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1.  Cytokine Profiles in Primary and Secondary Pulmonary Granulomas of Guinea Pigs with Tuberculosis 
The cytokine mRNA profiles of primary (arising from inhaled bacilli) and secondary (arising from hematogenous reseeding of the lung) granulomas from the lung lobes of bacillus Calmette-Guérin (BCG)-vaccinated and unimmunized guinea pigs challenged with virulent Mycobacterium tuberculosis by the pulmonary route were assessed in situ using laser capture microdissection (LCM) at 6 weeks after infection. The challenge dose chosen was so low that some lung lobes did not receive an implant from the airway. In unimmunized guinea pigs, some lobes contained either large, necrotic primary lesions or small, non-necrotic secondary lesions, or both. The lobes of BCG-vaccinated animals contained only non-necrotic primary tubercles, and no secondary lesions were visible. Real-time PCR analysis of the acquired RNA clearly demonstrated that primary tubercles from BCG-vaccinated guinea pigs were overwhelmed with mRNA from the anti-inflammatory cytokine, transforming growth factor (TGF)-β, with some IFN-γ and IL-12p40 mRNA. In contrast, primary lesions from unimmunized animals were dominated by proinflammatory TNF-α mRNA. The cytokine mRNA profile of secondary lesions from unimmunized animals was strikingly similar to the profile of primary lesions from BCG-vaccinated guinea pigs (i.e., a predominance of TGF-β mRNA with some IL-12p40 and IFN-γ mRNA), indicating that the lung lobes from which these lesions were retrieved had been naturally “vaccinated” by the time the bloodborne bacilli returned to the lung at 3 to 4 weeks after infection. Furthermore, cytokine mRNA analysis of splenic granulomas from nonvaccinated and vaccinated animals showed close resemblance to primary granulomas recovered from the lungs of the same animal, that is, high levels of TNF-α mRNA in unimmunized animals, and mostly TGF-β mRNA in BCG-vaccinated guinea pigs. Taken together, these data indicate that mycobacteria returning to the lungs of unimmunized guinea pigs 3 to 4 weeks after infection induce a local cytokine response that is fundamentally different from the response to inhaled bacilli and is reminiscent of the primary response in a vaccinated animal.
PMCID: PMC2274948  PMID: 18032570
guinea pig; vaccine; tuberculosis; granuloma; cytokine
2.  Molecular and Biochemical Characterization of Recombinant Guinea Pig Tumor Necrosis Factor-Alpha 
Mediators of Inflammation  2015;2015:619480.
Tumor necrosis factor alpha (TNF-α) is a cytokine which plays opposing roles in the context of infectious disease pathogenesis. TNF-α is essential for the development of a protective immune response to some pathogens, for example, Mycobacterium tuberculosis, by synergizing with other cytokines. However, exorbitant or uncontrolled TNF-α activity may also drive pathology and disease symptoms in many infectious diseases. In order to elucidate the beneficial and detrimental roles of TNF-α in tuberculosis (TB) and other diseases for which the guinea pig is the small animal model of choice, recombinant guinea pig (rgp)TNF-α has been produced using prokaryotic expression systems. However, it is unknown whether posttranslational modifications which cannot be made in the prokaryotic expression systems may be important for rgpTNF-α structure and function. Therefore, we carried out a comparative study by expressing rgpTNF-α in prokaryotic and eukaryotic expression systems and analyzed the eukaryotic-expressed rgpTNF-α for the presence of posttranslational modifications by subjecting it to NanoLC-MS/MS. We conclude that the eukaryotic-expressed rgpTNF-α lacks posttranslational modifications, and we found no significant difference in terms of the biological activity between prokaryotic- and eukaryotic-expressed rgpTNF-α. Taken together, results from our study show that a prokaryotic expression system can be used for generating large amounts of rgpTNF-α without concern for the biological integrity.
PMCID: PMC4427127  PMID: 25999670
3.  Prokaryotic Expression and In vitro Functional Analysis of IL-1 and MCP-1 from Guinea Pig 
Molecular biotechnology  2013;54(2):312-319.
