AdipoRon Attenuates Wnt Signaling by Reducing Cholesterol-Dependent Plasma Membrane Rigidity.

The increasing prevalence of adult and adolescent obesity and its associated risk of colorectal cancer reinforces the urgent need to elucidate the underlying mechanisms contributing to the promotion of colon cancer in obese individuals. Adiponectin is an adipose tissue-derived adipokine, whose levels are reduced during obesity. Both epidemiological and preclinical data indicate that adiponectin suppresses colon tumorigenesis. We have previously demonstrated that both adiponectin and AdipoRon, a small-molecule adiponectin receptor agonist, suppress colon cancer risk in part by reducing the number of Lgr5+ stem cells in mouse colonic organoids. However, the mechanism by which the adiponectin signaling pathway attenuates colon cancer risk remains to be addressed. Here, we have hypothesized that adiponectin signaling supports colonic stem cell maintenance through modulation of the biophysical properties of the plasma membrane (PM). Specifically, we investigated the effects of adiponectin receptor activation by AdipoRon on the biophysical perturbations linked to the attenuation of Wnt-driven signaling and cell proliferation as determined by LEF luciferase reporter assay and colonic organoid proliferation, respectively. Using physicochemical sensitive dyes, Di-4-ANEPPDHQ and C-laurdan, we demonstrated that AdipoRon decreased the rigidity of the colonic cell PM. The decrease in membrane rigidity was associated with a reduction in PM free cholesterol levels and the intracellular accumulation of free cholesterol in lysosomes. These results suggest that adiponectin signaling plays a role in modulating cellular cholesterol homeostasis, PM biophysical properties, and Wnt-driven signaling. These findings are noteworthy because they may in part explain how obesity drives colon cancer progression.

Remodelling of primary human CD4+ T cell plasma membrane order by n-3 PUFA.

Cell membrane fatty acids influence fundamental properties of the plasma membrane, including membrane fluidity, protein functionality, and lipid raft signalling. Evidence suggests that dietary n-3 PUFA may target the plasma membrane of immune cells by altering plasma membrane lipid dynamics, thereby regulating the attenuation of immune cell activation and suppression of inflammation. As lipid-based immunotherapy might be a promising new clinical strategy for the treatment of inflammatory disorders, we conducted in vitro and in vivo experiments to examine the effects of n-3 PUFA on CD4+ T cell membrane order, mitochondrial bioenergetics and lymphoproliferation. n-3 PUFA were incorporated into human primary CD4+ T cells phospholipids in vitro in a dose-dependent manner, resulting in a reduction in whole cell membrane order, oxidative phosphorylation and proliferation. At higher doses, n-3 PUFA induced unique phase separation in T cell-derived giant plasma membrane vesicles. Similarly, in a short-term human pilot study, supplementation of fish oil (4 g n-3 PUFA/d) for 6 weeks in healthy subjects significantly elevated EPA (20 : 5n-3) levels in CD4+ T cell membrane phospholipids, and reduced membrane lipid order. These results demonstrate that the dynamic reshaping of human CD4+ T cell plasma membrane organisation by n-3 PUFA may modulate down-stream clonal expansion.

Distinct Adipose Depots from Mice Differentially Respond to a High-Fat, High-Salt Diet.

BACKGROUND: Dietary factors such as high-sodium or high-fat (HF) diets have been shown to induce a proinflammatory phenotype. However, there is limited information with respect to how microenvironments of distinct intra-abdominal adipose depots respond to the combination of a high-salt, HF diet. OBJECTIVE: We tested the hypothesis that HF feeding would cause changes in distinct adipose depots, which would be further amplified by the addition of high salt to the diet. METHODS: Twenty-seven male C57BL6 mice were fed an HF diet (60% of kcal from fat), an HF + high-salt diet (4% wt:wt), a control diet [low-fat (LF);10% of kcal from fat], or an LF + high-salt diet for 12 wk. The main sources of fat in the diets were corn oil and lard. Adipokines in serum and released from adipose tissue organ cultures were measured by immunoassays. QIAGEN's Ingenuity Pathway Analysis was used to perform functional analysis of the RNA-sequencing data from distinct adipose depots. RESULTS: Diet-induced obesity resulted in a classical inflammatory phenotype characterized by increased concentrations of circulating inflammatory mediators (38-56%) and reduced adiponectin concentrations (27%). However, high-salt feeding did not exacerbate the HF diet-induced changes in adipokines and cytokines. Leptin and interleukin-6 were differentially released from adipose depots and HF feeding impaired adiponectin and resistin secretion across all 3 depots (34-48% and 45-83%, respectively). The addition of high salt to the HF diet did not further modulate secretion in cultured adipose tissue experiments. Although gene expression data from RNA sequencing indicated a >4.3-fold upregulation of integrin αX (Itgax) with HF feeding in all 3 depots, markers of cellular function were differentially expressed in response to diet across depots. CONCLUSION: Collectively, these findings highlight the role of distinct adipose depots in mice in the development of obesity and emphasize the importance of selecting specific depots to study the effects of therapeutic interventions on adipose tissue function.

