Necrosis plays a role in the concentration of mycobacterial antigens in granulomas from Mycobacterium bovis naturally infected cattle.

The dynamics that develop between cells and molecules in the host against infection by Mycobacterium bovis, leads to the formation of granulomas mainly present in the lungs and regional lymph nodes in cattle. Cell death is one of the main features in granuloma organization, however, it has not been characterized in granulomatous lesions caused by M. bovis. In this study we aimed to identify the profiles of cell death in the granuloma stages and its relationship with the accumulation of bacteria. We identified necrosis, activated caspase-3, LC3B/p62 using immunohistochemistry and digital pathology analysis on 484 granulomatous lesions in mediastinal lymph nodes from 23 naturally infected cattle. Conclusions: greater amounts of mycobacterial antigens were identified in granulomas from calves compared with adult cattle. The highest percentage of necrosis and quantity of mycobacterial antigens were identified in granuloma stages (III/IV) from adults. The LC3B/p62 profile was heterogeneous in granulomas between adults and calves. Our data suggest that necrosis is associated with a higher amount of mycobacterial antigens in the late stages of granuloma and the development of autophagy appears to play an heterogeneous effector response against infection in adults and calves. These results represent one of the first approaches in the identification of cell death in the four stages of granulomas in bovine tuberculosis.

100 years of Mycobacterium bovis bacille Calmette-Guérin.

Mycobacterium bovis bacille Calmette-Guérin (BCG), an experimental vaccine designed to protect cattle from bovine tuberculosis, was administered for the first time to a newborn baby in Paris in 1921. Over the past century, BCG has saved tens of millions of lives and has been given to more humans than any other vaccine. It remains the sole tuberculosis vaccine licensed for use in humans. BCG provides long-lasting strong protection against miliary and meningeal tuberculosis in children, but it is less effective for the prevention of pulmonary tuberculosis, especially in adults. Evidence mainly from the past two decades suggests that BCG has non-specific benefits against non-tuberculous infections in newborn babies and in older adults, and offers immunotherapeutic benefit in certain malignancies such as non-muscle invasive bladder cancer. However, as a live attenuated vaccine, BCG can cause localised or disseminated infections in immunocompromised hosts, which can also occur following intravesical installation of BCG for the treatment of bladder cancer. The legacy of BCG includes fundamental discoveries about tuberculosis-specific and non-specific immunity and the demonstration that tuberculosis is a vaccine-preventable disease, providing a foundation for new vaccines to hasten tuberculosis elimination.

Identifying Bacterial and Host Factors Involved in the Interaction of Mycobacterium bovis with the Bovine Innate Immune Cells.

Bovine tuberculosis is an important animal and zoonotic disease caused by Mycobacterium bovis. The innate immune response is the first line of defense against pathogens and is also crucial for the development of an efficient adaptive immune response. In this study we used an in vitro co-culture model of antigen presenting cells (APC) and autologous lymphocytes derived from peripheral blood mononuclear cells to identify the cell populations and immune mediators that participate in the development of an efficient innate response capable of controlling the intracellular replication of M. bovis. After M. bovis infection, bovine immune cell cultures displayed upregulated levels of iNOS, IL-22 and IFN-γ and the induction of the innate immune response was dependent on the presence of differentiated APC. Among the analyzed M. bovis isolates, only a live virulent M. bovis isolate induced an efficient innate immune response, which was increased upon stimulation of cell co-cultures with the M. bovis culture supernatant. Moreover, we demonstrated that an allelic variation of the early secreted protein ESAT-6 (ESAT6 T63A) expressed in the virulent strain is involved in this increased innate immune response. These results highlight the relevance of the compounds secreted by live M. bovis as well as the variability among the assessed M. bovis strains to induce an efficient innate immune response.

Development of a lateral flow immunochromatography test for the rapid detection of bovine tuberculosis.

