RESUMO
Vaccination for the control of bovine tuberculosis (bTB) in cattle is not currently used within any international control program, and is illegal within the EU. Candidate vaccines, based upon Mycobacterium bovis bacillus Calmette-Guérin (BCG) all interfere with the action of the tuberculin skin test, which is used to determine if animals, herds and countries are officially bTB-free. New diagnostic tests that Differentiate Infected from Vaccinated Animals (DIVA) offer the potential to introduce vaccination within existing eradication programs. We use within-herd transmission models estimated from historical data from Great Britain (GB) to explore the feasibility of such supplemental use of vaccination. The economic impact of bovine Tuberculosis for farmers is dominated by the costs associated with testing, and associated restrictions on animal movements. Farmers' willingness to adopt vaccination will require vaccination to not only reduce the burden of infection, but also the risk of restrictions being imposed. We find that, under the intensive sequence of testing in GB, it is the specificity of the DIVA test, rather than the sensitivity, that is the greatest barrier to see a herd level benefit of vaccination. The potential negative effects of vaccination could be mitigated through relaxation of testing. However, this could potentially increase the hidden burden of infection within Officially TB Free herds. Using our models, we explore the range of the DIVA test characteristics necessary to see a protective herd level benefit of vaccination. We estimate that a DIVA specificity of at least 99.85% and sensitivity of >40% is required to see a protective benefit of vaccination with no increase in the risk of missed infection. Data from experimentally infected animals suggest that this target specificity could be achieved in vaccinates using a cocktail of three DIVA antigens while maintaining a sensitivity of 73.3% (95%CI: 61.9, 82.9%) relative to post-mortem detection.
Assuntos
Modelos Imunológicos , Mycobacterium bovis/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose Bovina , Vacinação/estatística & dados numéricos , Criação de Animais Domésticos/legislação & jurisprudência , Animais , Bovinos , Biologia Computacional , Imunidade Coletiva , Legislação Veterinária , Vacinas contra a Tuberculose/administração & dosagem , Tuberculose Bovina/epidemiologia , Tuberculose Bovina/imunologia , Tuberculose Bovina/prevenção & controle , Reino Unido , Vacinação/veterináriaRESUMO
During adaptive immunity to pathogens, dendritic cells (DCs) capture, kill, process, and present microbial Ags to T cells. Ag presentation is accompanied by DC maturation driven by appropriate costimulatory signals. However, current understanding of the intricate regulation of these processes remains limited. Cryptococcus gattii, an emerging fungal pathogen in the Pacific Northwest of Canada and the United States, fails to stimulate an effective immune response in otherwise healthy hosts leading to morbidity or death. Because immunity to fungal pathogens requires intact cell-mediated immunity initiated by DCs, we asked whether C. gattii causes dysregulation of DC functions. C. gattii was efficiently bound and internalized by human monocyte-derived DCs, trafficked to late phagolysosomes, and killed. Yet, even with this degree of DC activation, the organism evaded pathways leading to DC maturation. Despite the ability to recognize and kill C. gattii, immature DCs failed to mature; there was no increased expression of MHC class II, CD86, CD83, CD80, and CCR7, or decrease of CD11c and CD32, which resulted in suboptimal T cell responses. Remarkably, no increase in TNF-α was observed in the presence of C. gattii. However, addition of recombinant TNF-α or stimulation that led to TNF-α production restored DC maturation and restored T cell responses. Thus, despite early killing, C. gattii evades DC maturation, providing a potential explanation for its ability to infect immunocompetent individuals. We have also established that DCs retain the ability to recognize and kill C. gattii without triggering TNF-α, suggesting independent or divergent activation pathways among essential DC functions.
Assuntos
Imunidade Adaptativa/imunologia , Diferenciação Celular/imunologia , Criptococose/imunologia , Criptococose/patologia , Cryptococcus gattii/imunologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Evasão da Resposta Imune/imunologia , Células Cultivadas , Criptococose/microbiologia , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus gattii/patogenicidade , Células Dendríticas/microbiologia , Humanos , Imunofenotipagem , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Multicomponent, community-based programs aiming to improve health behaviors in youth are needed but can be challenging to implement. Research is needed to better understand the factors that facilitate and inhibit effective implementation of these programs especially for youth at increased risk of health disparities. This study aimed to identify and explore the implementation determinants and outcomes of a multicomponent health intervention conducted from 2021 to 2022 for middle school students living in underserved communities in Philadelphia, PA, USA. Mixed methods approaches, including self-report surveys and semi-structured qualitative interviews, were administered to 18 members of the implementation staff at the end of the program, including coaches (n = 7), assistant coaches (n = 2), school champions (n = 6), administrative leaders (n = 2), and a school district administrator (n = 1). Survey and interview questions were guided by the Consolidated Framework for Implementation Research (CFIR), and interviews were thematically coded following transcription based on 26 CFIR constructs. Innovation source, evidence strength and quality, cosmopolitanism, and the personal attributes of individuals were key constructs associated with implementation effectiveness. Data revealed three multidimensional themes that highlighted broader challenges influencing implementation: (i) broad consensus, different interpretations, (ii) staffing challenges, and (iii) continuity is key. The need for the program was clearly recognized and overall belief in the purpose of the intervention was strong among key program staff and administration. However, issues including limited engagement with training, staffing turnover, and the rotational programming design hindered implementation. Future projects aiming to implement multicomponent after-school time interventions must ensure a consistent vision among partners and continuity in program delivery.
