Anti-inflammatory effects of resveratrol occur via inhibition of lipopolysaccharide-induced NF-κB activation in Caco-2 and SW480 human colon cancer cells.

Resveratrol, a polyphenol abundantly found in grapes and red wine, exhibits beneficial health effects due to its anti-inflammatory properties. In the present study, we evaluated the effect of resveratrol on inflammatory responses induced by lipopolysaccharide (LPS) treatment of human intestinal Caco-2 and SW480 cell lines. In the LPS-treated intestinal cells, resveratrol dose-dependently inhibited the expression of inducible NO synthase (iNOS) mRNA as well as protein expression, resulting in a decreased production of NO. In addition, Toll-like receptor-4 expression was significantly diminished in LPS-stimulated cells after resveratrol pre-treatment. To investigate the mechanisms by which resveratrol reduces NO production and iNOS expression, we examined the activation of inhibitor of κB (IκB) in LPS-stimulated intestinal cells. Results demonstrated that resveratrol inhibited the phosphorylation, as well as the degradation, of the IκB complex. Overall, these results show that resveratrol is able to reduce LPS-induced inflammatory responses by intestinal cells, interfering with the activation of NF-κB-dependent molecular mechanisms.

First identification of Toll-like receptor-4 in avian brain: evolution of lipopolysaccharide recognition and inflammation-dependent responses.

In this work, we examine the effects of lipopolysaccharide (LPS) treatment on nerve cells of chick embryo used as a universal avian model. We demonstrate that LPS leads to a dramatic cell loss in primary cultures of both glia and neurons, isolated from chick embryos. Toxic effects appear to be mediated by the Toll-like receptor (TLR)-4 complex, expressed in both glial and neuronal cells, since after TLR-4 silencing by RNA interference experiments LPS-induced cytotoxicity was prevented. The role of nitric oxide in LPS-induced cell damage has also been investigated. These results demonstrate, for the first time in avian nerve cells, the surface expression of TLR-4 and its role as a pattern recognition receptor involved in LPS-induced cell responses in a similar manner to that observed in mammals.

Chemokine receptor-related viral protein products.

Some Herpes-, Pox- and Irido-virus genes (and the controversial Stealth virus gene) share significant nucleotide sequences with vertebrate chemokine receptors (CKR) genes. In some instances the viral reading frame is the same as in the CKRs, giving rise to similar protein products. In other cases the reading frame is different and the viral protein product is not CKR-like. In yet other instances the segmental alignments between CKR genes and viral genes are more limited. In this article we discuss in detail only the more highly significant alignments. We propose the hypothesis that both CKR and CKR-like viral genes originated from a common ancestral gene. This older ancestor may have differentiated into two sequences, one giving rise to the group of extant CKR genes with relatively low levels of similarity with viruses, and the other to the other extant CKRs and the CKR-like viral products. The two extant proteins of the CKR and viral groups which share the maximum amino acid identities are the human CCR3 and the E1 of the Equid herpes virus 2, with a continuous alignment coverage of 73% of the viral molecule. It is thus proposed that the ancestral sequence giving rise to both CKRs and CKR-like viral products may have been similar to the extant human CCR3 and E1 Equid herpes virus 2.

Giardia and Cryptosporidium in inflowing water and harvested shellfish in a lagoon in Southern Italy.

Giardia and Cryptosporidium spp. are important enteric protozoan pathogens for humans and animals, and have been found to contaminate water as well as edible shellfish all over the world. This is the first study to simultaneously investigate the presence of Giardia and Cryptosporidium in inflowing water and harvested shellfish in a geographically closed environment (Varano Lagoon, Southern Italy). Samples of treated wastewater were collected each month - at the outlet from the treatment plant, and downstream at the inlet into the lagoon - from the channels flowing into the Lagoon, together with specimens of Ruditapes decussatus and Mytilus galloprovincialis from shellfish-farms on the same lagoon. Giardia cysts were found by immunofluorescence (IF) microscopy in 16 out of 21 samples of treated wastewater and in 7 out of 21 samples from downstream water channels, and viable cysts were also detected by a beta-giardin RT-PCR. G. duodenalis Assemblages A and B were identified by small ribosomal subunit (18S-rDNA) and triosephosphate isomerase (tpi)-PCR, followed by sequencing. Cryptosporidium oocysts were found by IF in 5 out of 21 wastewater samples, and in 8 out of 21 samples from water channels. Molecular analysis identified the zoonotic species Cryptosporidium parvum by oocyst wall protein (COWP)-PCR and sequencing. Higher concentrations of Giardia cysts than Cryptosporidium oocysts were registered in almost all wastewater and water samples. IF and molecular testing of shellfish gave negative results for both protozoa. Wastewaters carrying Giardia and Cryptosporidium (oo)cysts are discharged into the Lagoon; however, the shellfish harvested in the same environment were found to be unaffected, thus suggesting that physical, ecological and climatic conditions may prevent contamination of harvested shellfish.

f-Met-Leu-Phe stimulates nitric oxide production in chick embryo neurons: the role of NF-kB.