Detection of specific antibodies would be a useful test strategy for bovine tuberculosis (bTB) as a complement to the single skin test. We developed a lateral flow immunochromatography (LFIC) test for rapid bTB detection based on the use of a conjugate of gold nanoparticles with a recombinant G protein. After evaluating 3 Mycobacterium bovis (MB) antigens: ESAT-6, CFP-10 and MPB83 for the control line, we selected MPB83 given it was the most specific. The performance of the test was analyzed with 820 bovine sera, 40 sera corresponding to healthy animals, 5 sera from animals infected with Mycobacterium avium subsp. paratuberculosis (MAP) and 775 sera of animals from herds with bTB. All these sera were also submitted to a validated bTB-ELISA using whole-cell antigen from MB. From the 775 sera of animals from herds with bTB, 87 sera were positive by the bTB-ELISA, 45 were positive by LFIC and only 5 animals were positives by skin test (TST). To confirm bTB infection in the group of TST (-), bTB-ELISA (+) and LFIC (+) animals, we performed postmortem examination in 15 randomly selected animals. Macroscopically, these 15 animals had numerous small and large yellow-white granulomas, characteristic of bTB, and the infection was subsequently confirmed by PCR in these tissues with lesions (gold standard). No false positive test result was detected with the developed LFIC either with the sera from healthy animals or from animals infected with MAP demonstrating that it can be a useful technique for the rapid identification of animals infected with bTB.

TNFRp75-dependent immune regulation of alveolar macrophages and neutrophils during early Mycobacterium tuberculosis and Mycobacterium bovis BCG infection.

TNF signalling through TNFRp55 and TNFRp75, and receptor shedding is important for immune activation and regulation. TNFRp75 deficiency leads to improved control of Mycobacterium tuberculosis (M. tuberculosis) infection, but the effects of early innate immune events in this process are unclear. We investigated the role of TNFRp75 on cell activation and apoptosis of alveolar macrophages and neutrophils during M. tuberculosis and M. bovis BCG infection. We found increased microbicidal activity against M. tuberculosis occurred independently of IFNy and NO generation, and displayed an inverse correlation with alveolar macrophages (AMs) apoptosis. Both M. tuberculosis and M. bovis BCG induced higher expression of MHC-II in TNFRp75-/- AMs; however, M bovis BCG infection did not alter AM apoptosis in the absence of TNFRp75. Pulmonary concentrations of CCL2, CCL3 and IL-1ß were increased in TNFRp75-/- mice during M, bovis BCG infection, but had no effect on neutrophil responses. Thus, TNFRp75-dependent regulation of mycobacterial replication is virulence dependent and occurs independently of early alveolar macrophage apoptosis and neutrophil responses.

Cellular and Cytokine Responses in the Granulomas of Asymptomatic Cattle Naturally Infected with Mycobacterium bovis in Ethiopia.

Cell (CD3+ T cell and CD68+ macrophages), cytokine (interferon gamma-positive [IFN-γ+] and tumor necrosis factor alpha-positive [TNF-α+]), and effector molecule (inducible nitric oxide synthase-positive [iNOS+]) responses were evaluated in the lymph nodes and tissues of cattle naturally infected with Mycobacterium bovis Detailed postmortem and immunohistochemical examinations of lesions were performed on 16 cows that were positive by the single intradermal cervical comparative tuberculin (SICCT) test and that were identified from dairy farms located around the city of Addis Ababa, Ethiopia. The severity of the gross lesion was significantly higher (P = 0.003) in M. bovis culture-positive cows (n = 12) than in culture-negative cows (n = 4). Immunohistochemical techniques showed that in culture-positive cows, the mean immunolabeling fraction of CD3+ T cells decreased as the stage of granuloma increased from stage I to stage IV (P < 0.001). In contrast, the CD68+ macrophage, IFN-γ+, TNF-α+, and iNOS+ immunolabeling fractions increased from stage I to stage IV (P < 0.001). In the early stages, culture-negative cows showed a significantly higher fraction of CD68+ macrophage (P = 0.03) and iNOS+ (P = 0.007) immunolabeling fractions than culture-positive cows. Similarly, at advanced granuloma stages, culture-negative cows demonstrated significantly higher mean proportions of CD3+ T cells (P < 0.001) than culture-positive cows. Thus, this study demonstrates that, following natural infection of cows with M. bovis, as the stage of granuloma increases from stage I to stage IV, the immunolabeling fraction of CD3+ cells decreases, while the CD68+ macrophage, IFN-γ+, TNF-α+, and iNOS+ immunolabeling fractions increases.