There is a pressing need for effective interventions to improve health behaviors in youth and schools are ideal settings for this work. The needs are even greater in underresourced school settings where youth have limited access to sports and physical activity. However, implementing multicomponent sports-based programs can be challenging and this leads to inconsistencies in program delivery and effectiveness. Therefore, understanding the factors that support or inhibit program implementation in schools is of great public health importance. This study aimed to evaluate the factors impacting the implementation of an after-school sport-based intervention for youth at five middle schools in Philadelphia, PA, USA. Interviews and surveys were conducted with all programming staff at the end of the program, guided by the Consolidated Framework for Implementation Research (CFIR). Program staff highlighted the need for and importance of the program, but also identified that staffing and training, variability in approaches used, and a lack of consistent support for students were key issues that affected program success. Future programs should focus efforts and resources on hiring and training staff and providing support and consistency for youth to improve the implementation of these important programs in school settings.
RESUMO
Bovine tuberculosis is an infectious disease of global significance that remains endemic in many countries. Mycobacterium bovis infection in cattle is characterized by a cell-mediated immune response (CMI) that precedes humoral responses, however the timing and trajectories of CMI and antibody responses determined by newer generation assays remain undefined. Here we used defined-antigen interferon-gamma release assays (IGRA) and an eleven-antigen multiplex ELISA (Enferplex TB test) alongside traditional tuberculin-based IGRA and IDEXX M. bovis antibody tests to assess immune trajectories following experimental M. bovis infection of cattle. The results show CMI responses developed as early as two-weeks post-infection, with all infected cattle testing positive three weeks post-infection. Interestingly, 6 of 8 infected animals were serologically positive with the Enferplex TB assay as early as 4 weeks post-infection. As expected, application of the tuberculin skin test enhanced subsequent serological reactivity. Infrequent M. bovis faecal shedding was observed but was uncorrelated with observed immune trajectories. Together, the results show that early antibody responses to M. bovis infection are detectable in some individuals and highlight an urgent need to identify biomarkers that better predict infection outcomes, particularly for application in low-and-middle income countries where test-and-slaughter based control methods are largely unfeasible.
Assuntos
Mycobacterium bovis , Tuberculose Bovina , Humanos , Animais , Bovinos , Interferon gama , Tuberculose Bovina/diagnóstico , Teste Tuberculínico/veterinária , Imunidade CelularRESUMO
THE PROBLEM: Ante-mortem diagnosis of Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is normally achieved through faecal culture, PCR, or serological tests, but agreement as to which samples are positive for Johne's disease is often poor and sensitivities are low, particularly in early-stage infections. The potential solution: Mycobacterial cells contain very complex characteristic mixtures of mycolic acid derivatives that elicit antibodies during infection; this has been used to detect infections in humans. Here, we explore its application in providing an assay differentiating infected from vaccinated animals (DIVA assay) for Johne's disease in cattle. METHOD: Antibody responses to different classes of mycolic acid derivatives were measured using ELISA for serum from cattle positive for MAP by both faecal PCR and commercial serum ELISA, or just by PCR, and from animals from herds with no history of Johne's disease, bovine tuberculosis reactors, BCG-vaccinated, BCG-vaccinated and M. bovis-infected, and Gudair-vaccinated animals. RESULTS: The best-performing antigens, ZAM295 and ST123-the latter a molecule present in the cells of MAP but not of Mycobacterium bovis-achieved a sensitivity of 75% and 62.5%, respectively, for serum from animals positive by both faecal PCR and a commercial MAP serum ELISA, at a specificity of 94% compared to 80 no-history negatives. Combining the results of separate assays with two antigens (ST123 and JRRR121) increased the sensitivity/specificity to 75/97.5%. At the same cut-offs, animals vaccinated with Gudair or BCG vaccines and bTB reactors showed a similar specificity. The specificity in BCG-vaccinated but M. bovis-infected animals dropped to 85%. Combining the results of two antigens gave a sensitivity/specificity of 37.5/97.5% for the full set of 80 PCR-positive samples, detecting 30 positives compared 16 for IDEXX. CONCLUSION: Serum ELISA using synthetic lipids distinguishes effectively between MAP-negative cattle samples and those positive by both PCR and a commercial MAP serodiagnostic, without interference by Gudair or BCG vaccination. It identified almost twice as many PCR positives as the commercial serodiagnostic, offering the possibility of earlier detection of infection.