Molecular and biochemical characterization of recombinant guinea pig tumor necrosis factor-alpha.

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.

n-3 PUFAs reduce T-helper 17 cell differentiation by decreasing responsiveness to interleukin-6 in isolated mouse splenic CD4⁺ T cells.

Cluster of differentiation 4(+) (CD4(+)) effector T-cell subsets [e.g., T-helper (Th) 1 and Th17] are implicated in autoimmune and inflammatory disorders such as multiple sclerosis, psoriasis, and rheumatoid arthritis. Interleukin (IL)-6 is a pleiotropic cytokine that induces Th17 polarization via signaling through the membrane-bound transducer glycoprotein 130 (GP130). Previously, we demonstrated that n-3 (ω-3) polyunsaturated fatty acids (PUFAs) reduce CD4(+) T-cell activation and differentiation into pathogenic Th17 cells by 25-30%. Here we report that n-3 PUFAs alter the response of CD4(+) T cells to IL-6 in a lipid raft membrane-dependent manner. Naive splenic CD4(+) T cells from fat-1 transgenic mice exhibited 30% lower surface expression of the IL-6 receptor. This membrane-bound receptor is known to be shed during cellular activation, but the release of soluble IL-6 receptor after treatment with anti-CD3 and anti-CD28 was not changed in the CD4(+) T cells from fat-1 mice, suggesting that the decrease in surface expression was not due to ectodomain release. We observed a significant 20% decrease in the association of GP130 with lipid rafts in activated fat-1 CD4(+) T cells and a 35% reduction in GP130 homodimerization, an obligate requirement for downstream signaling. The phosphorylation of signal transducer and activator of transcription 3 (STAT3), a downstream target of IL-6-dependent signaling, was also decreased by 30% in response to exogenous IL-6 in fat-1 CD4(+) T cells. Our results suggest that n-3 PUFAs suppress Th17 cell differentiation in part by reducing membrane raft-dependent responsiveness to IL-6, an essential polarizing cytokine.

Dietary fish oil and DHA down-regulate antigen-activated CD4+ T-cells while promoting the formation of liquid-ordered mesodomains.

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.

Dietary n-3 polyunsaturated fatty acids (PUFA) decrease obesity-associated Th17 cell-mediated inflammation during colitis.

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.

Molecular cloning and expression of the IL-10 gene from guinea pigs.