PP2Ac Modulates AMPK-Mediated Induction of Autophagy in Mycobacterium bovis-Infected Macrophages.

Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis in cattle population across the world. Human beings are at equal risk of developing tuberculosis beside a wide range of M. bovis infections in animal species. Autophagic sequestration and degradation of intracellular pathogens is a major innate immune defense mechanism adopted by host cells for the control of intracellular infections. It has been reported previously that the catalytic subunit of protein phosphatase 2A (PP2Ac) is crucial for regulating AMP-activated protein kinase (AMPK)-mediated autophagic signaling pathways, yet its role in tuberculosis is still unclear. Here, we demonstrated that M. bovis infection increased PP2Ac expression in murine macrophages, while nilotinib a tyrosine kinase inhibitor (TKI) significantly suppressed PP2Ac expression. In addition, we observed that TKI-induced AMPK activation was dependent on PP2Ac regulation, indicating the contributory role of PP2Ac towards autophagy induction. Furthermore, we found that the activation of AMPK signaling is vital for the regulating autophagy during M. bovis infection. Finally, the transient inhibition of PP2Ac expression enhanced the inhibitory effect of TKI-nilotinib on intracellular survival and multiplication of M. bovis in macrophages by regulating the host's immune responses. Based on these observations, we suggest that PP2Ac should be exploited as a promising molecular target to intervene in host-pathogen interactions for the development of new therapeutic strategies towards the control of M. bovis infections in humans and animals.

A defined antigen skin test for the diagnosis of bovine tuberculosis.

Bovine tuberculosis (bTB) is a major zoonotic disease of cattle that is endemic in much of the world, limiting livestock productivity and representing a global public health threat. Because the standard tuberculin skin test precludes implementation of Bacille Calmette-Guérin (BCG) vaccine-based control programs, we here developed and evaluated a novel peptide-based defined antigen skin test (DST) to diagnose bTB and to differentiate infected from vaccinated animals (DIVA). The results, in laboratory assays and in experimentally or naturally infected animals, demonstrate that the peptide-based DST provides DIVA capability and equal or superior performance over the extant standard tuberculin surveillance test. Together with the ease of chemical synthesis, quality control, and lower burden for regulatory approval compared with recombinant antigens, the results of our studies show that the DST considerably improves a century-old standard and enables the development and implementation of critically needed surveillance and vaccination programs to accelerate bTB control.

Characterization of γδ T Cell Effector/Memory Subsets Based on CD27 and CD45R Expression in Response to Mycobacterium bovis Infection.

Tuberculosis (TB) remains a leading cause of death from infectious diseases worldwide. Mycobacterium bovis is the causative agent of bovine TB and zoonotic TB infection. γδ T cells are known to participate in the immune control of mycobacterial infections. Data in human and nonhuman primates suggest that mycobacterial infection regulates memory/effector phenotype and adaptive immune functions of γδ T cells. To date, the impact of M. bovis infection on bovine γδ T cells and their effector and memory differentiation remains unknown. In this study, we show that circulating γδ T cells from M. bovis-infected cattle can be differentiated based on the expression of CD27, which is indicative of their capacity to respond to virulent M. bovis infection: CD27+ γδ T cells proliferated in response to M. bovis Ag and, thus, may comprise the adaptive γδ T cell compartment in cattle. We further show that bovine M. bovis-specific γδ T cells express surface markers characteristic of central memory T cells (CD45R-CD27+CD62Lhi) and that M. bovis-specific CD4 and γδ T cells both upregulate the expression of the tissue-homing receptors CXCR3 and CCR5 during infection. Our studies contribute significantly to our understanding of γδ T cell differentiation during TB infection and provide important insights into the link between phenotypic and functional subsets in the bovine. Accurate characterization of γδ T cell effector and memory-like responses induced during mycobacterial infection will contribute to improved strategies for harnessing the γδ T cell response in protection against TB for humans and animals.