RESUMO
Bacillus Calmette-Guérin (BCG) Danish strain 1331 (CattleBCG) is currently the lead vaccine candidate for the control of bovine tuberculosis (TB) in cattle in GB, where prior vaccination has shown to result in a significant reduction in bovine TB pathology induced by infection with Mycobacterium bovis (M. bovis). A critical knowledge gap in our understanding of CattleBCG is the duration of immunity post vaccination at the minimum intended vaccine dose. To this end, we performed an experiment where calves were vaccinated with a targeted dose of 106 CFU and, after a period of 52 weeks, experimentally infected with M. bovis. Post mortem examination performed 13 weeks after infection revealed a statistically significant reduction in the severity of TB pathology in the CattleBCG vaccinated group compared with the unvaccinated control group. Additionally, this study allowed us to further assess the diagnostic performance of a defined antigen DIVA reagent (DST-F) developed to detect infected amongst vaccinated animals. Our results demonstrate that when used in a skin test format, DST-F showed high specificity (100 %) in BCG-vaccinated animals when tested prior to infection, whilst detecting all infected animals when re-tested after infection. Furthermore, we also present results supporting the use of the DST-F reagent in an interferon-gamma release assay. In conclusion, the results of this study demonstrate a 52-week duration of immunity following administration of a minimum dose of CattleBCG. This evidence will be a fundamental component in our efforts to apply for UK marketing authorisation to enable vaccination of cattle as a significant additional control measure in the ongoing fight against bovine TB in GB.
Assuntos
Mycobacterium bovis , Tuberculose Bovina , Animais , Bovinos , Vacina BCG , Tuberculose Bovina/prevenção & controle , Tuberculose Bovina/microbiologia , Vacinação/veterinária , Vacinação/métodos , DinamarcaRESUMO
COVID-19 significantly impacted physical activity among high-risk youth. Camp from Home, a digitally enhanced home-based intervention, was developed to address physical activity disparities among middle school youth during COVID-19. Camp from Home enrolled 62 youth in 54 families from five schools in Philadelphia during the summer of 2020. The 6-week intervention comprised of (1) three home deliveries of "activity kits" including exercise equipment and activity booklets, (2) asynchronous sport and exercise videos posted to a private YouTube channel, and (3) supportive text-messages from health coaches. YouTube analytics and self-report surveys completed by parents and youth at baseline and at the end of programming were used to assess engagement, acceptability, and preliminary efficacy. Youth participants were 12.4 (1.2) years, 38.7% female and 90.3% Black/African American. At follow-up, 41 parents (75.9%) and 34 youth (54.8%) completed measures. Youth self-reported increases in self-efficacy (ΔM(sd) = 0.4(1.0), p = .03) and physical activity (ΔM(sd) = 4.2(7.9), p = .004), despite suboptimal engagement in digital program components. Overall, participants highly rated the program. Activity kits and text-messages from health coaches were rated as most helpful. Most parents (95.1%) and youth (83.8%) expressed interested in participating again in the future. A 6-week digitally enhanced, home-based physical activity intervention was acceptable and feasible among parents and youth during the summer of 2020, with youth reporting improvements in self-efficacy and physical activity. Summer programs are critical for reducing disparities in physical activity and hold potential for addressing key barriers for high-risk youth even outside the context of COVID-19.
