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.

n-3 polyunsaturated fatty acids suppress CD4(+) T cell proliferation by altering phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] organization.

The mechanisms by which n-3 polyunsaturated fatty acids (n-3 PUFA), abundant in fish oil, exert their anti-inflammatory effects have not been rigorously defined. We have previously demonstrated that n-3 PUFA decrease the amount of phosphatidylinositol-(4,5)-bisphosphate, [PI(4,5)P2], in CD4(+) T cells, leading to suppressed actin remodeling upon activation. Since discrete pools of PI(4,5)P2 exist in the plasma membrane, we determined whether n-3 PUFA modulate spatial organization of PI(4,5)P2 relative to raft and non-raft domains. We used Förster resonance energy transfer (FRET) to demonstrate that lipid raft mesodomains in the plasma membrane of CD4(+) T cells enriched in n-3 PUFA display increased co-clustering of Lck(N10) and LAT(ΔCP), markers of lipid rafts. CD4(+) T cells enriched in n-3 PUFA also exhibited a depleted plasma membrane non-raft PI(4,5)P2 pool as detected by decreased co-clustering of Src(N15), a non-raft marker, and PH(PLC-δ), a PI(4,5)P2 reporter. Incubation with exogenous PI(4,5)P2 rescued the effects on the non-raft PI(4,5)P2 pool, and reversed the suppression of T cell proliferation in CD4(+) T cells enriched with n-3 PUFA. Furthermore, CD4(+) T cells isolated from mice fed a 4% docosahexaenoic acid (DHA)-enriched diet exhibited a decrease in the non-raft pool of PI(4,5)P2, and exogenous PI(4,5)P2 reversed the suppression of T cell proliferation. Finally, these effects were not due to changes to post-translational lipidation, since n-3 PUFA did not alter the palmitoylation status of signaling proteins. These data demonstrate that n-3 PUFA suppress T cell proliferation by altering plasma membrane topography and the spatial organization of PI(4,5)P2.

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.

Antagonizing arachidonic acid-derived eicosanoids reduces inflammatory Th17 and Th1 cell-mediated inflammation and colitis severity.

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.

n3 PUFAs reduce mouse CD4+ T-cell ex vivo polarization into Th17 cells.

Little is known about the impact of n3 (ω3) PUFAs on polarization of CD4(+) T cells into effector subsets other than Th1 and Th2. We assessed the effects of dietary fat [corn oil (CO) vs. fish oil (FO)] and fermentable fiber [cellulose (C) vs. pectin (P)] (2 × 2 design) in male C57BL/6 mice fed CO-C, CO-P, FO-C, or FO-P diets for 3 wk on the ex vivo polarization of purified splenic CD4(+) T cells (using magnetic microbeads) into regulatory T cells [Tregs; forkhead box P3 (Foxp3(+)) cells] or Th17 cells [interleukin (IL)-17A(+) and retinoic acid receptor-related orphan receptor (ROR) γτ(+) cells] by flow cytometry. Treg polarization was unaffected by diet; however, FO independently reduced the percentage of both CD4(+) IL-17A(+) (P < 0.05) and CD4(+) RORγτ(+) cells (P < 0.05). Moreover, expression of another critical Th17-cell-related transcription factor, signal transducer and activator of transcription 3, was reduced by FO. Dietary FO reduced the surface expression of both IL-6R and IL-23R on polarized Th17 cells (P ≤ 0.05), thus interfering with the promotive effects of these critical cytokines on Th17 polarization. Additionally, C57BL/6 mice fed diets enriched in eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or DHA + EPA similarly reduced Th17-cell polarization in comparison to CO by reducing expression of the Th17-cell signature cytokine (IL-17A; P = 0.0015) and transcription factor (RORγτ P = 0.02), whereas Treg polarization was unaffected. Collectively, these data show that n3 PUFAs exert a direct effect on the development of Th17 cells in healthy mice, implicating a novel n3 PUFA-dependent, anti-inflammatory mechanism of action via the suppression of the initial development of this inflammatory T-cell subset.

n-3 polyunsaturated fatty acids suppress phosphatidylinositol 4,5-bisphosphate-dependent actin remodelling during CD4+ T-cell activation.

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.

Long-term results of a total knee prosthesis utilising an all polyethylene tibial component.

INTRODUCTION: The aim of this study was to assess the long-term performance of a cemented total knee replacement utilising an All Polyethylene Tibial (APT) component and in addition to perform an engineering analysis of any failures to help refine surgical technique. MATERIALS AND METHODS: A total of 26 patients had a total knee replacement performed using a cemented Depuy Press Fit Condylar (PFC) APT component and a cruciate retaining femoral component. At final review all patients were assessed using The Knee Society Score together with radiographs. An engineering analysis simulated loading conditions of the implants that failed and these were compared with the performance of a modular metal-backed Tibial (MBT) component. RESULTS: A total of 20 patients were reviewed at mean time of 116 months following surgery. Knee Society Knee Scores and Function Scores in this cohort were 84/100 and 58/100, respectively. Two patients required revision for tibial component failure. Pre-operatively both had valgus deformities and in each case the tibial tray had been lateralised leaving a gap on the medial side where the APT component had no rigid support. The engineering analysis demonstrated that the volume of highly strained cancellous bone was greater in the APT design compared with the MBT design when a model with a 3 mm medial gap was loaded. The stiffer MBT base plate acted more rigidly and shielded the stress applied to the proximal tibial cancellous bone. CONCLUSION: The APT component demonstrated satisfactory clinical and radiographic performance at long-term follow up. Appropriate cortical support of the APT component is important. The implant should be used with a degree of caution in patients with severe deformities and osteoporosis.

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.

