated vaccine of A. marginale produced in vitro, and evaluate the toxic effects of the MWNT on renal and hepatic function. rMSP1a was covalently linked to MWNT. Inactivated vaccine (AmUFMG2) was produced by cultivating A. marginale in IDE8 cells. Twenty-four Holstein calves were divided (four groups) and immunized subcutaneously with PBS and non-carboxylated MWNT (control, G1), AmUFMG2 (G2), MWNT+rMSP1a (G3), and AmUFMG2 with MWNT+rMSP1a (G4). Blood samples were collected for total leukocyte counts, biochemical profiling and evaluation of the cellular and humoral response. Immunization with MWNT+rMSP1a induced increase in the total number of leukocytes, NK cells, in the lymphocyte populations and higher levels of antibodies compared to calves immunized only with AmUFMG2. Furthermore, MWNT did not induce changes in the biochemical profile. These data indicate that MWNT+rMSP1a were able to induce the immune responses more efficiently than AmUFMG2 alone, without generating toxicity.
Resumo Vacinação contra Anaplasma marginale tem sido considerada uma importante estratégia de controle da anaplasmose bovina. Recentemente, camundongos imunizados com rMSP1a funcionalizada à nanotubos de carbono (MWNT) apresentaram resposta imune significante, gerando nova possibilidade para o uso da vacina inativada. O objetivo desse estudo foi investigar a resposta celular e humoral em bezerros imunizados com MWNT+rMSP1a, associado com a vacina inativada de A. marginale produzida in vitro, e avaliar os efeitos tóxicos dos MWNT nas funções hepática e renal. rMSP1 a foi ligada covalentemente aos MWNT. Vacina inativada (AmUFMG2) foi produzida através do cultivo de A. marginale em células IDE8. Vinte e quatro bezerros Holandeses foram divididos (quatro grupos) e imunizados subcutaneamente com: PBS e MWNT não-carboxilados (controle, G1), AmUFMG2 (G2), MWNT+rMSP1 a (G3), e AmUFMG2 com MWNT+rMSP1a (G4). Amostras de sangue foram coletadas para contagem de leucócitos, perfil bioquímico e avaliação da resposta celular e humoral. Imunização com MWNT+rMSP1a induziu aumento dos leucócitos totais, células NK, na população de linfócitos e altos níveis de anticorpos comparado com animais imunizados apenas com AmUFMG2. Além disso, MWNT não induziu alterações no perfil bioquímico. Esses dados indicam que MWNT+rMSP1a foram capazes de induzir eficientemente a resposta imune comparado com AmUFMG2 sozinho, sem gerar toxicidade.
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Animales , Bovinos , Portadores de Fármacos , Vacunas Bacterianas/inmunología , Enfermedades de los Bovinos/microbiología , Enfermedades de los Bovinos/prevención & control , Nanotubos de Carbono , Anaplasma marginale/inmunología , Inmunogenicidad Vacunal , Anaplasmosis/prevención & control , Inmunidad Humoral , Inmunidad CelularRESUMEN
Vaccination against Anaplasma marginale has been considered an important control strategy for bovine anaplasmosis. Recently, mice immunized with rMSP1 a linked to carbon nanotubes (MWNT) showed significant immune responses, generating a new possibility for use of an inactivated vaccine. The objective of this study was to investigate the cellular and humoral responses in calves immunized with MWNT+rMSP1a , associated with inactivated vaccine of A. marginale produced in vitro, and evaluate the toxic effects of the MWNT on renal and hepatic function. rMSP1a was covalently linked to MWNT. Inactivated vaccine (AmUFMG2) was produced by cultivating A. marginale in IDE8 cells. Twenty-four Holstein calves were divided (four groups) and immunized subcutaneously with PBS and non-carboxylated MWNT (control, G1), AmUFMG2 (G2), MWNT+rMSP1a (G3), and AmUFMG2 with MWNT+rMSP1a (G4). Blood samples were collected for total leukocyte counts, biochemical profiling and evaluation of the cellular and humoral response. Immunization with MWNT+rMSP1a induced increase in the total number of leukocytes, NK cells, in the lymphocyte populations and higher levels of antibodies compared to calves immunized only with AmUFMG2. Furthermore, MWNT did not induce changes in the biochemical profile. These data indicate that MWNT+rMSP1a were able to induce the immune responses more efficiently than AmUFMG2 alone, without generating toxicity.
