New Insights into the Roles of Long Polar Fimbriae and Stg Fimbriae in Salmonella Interactions with Enterocytes and M Cells.

Salmonella enterica serovar Typhi causes the systemic disease typhoid fever. After ingestion, it adheres to and invades the host epithelium while evading the host innate immune response, causing little if any inflammation. Conversely, Salmonella enterica serovar Typhimurium causes gastroenteritis in humans and thrives in the inflamed gut. Upon entering the host, S Typhimurium preferentially colonizes Peyer's patches, a lymphoid organ in which microfold cells (M cells) overlay an arrangement of B cells, T cells, and antigen-presenting cells. Both serovars can adhere to and invade M cells and enterocytes, and it has been assumed that S Typhi also preferentially targets M cells. In this study, we present data supporting the alternative hypothesis that S Typhi preferentially targets enterocytes. Using a tissue culture M cell model, we examined S Typhi strains with a deletion in the stg fimbriae. The stg deletion resulted in increased adherence to M cells and, as expected, decreased adherence to Caco-2 cells. Adherence to M cells could be further enhanced by introduction of the long polar fimbriae (Lpf), which facilitate adherence of S Typhimurium to M cells. Deletion of stg and/or introduction of lpf enhanced M cell invasion as well, leading to significant increases in secretion of interleukin 8. These results suggest that S Typhi may preferentially target enterocytes in vivo.

A cellular model of amyloid precursor protein processing and amyloid-ß peptide production.

BACKGROUND: A hallmark pathologic feature of Alzheimer's disease (AD) is accumulation of neuritic senile plaques in the brain parenchyma. Neurotoxic plaque cores are composed predominantly of amyloid-ß (Aß) peptides of 40 and 42 amino acids in length, formed by sequential cleavage of amyloid precursor protein (APP) by ß-, and γ-secretases. There is a great interest in approaches to modulate Aß peptide production and develop therapeutic interventions to reduce Aß levels to halt or slow the progression of neurodegeneration. NEW METHOD: We characterized and present the BE(2)-M17 human neuroblastoma cell line as a novel in vitro model of the APP-cleavage cascade to support future (1) functional studies of molecular regulators in Aß production, and (2) high-throughput screening assays of new pharmacotherapeutics. RESULTS: In BE(2)-M17 cells, both RNA (i.e., RT-PCR, RNA sequencing) and protein analyses (i.e., Western blots, ELISA), show endogenous expression of critical components of the amyloidogenic pathway, APP-cleavage intermediates CTF83 and CTF99, and final cleavage products Aß40 and Aß42. We further report effects of retinoic acid-mediated differentiation on morphology and gene expression in this cell line. COMPARISON WITH EXISTING METHOD(S): In contrast to primary isolates or other cell lines reported in current literature, BE(2)-M17 not only sustains baseline expression of the full contingent of APP-processing components, but also remains stably adherent during culture, facilitating experimental manipulations. CONCLUSIONS: Our evidence supports the use of BE(2)-M17 as a novel, human, cell-based model of the APP processing pathway that offers a potential streamlined approach to dissect molecular functions of endogenous regulatory pathways, and perform mechanistic studies to identify modulators of Aß production.

A chemical analog of curcumin as an improved inhibitor of amyloid Abeta oligomerization.

Amyloid-like plaques are characteristic lesions defining the neuropathology of Alzheimer's disease (AD). The size and density of these plaques are closely associated with cognitive decline. To combat this disease, the few therapies that are available rely on drugs that increase neurotransmission; however, this approach has had limited success as it has simply slowed an imminent decline and failed to target the root cause of AD. Amyloid-like deposits result from aggregation of the Aß peptide, and thus, reducing amyloid burden by preventing Aß aggregation represents an attractive approach to improve the therapeutic arsenal for AD. Recent studies have shown that the natural product curcumin is capable of crossing the blood-brain barrier in the CNS in sufficient quantities so as to reduce amyloid plaque burden. Based upon this bioactivity, we hypothesized that curcumin presents molecular features that make it an excellent lead compound for the development of more effective inhibitors of Aß aggregation. To explore this hypothesis, we screened a library of curcumin analogs and identified structural features that contribute to the anti-oligomerization activity of curcumin and its analogs. First, at least one enone group in the spacer between aryl rings is necessary for measureable anti-Aß aggregation activity. Second, an unsaturated carbon spacer between aryl rings is essential for inhibitory activity, as none of the saturated carbon spacers showed any margin of improvement over that of native curcumin. Third, methoxyl and hydroxyl substitutions in the meta- and para-positions on the aryl rings appear necessary for some measure of improved inhibitory activity. The best lead inhibitors have either their meta- and para-substituted methoxyl and hydroxyl groups reversed from that of curcumin or methoxyl or hydroxyl groups placed in both positions. The simple substitution of the para-hydroxy group on curcumin with a methoxy substitution improved inhibitor function by 6-7-fold over that measured for curcumin.