Assuntos
COVID-19 , Humanos , Feminino , Adolescente , Masculino , COVID-19/prevenção & controle , Exercício Físico , Instituições AcadêmicasRESUMO
The single and comparative intradermal tuberculin tests (SITT and CITT) are official in vivo tests for bovine tuberculosis (TB) diagnosis using bovine and avian purified protein derivatives (PPD-B and PPD-A). Infection with bacteria other than Mycobacterium tuberculosis complex (MTC) can result in nonspecific reactions to these tests. We evaluated the performance of the skin test with PPDs and new defined antigens in the guinea pig model. A standard dose (SD) of Rhodococcus equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis, M. avium subsp. avium, M. avium subsp. hominissuis, M. scrofulaceum, M. persicum, M. microti, M. caprae and M. bovis, and a higher dose (HD) of M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis were tested using PPD-B, PPD-A, P22, ESAT-6-CFP-10-Rv3615c peptide cocktail long (PCL) and fusion protein (FP). The SD of R. equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare and M. avium subsp. paratuberculosis did not cause any reactions. The HD of M. nonchromogenicum, M. monacense, M. intracellulare, and M. avium subsp. paratuberculosis and the SD of M. avium subsp. hominissuis, M. scrofulaceum and M. persicum, caused nonspecific reactions (SIT). A CITT interpretation would have considered M. avium complex and M. scrofulaceum groups negative, but not all individuals from M. nonchromogenicum HD, M. monacense HD and M. persicum SD groups. Only animals exposed to M. bovis and M. caprae reacted to PCL and FP. These results support the advantage of complementing or replacing PPD-B to improve specificity without losing sensitivity.
Assuntos
Mycobacterium , Paratuberculose , Tuberculose Bovina , Animais , Cobaias , Bovinos , Tuberculina , Tuberculose Bovina/diagnóstico , Antígenos , Teste TuberculínicoRESUMO
Mycobacterial lipids have long been known to modulate the function of a variety of cells of the innate immune system. Here, we report the extraction and characterisation of polar and apolar free lipids from Mycobacterium bovis AF 2122/97 and identify the major lipids present in these fractions. Lipids found included trehalose dimycolate (TDM) and trehalose monomycolate (TMM), the apolar phthiocerol dimycocersates (PDIMs), triacyl glycerol (TAG), pentacyl trehalose (PAT), phenolic glycolipid (PGL), and mono-mycolyl glycerol (MMG). Polar lipids identified included glucose monomycolate (GMM), diphosphatidyl glycerol (DPG), phenylethanolamine (PE) and a range of mono- and di-acylated phosphatidyl inositol mannosides (PIMs). These lipid fractions are capable of altering the cytokine profile produced by fresh and cultured bovine monocytes as well as monocyte derived dendritic cells. Significant increases in the production of IL-10, IL-12, MIP-1ß, TNFα and IL-6 were seen after exposure of antigen presenting cells to the polar lipid fraction. Phenotypic characterisation of the cells was performed by flow cytometry and significant decreases in the expression of MHCII, CD86 and CD1b were found after exposure to the polar lipid fraction. Polar lipids also significantly increased the levels of CD40 expressed by monocytes and cultured monocytes but no effect was seen on the constitutively high expression of CD40 on MDDC or on the levels of CD80 expressed by any of the cells. Finally, the capacity of polar fraction treated cells to stimulate alloreactive lymphocytes was assessed. Significant reduction in proliferative activity was seen after stimulation of PBMC by polar fraction treated cultured monocytes whilst no effect was seen after lipid treatment of MDDC. These data demonstrate that pathogenic mycobacterial polar lipids may significantly hamper the ability of the host APCs to induce an appropriate immune response to an invading pathogen.
Assuntos
Citocinas/genética , Imunidade Inata , Lipídeos/farmacologia , Mycobacterium bovis/imunologia , Tuberculose Bovina/imunologia , Animais , Antígenos de Superfície/metabolismo , Bovinos , Proliferação de Células , Citocinas/metabolismo , Células Dendríticas/imunologia , Regulação para Baixo , Monócitos/microbiologia , Linfócitos T/metabolismo , Tuberculose Bovina/microbiologiaRESUMO
The aim of this study was to evaluate the validity of the SpO2/FiO2 diagram in estimating gas exchange in horses under general anaesthesia. In this prospective, controlled clinical study were included 10 horses under general anaesthesia. FiO2 was progressively reduced with the following steps: 0.6, 0.4, 0.3 and 0.21; SpO2 was recorded at each step. An arterial blood sample was collected at the steps of 1.0 and 0.21, to calculate intrapulmonary shunt with the Fshunt formula. The Fshunt value calculated at 0.21 FiO2 was defined as "Fshunt 0.21", the one calculated at 1.0 FiO2 as "Fshunt 1.0". The FiO2 vs SpO2 data points were analyzed using a computer algorithm which uses the haemoglobin and a fixed value for arterial-venous oxygen difference of 3.5 mL/dL. The algorithm estimates a shunt value fitting the obtained data with an ideal SpO2/FiO2 curve. The value of shunt (Sshunt) was considered for the study. Correlation between "Fshunt 1.0", "Fshunt0.21" and SShunt was determined using the Spearman Rank Correlation Coefficient test, the analysis of the regression curve and the coefficient of determination (r2). Values of P < .05 were considered statistically significant. A significant and strong correlation (P = .0069; r = 0.839; r2=0.6194) and a significant and moderate correlation (P = .0443; r = 0.644; r2=0.2336) was found between Sshunt and "Fshunt 1.0" and between Sshunt and "Fshunt 0.21", respectively. The SpO2/FiO2 diagram proved to be a useful and non-invasive tool to characterize gas exchange in horses under general anaesthesia and mechanical ventilation.
