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1.  Dietary fish oil and DHA down-regulate antigen-activated CD4+ T-cells while promoting the formation of liquid-ordered mesodomains 
The British journal of nutrition  2013;111(2):254-260.
We have demonstrated previously that n-3 PUFA endogenously produced by fat-1 transgenic mice regulate CD4+ T-cell function by affecting the formation of lipid rafts, liquid-ordered mesodomains in the plasma membrane. In the present study, we tested the effects of dietary sources of n-3 PUFA, i.e. fish oil (FO) or purified DHA, when compared with an n-6 PUFA-enriched maize oil control diet in DO11.10 T-cell receptor transgenic mice. Dietary n-3 PUFA were enriched in CD4+ T-cells, resulting in the increase of the n-3:n-6 ratio. Following antigen-specific CD4+ T-cell activation by B-lymphoma cells pulsed with the ovalbumin 323–339 peptide, the formation of liquid-ordered mesodomains at the immunological synapse relative to the whole CD4+ T-cell, as assessed by Laurdan labelling, was increased (P<0·05) in the FO-fed group. The FO diet also suppressed (P<0·05) the co-localisation of PKCθ with ganglioside GM1 (monosialotetrahexosylganglioside), a marker for lipid rafts, which is consistent with previous observations. In contrast, the DHA diet down-regulated (P<0·05) PKCθ signalling by moderately affecting the membrane liquid order at the immunological synapse, suggesting the potential contribution of the other major n-3 PUFA components of FO, including EPA.
PMCID: PMC4327854  PMID: 23962659
n-3 PUFA; DHA; Fish oil; Lipid rafts; CD4+ T-cells
2.  Immunogenicity of an Electron Beam Inactivated Rhodococcus equi Vaccine in Neonatal Foals 
PLoS ONE  2014;9(8):e105367.
Rhodococcus equi is an important pathogen of foals that causes severe pneumonia. To date, there is no licensed vaccine effective against R. equi pneumonia of foals. The objectives of our study were to develop an electron beam (eBeam) inactivated vaccine against R. equi and evaluate its immunogenicity. A dose of eBeam irradiation that inactivated replication of R. equi while maintaining outer cell wall integrity was identified. Enteral administration of eBeam inactivated R. equi increased interferon-γ production by peripheral blood mononuclear cells in response to stimulation with virulent R. equi and generated naso-pharyngeal R. equi-specific IgA in newborn foals. Our results indicate that eBeam irradiated R. equi administered enterally produce cell-mediated and upper respiratory mucosal immune responses, in the face of passively transferred maternal antibodies, similar to those produced in response to enteral administration of live organisms (a strategy which previously has been documented to protect foals against intrabronchial infection with virulent R. equi). No evidence of adverse effects was noted among vaccinated foals.
PMCID: PMC4143214  PMID: 25153708
3.  Antagonizing Arachidonic Acid-Derived Eicosanoids Reduces Inflammatory Th17 and Th1 Cell-Mediated Inflammation and Colitis Severity 
Mediators of Inflammation  2014;2014:917149.
During colitis, activation of two inflammatory T cell subsets, Th17 and Th1 cells, promotes ongoing intestinal inflammatory responses. n-6 polyunsaturated fatty acid- (PUFA-) derived eicosanoids, such as prostaglandin E2 (PGE2), promote Th17 cell-mediated inflammation, while n-3 PUFA antagonize both Th17 and Th1 cells and suppress PGE2 levels. We utilized two genetic mouse models, which differentially antagonize PGE2 levels, to examine the effect on Th17 cells and disease outcomes in trinitrobenzene sulfonic acid- (TNBS-) induced colitis. Fat-1 mice contain the ω3 desaturase gene from C. elegans and synthesize n-3 PUFA de novo, thereby reducing the biosynthesis of n-6 PUFA-derived eicosanoids. In contrast, Fads1 Null mice contain a disrupted Δ5 desaturase gene and produce lower levels of n-6 PUFA-derived eicosanoids. Compared to Wt littermates, Fat-1 and Fads1 Null mice exhibited a similar colitic phenotype characterized by reduced colonic mucosal inflammatory eicosanoid levels and mRNA expression of Th17 cell markers (IL-17A, RORγτ, and IL-23), decreased percentages of Th17 cells and, improved colon injury scores (P ≤ 0.05). Thus, during colitis, similar outcomes were obtained in two genetically distinct models, both of which antagonize PGE2 levels via different mechanisms. Our data highlight the critical impact of n-6 PUFA-derived eicosanoids in the promotion of Th17 cell-mediated colonic inflammation.