Inhibition of nuclear factor kappaB activation and cyclooxygenase-2 expression by aqueous extracts of Hispanic medicinal herbs.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a primary choice of therapy for diseases with a chronic inflammatory component. Unfortunately, long-term NSAID therapy is often accompanied by severe side effects, including cardiovascular and gastrointestinal complications. Because of this, there is critical need for identification of new and safer treatments for chronic inflammation to circumvent these side effects. Inflammatory diseases have been successfully remedied with natural herbs by many cultures. To better understand the potential of natural herbs in treating chronic inflammation and to identify their mechanism of action, we have evaluated the anti-inflammatory activities of 20 medicinal herbs commonly used in the Hispanic culture. We have established a standardized method for preparing aqueous extracts (teas) from the selected medicinal herbs and screened for inhibition of tumor necrosis factor-alpha-induced activation of nuclear factor kappaB (NF-kappaB), which is the central signaling pathway of the inflammatory response. A number of herbal teas were identified that exhibited significant anti-inflammatory activity. In particular, tea from the herb commonly called laurel was found to be an especially potent inhibitor of NF-kappaB-dependent cyclooxygenase-2 gene expression and prostaglandin E(2) production in cultured murine macrophages. These findings indicate that laurel tea extract contains potent anti-inflammatory compounds that function by inhibiting the major signal transduction pathway responsible for inducing an inflammatory event. Based on these results, laurel may represent a new, safe therapeutic agent for managing chronic inflammation.

A Jurkat transcriptional reporter cell line for high-throughput analysis of the nuclear factor-kappaB signaling pathway.

The transcription factor, Nuclear Factor-kappaB (NF-kappaB), regulates many genes involved in host immunity and cell survival. Unregulated NF-kappaB activity has been linked to many chronic inflammatory diseases and is an important target for the identification of inhibitors to better manage these disorders. We present a novel screening system to identify NF-kappaB inhibitors that combines sensitive fluorescence detection with medium- to high-throughput flow cytometry (HyperCyt). To validate this approach, we quantified the activation of NF-kappaB by standard flow cytometry and the HyperCyt platform. Results were comparable with regard to EC(50) values for TNFalpha-mediated activation; however, the HyperCyt platform provided more sensitive signal detection and a greater linear range for detection. To demonstrate the usefulness of this screening tool, we identified a novel inhibitor of NF-kappaB activation from a resveratrol-based chemical library. The inhibition of NF-kappaB activation by analog 6q (IC(50) = 19 microm) showed a 3.7-fold improvement over that of resveratrol (IC(50) approximately 70 microm).

Substituted trans-stilbenes can inhibit or enhance the TPA-induced up-regulation of activator protein-1.

BACKGROUND: The activator protein-1 (AP-1) family of transcription factors contributes to regulation of numerous genes involved in proliferation, apoptosis, and tumorigenesis. A wide array of stimuli can activate AP-1, including pro-inflammatory cytokines, growth factors, tumor promoters and stress. Numerous plant polyphenols have been shown to inhibit the activation of AP-1, which often is ascribed to the anti-oxidant properties of these natural products. METHODS: In the present study, a library of substituted trans-stilbenes, including polyphenols, was screened for activity against the TPA-induced activation of AP-1 using the Panomics AP-1 Reporter 293 Stable Cell Line, which is designed for screening potential inhibitors or activators. RESULTS: Several trans-stilbenes were identified that inhibit TPA-induced activation of AP-1, with IC50 values as low as 0.5 microM. Moreover, some other trans-stilbenes were able to enhance the effects of TPA 2 to 3-fold. Many of the trans-stilbenes identified as inhibitors or enhancers are devoid of anti-oxidant properties. CONCLUSION: The ability of trans-stilbenes to inhibit or enhance the effects of TPA does not depend upon their anti-oxidant properties.

Curcumin and resveratrol inhibit nuclear factor-kappaB-mediated cytokine expression in adipocytes.