Assuntos
Anestesia Geral , Oxigênio , Anestesia Geral/veterinária , Animais , Cavalos , Consumo de Oxigênio , Estudos Prospectivos , Respiração Artificial/veterináriaRESUMO
Bacillus Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis (M. bovis), is the lead candidate vaccine for control of bovine tuberculosis (TB) in cattle. However, BCG vaccination sensitises cattle to bovine tuberculin, thus compromising the use of the current bovine TB surveillance tests. To address this, we have developed a diagnostic skin test that is not compromised by BCG vaccination and is able to detect BCG vaccinated animals that subsequently develop bovine TB following exposure to M. bovis. Building on previous work using 'in house' formulated protein cocktail reagents, we herein present test performance data for a single fusion protein (DST-F) containing the mycobacterial antigens ESAT-6, CFP-10 and Rv3615c formulated as a 'ready to use' reagent by a commercial manufacturer. Our results demonstrate that, unlike tuberculin reagents, a diagnostic skin test using DST-F maintained high specificity in BCG vaccinated animals. Furthermore, the DST-F skin test demonstrated a high relative sensitivity in identifying M. bovis infected animals, including those where BCG vaccination failed to prevent bovine TB pathology following experimental exposure to M. bovis. The DST-F is currently undergoing field trials in Great Britain to support its licensure and commercialisation.
Assuntos
Mycobacterium bovis , Tuberculose Bovina , Animais , Antígenos de Bactérias , Vacina BCG , Bovinos , Indicadores e Reagentes , Testes Cutâneos , Tuberculina , Tuberculose Bovina/diagnóstico , Tuberculose Bovina/prevenção & controle , Vacinação/veterináriaRESUMO
Bovine tuberculosis (bTB) challenges intensive dairy production in Ethiopia and implementation of the test and slaughter control strategy is not economically acceptable in the country. Vaccination of cattle with Bacillus Calmette-Guerin (BCG) could be an important adjunct to control, which would require a diagnostic test to differentiate Mycobacterium bovis (M. bovis)-infected and BCG-vaccinated animals (DIVA role). This study describes an evaluation of a DIVA skin test (DST) that is based on a cocktail (DSTc) or fusion (DSTf) of specific (ESAT-6, CFP-10 and Rv3615c) M. bovis proteins in Zebu-Holstein-Friesians crossbred cattle in Ethiopia. The study animals used were 74 calves (35 BCG vaccinated and 39 unvaccinated) aged less than 3 weeks at the start of experiment and 68 naturally infected 'TB reactor' cows. Six weeks after vaccination, the 74 calves were tested with the DSTc and the single intradermal cervical comparative tuberculin (SICCT) test. The TB reactor cows were tested with the DSTc and the SICCT test. Reactions to the DSTc were not observed in BCG-vaccinated and unvaccinated calves, while SICCT test reactions were detected in vaccinated calves. DSTc reactions were detected in 95.6% of the TB reactor cows and single intradermal tuberculin positive reactions were found in 98.2% (95% confidence interval, CI, 92.1-100%). The sensitivity of the DSTc was 95.6% (95% CI, 87.6-99.1%), and significantly (p < .001) higher than the sensitivity (75%, 95% CI, 63.0-84.7%) of the SICCT test at 4 mm cut-off. DSTf and DSTc reactions were correlated (r = 0.75; 95% CI = 0.53-0.88). In conclusion, the DSTc could differentiate M. bovis-infected from BCG-vaccinated cattle in Ethiopia. DST had higher sensitivity than the SICCT test. Hence, the DSTc could be used as a diagnostic tool for bTB if BCG vaccination is implemented for the control of bTB in Ethiopia and other countries.