The Guinea pig (Cavia porcellus) is an excellent animal model for studying human tuberculosis (TB) and also for a number of other infectious and non-infectious diseases. One of the major roadblocks in effective utilization of this animal model is the lack of readily available immunological reagents. In order to address this issue, guinea pig interleukin 1 beta (IL-1β) and monocyte chemo attractant protein-1 (MCP-1) were efficiently cloned and expressed in a prokaryotic expression vector (pET-30a) and the expressed proteins in soluble form from both the genes were confirmed by N-terminal sequencing. The biological activity of recombinant guinea pig IL-1β was demonstrated by its ability to drive proliferation in thymocytes and the recombinant guinea pig MCP-1 exhibited chemotactic activity for guinea pig resident peritoneal macrophages. These biologically active recombinant guinea pig proteins will facilitate an in-depth understanding of the role they play in the immune responses of the guinea pig to TB and other diseases.
PMCID: PMC3594633  PMID: 22744745
assays; guinea pig; IL-1β; MCP-1; proteins
4.  Guinea pig neutrophil-macrophage interactions during infection with Mycobacterium tuberculosis 
We examined the ability of recombinant guinea pig IL-8 (CXCL8) to activate neutrophils upon infection with virulent M. tuberculosis. Using a Transwell insert culture system, contact-independent cell cultures were studied in which rgpIL-8-treated neutrophils were infected with virulent M. tuberculosis in the upper well, and AM were cultured in the lower well. IL-1β and TNF-α mRNA expression was significantly up-regulated by AM. Neutralizing anti-rgpTNF-α polyclonal antibody abrogated the response of AM to supernatants from the rgpIL-8 treated, infected neutrophils, while an anti-rgpIL-8 polyclonal antibody had no effect. This suggests that TNF-α produced by rgpIL-8 treated, infected neutrophils may play an important role in the activation of AM in the early response of the host against M. tuberculosis infection. Significant induction of apoptosis in M. tuberculosis-infected neutrophils was observed as compared to the uninfected neutrophils. Feeding of infected, apoptotic neutrophils to AM induced a significant up-regulation of TNF-α and IL-1β mRNA compared to AM exposed to staurosporine-treated apoptotic neutrophils. Suppressed intracellular mycobacterial growth was also seen in AM fed with infected, apoptotic neutrophils as compared to the AM infected with M. tuberculosis H37Rv alone. Taken together, these data suggest that neutrophil-macrophage interactions may contribute to host defense against M. tuberculosis infection.
PMCID: PMC2952707  PMID: 20685396
Neutrophils; alveolar macrophages; IL-8; tuberculosis; guinea pig
5.  Incorporation of a Dietary Omega 3 Fatty Acid Impairs Murine Macrophage Responses to Mycobacterium tuberculosis 
PLoS ONE  2010;5(5):e10878.
Beside their health benefits, dietary omega 3 polyunsaturated fatty acids (n-3 PUFA) might impair host resistance to Mycobacterium tuberculosis (Mtb) by creating an immunosuppressive environment. We hypothesized that incorporation of n-3 PUFA suppresses activation of macrophage antimycobacterial responses and favors bacterial growth, in part, by modulating the IFNγ-mediated signaling pathway.
Methodology/Principal Findings
Murine macrophage-like J774A.1 cells were incubated with bovine serum albumin (BSA)-conjugated docosahexaenoic acid (DHA; 22:6n-3) or BSA alone, activated with recombinant IFNγ, and infected with a virulent strain (H37Rv) of M. tuberculosis. The fatty acid composition of macrophage membranes was modified significantly by DHA treatment. DHA-treated macrophages were less effective in controlling intracellular mycobacteria and showed impaired oxidative metabolism and reduced phagolysosome maturation. Incorporation of DHA resulted in defective macrophage activation, as characterized by reduced production of pro-inflammatory cytokines (TNFα, IL-6 and MCP-1), and lower expression of co-stimulatory molecules (CD40 and CD86). DHA treatment impaired STAT1 phosphorylation and colocalization of the IFNγ receptor with lipid rafts, without affecting surface expression of IFNγ receptor.
We conclude that DHA reduces the ability of J774A.1 cells to control M. tuberculosis in response to activation by IFNγ, by modulation of IFNγ receptor signaling and function, suggesting that n-3 PUFA-enriched diets may have a detrimental effect on host immunity to tuberculosis.