The Guinea pig (Cavia porcellus) is one of the most relevant small animals for modeling human tuberculosis (TB) in terms of susceptibility to low dose aerosol infection, the organization of granulomas, extrapulmonary dissemination and vaccine-induced protection. It is also considered to be a gold standard for a number of other infectious and non-infectious diseases; however, this animal model has a major disadvantage due to the lack of readily available immunological reagents. In the present study, we successfully cloned a cDNA for the critical Th2 cytokine, interleukin-10 (IL-10), from inbred Strain 2 guinea pigs using the DNA sequence information provided by the genome project. The complete open reading frame (ORF) consists of 537 base pairs which encodes a protein of 179 amino acids. This cDNA sequence exhibited 87% homology with human IL-10. Surprisingly, it showed only 84% homology with the previously published IL-10 sequence from the C4-deficient (C4D) guinea pig, leading us to clone IL-10 cDNA from the Hartley strain of guinea pig. The IL-10 gene from the Hartley strain showed 100% homology with the IL-10 sequence of Strain 2 guinea pigs. In order to validate the only published IL-10 sequence existing in Genbank reported from C4D guinea pigs, genomic DNA was isolated from tissues of C4D guinea pigs. Amplification with various sets of primers showed that the IL-10 sequence reported from C4D guinea pigs contained numerous errors. Hence the IL-10 sequence that is being reported by us replaces the earlier sequence making our IL-10 sequence to be the first one accurate from guinea pig. Recombinant guinea pig IL-10 proteins were subsequently expressed in both prokaryotic and eukaryotic cells, purified and were confirmed by N-terminal sequencing. Polyclonal anti-IL-10 antibodies were generated in rabbits using the recombinant IL-10 protein expressed in this study. Taken together, our results indicate that the DNA sequence information provided by the genome project is useful to directly clone much needed cDNAs necessary to study TB in the guinea pig. The newly cloned guinea pig IL-10 cDNA and recombinant proteins will serve as valuable resources for immunological studies in the guinea pig model of TB and other diseases.

Immunomodulatory action of dietary fish oil and targeted deletion of intestinal epithelial cell PPARδ in inflammation-induced colon carcinogenesis.

The ligand-activated transcription factor peroxisome proliferator-activated receptor (PPAR)-δ is highly expressed in colonic epithelial cells; however, the role of PPARδ ligands, such as fatty acids, in mucosal inflammation and malignant transformation has not been clarified. Recent evidence suggests that the anti-inflammatory/chemoprotective properties of fish oil (FO)-derived n-3 polyunsaturated fatty acids (PUFAs) may be partly mediated by PPARδ. Therefore, we assessed the role of PPARδ in modulating the effects of dietary n-3 PUFAs by targeted deletion of intestinal epithelial cell PPARδ (PPARδ(ΔIEpC)). Subsequently, we documented changes in colon tumorigenesis and the inflammatory microenvironment, i.e., local [mesenteric lymph node (MLN)] and systemic (spleen) T cell activation. Animals were fed chemopromotive [corn oil (CO)] or chemoprotective (FO) diets during the induction of chronic inflammation/carcinogenesis. Tumor incidence was similar in control and PPARδ(ΔIEpC) mice. FO reduced mucosal injury, tumor incidence, colonic STAT3 activation, and inflammatory cytokine gene expression, independent of PPARδ genotype. CD8(+) T cell recruitment into MLNs was suppressed in PPARδ(ΔIEpC) mice. Similarly, FO reduced CD8(+) T cell numbers in the MLN. Dietary FO independently modulated MLN CD4(+) T cell activation status by decreasing CD44 expression. CD11a expression by MLN CD4(+) T cells was downregulated in PPARδ(ΔIEpC) mice. Lastly, splenic CD62L expression was downregulated in PPARδ(ΔIEpC) CD4(+) and CD8(+) T cells. These data demonstrate that expression of intestinal epithelial cell PPARδ does not influence azoxymethane/dextran sodium sulfate-induced colon tumor incidence. Moreover, we provide new evidence that dietary n-3 PUFAs attenuate intestinal inflammation in an intestinal epithelial cell PPARδ-independent manner.

n-3 Fatty acids uniquely affect anti-microbial resistance and immune cell plasma membrane organization.

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.

Dietary fish oil and curcumin combine to modulate colonic cytokinetics and gene expression in dextran sodium sulphate-treated mice.

Both fish oil (FO) and curcumin have potential as anti-tumour and anti-inflammatory agents. To further explore their combined effects on dextran sodium sulphate (DSS)-induced colitis, C57BL/6 mice were randomised to four diets (2 × 2 design) differing in fatty acid content with or without curcumin supplementation (FO, FO+2 % curcumin, maize oil (control, MO) or MO+2 % curcumin). Mice were exposed to one or two cycles of DSS in the drinking-water to induce either acute or chronic intestinal inflammation, respectively. FO-fed mice exposed to the single-cycle DSS treatment exhibited the highest mortality (40 %, seventeen of forty-three) compared with MO with the lowest mortality (3 %, one of twenty-nine) (P = 0·0008). Addition of curcumin to MO increased (P = 0·003) mortality to 37 % compared with the control. Consistent with animal survival data, following the one- or two-cycle DSS treatment, both dietary FO and curcumin promoted mucosal injury/ulceration compared with MO. In contrast, compared with other diets, combined FO and curcumin feeding enhanced the resolution of chronic inflammation and suppressed (P < 0·05) a key inflammatory mediator, NF-κB, in the colon mucosa. Mucosal microarray analysis revealed that dietary FO, curcumin and FO plus curcumin combination differentially modulated the expression of genes induced by DSS treatment. These results suggest that dietary lipids and curcumin interact to regulate mucosal homeostasis and the resolution of chronic inflammation in the colon.