BACKGROUND: Adipocytes express inflammatory mediators that contribute to the low-level, chronic inflammation found in obese subjects and have been linked to the onset of cardiovascular disorders and insulin resistance associated with type 2 diabetes mellitus. A reduction in inflammatory gene expression in adipocytes would be expected to reverse this low-level, inflammatory state and improve cardiovascular function and insulin sensitivity. The natural products, curcumin and resveratrol, are established anti-inflammatory compounds that mediate their effects by inhibiting activation of NF-kappaB signaling. In the present study, we examined if these natural products can inhibit NF-kappaB activation in adipocytes and in doing so reduce cytokine expression. METHODS: Cytokine (TNF-alpha, IL-1beta, IL-6) and COX-2 gene expression in 3T3-L1-derived adipocytes was measured by quantitative real-time PCR (qRT-PCR) with or without TNFalpha-stimulation. Cytokine protein and prostaglandin E2 (PGE2) expression were measured by ELISA. Effects of curcumin and resveratrol were evaluated by treating TNFalpha-stimulated adipocytes with each compound and 1) assessing the activation state of the NF-kappaB signaling pathway and 2) measuring inflammatory gene expression by qRT-PCR and ELISA. RESULTS: Both preadipocytes and differentiated adipocytes express the genes for TNF-alpha, IL-6, and COX-2, key mediators of the inflammatory response. Preadipocytes were also found to express IL-1beta; however, IL-1beta expression was absent in differentiated adipocytes. TNF-alpha treatment activated NF-kappaB signaling in differentiated adipocytes by inducing IkappaB degradation and NF-kappaB translocation to the nucleus, and as a result increased IL-6 (6-fold) and COX-2 (2.5-fold) mRNA levels. TNF-alpha also activated IL-1beta gene expression in differentiated adipocytes, but had no effect on endogenous TNF-alpha mRNA levels. No detectable TNFalpha or IL-1beta was secreted by adipocytes. Curcumin and resveratrol treatment inhibited NF-kappaB activation and resulted in a reduction of TNF-alpha, IL-1beta, IL-6, and COX-2 gene expression (IC50 = 2 muM) and a reduction of secreted IL-6 and PGE2 (IC50 ~ 20 muM). CONCLUSION: Curcumin and resveratrol are able to inhibit TNFalpha-activated NF-kappaB signaling in adipocytes and as a result significantly reduce cytokine expression. These data suggest that curcumin and resveratrol may provide a novel and safe approach to reduce or inhibit the chronic inflammatory properties of adipose tissue.

Role of adipocyte-derived lipoprotein lipase in adipocyte hypertrophy.

BACKGROUND: A major portion of available fatty acids for adipocyte uptake is derived from lipoprotein lipase (LPL)-mediated hydrolysis of circulating lipoprotein particles. In vivo studies aimed at identifying the precise role of adipocyte-derived LPL in fat storage function of adipose tissue have been unable to provide conclusive evidence due to compensatory mechanisms that activate endogenous fatty acid synthesis. To address this gap in knowledge, we have measured the effect of reducing adipocyte LPL expression on intracellular lipid accumulation using a well-established cultured model of adipocyte differentiation. METHODS: siRNA specific for mouse LPL was transfected into 3T3-L1 adipocytes. Expression of LPL was measured by quantitative real-time PCR and cell surface-associated LPL enzymatic activity was measured by colorimetric detection following substrate (p-nitrophenyl butyrate) hydrolysis. Apolipoprotein CII and CIII expression ratios were also measured by qRT-PCR. Intracellular lipid accumulation was quantified by Nile Red staining. RESULTS: During differentiation of 3T3-L1 pre-adipocytes, LPL mRNA expression increases 6-fold resulting in a 2-fold increase in cell surface-associated LPL enzymatic activity. Parallel to this increase in LPL expression, we found that intracellular lipids increased ~10-fold demonstrating a direct correlation between adipocyte-derived LPL expression and lipid storage. We next reduced LPL expression in adipocytes using siRNA transfections to directly quantify the contributions of adipocyte-derived LPL to lipid storage, This treatment reduced LPL mRNA expression and cell surface-associated LPL enzymatic activity to ~50% of non-treated controls while intracellular lipid levels were reduced by 80%. Exogenous addition of purified LPL (to restore extracellular lipolytic activity) or palmitate (as a source of free fatty acids) to siRNA-treated cells restored intracellular lipid levels to those measured for non-treated controls. We also found that adipocytes express apolipoprotein CII and CIII and, in addition, the apoCII/apoCIII ratio remains largely unchanged in cells with reduced LPL expression. CONCLUSION: We provide evidence that adipocyte-derived LPL is required for efficient fatty acid uptake and storage, and that adipocytes express their own source of apoCII and apoCIII for regulating extracellular LPL activity. These findings demonstrate that adipocytes are capable of producing the necessary enzymatic components and co-factors for efficient lipid storage independent of vascular sources.

Substituted trans-stilbenes, including analogues of the natural product resveratrol, inhibit the human tumor necrosis factor alpha-induced activation of transcription factor nuclear factor kappaB.