PMCID: PMC4127240  PMID: 25136149
4.  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
5.  n-3 Fatty acids uniquely affect anti-microbial resistance and immune cell plasma membrane organization 
Chemistry and physics of lipids  2011;164(7):626-635.
It is now well established that dietary lipids are incorporated into macrophage and T-cell membrane microdomains, altering their structure and function. Within cell membranes, there are specific detergent-resistant domains in which key signal transduction proteins are localized. These regions are classified as “lipid rafts”. Rafts are composed mostly of cholesterol and sphingolipids and therefore do not integrate well into the fluid phospholipid bilayers causing them to form microdomains. Upon cell activation, rafts compartmentalize signal-transducing molecules, thus providing an environment conducive to signal transduction. In this review, we discuss recent novel data describing the effects of n-3 PUFA on alterations in the activation and functions of macrophages and T-cells. We believe that the modifications in these two disparate immune cell types are linked by fundamentally similar changes in membrane lipid composition and transmembrane signaling functions. We conclude that the outcomes of n-3 PUFA-mediated immune cell alterations may be beneficial (e.g., anti-inflammatory) or detrimental (e.g., loss of microbial immunity) depending upon the cell type interrogated.
PMCID: PMC3183364  PMID: 21798252
Macrophage; tuberculosis; phagosome maturation; lipid rafts; polyunsaturated fatty acids; fish oil; fat-1 mice
6.  n – 3 polyunsaturated fatty acids suppress phosphatidylinositol 4,5-bisphosphate-dependent actin remodelling during CD4+ T-cell activation 
The Biochemical journal  2012;443(1):10.1042/BJ20111589.
n – 3 PUFA (polyunsaturated fatty acids), i.e. DHA (docosahexaenoic acid), found in fish oil, exhibit anti-inflammatory properties; however, the molecular mechanisms remain unclear. Since PtdIns(4,5)P2 resides in raft domains and DHA can alter the size of rafts, we hypothesized that PtdIns(4,5)P2 and downstream actin remodelling are perturbed by the incorporation of n – 3 PUFA into membranes, resulting in suppressed T-cell activation. CD4+ T-cells isolated from Fat-1 transgenic mice (membranes enriched in n – 3 PUFA) exhibited a 50% decrease in PtdIns(4,5)P2. Upon activation by plate-bound anti-CD3/anti-CD28 or PMA/ionomycin, Fat-1 CD4+ T-cells failed to metabolize PtdIns(4,5)P2. Furthermore, actin remodelling failed to initiate in Fat-1 CD4+ T-cells upon stimulation; however, the defect was reversed by incubation with exogenous PtdIns(4,5)P2. When Fat-1 CD4+ T-cells were stimulated with anti-CD3/anti-CD28-coated beads, WASP (Wiskott–Aldrich syndrome protein) failed to translocate to the immunological synapse. The suppressive phenotype, consisting of defects in PtdIns(4,5)P2 metabolism and actin remodelling, were recapitulated in CD4+ T-cells isolated from mice fed on a 4% DHA triacylglycerol-enriched diet. Collectively, these data demonstrate that n – 3 PUFA, such as DHA, alter PtdIns(4,5)P2 in CD4+ T-cells, thereby suppressing the recruitment of WASP to the immunological synapse, and impairing actin remodelling in CD4+ T-cells.
PMCID: PMC3814172  PMID: 22250985
actin remodelling; immunological synapse; n – 3 polyunsaturated fatty acid; PtdIns(4,5)P2; T-cell activation; Wiskott–Aldrich syndrome protein
7.  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
8.  Dietary n-3 Polyunsaturated Fatty Acids (PUFA) Decrease Obesity-Associated Th17 Cell-Mediated Inflammation during Colitis 
PLoS ONE  2012;7(11):e49739.