PMCID: PMC2878322  PMID: 20526363
6.  Neutralization of TNFα alters inflammation in guinea pig tuberculous pleuritis 
Previously, treatment with anti-gpTNFα antibody enhanced TNFα mRNA expression in pulmonary granulomas microdissected from non-vaccinated guinea pigs, and modified splenic granuloma architecture. In this study, pleural fluid, cells, and granulomatous tissues were collected 3, 5, and 8 days post-pleurisy induction in guinea pigs treated with anti-gpTNFα or normal serum control. Neutralizing TNFα reduced the percentage of macrophages in the pleural exudate while increasing the proportions of neutrophils and lymphocytes. Cell associated mycobacterial loads were increased in guinea pigs treated with anti-gpTNFα antibody. Cells from the pleural exudate in both treatment groups at day 3 expressed predominantly TNFα and IFNγ mRNA. By day 5, treatment with anti-gpTNFα antibody significantly reduced TNFα mRNA and increased TGFβ and iNOS mRNA expression, a transition which did not occur in the control group until day 8. TNFα mRNA overwhelmed the cytokine milieu of microdissected pleural granulomas in the control group at day 3 whereas TNFα, IFNγ, and TGFβ mRNA dominated the anti-gpTNFα-treated group. At day 8, granulomas from the control group began shifting towards an anti-inflammatory profile with increased levels of TGFβ mRNA. Neutralization of TNFα hastened the transition to an anti-inflammatory cytokine response in guinea pig pleural granulomas and exudate cells.
PMCID: PMC2744482  PMID: 19389482
7.  The Impact of Mouse Passaging of Mycobacterium tuberculosis Strains prior to Virulence Testing in the Mouse and Guinea Pig Aerosol Models 
PLoS ONE  2010;5(4):e10289.
It has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis and recombinant M. tuberculosis mutants might be reduced due to multiple in vitro passages, and that virulence might be augmented by passage of these strains through mice before quantitative virulence testing in the mouse or guinea pig aerosol models.
Methodology/Principal Findings
By testing three M. tuberculosis H37Rv samples, one deletion mutant, and one recent clinical isolate for survival by the quantitative organ CFU counting method in mouse or guinea pig aerosol or intravenous infection models, we could discern no increase in bacterial fitness as a result of passaging of M. tuberculosis strains in mice prior to quantitative virulence testing in two animal models. Surface lipid expression as assessed by neutral red staining and thin-layer chromatography for PDIM analysis also failed to identify virulence correlates.
These results indicate that animal passaging of M. tuberculosis strains prior to quantitative virulence testing in mouse or guinea pig models does not enhance or restore potency to strains that may have lost virulence due to in vitro passaging. It is critical to verify virulence of parental strains before genetic manipulations are undertaken and comparisons are made.
PMCID: PMC2858211  PMID: 20422019
8.  Role of the dosR-dosS Two-Component Regulatory System in Mycobacterium tuberculosis Virulence in Three Animal Models▿ † 
Infection and Immunity  2008;77(3):1230-1237.
The Mycobacterium tuberculosis dosR gene (Rv3133c) is part of an operon, Rv3134c-Rv3132c, and encodes a response regulator that has been shown to be upregulated by hypoxia and other in vitro stress conditions and may be important for bacterial survival within granulomatous lesions found in tuberculosis. DosR is activated in response to hypoxia and nitric oxide by DosS (Rv3132c) or DosT (Rv2027c). We compared the virulence levels of an M. tuberculosis dosR-dosS deletion mutant (ΔdosR-dosS [ΔdosR-S]), a dosR-complemented strain, and wild-type H37Rv in rabbits, guinea pigs, and mice infected by the aerosol route and in a mouse hollow-fiber model that may mimic in vivo granulomatous conditions. In the mouse and the guinea pig models, the ΔdosR-S mutant exhibited a growth defect. In the rabbit, the ΔdosR-S mutant did not replicate more than the wild type. In the hollow-fiber model, the mutant phenotype was not different from that of the wild-type strain. Our analyses reveal that the dosR and dosS genes are required for full virulence and that there may be differences in the patterns of attenuation of this mutant between the animal models studied.
PMCID: PMC2643651  PMID: 19103767
9.  Studies of a Ring-Cleaving Dioxygenase Illuminate the Role of Cholesterol Metabolism in the Pathogenesis of Mycobacterium tuberculosis 
PLoS Pathogens  2009;5(3):e1000344.