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Anaplasma marginale/inmunología , Anaplasmosis/prevención & control , Vacunas Bacterianas/inmunología , Enfermedades de los Bovinos/microbiología , Enfermedades de los Bovinos/prevención & control , Portadores de Fármacos , Inmunogenicidad Vacunal , Nanotubos de Carbono , Animales , Bovinos , Inmunidad Celular , Inmunidad HumoralRESUMEN
Resveratrol (RSV) is anti-inflammatory and neuroprotective, cross the blood-brain barrier (BBB) and has a safe profile. Besides, RSV modulates the expression of some miRNAs related to neurological disorders. Thus, we hypothesized that RSV can be neuroprotective in pneumococcal meningitis by modulating the global microRNA expression profile (miRNome). Eleven-day old rats were intracysternally infected with S. pneumoniae (~ 2 × 106 c.f.u.) and were orally administered with RSV (50 mg/kg) or vehicle in pre-treatment (before infection) or post-treatment schedules (3 and 18 h p.i.). At 24 h p.i., animals were euthanized and apoptotic cells were counted in the hippocampal dentate gyrus of the right brain hemispheres. The hippocampi from left hemispheres were used for cytokines and chemokines multiplex assay and miRNome profiling with TaqMan OpenArray Rodent MicroRNA. Infected rats treated with RSV had lower apoptotic scores and IL-1ß, CCL2, and CCL3 levels when compared to the infected group receiving placebo. Seven miRNAs were down regulated, and 18 were up regulated by pneumococcal acute meningitis. Thirty-seven miRNAs were down regulated, and three were up regulated (hsa-miR-15b-5p, hsa-miR-25-3p, hsa-miR-125b-5p) by the interaction between meningitis and RSV. Pathway enriched analysis revealed that meningitis and RSV modulate the expression of miRNAs targeting critical pathways related to the pathophysiology of bacterial meningitis. Nevertheless, hsa-miR-25-3p and hsa-miR-125b-5p target the transcription factor TEF-1, for which there are binding sites in Il-1ß, Ccl2, and Ccl3 genes. RSV is anti-inflammatory and neuroprotective in an infant rat model of pneumococcal meningitis and these positive effects involve the modulation of the hippocampal miRNome.
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Antiinflamatorios/uso terapéutico , Hipocampo/metabolismo , Meningitis Neumocócica/tratamiento farmacológico , Meningitis Neumocócica/genética , MicroARNs/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Resveratrol/uso terapéutico , Animales , Animales Recién Nacidos , Antiinflamatorios/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Quimiocina CCL2/metabolismo , Quimiocina CCL3/metabolismo , Análisis por Conglomerados , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Interleucina-1beta/metabolismo , Meningitis Neumocócica/patología , Meningitis Neumocócica/fisiopatología , MicroARNs/genética , Modelos Biológicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/farmacología , Ratas Wistar , Resveratrol/farmacología , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismoRESUMEN
Eosinophils are multifunctional cells that have cytotoxic proinflammatory activities and stimulate CD4+ T-cells in experimental models of allergy and parasitic infections. Eosinophils, when exposed to antigens, are activated, expressing the CD38/CD69 molecules and exhibited increased expression of major histocompatibility complex (MHC-II), CD80 and CD86, suggesting they play a role upon Toxocara canis antigen stimulation. In the present study, we evaluated the profile of eosinophils using conventional and image flow cytometry upon experimental T. canis infection. T. canis antigens induced a robust activation on this subset, contributing to the immune responses elicited in the experimental model for T. canis-associated visceral larva migrans syndrome. Data analysis demonstrated that, during murine T. canis infection, eosinophils from peripheral blood, spleen, and bone marrow presented upregulated expression of CD69/MHC-II/CD80/CD86. As opposed to splenic and bone marrow eosinophils, circulating eosinophils had increased expression of activation markers upon T. canis infection. The enhanced connectivity between eosinophils and T-cells in T. canis-infected mice in all three compartments (peripheral blood, spleen, and bone marrow) also supports the hypothesis that eosinophils may adopt a role during T. canis infection. Moreover, in vitro T. canis antigen stimulation resulted in activation and upregulation of co-stimulatory-related molecules by bone marrow-derived eosinophils. Our findings are evidence of activation and upregulation of important activation and co-stimulatory-related molecules in eosinophils and suggest a reshape of activation hierarchy toward eosinophils during experimental T. canis infection.