Clinical and experimental evidence suggests that obesity-associated inflammation increases disease activity during colitis, attributed in part to the effects of Th17 cells. Using a model of concurrent obesity and colitis, we monitored changes in critical immune cell subsets and inflammatory biomarker expression in three key tissues: visceral adipose tissue, colon (local inflammatory site) and spleen (systemic inflammatory site), and we hypothesized that n-3 PUFA would reduce the percentage of inflammatory immune cell subsets and suppress inflammatory gene expression, thereby improving the disease phenotype. Obesity was induced in C57BL/6 mice by feeding a high fat (HF) diet (59.2% kcal) alone or an isocaloric HF diet supplemented with fish oil (HF-FO) for 12 weeks. Colitis was induced via a 2.5% trinitrobenzene sulfonic acid (TNBS) enema. The HF-FO diet improved the obese phenotype by reducing i) serum hormone concentrations (leptin and resistin), ii) adipose tissue mRNA expression of inflammatory cytokines (MCP-1, IFNγ, IL-6, IL17F and IL-21) and iii) total (F4/80+ CD11b+) and inflammatory adipose tissue M1 (F4/80+ CD11c+) macrophage content compared to HF (P<0.05). In addition, the HF-FO diet reduced both colitis-associated disease severity and colonic mRNA expression of the Th17 cell master transcription factor (RORγτ) and critical cytokines (IL-6, IL-17A, IL-17F, IL-21, IL-23 and IFNγ) versus HF (P<0.05). Compared to HF, the percentage of both splenic Th17 and Th1 cells were reduced by the HF-FO group (P<0.05). Under ex vivo polarizing conditions, the percentage of HF-FO derived CD4+ T cells that reached Th17 cell effector status was suppressed (P = 0.05). Collectively, these results indicate that n-3 PUFA suppress Th1/Th17 cells and inflammatory macrophage subsets and reconfigure the inflammatory gene expression profile in diverse tissue sites in obese mice following the induction of colitis.
PMCID: PMC3500317  PMID: 23166761
9.  Natural infection of guinea pigs exposed to patients with highly drug-resistant tuberculosis 
A natural TB infection model using guinea pigs may provide useful information for investigating differences in transmission efficiency and establishment of active disease by clinical TB strains in a highly susceptible host under controlled environmental conditions. We sought to examine the capacity of naturally transmitted multidrug-resistant M. tuberculosis to establish infection and produce active disease in guinea pigs. Guinea pigs were continuously exposed for 4 months to the exhaust air of a 6-bed multidrug-resistant tuberculosis inpatient hospital ward in South Africa. Serial tuberculin skin test reactions were measured to determine infection. All animals were subsequently evaluated for histologic disease progression at necropsy. Although 75% of the 362 exposed guinea pigs had positive skin test reactions [≥6mm], only 12% had histopathologic evidence of active disease. Reversions (≥ 6 mm change) in skin test reactivity were seen in 22% of animals, exclusively among those with reactions of 6 to 13 mm. Only two of 86 guinea pigs with reversion had histological evidence of disease compared to 47% (31/66) of guinea pigs with large, non-reverting reactions. Immunosuppression of half the guinea pigs across all skin test categories did not significantly accelerate disease progression. In guinea pigs that reverted a skin test, a second positive reaction in 27 (33%) of them strongly suggested re-infection due to ongoing exposure. These results show that a large majority of guinea pigs naturally exposed to human-source strains of multidrug-resistant tuberculosis became infected, but that many resolved their infection and a large majority failed to progress to detectable disease.
PMCID: PMC3339040  PMID: 21478054
MDR/XDR tuberculosis; guinea pig model; natural infection; skin test reactivity
10.  Regulatory Activity of Polyunsaturated Fatty Acids in T-Cell Signaling 
Progress in lipid research  2010;49(3):250-261.