Assuntos
Doenças dos Bovinos , Mycobacterium bovis , Tuberculose Bovina , Animais , Vacina BCG , Bovinos , Etiópia , Feminino , Tuberculina , Teste Tuberculínico/veterinária , Tuberculose Bovina/diagnóstico , Tuberculose Bovina/prevenção & controle , Vacinação/veterináriaRESUMO
Tuberculin Purified Protein Derivatives (PPDs) exhibit multiple limitations: they are crude extracts from mycobacterial cultures with largely unknown active components; their production depends on culture of mycobacteria requiring expensive BCL3 production facilities; and their potency depends on the technically demanding guinea pig assay. To overcome these limitations, we developed a molecularly defined tuberculin (MDT) by adding further antigens to our prototype reagent composed of ESAT-6, CFP-10 and Rv3615c (DIVA skin test, DST). In vitro screening using PBMC from infected and uninfected cattle shortlisted four antigens from a literature-based list of 18 to formulate the MDT. These four antigens plus the previously identified Rv3020c protein, produced as recombinant proteins or overlapping synthetic peptides, were formulated together with the three DST antigens into the MDT to test cattle experimentally and naturally infected with M. bovis, uninfected cattle and MAP vaccinated calves. We demonstrated significant increases in MDT-induced skin responses compared to DST in infected animals, whilst maintaining high specificity in unvaccinated or MAP vaccinated calves. Further, MDT can also be applied in in vitro blood-based interferon-gamma release assays. Thus, MDT promises to be a robust diagnostic skin and blood test reagent overcoming some of the limitations of PPDs and warrants full validation.
Assuntos
Mycobacterium bovis/isolamento & purificação , Paratuberculose/prevenção & controle , Teste Tuberculínico/veterinária , Tuberculina/imunologia , Tuberculose Bovina/diagnóstico , Animais , Bovinos , Leucócitos Mononucleares , Masculino , Mycobacterium avium subsp. paratuberculosis/imunologia , Mycobacterium bovis/imunologia , Paratuberculose/microbiologia , Teste Tuberculínico/métodos , Tuberculose Bovina/imunologia , Tuberculose Bovina/microbiologia , Vacinação/veterináriaRESUMO
Results of previous studies utilizing bioinformatic approaches in antigen-mining experiments revealed that secreted proteins are among the most frequently recognized antigens from Mycobacterium bovis. Thus, we hypothesized that the analysis of secreted proteins is likely to reveal additional immunogenic antigens that can be used to increase the specificity of diagnostic tests or be suitable vaccination candidates for mycobacterial infections. To test this hypothesis, 382 pools of overlapping peptides spanning 119 M. bovis secreted and potentially secreted proteins were screened for the ability to stimulate a gamma interferon response in vitro using whole blood from tuberculin-positive reactor (TB reactor) cattle. Of the 119 proteins screened, 70 (59%) induced positive responses in the TB reactor animals to various degrees. Strikingly, all but one of the 15 ESAT-6 proteins tested were recognized by at least 30% of the TB reactor animals, with 12 of the 22 most commonly recognized antigens belonging to this protein family. Further analysis of these data demonstrated that there was no significant difference in immunogenicity between the ESAT-6 proteins that were components of potentially intact ESX secretory systems and those corresponding to additional partial esx loci. Importantly for vaccine design, antigenic epitopes in some highly conserved regions shared by numerous ESAT-6 proteins were identified. However, despite this considerable homology, peptide-mapping experiments also revealed that immunodominant peptides were located in regions of amino acid variability.
Assuntos
Antígenos de Bactérias/imunologia , Mycobacterium bovis/imunologia , Animais , Sangue/imunologia , Bovinos , Sequência Conservada/imunologia , Epitopos Imunodominantes/imunologia , Interferon gama/metabolismoRESUMO
NK cell cytotoxicity requires two positive signals for killing of tumors. Activation receptors induce polarization of the microtubule organization center and degranulation, while leukocyte function-associated antigen (LFA)-1 is required for conjugate formation and actin polymerization and under some circumstances may be sufficient for NK cell cytotoxicity. Although the receptor for direct killing of fungi is not known, CD18, the beta2 chain of LFA-1, binds components of the capsule and cell wall of the opportunistic pathogen Cryptococcus neoformans, namely the polysaccharides glucoronoxylomannan and galactoxylomannan. Herein, we also demonstrate that LFA-1 was concentrated in regions of the NK cell surface interacting with C. neoformans. Consequently, there was compelling evidence to hypothesize that NK cells would also use LFA-1 to recognize and kill C. neoformans. Using a combination of NK cell lines that did or did not express LFA-1 or by using a CD18-specific functional blocking antibody, we confirm that NK cell anti-tumor activity is critically dependent upon the expression of LFA-1. Duplicating the events of tumor cytotoxicity, NK cells form conjugates with cryptococcal targets, rearrange the cell cytoskeleton to develop an NK immunologic synapse and release perforin-containing granules; however, each of these events occurred independently of LFA-1. Furthermore, NK cell-mediated killing of C. neoformans was detectable in both NK cells pre-treated with CD18-blocking antibodies and in NK cells lacking cell surface LFA-1 expression. These results demonstrate that in the absence of LFA-1 expression, NK cells are fully capable of recognizing a target (C. neoformans) and retain all of the events required for cytotoxicity.