The Humoral Immune Response to BCG Vaccination.

Bacillus Calmette Guérin (BCG) is the only currently available vaccine against tuberculosis (TB), but it confers incomplete and variable protection against pulmonary TB in humans and bovine TB (bTB) in cattle. Insights into the immune response induced by BCG offer an underexploited opportunity to gain knowledge that may inform the design of a more efficacious vaccine, which is urgently needed to control these major global epidemics. Humoral immunity in TB and bTB has been neglected, but recent studies supporting a role for antibodies in protection against TB has driven a growing interest in determining their relevance to vaccine development. In this manuscript we review what is known about the humoral immune response to BCG vaccination and re-vaccination across species, including evidence for the induction of specific B cells and antibodies; and how these may relate to protection from TB or bTB. We discuss potential explanations for often conflicting findings and consider how factors such as BCG strain, manufacturing methodology and route of administration influence the humoral response. As novel vaccination strategies include BCG prime-boost regimens, the literature regarding off-target immunomodulatory effects of BCG vaccination on non-specific humoral immunity is also reviewed. Overall, reported outcomes to date are inconsistent, but indicate that humoral responses are heterogeneous and may play different roles in different species, populations, or individual hosts. Further study is warranted to determine whether a new TB vaccine could benefit from the targeting of humoral as well as cell-mediated immunity.

Kallikrein 12 Regulates Innate Resistance of Murine Macrophages against Mycobacterium bovis Infection by Modulating Autophagy and Apoptosis.

Mycobacterium bovis (M. bovis) is a member of the Mycobacterium tuberculosis (Mtb) complex causing bovine tuberculosis (TB) and imposing a high zoonotic threat to human health. Kallikreins (KLKs) belong to a subgroup of secreted serine proteases. As their role is established in various physiological and pathological processes, it is likely that KLKs expression may mediate a host immune response against the M. bovis infection. In the current study, we report in vivo and in vitro upregulation of KLK12 in the M. bovis infection. To define the role of KLK12 in immune response regulation of murine macrophages, we produced KLK12 knockdown bone marrow derived macrophages (BMDMs) by using siRNA transfection. Interestingly, the knockdown of KLK12 resulted in a significant downregulation of autophagy and apoptosis in M. bovis infected BMDMs. Furthermore, we demonstrated that this KLK12 mediated regulation of autophagy and apoptosis involves mTOR/AMPK/TSC2 and BAX/Bcl-2/Cytochrome c/Caspase 3 pathways, respectively. Similarly, inflammatory cytokines IL-1ß, IL-6, IL-12 and TNF-α were significantly downregulated in KLK12 knockdown macrophages but the difference in IL-10 and IFN-ß expression was non-significant. Taken together, these findings suggest that upregulation of KLK12 in M. bovis infected murine macrophages plays a substantial role in the protective immune response regulation by modulating autophagy, apoptosis and pro-inflammatory pathways. To our knowledge, this is the first report on expression and the role of KLK12 in the M. bovis infection and the data may contribute to a new paradigm for diagnosis and treatment of bovine TB.

Comparative Study of the Molecular Basis of Pathogenicity of M. bovis Strains in a Mouse Model.