n-3 polyunsaturated fatty acids (PUFA) are considered to be authentic immunosuppressors and appear to exert beneficial effects with respect to certain immune-mediated diseases. In addition to promoting T-helper 1 (Th1) cell to T-helper 2 (Th2) cell effector T-cell differentiation, n-3 PUFA may also exert anti-inflammatory actions by inducing apoptosis in Th1 cells. With respect to mechanisms of action, effects range from the modulation of membrane receptors to gene transcription via perturbation of a number of second messenger cascades. In this review, the putative targets of anti-inflammatory n-3 PUFA, activated during early and late events of T-cell activation will be discussed. Studies have demonstrated that these fatty acids alter plasma membrane micro-organization (lipid rafts) at the immunological synapse, the site where T-cells and antigen presenting cells (APC) form a physical contact for antigen initiated T-cell signaling. In addition, the production of diacylglycerol and the activation of different isoforms of protein kinase C (PKC), mitogen activated protein kinase (MAPK), calcium signaling, and nuclear translocation/activation of transcriptional factors, can be modulated by n-3 PUFA. Advantages and limitations of diverse methodologies to study the membrane lipid raft hypothesis, as well as apparent contradictions regarding the effect of n-3 PUFA on lipid rafts will be critically presented.
PMCID: PMC2872685  PMID: 20176053
11.  Pulmonary Immunization Using Antigen 85-B Polymeric Microparticles to Boost Tuberculosis Immunity 
The AAPS Journal  2010;12(3):338-347.
This study aims to evaluate immunization with polymeric microparticles containing recombinant antigen 85B (rAg85B) delivered directly to the lungs to protect against tuberculosis. rAg85B was expressed in Escherichia coli and encapsulated in poly(lactic-co-glycolic acid) microparticles (P-rAg85B). These were delivered as dry powders to the lungs of guinea pigs in single or multiple doses of homologous and heterologous antigens. Bacille Calmette–Guérin (BCG) delivered subcutaneously was employed as the positive control and as part of immunization strategies. Immunized animals were challenged with a low-dose aerosol of Mycobacterium tuberculosis (MTB) H37Rv to assess the extent of protection measured as reduction in bacterial burden (CFU) in the lungs and spleens of guinea pigs. Histopathological examination and morphometric analysis of these tissues were also performed. The heterologous strategy of BCG prime–P-rAg85B aerosol boosts appeared to enhance protection from bacterial infection, as indicated by a reduction in CFU in both the lungs and spleens compared with untreated controls. Although the CFU data were not statistically different from the BCG and BCG–BCG groups, the histopathological and morphometric analyses indicated the positive effect of BCG–P-rAg85B in terms of differences in area of tissue affected and number and size of granulomas observed in tissues. P-rAg85B microparticles appeared to be effective in boosting a primary BCG immunization against MTB infection, as indicated by histopathology and morphometric analysis. These encouraging observations are relevant to boosting adults previously immunized with BCG or exposed to MTB, commonly the case in the developing world, and should be followed by further assessment of an appropriate immunization protocol for maximum protection.
PMCID: PMC2895436  PMID: 20422340
antigen 85B; BCG; boost immunization; microparticles; pulmonary delivery
12.  n-3 polyunsaturated fatty acids - physiological relevance of dose 
n-3 polyunsaturated fatty acids (PUFA) are widely used for chemotheraphy/chemoprevention of chronic diseases. However, the molecular mechanism(s) by which the bioactive n-3 PUFA (eicosapentaenoic acid and docosahexaenoic acid) modulate effector pathways are not fully elucidated. Multiple experimental approaches, including use of animal models, cell lines, and human clinical trials, have been utilized to dissect the complex effectors. It is imperative to link these different experimental approaches together in order to interpret outcomes in the context of human physiology and pathophysiology. Unfortunately, the adoption of a broad array of model systems and a wide range of fatty acid exposures (i.e. doses) has made it difficult to interpret biological outcomes. Therefore, in this mini-review we discuss the impact of (a) molecular structure of bioactive fatty acids, (b) dose relevance relative to human consumption, (c) enrichment of fatty acids in sera and tissues following dietary intake, and (d) limitations of cell/tissue culture studies.