The transcription factor nuclear factor kappaB (NF-kappaB), which regulates expression of numerous antiinflammatory genes as well as genes that promote development of the prosurvival, antiapoptotic state is up-regulated in many cancer cells. The natural product resveratrol, a polyphenolic trans-stilbene, has numerous biological activities and is a known inhibitor of activation of NF-kappaB, which may account for some of its biological activities. Resveratrol exhibits activity against a wide variety of cancer cells and has demonstrated activity as a cancer chemopreventive against all stages, i.e., initiation, promotion, and progression. The biological activities of resveratrol are often ascribed to its antioxidant activity. Both antioxidant activity and biological activities of analogues of resveratrol depend upon the number and location of the hydroxy groups. In the present study, phenolic analogues of resveratrol and a series of substituted trans-stilbenes without hydroxy groups were compared with resveratrol for their abilities to inhibit the human tumor necrosis factor alpha-induced (TNF-alpha) activation of NF-kappaB, using the Panomics NF-kappaB stable reporter cell line 293/NF-kappaB-luc. A series of 75 compounds was screened to identify substituted trans-stilbenes that were more active than resveratrol. Dose-response studies of the most active compounds were carried out to obtain IC50 values. Numerous compounds were identified that were more active than resveratrol, including compounds that were devoid of hydroxy groups and were 100-fold more potent than resveratrol. The substituted trans-stilbenes that were potent inhibitors of the activation of NFkappaB generally did not exhibit antioxidant activity. The results from screening were confirmed using BV-2 microglial cells where resveratrol and analogues were shown to inhibit LPS-induced COX-2 expression.

Syndecan-1 mediates internalization of apoE-VLDL through a low density lipoprotein receptor-related protein (LRP)-independent, non-clathrin-mediated pathway.

BACKGROUND: Triacylglyerol-rich very low density lipoprotein (VLDL) particles are the primary carriers of fatty acids in the circulation and as such serve as a rich energy source for peripheral tissues. Receptor-mediated uptake of these particles is dependent upon prior association with apolipoprotein E (apoE-VLDL) and is brought about by cell surface heparan sulfate proteoglycans (HSPG) in some cell types and by the low density lipoprotein receptor-related protein (LRP) in others. Although LRP's role in apoE-VLDL uptake has been well studied, the identity of the HSPG family member that mediates apoE-VLDL uptake has not been established. We investigated if syndecan-1 (Syn-1), a transmembrane cell surface HSPG, is able to mediate the internalization of apoE-VLDL and examined the relationship between Syn-1 and LRP toward apoE-VLDL uptake. For this study, we used a human fibroblast cell line (GM00701) that expresses large amounts of LRP, but possesses no LDL receptor activity to eliminate its contributions toward apoE-VLDL uptake. RESULTS: Although LRP in these cells is fully active as established by substantial alpha2macroglobulin binding and internalization, uptake of apoE-VLDL is absent. Expression of human Syn-1 cDNA restored apoE-VLDL binding and uptake by these cells. Competition for this uptake with an LRP ligand-binding antagonist had little or no effect, whereas co-incubation with heparin abolished apoE-VLDL internalization. Depleting Syn-1 expressing cells of K+, to block clathrin-mediated endocytosis, showed no inhibition of Syn-1 internalization of apoE-VLDL. By contrast, treatment of cells with nystatin to inhibit lipid raft function, prevented the uptake of apoE-VLDL by Syn-1. CONCLUSION: These data demonstrate that Syn-1 is able to mediate apoE-VLDL uptake in human fibroblasts with little or no contribution from LRP and that the endocytic path taken by Syn-1 is clathrin-independent and relies upon lipid raft function. These data are consistent with previous studies demonstrating Syn-1 association with lipid raft domains.

TPA-induced up-regulation of activator protein-1 can be inhibited or enhanced by analogs of the natural product curcumin.

The activator protein-1 (AP-1) family of transcription factors, including the most common member c-Jun-c-Fos, participates in regulation of expression of numerous genes involved in proliferation, apoptosis, and tumorigenesis in response to a wide array of stimuli including pro-inflammatory cytokines, growth factors, stress, and tumor promoters. A number of plant polyphenols including curcumin, a yellow compound in the spice turmeric, have been shown to inhibit the activation of AP-1. Curcumin is a polyphenolic dienone that is potentially reactive as a Michael acceptor and also is a strong anti-oxidant. Multiple activities reported for curcumin, including inhibition of the stress-induced activation of AP-1, have been suggested to involve the anti-oxidant properties of curcumin. In the present study, a library of analogs of curcumin was screened for activity against the TPA-induced activation of AP-1 using the Panomics AP-1 Reporter 293 stable cell line which is designed for screening potential inhibitors. Numerous analogs were identified that were more active than curcumin, including analogs that were not anti-oxidants and analogs that were not Michael acceptors. Clearly, anti-oxidant activity or reactivity as a Michael acceptor is not an essential feature of active compounds. In addition, a number of analogs were identified that enhanced the TPA-induced activation of AP-1. The results from screening were confirmed using BV-2 microglial cells where curcumin and analogs were shown to inhibit LPS-induced COX-2 expression; analogs identified as more potent than curcumin in the screening assay were also more potent than curcumin in preventing COX-2 expression.