Assuntos
Antígenos CD18/imunologia , Cryptococcus neoformans/imunologia , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Neoplasias/imunologia , Actinas/imunologia , Actinas/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos CD18/metabolismo , Degranulação Celular/imunologia , Linhagem Celular Tumoral , Citoesqueleto/imunologia , Citoesqueleto/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/metabolismo , Perforina/imunologia , Perforina/metabolismo , Transdução de Sinais/imunologiaRESUMO
Whole blood based assays, particularly interferon gamma (IFN-γ) release assays (IGRAs), are used for the diagnosis of both bovine and human tuberculosis (TB). The aim of the current study was to evaluate a panel of cytokines and chemokines for potential use as diagnostic readouts indicative of Mycobacterium bovis (M. bovis) infection in cattle. A gene expression assay was used to determine the kinetics of the response to M. bovis purified protein derivative and a fusion protein consisting of ESAT-6, CFP10, and Rv3615c upon aerosol infection with â¼104 cfu of M. bovis. The panel of biomarkers included: IFN-γ, CXCL9, CXCL10, CCL2, CCL3, TNF-α, IL-1α, IL-1ß, IL-1Ra, IL-22, IL-21 and IL-13. Protein levels of IFN-γ, CXCL9, and CXCL10 were determined by ELISA. Findings suggest that CXCL9, CXCL10, IL-21, IL-13, and several acute phase cytokines may be worth pursuing as diagnostic biomarkers of M. bovis infection in cattle.
Assuntos
Doenças dos Bovinos/diagnóstico , Citocinas/genética , Imunidade Celular , Tuberculose Bovina/diagnóstico , Tuberculose Bovina/imunologia , Animais , Biomarcadores/sangue , Bovinos , Doenças dos Bovinos/sangue , Doenças dos Bovinos/imunologia , Quimiocina CXCL10/sangue , Quimiocina CXCL9/sangue , Citocinas/imunologia , Expressão Gênica , Interferon gama , Testes de Liberação de Interferon-gama , Masculino , Mycobacterium bovis , Tuberculose Bovina/sangueRESUMO
Bovine tuberculosis is an important animal health problem and the predominant cause of zoonotic tuberculosis worldwide. It results in serious economic burden due to losses in productivity and the cost of control programmes. Control could be greatly improved by the introduction of an efficacious cattle vaccine but the most likely candidate, BCG, has several limitations including variable efficacy. Augmentation of BCG with a subunit vaccine booster has been shown to increase protection but the selection of antigens has hitherto been left largely to serendipity. In the present study, we take a rational approach to identify the protective antigens of BCG, selecting a BCG transposon mutant library in naïve and BCG-vaccinated cattle. Ten mutants had increased relative survival in vaccinated compared to naïve cattle, consistent with loss of protective antigen targets making the mutants less visible to the BCG immune response. The immunogenicity of three putative protective antigens, BCG_0116, BCG_0205 (YrbE1B) and BCG_1448 (PPE20) was investigated using peptide pools and PBMCs from BCG vaccinated cattle. BCG vaccination induced PBMC to release elevated levels of IP10, IL-17a and IL-10 in response to all three antigens. Taken together, the data supports the further study of these antigens for use in subunit vaccines.
Assuntos
Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Vacina BCG/administração & dosagem , Imunogenicidade da Vacina , Leucócitos Mononucleares/imunologia , Mycobacterium tuberculosis/genética , Tuberculose Bovina/prevenção & controle , Vacinação/veterinária , Animais , Antígenos de Bactérias/imunologia , Vacina BCG/imunologia , Bovinos , Citocinas/imunologia , Citocinas/metabolismo , Elementos de DNA Transponíveis , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/microbiologia , Mutação , Mycobacterium tuberculosis/imunologia , Tuberculose Bovina/imunologia , Tuberculose Bovina/metabolismo , Tuberculose Bovina/microbiologiaRESUMO
NK cells, in addition to possessing antitumor and antiviral activity, exhibit perforin-dependent microbicidal activity against the opportunistic pathogen Cryptococcus neoformans. However, the factors controlling this response, particularly whether the pathogen itself provides an activation or rearming signal, are largely unknown. The current studies were performed to determine whether exposure to this fungus alters subsequent NK cell anticryptococcal activity. NK cells lost perforin and mobilized lysosome-associated membrane protein 1 to the cell surface following incubation with the fungus, indicating that degranulation had occurred. Despite a reduced perforin content during killing, NK cells acquired an enhanced ability to kill C. neoformans, as demonstrated using auxotrophs that allowed independent assessment of the killing of two strains. De novo protein synthesis was required for optimal killing; however, there was no evidence that a soluble factor contributed to the enhanced anticryptococcal activity. Exposure of NK cells to C. neoformans caused the cells to rearm, as demonstrated by increased perforin mRNA levels and enhanced loss of perforin when transcription was blocked. Degranulation alone was insufficient to provide the activation signal as NK cells lost anticryptococcal activity following treatment with strontium chloride. However, NK cells regained the activity upon prolonged exposure to C. neoformans, which is consistent with activation by the microbe. The enhanced cytotoxicity did not extend to tumor killing since NK cells exposed to C. neoformans failed to kill NK-sensitive tumor targets (K562 cells). These studies demonstrate that there is contact-mediated microbe-specific rearming and activation of microbicidal activity that are necessary for optimal killing of C. neoformans.