Development of therapeutic and prophylactic vaccine against Tuberculosis using monkey and transgenic mice models.

PURPOSE: BCG is not efficacious against M. tuberculosis (TB) in adult. Therefore, novel TB vaccines were established by using three kinds of animal models (cynomolgus monkey model which is the best animal model of human TB, IL-2R knock out SCID mice as a human immune model, and granulysin transgenic mouse). METHODS AND RESULTS: DNA vaccine expressing TB Hsp65 and IL-12 was delivered by the hemagglutinating virus of Japan (HVJ)-envelope. The BCG prime followed by Hsp65+IL-12/HVJ vaccine boost showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). In contrast, 33% of monkeys were alive in BCG alone group. Furthermore, the prolongation of survival period of the monkey was observed by the combination of BCG and DNA vaccine even when the boost was performed after long-term period (4month) from prime. This combination also improved the erythrocyte sedimentation rate (ESR), increased the body weight, and augmented the proliferation of PBL and IL-12 production at higher levels than BCG alone or saline. Furthermore, this vaccine exerted therapeutic efficacy in IL-2R knock out SCID-PBL/hu mice, which were transplanted with human T cells. Granulysin is an important defensive molecule expressed by human T cells and NK cells and has a cytolytic activity against microbes including Mycobacterium tuberculosis (TB) and tumors. Expression of 15kD (15K) granulysin protein and mRNA in CD8 positive T cells in the patients infected with drug sensitive (TB) or multi-drug resistant (MDR-TB) M. tuberculosis were lower than that in the healthy volunteers, suggesting that granulysin treatment might improve the tuberculous disease in human. Therefore, we established two kinds of granulysin transgenic mice (15K granulysin transgenic mice and 9K granulysin transgenic mice). It was demonstrated that 15K granulysin transgenic mice as well as 9K granulysin transgenic mice exerted in vivo anti-TB effect, including the decrease of the number of TB and augmentation of the CTL activity. These are the first findings which demonstrate in vivo effects of 15K granulysin and 9K granulysin against TB infection. Moreover, DNA vaccine expressing 15K granulysin showed a therapeutic activity against TB in mice. CONCLUSION: These data indicate that monkey, IL-2R gene-knock out SCID-PBL/hu and granulysin transgenic mice models provide useful tools for the development of novel vaccines (HVJ-Envelope/Hsp65 DNA + IL-12 DNA vaccine and granulysin vaccine) against TB.

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 Mycobacterium 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-1ß and TNF-α mRNA expression was significantly upregulated 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-1ß 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.

Incorporation of a dietary omega 3 fatty acid impairs murine macrophage responses to Mycobacterium tuberculosis.

BACKGROUND: 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 IFNgamma-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 IFNgamma, 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 (TNFalpha, IL-6 and MCP-1), and lower expression of co-stimulatory molecules (CD40 and CD86). DHA treatment impaired STAT1 phosphorylation and colocalization of the IFNgamma receptor with lipid rafts, without affecting surface expression of IFNgamma receptor. CONCLUSIONS/SIGNIFICANCE: We conclude that DHA reduces the ability of J774A.1 cells to control M. tuberculosis in response to activation by IFNgamma, by modulation of IFNgamma receptor signaling and function, suggesting that n-3 PUFA-enriched diets may have a detrimental effect on host immunity to tuberculosis.

Pulmonary immunization using antigen 85-B polymeric microparticles to boost tuberculosis immunity.

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.

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.

Transgenic mice enriched in omega-3 fatty acids are more susceptible to pulmonary tuberculosis: impaired resistance to tuberculosis in fat-1 mice.