N-formyl-methionyl-leucyl-phenylalanine (fMLP) is a major chemotactic factor produced by Escherichia coli and other Gram-negative bacteria. In avian models the fMLP effects and the possible expression of FPRs have been poorly investigated. This report demonstrates that fMLP stimulation in vitro is able to elicit significant cellular responses from 10-day chick embryo nerve cells. Cell treatment with 10(-7) M fMLP at 37 degrees C induces a dramatic increase of nitric oxide (NO) production, after 24, 48, and 72 h, respectively. After 72 h of treatment with 10(-7) M fMLP the maximum nuclear translocation of the NF-kB complex protein p65 is visible, corresponding to the greatest NO production. In this context, 72 h of fMLP stimulation lead to a marked expression of the antiapoptotic protein Bcl-2, involved in cell survival. This suggests that activation of the NF-kB complex plays a protective role in chick neuronal cells treated with fMLP, confirmed by the significant neuronal cells degeneration observed after NF-kB inhibition with the specific inhibitor, TPCK. Overall, these data suggest a possible protective mechanism displayed by neurons against toxic molecules, like NO, released after cell exposure to bacterial products.

CD14 major role during lipopolysaccharide-induced inflammation in chick embryo cardiomyocytes.

CD14 is a surface differentiation antigen that functions as a receptor for bacterial lipopolysaccharide. The cellular signaling events that lead to lipopolysaccharide-induced production of inflammatory mediators are the primary cause of myocardial dysfunction observed in sepsis. Here, we evaluated the role of CD14 in chick embryo cardiomyocytes stimulated with lipopolysaccharide. CD14 expression was detected by confocal laser microscopy observation and by immunoblotting analysis. Moreover, we provided evidence for CD14-dependent functional responses of lipopolysaccharide-stimulated cardiomyocytes in terms of tumor necrosis factor (TNF)-alpha and nitric oxide (NO) production. Attenuated TNF-alpha and NO secretion, following anti-CD14 treatment of cardiomyocytes, suggested a role for this receptor in lipopolysaccharide-mediated cell responses. We also evidenced that labeled lipopolysaccharide was internalized and localized next to the Golgi complex, at the level of lysosomes, and in the perinuclear zone. The intracytoplasmatic transport seems to depend on the contractile apparatus, because cell pretreatment with cytochalasin D prevented lipopolysaccharide internalization and reduced both TNF-alpha and NO release. Lipopolysaccharide internalization was dependent on CD14 receptor, since anti-CD14 pre-treatment prevented endotoxin uptake by cardiomyocytes. Results demonstrated: (1) CD14 is expressed on the surface membrane of cardiomyocytes; (2) CD14 is involved in cytoskeletal dependent lipopolysaccharide internalization at specific cytoplasmatic locations; (3) CD14 plays a role in lipopolysaccharide-mediated responses by cardiomyocytes after lipopolysaccharide internalization.

Caspase-dependent apoptosis of the HCT-8 epithelial cell line induced by the parasite Giardia intestinalis.

Giardia intestinalis is a flagellated protozoan which causes enteric disease worldwide. Giardia trophozoites infect epithelial cells of the proximal small intestine and can cause acute or chronic diarrhea. The mechanism of epithelial injury in giardiasis remains unknown. A number of enteric pathogens, including protozoan parasites, are able to induce enterocyte apoptosis. The aim of this work was to assess whether G. intestinalis strain WB clone C6 is able to induce apoptosis in the human intestinal epithelial cell line HCT-8, and to investigate the role of caspases in this process. Results demonstrated that the parasite induces cell apoptosis, as confirmed by DNA fragmentation analysis, detection of active caspase-3 and degradation of the caspase-3 substrate PARP [poly(ADP-ribose) polymerase]. Furthermore, G. intestinalis infection induces activation of both the intrinsic and the extrinsic apoptotic pathways, down-regulation of the antiapoptotic protein Bcl-2 and up-regulation of the proapoptotic Bax, suggesting a possible role for caspase-dependent apoptosis in the pathogenesis of giardiasis.

Modulation of NF-κB activation by resveratrol in LPS treated human intestinal cells results in downregulation of PGE2 production and COX-2 expression.