It is widely accepted that different strains of Mycobacterium tuberculosis have variable degrees of pathogenicity and induce different immune responses in infected hosts. Similarly, different strains of Mycobacterium bovis have been identified but there is a lack of information regarding the degree of pathogenicity of these strains and their ability to provoke host immune responses. Therefore, in the current study, we used a mouse model to evaluate various factors involved in the severity of disease progression and the induction of immune responses by two strains of M. bovis isolated from cattle. Mice were infected with both strains of M. bovis at different colony-forming unit (CFU) via inhalation. Gross and histological findings revealed more severe lesions in the lung and spleen of mice infected with M. bovis N strain than those infected with M. bovis C68004 strain. In addition, high levels of interferon-γ (IFN-γ), interleukin-17 (IL-17), and IL-22 production were observed in the serum samples of mice infected with M. bovis N strain. Comparative genomic analysis showed the existence of 750 single nucleotide polymorphisms and 145 small insertions/deletions between the two strains. After matching with the Virulence Factors Database, mutations were found in 29 genes, which relate to 17 virulence factors. Moreover, we found an increased number of virulent factors in M. bovis N strain as compared to M. bovis C68004 strain. Taken together, our data reveal that variation in the level of pathogenicity is due to the mutation in the virulence factors of M. bovis N strain. Therefore, a better understanding of the mechanisms of mutation in the virulence factors will ultimately contribute to the development of new strategies for the control of M. bovis infection.

The expression of Indoleamine 2, 3-dioxygenase (IDO) is reduced in granulomas from BCG vaccinated cattle compared to granulomas from unvaccinated controls after experimental challenge with Mycobacterium bovis.

Bovine tuberculosis (bTB), mainly caused by Mycobacterium bovis (M. bovis), is a major economic disease of livestock worldwide. Vaccination is considered as a potentially sustainable adjunct to the current control strategy. Cattle vaccination with the live attenuated M. bovis bacillus Calmette-Guerin (BCG) confers variable protection; the reasons for this variability are not understood. Indoleamine 2, 3-dioxygenase (IDO), through the catalysis of tryptophan, is thought to have an immunoregulatory role in the immune response to Mycobacterium tuberculosis (M. tuberculosis). In this work, we used immunohistochemistry and digital image analysis to evaluate the presence of IDO in granulomas at different stages of development in cattle that had been BCG-vaccinated or not and then challenged with M. bovis. Our results show that the expression of IDO in granulomas from non-vaccinated M. bovis challenged animals is higher than in granulomas from BCG-vaccinated M. bovis challenged animals. Thus, it is possible that vaccination with BCG prevents the induction of what are thought to be host immunosuppressive pathways by M. bovis, which contribute to pathology during the disease.

Endonuclease G takes part in AIF-mediated caspase-independent apoptosis in Mycobacterium bovis-infected bovine macrophages.

Mycobacterium bovis, the causative agent of bovine tuberculosis encodes different virulence mechanisms to survive inside of host cells. One of the possible outcomes in this host-pathogen interaction is cell death. Previous results from our group showed that M. bovis induces a caspase-independent apoptosis in bovine macrophages with the possible participation of apoptosis inducing factor mitochondria associated 1 (AIFM1/AIF), a flavoprotein that functions as a cell-death regulator. However, contribution of other caspase-independent cell death mediators in M. bovis-infected macrophages is not known. In this study, we aimed to further characterize M. bovis-induced apoptosis, addressing Endonuclease G (Endo G) and Poly (ADP-ribose) polymerase 1 (PARP-1). In order to accomplish our objective, we infected bovine macrophages with M. bovis AN5 (MOI 10:1). Analysis of M. bovis-infected nuclear protein extracts by immunoblot, identified a 15- and 43-fold increase in concentration of mitochondrial proteins AIF and Endo G respectively. Interestingly, pretreatment of M. bovis-infected macrophages with cyclosporine A, a mitochondrial permeability transition pore inhibitor, abolished AIF and Endo G nuclear translocation. In addition, it also decreased macrophage DNA fragmentation to baseline and caused a 26.2% increase in bacterial viability. We also demonstrated that PARP-1 protein expression in macrophages did not change during M. bovis infection. Furthermore, pretreatment of M. bovis-infected bovine macrophages with 3-aminobenzamide, a PARP-1 inhibitor, did not change the proportion of macrophage DNA fragmentation. Our results suggest participation of Endo G, but not PARP-1, in M. bovis-induced macrophage apoptosis. To the best of our knowledge this is the first report associating Endo G with caspase-independent apoptosis induced by a member of the Mycobacterium tuberculosis complex.