Asunto(s)
Eosinófilos/inmunología , Fenotipo , Toxocara canis/inmunología , Toxocariasis/inmunología , Toxocariasis/parasitología , Animales , Antígenos Helmínticos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Biomarcadores , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Eosinófilos/metabolismo , Perfilación de la Expresión Génica/métodos , Inmunofenotipificación , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Monocitos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Toxocariasis/genética , Toxocariasis/metabolismoRESUMEN
Background: Over the years, the most commonly used extenders for semen cryopreservation contain egg yolk as cryoprotectant. However, more recent studies have used the low density lipoproteins, extract of hen egg yolk which is responsible for the cryoprotective effect. Nevertheless, little was known about its required minimum concentration as well as its interaction with other extra cellular cryoprotectants, like skimmed milk. The present study aimed at investigating the effect of replacing whole egg yolk by adding low density lipoproteins at low concentrations, in TES-Tris-skim milk based extender, on the post-thaw quality of buffalo bull sperm.Materials, Methods & Results: Eighteen ejaculates were collected from six buffalo bulls and diluted with TES-Tris-skim milk based extender containing LDL, extracted from hen egg yolks, at the concentrations of 2%, 4%, 8% and 14%, against a control extender containing 20% fresh egg yolk. After semen collection, analyses of subjective motility, vigor, force tourbillon, sperm concentration (Neubauer chamber) and sperm morphology (phase contrast microscopy) were performed. The diluted semen was packaged in 0.25 mL straws, and cooling was performed on computerized machine (TK 4000®), using a cooling rate of -0.25°C/min to 5°C. Semen was kept in balance at 5°C for 4 h. The straws were frozen in an ice chest, kept at 5 cm from the surface of liquid nitrogen for 20 min and then immersed in liquid nitrogen. The samples were kept in cryogenic container until thawing. Post-thaw kinetic parameters during incubation at 37°C (CASA), sperm membrane integrity (SYBR-14/PI), membrane functionality (hypo-osmotic swelling test) and DNA fragmentation (%DFI - SCSA) were evaluated after thawing. Immediately post-thaw, total motility was higher in the control (56.53 ± 9.73) than in the tested extenders; however, after 30 min the difference was no longer detected.[ ]
Asunto(s)
Masculino , Animales , Bovinos , Criopreservación/métodos , Criopreservación/veterinaria , Lipoproteínas LDL/análisis , Preservación de Semen/métodos , Preservación de Semen/veterinaria , Citometría de Flujo/veterinaria , Motilidad Espermática , Proteínas del Huevo/análisisRESUMEN
Background: Over the years, the most commonly used extenders for semen cryopreservation contain egg yolk as cryoprotectant. However, more recent studies have used the low density lipoproteins, extract of hen egg yolk which is responsible for the cryoprotective effect. Nevertheless, little was known about its required minimum concentration as well as its interaction with other extra cellular cryoprotectants, like skimmed milk. The present study aimed at investigating the effect of replacing whole egg yolk by adding low density lipoproteins at low concentrations, in TES-Tris-skim milk based extender, on the post-thaw quality of buffalo bull sperm.Materials, Methods & Results: Eighteen ejaculates were collected from six buffalo bulls and diluted with TES-Tris-skim milk based extender containing LDL, extracted from hen egg yolks, at the concentrations of 2%, 4%, 8% and 14%, against a control extender containing 20% fresh egg yolk. After semen collection, analyses of subjective motility, vigor, force tourbillon, sperm concentration (Neubauer chamber) and sperm morphology (phase contrast microscopy) were performed. The diluted semen was packaged in 0.25 mL straws, and cooling was performed on computerized machine (TK 4000®), using a cooling rate of -0.25°C/min to 5°C. Semen was kept in balance at 5°C for 4 h. The straws were frozen in an ice chest, kept at 5 cm from the surface of liquid nitrogen for 20 min and then immersed in liquid nitrogen. The samples were kept in cryogenic container until thawing. Post-thaw kinetic parameters during incubation at 37°C (CASA), sperm membrane integrity (SYBR-14/PI), membrane functionality (hypo-osmotic swelling test) and DNA fragmentation (%DFI - SCSA) were evaluated after thawing. Immediately post-thaw, total motility was higher in the control (56.53 ± 9.73) than in the tested extenders; however, after 30 min the difference was no longer detected.[ ](AU)
Asunto(s)
Animales , Masculino , Bovinos , Criopreservación/métodos , Criopreservación/veterinaria , Lipoproteínas LDL/análisis , Preservación de Semen/métodos , Preservación de Semen/veterinaria , Proteínas del Huevo/análisis , Citometría de Flujo/veterinaria , Motilidad EspermáticaRESUMEN
The study aimed at identifying biomarkers of immune response elicited by non-adjuvanted-(NAV) and adjuvanted-(AV) H1N1(pdm09) vaccines. The results showed that despite both vaccines elicited similar levels of anti-H1N1 antibodies at day30 after vaccination, higher reactivity was observed in AV at day180. While AV induced early changes in cell-surface molecules on monocytes, CD4+, CD8+ T-cells and B-cells, NAV triggered minor changes, starting later on at day3. Furthermore, AV induced a late and persistent increase in TLR gene expression after day3, except for tlr4, while NAV displayed earlier but transient tlr3/4/7/9 up-regulation. Contrasting with NAV, prominent chemokine gene expression (cxcl8,cxcl9,ccl5) and a broad spectrum up-regulation of plasmatic biomarkers (CXCL8,IL-6,IL-1ß,IL-12,IL-10) was evident in AV, which showed a major involvement of TNF and IL-10. Similarly, AV induced a robust IL-10-modulated proinflammatory storm, with early and persistent involvement of TNF-α/IL-12/IFN-γ axis derived from NK-cells, CD4+ and CD8+ T-cells along with promiscuous production of IL-4/IL-5/IL-13. Conversely, NAV promotes a concise and restricted intracytoplasmic chemokine/cytokine response, essentially mediated by TNF-α and IL-4, with late IL-10 production by CD8+ T-cells. Systems biology approach underscored that AV guided the formation of an imbricate network characterized by a progressive increase in the number of neighborhood connections amongst innate and adaptive immunity. In AV, the early cross-talk between innate and adaptive immunity, followed by the triad NK/CD4+/CD8+ T-cells at day3, sponsored a later/robust biomarker network. These findings indicate the relevance of adjuvanted vaccination to orchestrate broad, balanced and multifactorial cellular immune events that lead ultimately to a stronger H1N1 humoral immunity.