Resveratrol is a natural phytoalexin present in a variety of plant species, such as grapes and red wine, that is well known for its anti-inflammatory effects. In addition, a cancer chemotherapeutic activity of resveratrol has been described. Here we evaluated the effect of resveratrol on COX-2 and prostaglandin E(2) production in human intestinal cells Caco-2 cells treated with lipopolysaccharide (LPS). Resveratrol concentration-dependently inhibited the expression of COX-2 mRNA in the LPS-treated cells, as well as protein expression, resulting in a decreased production of PGE(2). In order to investigate the mechanisms through which resveratrol exhibited these anti-inflammatory effects, we examined the activation of IκB in LPS-stimulated intestinal cells. Results demonstrated that resveratrol inhibited the translocation of NF-κB p65 subunits from the cytosol to the nucleus, which correlated with its inhibitory effects on IκBα phosphorylation and degradation. These results suggest that the down-regulation of COX-2 and PGE(2) by resveratrol may be related to NF-κB inhibition through the negative regulation of IKK phosphorylation in intestinal cells.

Lipopolysaccharide (LPS) of helicobacter modulates cellular DNA repair systems in intestinal cells.

The epithelium of the intestinal tract is exposed to a variety of genotoxic agents, both exogenous and endogenous, that can injure nuclear and mitochondrial DNA. DNA damage can be repaired by a series of DNA repair enzymes, while defects in this system will make these cells once more susceptible to malignant transformation or cell death. Recent studies suggest that intestinal bacteria may contribute to induce inflammation in individuals afflicted by inflammatory bowel disease (IBD), increasing the risk of developing colon cancer. Accumulating evidence suggests that Helicobacter organisms are linked to IBD as well as to gastric and colon cancer. Therefore, the focus of this study was to evaluate the effect of lipopolysaccharide (LPS) isolated from Helicobacter on modulating the DNA repair system. We used an in vitro model represented by two colon carcinoma cell lines, the DNA repair-proficient SW480 and the DNA repair-deficient LoVo, and transfected with a UVC-irradiated psV-beta-galactosidase plasmid. We observed that LPS, by upregulating the expression of inducible nitric oxide (NO), leads to an increased NO release, demonstrating that LPS is able to interfere with the DNA repair machinery of intestinal cells, thus increasing the risk of permanent genotoxic effects.

Expression of UDP-glucuronosyltransferase 1A6 isoform in Caco-2 cells stimulated with lipopolysaccharide.

Glucuronidation is an important metabolic process of detoxification in all vertebrates. The reaction is catalyzed by a multigene family of UDP-glucuronosyltransferases (UGTs) able to convert many xenobiotics and endobiotics (hydrophobic substances) to inactive, water-soluble glucuronides. The UGTs play a protective role, facilitating the elimination of potentially toxic metabolites via urine, bile and feces; therefore, impairment of UGTs may have important toxicological consequences. The regulation of UGTs during bacterial infection or inflammation is not well described. In this study, we investigated the in vitro effect of lipopolysaccharide (LPS) on the expression of the UGT1A6 isoform in human colon carcinoma Caco-2 cells. Results demonstrated a significant down-regulation of UGT1A6 expression, both in terms of mRNA and protein levels, and a reduced UGT activity after LPS exposure of cell cultures, suggesting a role for endotoxins on UGT regulation mechanisms.

Inflammatory responses in embryonal cardiomyocytes exposed to LPS challenge: an in vitro model of deciphering the effects of LPS on the heart.

This study is focused on the links between the major products of inflammation and cell damage induced by the administration of lipopolysaccharide (LPS) from Salmonella typhimurium in embryonal cardiomyocytes. LPS treatment for 72 hours induced transcription factor NF-kappaB activation, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expression, nitric oxide (NO) and tumor necrosis factor (TNF)-alpha release. Moreover, LPS administration induced a significant cell loss, reversed by the NO-synthases inhibitor, suggesting a relationship between cell damage and iNOS-dependent NO overproduction. Cell death was reversed by the specific NF-kappaB inhibitor, TPCK, whereas COX-2 specific inhibitor determined an increase of cell damage in terms of apoptosis, as observed by YO-PRO immunostaining, DNA laddering analysis and caspase-3 activation. Overall these findings evidenced a selective role for NF-kappaB in mediating NO-induced cell damage and a protective action by COX-2 in LPS-treated embryonal cardiomyocytes. The reflection of these experiments on human cardiac pathology will be discussed.

The HIV-1 Rev binding family of proteins: the dog proteins as a study model.