BACKGROUND. Besides their health benefits, dietary omega-3 fatty acids (n-3 PUFAs) can impair host resistance to intracellular pathogens. Previously, we and others have showed that n-3 PUFA-treated macrophages poorly control Mycobacterium tuberculosis infection in vitro. METHODS. Wild-type and fat-1 transgenic mice were infected with virulent H37Rv M. tuberculosis via the aerosol route. We evaluated bacteriological and histopathological changes in lungs, as well as differences in activation and antimycobacterial capacity in primary macrophages ex vivo. RESULTS. fat-1 mice were more susceptible to tuberculosis, as demonstrated by higher bacterial loads and less robust inflammatory responses in lungs. Macrophages obtained from fat-1 mice were more readily infected with M. tuberculosis in vitro, compared with wild-type macrophages. This impaired bacterial control in cells from fat-1 mice correlated with reduced proinflammatory cytokine secretion, impaired oxidative metabolism, and diminished M. tuberculosis-lysotracker colocalization within phagosomes. CONCLUSIONS. We showed that endogenous production of n-3 PUFAs in fat-1 mice increases their susceptibility to tuberculosis, which could be explained in part by diminished activation and antimycobacterial responses in cells from fat-1 mice. These data suggest that n-3 PUFA-supplemented diets might have a detrimental effect on immunity to M. tuberculosis and raise concerns regarding the safety of omega-3 dietary supplementation in humans.

Ultraviolet radiation reduces resistance to Mycobacterium tuberculosis infection in BCG-vaccinated guinea pigs.

Ultraviolet radiation (UVR) in sunlight suppresses anti-microbial immunity and may impair vaccine inefficacy. To investigate whether UVR reduces the protective efficacy of BCG vaccine, guinea pigs were exposed to a single dose of 5.6, 11.2, or 22.5 kJ/m(2) of UVR either 3 days before or 3 or 28 days after BCG vaccination and then challenged by the aerosol route with virulent Mycobacterium tuberculosis 6 weeks later. Pre-challenge skin test responses to purified protein derivative (PPD) were significantly reduced in the UV-irradiated animals. Similarly, exposure to UVR reduced the proliferation of spleen cells to PPD, altered cytokine mRNA expression in spleen and lung digest cells, and increased the colony forming units (CFU) in the lungs of M. tuberculosis-infected guinea pigs at 5 weeks post-infection. Spleen IFN-gamma, TNF-alpha, IL-10 and IL-12p40 mRNA levels were higher when exposed to 5.6 kJ/m(2) UVR, while the levels were significantly reduced at 11.2 and 22.5 kJ/m(2). Lung cell IL-10 mRNA expression increased at all UVR doses. Thus, UVR exposure a few days before or after BCG vaccination at a distant unirradiated site impaired vaccine-induced resistance against virulent M. tuberculosis in guinea pigs possibly by altering cytokine responses in a UVR dose-dependent manner.

The Yin-Yang of TNFalpha in the guinea pig model of tuberculosis.

Tumour necrosis factor alpha (TNF-alpha) is a prototypic pro-inflammatory cytokine that has a central role in the initial host response to Mycobacterium tuberculosis infection. It is a key player in granuloma formation, macrophage activation, bacterial killing, and pathology in vivo. However, the exact mechanism has not been completely understood. This review summarizes the TNFalpha data acquired from the 'gold standard' guinea pig animal model of tuberculosis. While production of TNFalpha is widely accepted as beneficial to the host response, we have found that this hypothesis is just one side of the story. TNFalpha can up-regulate and down-regulate some key pro-inflammatory cytokines (IFNgamma, IL-12p40) and differentially modulate macrophage activation and intracellular bacterial growth. Neutralization of TNFalpha in vivo allows an antiinflammatory TGFbeta-mediated response to develop. Furthermore, BCG vaccination modulates TNFalpha responses directly in the pulmonary granulomas to reduce tissue damage. The bipolar nature of TNFalpha should be considered as knowledge of this critical molecule continues to grow.

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-kappaB (NF-kappaB) suppression; (ii) suppression of arachidonic acid-cyclooxygenase-derived eicosanoids, primarily prostaglandin E(2)-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.