PMCID: PMC2875929  PMID: 20188532
13.  Novel Prophylactic Vaccine Using a Prime-Boost Method and Hemagglutinating Virus of Japan-Envelope against Tuberculosis 
Objective. Mycobacterium tuberculosis infection is a major global threat to human health. The only tuberculosis (TB) vaccine currently available is bacillus Calmette-Guérin (BCG), although it has no efficacy in adults. Therefore, the development of a novel vaccine against TB for adults is desired. Method. A novel TB vaccine expressing mycobacterial heat shock protein 65 (HSP65) and interleukin-12 (IL-12) delivered by the hemagglutinating virus of Japan- (HVJ)- envelope was evaluated against TB infection in mice. Bacterial load reductions and histopathological assessments were used to determine efficacy. Results. Vaccination by BCG prime with IgHSP65+murine IL-12/HVJ-envelope boost resulted in significant protective efficacy (>10, 000-fold versus BCG alone) against TB infection in the lungs of mice. In addition to bacterial loads, significant protective efficacy was demonstrated by histopathological analysis of the lungs. Furthermore, the vaccine increased the number of T cells secreting IFN-γ. Conclusion. This vaccine showed extremely significant protection against TB in a mouse model, consistent with results from a similar paper on cynomolgus monkeys. The results suggest that further development of the vaccine for eventual testing in clinical trials may be warranted.
PMCID: PMC3061297  PMID: 21437226
14.  Dietary docosahexaenoic and eicosapentaenoic acid: Emerging mediators of inflammation✩ 
The inflammatory response is designed to help fight and clear infection, remove harmful chemicals, and repair damaged tissue and organ systems. Although this process, in general, is protective, the failure to resolve the inflammation and return the target tissue to homeostasis can result in disease, including the promotion of cancer. A plethora of published literature supports the contention that dietary n-3 polyunsaturated fatty acids (PUFA), and eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in particular, are important modulators of a host's inflammatory/immune responses. The following review describes a mechanistic model that may explain, in part, the pleiotropic anti-inflammatory and immunosuppressive properties of EPA and DHA. In this review, we focus on salient studies that address three overarching mechanisms of n-3 PUFA action: (i) modulation of nuclear receptor activation, i.e., nuclear factor-κB (NF-κB) suppression; (ii) suppression of arachidonic acid–cyclooxygenase-derived eicosanoids, primarily prostaglandin E2-dependent signaling; and (iii) alteration of the plasma membrane micro-organization (lipid rafts), particularly as it relates to the function of Toll-like receptors (TLRs), and T-lymphocyte signaling molecule recruitment to the immunological synapse (IS). We propose that lipid rafts may be targets for the development of n-3 PUFA-containing dietary bioactive agents to down-modulate inflammatory and immune responses and for the treatment of autoimmune and chronic inflammatory diseases.
PMCID: PMC2755221  PMID: 19502020
15.  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
16.  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
17.  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
18.  n-3 polyunsaturated fatty acids suppress the localization and activation of signaling proteins at the immunological synapse in murine CD4+ T cells by affecting lipid raft formation1 
The molecular properties of immunosuppressive n-3 polyunsaturated fatty acids (PUFA) have not been fully elucidated. Using CD4+ T cells from wild type control and fat-1 transgenic mice (enriched in n-3 PUFA), we show that membrane raft accumulation assessed by Laurdan (6-dodecanoyl-2-dimethyl aminonaphthalene) labeling was enhanced in fat-1 cells following immunological synapse (IS) formation by CD3-specific Ab expressing hybridoma cells. However, the localization of PKCθ, PLCγ-1 and F-actin into the IS was suppressed. In addition, both the phosphorylation status of PLCγ-1 at the IS and cell proliferation as assessed by CFSE labeling and [3H]-thymidine incorporation were suppressed in fat-1 cells. These data imply that lipid rafts may be targets for the development of dietary agents for the treatment of autoimmune and chronic inflammatory diseases.
PMCID: PMC2597670  PMID: 18941214
T cells; Signal Transduction; Cell Activation; fat-1; immunological synapse; lipid rafts; nutrition
19.  Chemotherapeutic Properties of n-3 Polyunsaturated Fatty Acids - Old Concepts and New Insights 
Over the past several decades, data from both experimental animal studies and human clinical trials have shown that dietary n-3 polyunsaturated fatty acids (PUFA) exhibit anti-inflammatory bioactive properties, compared to n-6 PUFA. Collectively, these studies have identified multiple mechanisms by which n-3 PUFA affect immune cell responses. In this review, we discuss the putative targets of anti-inflammatory n-3 PUFA, specifically, cytokine production, antagonism of n-6 PUFA metabolism, binding to nuclear receptors as ligands, and the alteration of signaling protein acylation. In addition, we investigate the effect of n-3 PUFA on the coalescence of lipid rafts, specialized signaling platforms in the plasma membrane.
