Cytokine-stimulated IκBα degradation is impaired in HT-29 and primary intestinal epithelial cells. To gain more insight into the mechanism of this defect, we dissected cytokine-induced NF-κB signaling pathway in HT-29 cells. IL-1β and TNF, alone or in combination with IFNγ, failed to induce IκBα or IκBβ degradation in HT-29 cells. Despite similar 125I-IL-1β binding, HT-29 cells displayed no IRAK degradation, a 75% reduction of IKK activity, and decreased IκBα phosphorylation, NF-κB DNA binding activity and IL-8 mRNA accumulation in response to IL-1β compared to Caco-2 cells. Selective activation of NF-κB pathway by adenoviral delivery of NF-κB-inducing kinase (Ad5NIK) or IKKβ (Ad5IKKβ) strongly activated IKK activity (>20 fold) in HT-29 cells with concomitant endogenous IκBα serine 32 phosphorylation and total IκBα degradation. In addition, NF-κB DNA binding activity and IL-8 secretion is higher in Ad5NIK-infected than in IL-1β-stimulated HT-29 cells. These data show that altered NF-κB signaling is associated with impaired stimulation of an upstream IKK activator.

Bacterial lipopolysaccharide (LPS) stimulates Kupffer cells and participates in the pathogenesis of alcohol-induced liver injury. However, it is unknown whether LPS directly affects hepatic stellate cells (HSCs), the main fibrogenic cell type in the injured liver. This study characterizes LPS-induced signal transduction and proinflammatory gene expression in activated human HSCs. Culture-activated HSCs and HSCs isolated from patients with hepatitis C virus-induced cirrhosis express LPS-associated signaling molecules, including CD14, toll-like receptor (TLR) 4, and MD2. Stimulation of culture-activated HSCs with LPS results in a rapid and marked activation of NF-kappaB, as assessed by in vitro kinase assays for IkappaB kinase (IKK), IkappaBalpha steady-state levels, p65 nuclear translocation, NF-kappaB-dependent luciferase reporter gene assays, and electrophoretic mobility shift assays. Lipid A induces NF-kappaB activation in a similar manner. Both LPS- and lipid A-induced NF-kappaB activation is blocked by preincubation with either anti-TLR4 blocking antibody (HTA125) or Polymyxin B. Lipid A induces NF-kappaB activation in HSCs from TLR4-sufficient (C3H/OuJ) mice but not from TLR4-deficient (C3H/HeJ) mice. LPS also activates c-Jun N-terminal kinase (JNK), as assessed by in vitro kinase assays. LPS up-regulates IL-8 and MCP-1 gene expression and secretion. LPS-induced IL-8 secretion is completely inhibited by the IkappaB super repressor (Ad5IkappaB) and partially inhibited by a specific JNK inhibitor, SP600125. LPS also up-regulates cell surface expression of ICAM-1 and VCAM-1. In conclusion, human activated HSCs utilize components of TLR4 signal transduction cascade to stimulate NF-kappaB and JNK and up-regulate chemokines and adhesion molecules. Thus, HSCs are a potential mediator of LPS-induced liver injury.

Pathogenic and enteroinvasive bacteria have been shown to trigger the lkappaB/NF-kappaB transcriptional system and proinflammatory gene expression in epithelial cells. In this study, we investigated the molecular mechanism of the commensal Gram-negative Bacteroides vulgatus-induced NF-kappaB signal transduction in intestinal epithelial cells (IEC). We report that B. vulgatus induced interleukin-1 receptor-associated kinase-1 degradation, IkappaBalpha phosphorylation/degradation, RelA and Akt phosphorylation, as well as NF-kappaB DNA binding and NF-kappaB transcriptional activity in rat non-transformed IEC-6 cells. B. vulgatus- but not interleukin-1beta-mediated NF-kappaB transcriptional activity was inhibited by dominant negative (dn) toll-like receptor 4. Of importance, B. vulgatus induced IkappaBalpha phosphorylation/degradation and IKKalpha/beta and RelA phosphorylation in primary IEC derived from germ-free or mono-associated HLA-B27 transgenic and wild type rats, demonstrating the physiological relevance of non-pathogenic bacterial signaling in IEC. Adenoviral delivery of dn IKKbeta or treatment with wortmannin inhibited B. vulgatus-induced endogenous RelA Ser-536 and GST-p65TAD (Ser-529/Ser-536) phosphorylation as well as NF-kappaB transcriptional activity in IEC-6 cells, suggesting a critical role of IKKbeta and phosphatidylinositol 3-kinase/Akt in bacteria-induced RelA phosphorylation and NF-kappaB activation. Interestingly, B. vulgatus-induced IkappaBalpha degradation and NF-kappaB transcriptional activity in IEC transwell cultures were inhibited in the presence of lymphocytes. We propose that non-pathogenic B. vulgatus activates the NF-kappaB signaling pathway through both IkappaB degradation and RelA phosphorylation but that immune cells mediate tolerance of IEC to this commensal bacteria.