Th17 cell accumulation is decreased during chronic experimental colitis by (n-3) PUFA in Fat-1 mice.

During colon inflammation, Th17 cells and immunosuppressive regulatory T cells (Treg) are thought to play promotive and preventative roles, respectively. Dietary (n-3) PUFA favorably modulate intestinal inflammation in part by downregulating T-cell activation and functionality. We used the Fat-1 mouse, a genetic model that synthesizes long-chain (n-3) PUFA de novo, to test the hypothesis that (n-3) PUFA protect against colonic inflammation by modulating the polarization of Treg and Th17 cells during colitis. Male and female wild-type (WT) and Fat-1 mice were administered dextran sodium sulfate (DSS) in the drinking water (2.5%) to induce acute (5 d DSS) or chronic (3 cycles DSS) colitis and the percentage of Treg and Th17 cells residing locally [colonic lamina propria (cLP)] and systemically (spleen) was determined by flow cytometry. The percentage of Treg in either tissue site was unaffected by genotype (P > 0.05); however, during chronic colitis, the percentage of Th17 cells residing in both the spleen and cLP was lower in Fat-1 mice compared to WT mice (P < 0.05). Colonic mucosal mRNA expression of critical Th17 cell cytokines and chemokine receptors (IL-17F, IL-21, and CCR6) were lower, whereas expression of the Th17 cell suppressive cytokine, IL-27, was greater in Fat-1 mice compared to WT mice during chronic colitis (P < 0.05). Moreover, colon histological scores were improved in Fat-1 mice (P < 0.05). Collectively, these results demonstrate for the first time, to our knowledge, that (n-3) PUFA can modulate the colonic mucosal microenvironment to suppress Th17 cell accumulation and inflammatory damage following the induction of chronic colitis.

n-3 polyunsaturated fatty acids suppress mitochondrial translocation to the immunologic synapse and modulate calcium signaling in T cells.

Recent studies indicate that the process of Ag presentation induces cytoskeleton-dependent mitochondrial redistribution to the immediate vicinity of the immunologic synapse (IS). This redistribution of mitochondria to the IS in T cells is necessary to maintain Ca(2+) influx and Th cell activation. Recently, we demonstrated that n-3 polyunsaturated fatty acids (PUFAs) suppress the localization and activation of signaling proteins at the IS. Therefore, we hypothesized that n-3 PUFAs suppress CD4(+) T cell mitochondrial translocation during the early stages of IS formation and downmodulate Ca(2+)-dependent Th cell activation. CD4(+) cells derived from fat-1 mice, a transgenic model that synthesizes n-3 PUFA from n-6 PUFA, were cocultured with anti-CD3-expressing hybridoma cells (145-2C11) for 15 min at 37 degrees C, and mitochondrial translocation to the IS was assessed by confocal microscopy. Fat-1 mice exhibited a significantly (p < 0.05) reduced percentage of T cells with mitochondria which translocated to the IS; fat-1 (30%) versus wild type control (82%). Regarding the effect on the mitochondrial-to-cytosolic Ca(2+) ratio, wild type cells showed significant increases at the IS (71%) and total cell (60%) within 30 min of IS formation. In contrast, fat-1 CD4(+) T cells remained at basal levels following the IS formation. A similar blunting of the mitochondrial-to-cytosolic Ca(2+) ratio was observed in wild type cells that were coincubated with inhibitors of the mitochondrial uniporter, RU360 or calcium release-activated Ca(2+) (CRAC) channels, BTP2. These observations provide evidence that n-3 PUFAs modulate Th cell activation by limiting mitochondrial translocation to the IS and reducing Ca(2+) entry.

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.

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.

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.

Novel therapeutic vaccine: granulysin and new DNA vaccine against Tuberculosis.

PURPOSE: Multi-drug resistant (MDR) Mycobacterium Tuberculosis (M.TB) is a big problem in the world. We have developed novel TB therapeutic vaccines. METHODS AND RESULTS: DNA vaccine expressing mycobacterial heat shock protein 65 and IL-12 was delivered by the hemagglutinating virus of Japan (HVJ)-envelope. M. TB, MDR-TB or extremenly drug resistant (XDR-TB) was injected i.v. into DBA/1 mice, and treated with the vaccine three times. This HVJ-E/Hsp65DNA+IL-12DNA vaccine provided strong therapeutic efficacy against MDR-TB and XDR-TB (prolongation of survival time and the decrease in the number of TB) in mice. Therapeutic effect of this vaccine on TB infection was also demonstrated in chronic TB infection murine model using aerosol infection intratracheally. On the other hand, granulysin protein produced from CTL has lethal activity against TB. Granulysin protein vaccine also exerted strong therapeutic effect. Furthermore, we extended our studies to monkey model, which is currently the best animal model of human TB. Hsp65DNA+IL-12 DNA vaccine exerted strong therapeutic efficacy (100% survival and augmentation of immune responses) in the TB-infected monkeys. In contrast, the survival of the saline control group was 60% at 16 week post-challenge. HVJ-Envelope/HSP65 DNA+IL-12 DNA vaccine increased the body weight of TB-infected monkeys, improved the erythrocyte sedimentation rate, and augmentated the immune responses (proliferation of PBL and IL-2 production). The enhancement of IL-2 production from monkeys treated with this vaccine was correlated with the therapeutic efficacy of the vaccine. CONCLUSION: These data indicate that novel vaccines might be useful against TB including XDR-TB and MDR-TB for human therapeutic clinical trials.

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.

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 formation.

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 protein kinase Ctheta, phospholipase Cgamma-1, and F-actin into the IS was suppressed. In addition, both the phosphorylation status of phospholipase Cgamma-1 at the IS and cell proliferation as assessed by CFSE labeling and [(3)H]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.