Mycobacterium tuberculosis, the etiological agent of TB, possesses a cholesterol catabolic pathway implicated in pathogenesis. This pathway includes an iron-dependent extradiol dioxygenase, HsaC, that cleaves catechols. Immuno-compromised mice infected with a ΔhsaC mutant of M. tuberculosis H37Rv survived 50% longer than mice infected with the wild-type strain. In guinea pigs, the mutant disseminated more slowly to the spleen, persisted less successfully in the lung, and caused little pathology. These data establish that, while cholesterol metabolism by M. tuberculosis appears to be most important during the chronic stage of infection, it begins much earlier and may contribute to the pathogen's dissemination within the host. Purified HsaC efficiently cleaved the catecholic cholesterol metabolite, DHSA (3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione; kcat/Km = 14.4±0.5 µM−1 s−1), and was inactivated by a halogenated substrate analogue (partition coefficient<50). Remarkably, cholesterol caused loss of viability in the ΔhsaC mutant, consistent with catechol toxicity. Structures of HsaC:DHSA binary complexes at 2.1 Å revealed two catechol-binding modes: bidentate binding to the active site iron, as has been reported in similar enzymes, and, unexpectedly, monodentate binding. The position of the bicyclo-alkanone moiety of DHSA was very similar in the two binding modes, suggesting that this interaction is a determinant in the initial substrate-binding event. These data provide insights into the binding of catechols by extradiol dioxygenases and facilitate inhibitor design.
Author Summary
Mycobacterium tuberculosis, the etiological agent of TB, is the most devastating infectious agent of mortality worldwide: it is carried by one-third of all humans and kills nearly two million people annually. Recent work has established that the pathogen metabolizes cholesterol, although the role of this metabolism in pathogenesis remains unclear. In the current study, we demonstrate that HsaC is a key enzyme in the cholesterol catabolic pathway and that it can be inactivated by compounds that resemble its substrate. Using molecular genetic approaches, we demonstrated that the enzyme is essential for the growth of M. tuberculosis on cholesterol and that a lack of this enzyme impairs the survival of the pathogen in each of two animal models. These studies provide definitive evidence that M. tuberculosis metabolizes cholesterol during infection and that this metabolism occurs during the early stages of infection. The oxygen-utilizing enzymes of the cholesterol catabolic pathway, of which HsaC is but one example, are intriguing potential chemotherapeutic targets, as their inhibition can lead to toxic metabolites, including reactive oxygen species. Overall, our study combines a variety of approaches to provide novel insights into a disease of global importance and into the mechanism of an interesting class of enzymes.
PMCID: PMC2652662  PMID: 19300498
10.  Altered cellular infiltration and cytokine levels during early Mycobacterium tuberculosis sigC mutant infection are associated with late-stage disease attenuation and milder immunopathology in mice 
BMC Microbiology  2008;8:151.
Mouse virulence assessments of certain Mycobacterium tuberculosis mutants have revealed an immunopathology defect in which high tissue CFU counts are observed but the tissue pathology and lethality are reduced. M. tuberculosis mutants which grow and persist in the mouse lungs, but have attenuated disease progression, have the immunopathology (imp) phenotype. The antigenic properties of these strains may alter the progression of disease due to a reduction in host immune cell recruitment to the lungs resulting in disease attenuation and prolonged host survival.
In this study we focused on the mouse immune response to one such mutant; the M. tuberculosis ΔsigC mutant. Aerosol infection of DBA/2 and SCID mice with the M. tuberculosis ΔsigC mutant, complemented mutant and wild type strain showed proliferation of mutant bacilli in mouse lungs, but with decreased inflammation and mortality in DBA/2 mice. SCID mice shared the same phenotype as the DBA/2 mice in response to the ΔsigC mutant, however, they succumbed to the infection faster. Bronchoalveolar lavage (BAL) fluid analysis revealed elevated numbers of infiltrating neutrophils in the lungs of mice infected with wild type and complemented ΔsigC mutant strains but not in mice infected with the ΔsigC mutant. In addition, DBA/2 mice infected with the ΔsigC mutant had reduced levels of TNF-α, IL-1β, IL-6 and IFN-γ in the lungs. Similarly, there was a reduction in proinflammatory cytokines in the lungs of SCID mice. In contrast to the mouse model, the ΔsigC mutant had reduced initial growth in guinea pig lungs. A possible mechanism of attenuation in the ΔsigC mutant may be a reduction in neutrophilic-influx in the alveolar spaces of the lungs, and decreased proinflammatory cytokine secretion. In contrast to mouse data, the M. tuberculosis ΔsigC mutant proliferates slowly in guinea pig lungs, a setting characterized by caseating necrosis.
Our observations suggest that the immunopathology phenotype is associated with the inability to trigger a strong early immune response, resulting in disease attenuation. While macrophages and T cells have been shown to be important in containing M. tuberculosis disease our study has shown that neutrophils may also play an important role in the containment of this organism.
PMCID: PMC2564956  PMID: 18798983

Results 1-10 (10)