In this study, we examined the role of the nuclear factor-kappaB (NF-kappaB)-inducing kinase (NIK) in distinct signaling pathways leading to NF-kappaB activation. We show that a dominant-negative form of NIK (dnNIK) delivered by adenoviral (Ad5dnNIK) vector inhibits Fas-induced IkappaBalpha phosphorylation and NF-kappaB-dependent gene expression in HT-29 and HeLa cells. Interleukin (IL)-1beta- and tumor necrosis factor-alpha (TNF-alpha)-induced NF-kappaB activation and kappaB-dependent gene expression are inhibited in HeLa cells but not in Ad5dnNIK-infected HT-29 cells. Moreover, Ad5dnNIK failed to sensitize HT-29 cells to TNF-alpha-induced apoptosis at an early time point. However, cytokine- and Fas-induced signals to NF-kappaB are finally integrated by the IkappaB kinase (IKK) complex, since IkappaBalpha phosphorylation, NF-kappaB DNA binding activity, and IL-8 gene expression were strongly inhibited in HT-29 and HeLa cells overexpressing dominant-negative IKKbeta (Ad5dnIKKbeta). Our findings support the concept that cytokine signaling to NF-kappaB is redundant at the level of NIK. In addition, this study demonstrates for the first time the critical role of NIK and IKKbeta in Fas-induced NF-kappaB signaling cascade.

The function of nuclear factor (NF)-kappaB within the developing and mature CNS is controversial. We have generated transgenic mice to reveal NF-kappaB transcriptional activity in vivo. As expected, constitutive NF-kappaB activity was observed within immune organs, and tumor necrosis factor-inducible NF-kappaB activity was present in mesenchymal cells. Intriguingly, NF-kappaB activity was also prominent in the CNS throughout development, especially within neocortex, olfactory bulbs, amygdala, and hippocampus. NF-kappaB in the CNS was restricted to neurons and blocked by overexpression of dominant-negative NF-kappaB-inducible kinase or the IkappaBalphaM super repressor. Blocking endogenous neuronal NF-kappaB activity in cortical neurons using recombinant adenovirus induced neuronal death, whereas induction of NF-kappaB activity increased levels of anti-apoptotic proteins and was strongly neuroprotective. Together, these data demonstrate a physiological role for NF-kappaB in maintaining survival of central neurons.

Thirty-eight typical grapevine varieties of the Apulia region, Southern Italy, were genotyped at 6 microsatellite loci (VVS2, VVMD5, VVMD7, VrZAG47, VrZAG62, VrZAG79) with the aim to find synonymy and to confirm some pedigrees reported in literature. Microsatellites were amplified by PCR with P-33-ATP labelled primers and alleles separated by electrophoresis using 6 % acrylamide sequencing gels. The results confirm the high information level of the selected microsatellites. The number of alleles ranged from 7 to 11, producing up to 23 different combination patterns. The observed heterozygosity varied between 81.6 and 94.7 %, the discrimination power between 0.888 and 0.939, and the probability of identity was as low as 0.06-0.12. All cultivars of the study were discriminated from each other, except Regina (syn. Afuz Ali) and Mennavacca, which had the same profile. Finally, we were able to confirm the parentage of Victoria (Cardinal x Afuz Ali) and Matilde (Italia x Cardinal).