Complement Dependent and Independent Interaction Between Bovine Conglutinin and Mycobacterium bovis BCG: Implications in Bovine Tuberculosis.

Bovine conglutinin, the first animal collectin to be discovered, is structurally very similar to Surfactant Protein D (SP-D). SP-D is known to interact with Mycobacterium tuberculosis, and the closely-related M. bovis, the causative agent of bovine tuberculosis. We speculated that due to the overall similarities between conglutinin and SP-D, conglutinin is likely to have a protective influence in bovine tuberculosis. We set out to investigate the role of conglutinin in host-pathogen interaction during mycobacterial infection. We show here that a recombinant truncated form of conglutinin (rfBC), composed of the neck and C-type lectin domains, binds specifically and in a dose-dependent manner to the model organism Mycobacterium bovis BCG. rfBC showed a significant direct bacteriostatic effect on the growth of M. bovis BCG in culture. In addition, rfBC inhibited the uptake of M. bovis BCG by THP-1 macrophages (human monocyte lineage cell line) and suppressed the subsequent pro-inflammatory response. Conglutinin is well-known as a binder of the complement activation product, iC3b. rfBC was also able to inhibit the uptake of complement-coated M. bovis BCG by THP-1 macrophages, whilst modulating the pro-inflammatory response. It is likely that rfBC inhibits the phagocytosis of mycobacteria by two distinct mechanisms: firstly, rfBC interferes with mannose receptor-mediated uptake by masking lipoarabinomannan (LAM) on the mycobacterial surface. Secondly, since conglutinin binds iC3b, it can interfere with complement receptor-mediated uptake via CR3 and CR4, by masking interactions with iC3b deposited on the mycobacterial surface. rfBC was also able to modulate the downstream pro-inflammatory response in THP-1 cells, which is important for mobilizing the adaptive immune response, facilitating containment of mycobacterial infection. In conclusion, we show that conglutinin possesses complement-dependent and complement-independent anti-mycobacterial activities, interfering with both known mechanisms of mycobacterial uptake by macrophages. As mycobacteria are specialized intracellular pathogens, conglutinin may inhibit M. bovis and M. tuberculosis from establishing an intracellular niche within macrophages, and thus, negatively affect the long-term survival of the pathogen in the host.

Pathway analysis of differentially expressed genes in Mycobacterium bovis challenged bovine macrophages.

The immune signalling genes during the challenge of bovine macrophages with bacterial products derived from tuberculosis causing bacteria in cattle were investigated in the present study. An in-vitro cell culture model of bovine monocyte-derived macrophages were challenged to Mycobacterium bovis. Macrophages from healthy and already infected animals can both be fully activated during M. bovis infection. Analysis of mRNA abundance in peripheral blood mononuclear cells from M. bovis infected and non-infected cattle were performed as a controls. Cells of treatment were challenged after six days for six hours incubation at 37 °C, with 5% CO2, to total RNA was extracted then cDNA labelling, hybridization and scanning for microarray methods have been developed for microarray based immune related gene expression analysis. The differential expressions twenty genes (IL1, CCL3, CXCR4, TNF, TLR2, IL12, CSF3, CCR5, CCR3, MAPT, NFKB1, CCL4, IL6, IL2, IL23A, CCL20, IL8, CXCL8, TRIP10, CXCL2 and IL1B) implicated in M. bovis response were examined Agilent Bovine_GXP_8 × 60 K microarray platform. Cells of treatment were challenged after six days for six hours incubation then pathways analysis of Toll like receptor and Chemokine signalling pathway study of responsible genes in bovine tuberculosis. The PBMC from M. bovis infected cattle exhibit different transcriptional profiles compared with PBMC from healthy control animals in response to M. bovis antigen stimulation, providing evidence of a novel genes expression program due to M. bovis exposure. It will guide future studies, regarding the complex macrophage specific signalling pathways stimulated upon phagocytosis of M. bovis and role of signalling pathways in creating the host immune response to cattle tuberculosis.