PMCID: PMC2759763  PMID: 19823600
Diet; n-3 PUFA; chronic inflammation; CD4+ T-cells
20.  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
21.  fat-1 transgene expression prevents cell culture-induced loss of membrane n-3 fatty acids in activated CD4+ T-cells☆ 
In order to evaluate the effects of fatty acids on immune cell membrane structure and function, it is often necessary to maintain cells in culture. However, cell culture conditions typically reverse alterations in polyunsaturated fatty acid (PUFA) composition achieved by dietary lipid manipulation. Therefore, we hypothesized that T-cells from transgenic mice expressing the Caenorhabditis elegans n-3 desaturase (fat-1) gene would be resistant to the culture-induced loss of n-3 PUFA and, therefore, obviate the need to incorporate fatty acids or homologous serum into the medium. CD4+ T-cells were isolated from (i) control wild type (WT) mice fed a safflower oil-n-6 PUFA enriched diet (SAF) devoid of n-3 PUFA, (ii) fat-1 transgenic mice (enriched with endogenous n-3 PUFA) fed a SAF diet, or (iii) WT mice fed a fish oil (FO) based diet enriched in n-3 PUFA. T-cell phospholipids isolated from WT mice fed FO diet (enriched in n-3 PUFA) and fat-1 transgenic mice fed a SAF diet (enriched in n-6 PUFA) were both enriched in n-3 PUFA. As expected, the mol% levels of both n-3 and n-6 PUFA were decreased in cultures of CD4+ T-cells from FO-fed WT mice after 3 d in culture. In contrast, the expression of n-3 desaturase prevented the culture-induced decrease of n-3 PUFA in CD4+ T-cells from the transgenic mice. Carboxyfluorescein succinidyl ester (CFSE) -labeled CD4+ T-cells from fat-1/SAF vs. WT/SAF mice stimulated with anti-CD3 and anti-CD28 for 3 d, exhibited a reduced (P<0.05) number of cell divisions. We conclude that fat-1-containing CD4+ T-cells express a physiologically relevant, n-3 PUFA enriched, membrane fatty acid composition which is resistant to conventional cell culture-induced depletion.
PMCID: PMC2718534  PMID: 18977126
n-3 fatty acid desaturase; Fish oil; Phospholipids; Lymphocyte
22.  Intranasal Mucosal Boosting with an Adenovirus-Vectored Vaccine Markedly Enhances the Protection of BCG-Primed Guinea Pigs against Pulmonary Tuberculosis 
PLoS ONE  2009;4(6):e5856.
Recombinant adenovirus-vectored (Ad) tuberculosis (TB) vaccine platform has demonstrated great potential to be used either as a stand-alone or a boost vaccine in murine models. However, Ad TB vaccine remains to be evaluated in a more relevant and sensitive guinea pig model of pulmonary TB. Many vaccine candidates shown to be effective in murine models have subsequently failed to pass the test in guinea pig models.
Methods and Findings
Specific pathogen-free guinea pigs were immunized with BCG, AdAg85A intranasally (i.n), AdAg85A intramuscularly (i.m), BCG boosted with AdAg85A i.n, BCG boosted with AdAg85A i.m, or treated only with saline. The animals were then infected by a low-dose aerosol of M. tuberculosis (M.tb). At the specified times, the animals were sacrificed and the levels of infection in the lung and spleen were assessed. In separate studies, the long-term disease outcome of infected animals was monitored until the termination of this study. Immunization with Ad vaccine alone had minimal beneficial effects. Immunization with BCG alone and BCG prime-Ad vaccine boost regimens significantly reduced the level of M.tb infection in the tissues to a similar extent. However, while BCG alone prolonged the survival of infected guinea pigs, the majority of BCG-immunized animals succumbed by 53 weeks post-M.tb challenge. In contrast, intranasal or intramuscular Ad vaccine boosting of BCG-primed animals markedly improved the survival rate with 60% of BCG/Ad i.n- and 40% of BCG/Ad i.m-immunized guinea pigs still surviving by 74 weeks post-aerosol challenge.