Various proteins that are required for the building of new complete human immunodeficiency type 1 virions (HIV-1) are coded by unspliced or partly spliced virus-derived mRNAs. HIV-1 has developed special strategies for moving these mRNAs to the cytoplasm to be translated. In the nucleus of the infected cell the virus-derived protein Regulator of expression of viral proteins (Rev) can bind both the viral intron-containing mRNAs and the cellular co-factor HIV-1 Rev binding protein (HRB) and this complex may be shuttled through the nuclear pores. HRB genes have been relatively well conserved during evolution, from Drosophila to humans. However, as a consequence of reading-frame shifts due to nt insertions/deletions, the protein products generated may differ considerably from the prototypal HRB protein, which comprises one Arf-GAP zinc finger domain, several Phenylalanine-Glycine (FG) motifs and four Asparagine-Proline-Phenylalanine (NPF) motifs. This variability is best exemplified by four HRB proteins of the dog, which are discussed here in more detail. The hypothesis is advanced that atypical HRB proteins may not be able to bind Rev and possibly have other, still undetermined, functions. Since the cellular co-factor HRB is essential for viral replication and spread but is not required for cell viability and main bodily functions, it might be an attractive candidate for anti-HIV-1 drug targeting.

Canine leishmaniasis in Southern Italy: a role for nitric oxide released from activated macrophages in asymptomatic infection?

BACKGROUND: Human and canine leishmaniasis (CanL) by Leishmania infantum is endemic in Italy, with a high percentage of infected asymptomatic animals. However, the immune response mechanisms underlying the clinical presentation of CanL have not been fully investigated. Among leishmanicidal molecules produced by activated macrophages, nitric oxide (NO) produced by an inducible NO synthase seems to play an important protective role, but no conclusive data are available. Therefore, NO released by cultured macrophages from dogs with natural Leishmania infection living in an endemic area for CanL was evaluated. METHODS: On the basis of one year's clinical and laboratory follow-up, 22 dogs infected by Leishmania infantum were identified and grouped as: asymptomatic dogs (n = 13) and dogs with symptoms of leishmaniasis (n = 9). Each animal was bled twice at 4-month intervals and macrophage and lymphocyte cultures were obtained from peripheral blood mononuclear cells. Supernatants of L. infantum-infected macrophage cultures, with or without addition of autologous lymphocytes, were assayed for NO production by Griess reaction for nitrites. RESULTS: In the first months of the infection the levels of NO in supernatants of Leishmania-infected macrophages were higher in symptomatic than in asymptomatic dogs, but they were significantly increased in the latter group eight months after the diagnosis of infection. Furthermore, NO release significantly decreased in the presence of autologous lymphocytes in both groups of animals. CONCLUSION: These results suggest that NO may be involved in the long-term protection of dogs against natural Leishmania infection and in the clinical presentation of canine leishmaniasis in the Mediterranean area.

Toll-like receptor-positive cells and recognition of pathogens: how human myeloid dendritic cells respond to in vitro infection with Leishmania infantum.

Dendritic cells (DCs), instructed by the priming signals from microbial factors, can produce interleukin (IL)-12p70 and promote T helper (Th)1 proliferation and interferon (IFN)-gamma production. This event seems to be critical for the containment of infections caused by intracellular pathogens, even including Leishmania infection. In the present in vitro study we have investigated: 1) phagocytic capacities and IL-12 production by human monocyte-derived DCs and macrophages (MØs), infected with Leishmania infantum promastigotes; 2) IFN-gamma production by human CD4+ T cells co-incubated with DCs or macrophages pulsed with live promastigotes. Monocyte-derived myeloid DCs and MØs from healthy donors were infected with live metacyclic Leishmania infantum (MON-1) promastigotes, previously opsonized with 5% autologous serum, at 1:4 cell/parasite ratio. Percentage and index of phagocytosis were calculated after 2, 24 and 48 h of incubation. IL-12 production was evaluated by an ELISA in supernatants from 48 h Leishmania-infected or lipopolysaccharides (LPS)-stimulated DCs and MØs, also in the presence of phytohemagglutinin-activated or inactivated CD4+ T cells. For IFN-gamma production, CD4+ T cells were repeatedly stimulated with DCs or MØs, pulsed with live Leishmania promastigotes or activated with LPS. The number of IFN-gamma-secreting cells was evaluated by an ELISpot assay. Results showed that MØs have a higher phagocytic capacity towards L. infantum promastigotes than DCs. Moreover, unlike MØs, Leishmania-infected DCs were able to release IL-12p70; this production significantly increased in the presence of activated CD4+ T cells. Finally, DCs pulsed with live parasites and added to autologous CD4+ T cells induced a higher number of IFN-gamma-secreting cells than MØs, thus indicating their ability to polarize Th cells toward the Th1 subset. These data indicate that DCs are able to promote protective Th1 immune responses in our experimental model of Leishmania infantum infection, thus representing the grounds for initiating immunoterapeutic and vaccinal strategies.