Measuring bovine γδ T cell function at the site of Mycobacterium bovis infection.

Bovine γδ T cells are amongst the first cells to accumulate at the site of Mycobacterium bovis infection; however, their role in the developing lesion remains unclear. We utilized transcriptomics analysis, in situ hybridization, and a macrophage/γδ T cell co-culture system to elucidate the role of γδ T cells in local immunity to M. bovis infection. Transcriptomics analysis revealed that γδ T cells upregulated expression of several novel, immune-associated genes in response to stimulation with M. bovis antigen. BCG-infected macrophage/γδ T cell co-cultures confirmed the results of our RNAseq analysis, and revealed that γδ T cells from M. bovis-infected animals had a significant impact on bacterial viability. Analysis of γδ T cells within late-stage M. bovis granulomas revealed significant expression of IFN-γ and CCL2, but not IL-10, IL-22, or IL-17. Our results suggest γδ T cells influence local immunity to M. bovis through cytokine secretion and direct effects on bacterial burden.

Immunotherapeutic effect of BCG-polysaccharide nucleic acid powder on Mycobacterium tuberculosis-infected mice using microneedle patches.

Polysaccharide nucleic acid fractions of bacillus Calmette-Guérin, termed BCG-PSN, have traditionally been used as immunomodulators in the treatment of dermatitis and allergic diseases. While the sales of injectable BCG-PSN have shown steady growth in recent years, no reports of using BCG-PSN powder or its immunotherapeutic effects exist. Here, BCG-PSN powder was applied directly to the skin to evaluate the immunotherapeutic effects on mice infected with Mycobacterium tuberculosis (MTB). In total, 34 µg of BCG-PSN powder could be loaded into a microneedle patch (MNP). Mice receiving BCG-PSN powder delivered via MNP exhibited significantly increased IFN-γ and TNF-α production in peripheral blood CD4 + T cells and improved pathological changes in their lungs and spleens compared to control group mice. The immunotherapeutic effect of BCG-PSN powder delivered via MNP was better than that delivered via intramuscular injection to some extent. Furthermore, MNPs eliminate the side effects of syringes, and this study demonstrated that BCG-PSN can be clinically administrated in powder form.

Immunohistochemical detection of pro-inflammatory and anti-inflammatory cytokines in granulomas in cattle with natural Mycobacterium bovis infection.

Cellular immune response was evaluated in lymph nodes and lung with different granulomatous lesions from cattle naturally infected with Mycobacterium bovis. For this purpose, we assessed pro-inflammatory and anti-inflammatory cytokines by immunohistochemical assays. Immunoreaction was observed for all the cytokines analyzed. Fourteen animals displayed advanced stage IV granulomas, with intense immunoreactivity to IFN-γ and TGF-ß in areas of caseous necrosis, macrophages and lymphocytes. Seven animals showed stage III granuloma, with high immunoreactivity to IFN-γ (average of 44.5% immunoreactive cells) and moderate to TNF-α and to the anti-inflammatory cytokines IL-10 and TGF-ß, in relation to the proliferation of fibroblasts in granuloma periphery We found satellite stage I granulomas in 4 bovines and stage II granulomas in 2 bovines, which exhibited low immunostaining response (-13%). Cytokine expression in stage III and IV granulomas was significant, with predominance of immunoreactivity to IFN-γ, thus suggesting a strong, longstanding local immune response mediated by macrophages and epithelioid cells. In addition, these two stages displayed lower reactivity to IL-10; which suggests a deficit of anti-inflammatory cytokines, suppressed immunity and persistence of the infection. High expression of TGF-ß could indicate a chronic process with greater tissue damage and fibrosis. Numerous bacilli observed in necrotic areas in stage III and IV granulomas with low expression of IL-1ß suggest failure in the immune response with bacterial multiplication. In this study, evidence of in situ presence of cytokines demonstrates these cytokines are involved in the development and evolution of bovine tuberculosis granulomas.