Assuntos
Cryptococcus neoformans/imunologia , Células Matadoras Naturais/imunologia , Viabilidade Microbiana , Perforina/biossíntese , Degranulação Celular , Linhagem Celular , Contagem de Colônia Microbiana , Humanos , RNA Mensageiro/biossínteseRESUMO
Human studies have identified the potential of measuring Mycobacterium tuberculosis specific IFN-γ and/or IL-2 secreting T cell subsets to distinguish different clinical stages of human tuberculosis (TB). To assess these functional T cell subsets in different states of bovine TB we have established a bovine dual IFN-γ/IL-2 fluorescence-immunospot (FluoroSpot) assay and analysed the frequencies of Mycobacterium bovis (M. bovis) specific IL-2 and/or IFN-γ producing cells in PBMC from 30 cattle naturally infected with M. bovis. Depending on their post mortem results the animals were grouped in 22 cattle with visible lesions (VL) and 8 cattle without visible lesions (NVL). In response to bovine tuberculin purified protein derivative (PPD-B) the frequencies of cytokine producing cells and proportions of IL-2 single producers were significantly higher in VL compared to NVL while PWM-induced cytokine responses were similar between the two groups. Dual IL-2+IFN-γ+ T cells could be identified as the largest PPD-B responsive T cell subset in both cattle groups. In conclusion, our FluoroSpot is a valid method to enumerate individual antigen-specific IFN-γ+ and IL-2+ T cell subsets ex vivo. The greater levels of single IL-2 producing T cells associated with the presence of pathology could be a potential biomarker for active TB in cattle.
Assuntos
ELISPOT/veterinária , Fluorescência , Interferon gama/imunologia , Interleucina-2/imunologia , Tuberculose Bovina/diagnóstico , Tuberculose Bovina/imunologia , Animais , Antígenos de Bactérias/imunologia , Bovinos , Cor , Mycobacterium bovis/imunologia , Subpopulações de Linfócitos T/imunologiaRESUMO
Despite the introduction of highly active antiretroviral therapy, dementia caused by human immunodeficiency virus-1 (HIV-1) infection remains a devastating and common neurological disorder. Although the mechanisms governing neurodegeneration during HIV-1 infection remain uncertain, the HIV-1 accessory protein, viral protein R (Vpr), has been proposed as a neurotoxic protein. Herein, we report that Vpr protein and transcript were present in the brains of HIV-infected persons. Moreover, soluble Vpr caused neuronal apoptosis, involving cytochrome c extravasation, p53 induction, and activation of caspase-9 while exerting a depressive effect on whole-cell currents in neurons (p < 0.05), which was inhibited by iberiotoxin. Vpr-activated glial cells secreted neurotoxins in a concentration-dependent manner (p < 0.001). Transgenic (Tg) mice expressing Vpr in brain monocytoid cells displayed the transgene principally in the basal ganglia (p < 0.05) and cerebral cortex (p < 0.01) compared with hindbrain expression. Vpr was released from cultured transgenic macrophages, which was cytotoxic to neurons and was blocked by anti-Vpr antibody (p < 0.05). Neuronal injury was observed in Tg animals compared with wild-type littermates, chiefly affecting GAD65 (p < 0.01) and vesicular acetylcholine transferase (p < 0.001) immunopositive neuronal populations in the basal ganglia. There was also a loss of subcortical synaptophysin (p < 0.001) immunoreactivity as well as an increase in activated caspase-3, which was accompanied by a hyperexcitable neurobehavioral phenotype (p < 0.05). Thus, HIV-1 Vpr caused neuronal death through convergent pathogenic mechanisms with ensuing in vivo neurodegeneration, yielding new insights into the mechanisms by which HIV-1 injures the nervous system.