Boosting, particularly via the intranasal mucosal route, with AdAg85A vaccine is able to significantly enhance the long-term survival of BCG-primed guinea pigs following pulmonary M.tb challenge. Our results thus support further evaluation of this viral-vectored TB vaccine in clinical trials.
PMCID: PMC2689939  PMID: 19516906
23.  Reduced colitis-associated colon cancer in fat-1 (n-3 fatty acid desaturase) transgenic mice 
Cancer research  2008;68(10):3985-3991.
Bioactive food components containing n-3 polyunsaturated fatty acids (PUFA) modulate multiple determinants which link inflammation to cancer initiation and progression. Therefore, in this study, fat-1 transgenic mice which convert endogenous n-6 PUFA to n-3 PUFA in multiple tissues, were injected with azoxymethane followed by 3 cycles of dextran sodium sulphate (DSS) to induce colitis-associated cancer. fat-1 mice exhibited a reduced number of colonic adenocarcinomas per mouse (1.05±0.29 vs 2.12±0.51, p=0.033), elevated apoptosis (p=0.03) and a decrease in n-6 PUFA-derived eicosanoids, compared to wild type (wt) mice. To determine whether the chemoprotective effects of n-3 PUFA could be attributed to its pleiotropic anti-inflammatory properties, colonic inflammation and injury scores were evaluated 5 d after DSS exposure followed by either a 3 d or 2 wk recovery period. There was no effect of n-3 PUFA at 3 d. However, following a 2 wk recovery period, colonic inflammation and ulceration scores returned to pretreatment levels compared to 3 d recovery only in fat-1 mice. For the purpose of examining the specific reactivity of lymphoid elements in the intestine, CD3+ T cells, CD4+ T helper cells and macrophages from colonic lamina propria were quantified. Comparison of 3 d vs 2 wk recovery time points revealed that fat-1 mice exhibited decreased (p<0.05) CD3+, CD4+ T helper, and macrophage cell numbers per colon as compared to wt mice. These results suggest that the anti-tumorigenic effect of n-3 PUFA may be mediated in part via its anti-inflammatory properties.
PMCID: PMC2648804  PMID: 18483285
inflammation; chemoprevention; dextran sodium sulphate; tumor promotion
24.  Mechanisms by which docosahexaenoic acid and related fatty acids reduce colon cancer risk and inflammatory disorders of the intestine 
Chemistry and physics of lipids  2008;153(1):14-23.
A growing body of epidemiological, clinical, and experimental evidence has underscored both the pharmacological potential and the nutritional value of dietary fish oil enriched in very long chain n-3 PUFAs such as docosahexaenoic acid (DHA, 22:6, n-3) and eicosapentaenoic acid (EPA, 20:5, n-3). The broad health benefits of very long chain n-3 PUFAs and the pleiotropic effects of dietary fish oil and DHA have been proposed to involve alterations in membrane structure and function, eicosanoid metabolism, gene expression and the formation of lipid peroxidation products, although a comprehensive understanding of the mechanisms of action has yet to be elucidated. In this review, we present data demonstrating that DHA selectively modulates the subcellular localization of lipidated signaling proteins depending on their transport pathway, which may be universally applied to other lipidated protein trafficking. An interesting possibility raised by the current observations is that lipidated proteins may exhibit different subcellular distribution profiles in various tissues, which contain a distinct membrane lipid composition. In addition, the current findings clearly indicate that subcellular localization of proteins with a certain trafficking pathway can be subjected to selective regulation by dietary manipulation. This form of regulated plasma membrane targeting of a select subset of upstream signaling proteins may provide cells with the flexibility to coordinate the arrangement of signaling translators on the cell surface. Ultimately, this may allow organ systems such as the colon to optimally decode, respond, and adapt to the vagaries of an ever-changing extracellular environment.
PMCID: PMC2430411  PMID: 18346463
protein targeting; trafficking; fish oil; chemoprevention; colon cancer
25.  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

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