The neonatal Fc receptor FcRn functions in rodents to transfer maternal gammaglobulins (IgG) from mother to young via the neonatal intestine.In humans most of this transfer is presumed to occur in utero via the placenta.The fetus swallows amniotic fluid that contains immunogiobulins but it is unknown whether this transfer also occurs via the fetal intestine.Human FcRn has been identified in the human syncitiotrophoblast and plays a role in the materno-fetal transfer of antibody.It is also expressed in adult intestine and may be important in IgG catabolism.Objective: We hypothesize that the FcRn receptor is expressed in the human fetal intestine and may play a role in IgG transport from the amniotic fluid/colostrum into the fetal circulation.We wish to demonstrate expression of the FcRn 1) along different segments of the human fetal intestine and 2) in a human non-malignant fetal intestinal epithelial cell line (H4).Methods: Lysates were prepared from human fetal stomach, small intestine and colon from a 22 week gestation fetus and from H4 cells.Caco-2 cells and T-84 cells were used as positive controls.Prepared lysates were run on Western blots and were probed with anti-FcRn antibodies.Bound antibody was detected by HRP-conjugated secondary antibody and enhanced chemiluminescence.FcRn expression was normalized against GAPDH and quantitated by densitometry.Immunostaining of human fetal intestine, neonatal mouse intestine (+ contro[), H4 cells and T-B4 ceils (+ control), was done with a rabbit anti-human FcRn antibody and detected with the appropriate conjugated secondary antibody for microscopy.Results: The FcRn heavy chain was detected from the fetal intestine and H4 cells on Western blot analysis.On immunostaining, a subapical staining pattern for FcRn was seen on human fetal intestine.The H4 cells also stained positive for FcRn.Conclusion: FcRn expression is demonstrated in the human fetal intestine and in the human non-malignant fetal intestinal epithelial cell line (H4) suggesting that FcRn may play a role in the uptake and transport of IgG in the fetus.

BACKGROUND AND AIMS:Cyclooxygenase (COX)-2 is up-regulated in most colonic cancers and in inflammatory bowel disease in which tumor necrosis factor (TNF)-alpha is believed to play a central role. There has been recent speculation on the activation of phosphatidylinositol 3-kinase (PI 3-kinase) by TNF-alpha and its role in the regulation of genes controlled by NF-kappaB. We investigated the regulatory role of PI 3-kinase on COX-2 expression in colonic epithelial cells.METHODS:In HT-29 and Caco-2 colonic epithelial cells, COX-2 expression was induced by either TNF-alpha or interleukin (IL)-1alpha as observed by Northern and Western analyses. COX-2 activity was assessed by measuring prostaglandin E(2) (PGE2) production by enzyme-linked immunosorbent assay. NF-kappaB binding activity was assessed by electrophoretic mobility shift assay. PI 3-kinase activity was measured by quantifying the accumulation of PI 3-kinase-dependent D-3 lipid products by high-performance liquid chromatography.RESULTS:The PI 3-kinase inhibitor wortmannin up-regulated induced COX-2 expression in a concentration-dependent manner in both HT-29 and Caco-2 cells. An alternative PI 3-kinase inhibitor, LY294002, caused up-regulation of induced COX-2 messenger RNA (mRNA) in HT-29 cells at concentrations of < or =1 micromol/L. IL-4 and IL-13, which are known to activate PI 3-kinase, down-regulated HT-29 COX-2 mRNA, protein, and PGE2 production. NF-kappaB binding activity was unaltered by PI 3-kinase inhibition in HT-29 cells, in which TNF-alpha was shown to activate PI 3-kinase directly.CONCLUSIONS:COX-2 is negatively regulated by PI 3-kinase; we propose that the inhibitory effect of IL-4 and IL-13 is mediated via a PI 3-kinase-dependent pathway. This mechanism does not appear to involve NF-kappaB because PI 3-kinase inhibition did not alter NF-kappaB binding activity. TNF-alpha can activate PI 3-kinase directly in addition to inducing COX-2.

Background: Histamine exerts a broad range of immediate proinflammatory effects in the body.There is growing evidence that mast cells, histamine and other immunomediators associated with a TH2-response may playa role in the pathogenesis of IBD.This study was designed to investigate whether loratadine (10 mg/die), a non-sedating HI-receptor antagonist, may have a positive effect on the course of Ue.Methods: 16 UC patients (CAl> 4) were randomly assigned to receive either prednisolone/5ASAJloratadine or prednisolone/5ASNplacebo over 6 months.In both groups, a standardized conventional steroid tapering regime (starting with 60 mg/day) over 12-16 weeks was used and the dose of 5ASA (3g/day) kept stable during the study period.Clinical activity, cumulative steroid dose, CRP, urine methylhistamine and orosomucoid were recorded at I, 2, 4 and 6 months.Results: After month 2, clinical activity was found to be slightly lower in the loratadine-treated group than in the placebo group.The most impressive effect of loratadine was seen at the cumulative steroid dose.After 2, 4 and 6 months loratadine-treated patients had clearly lower mean cumulative prednisolone doses (23.2, 26.0 and 28.5mg/kg) than untreated patients (24.9, 33.7 and 40.8 mg/kg).In addition, at the same time points C-reactive protein levels were also lower in treated (0.3, 0.37 and 0.83 mg/dl) than untreated UC-patients (0.78, 1.09 and 1.67mg/dl), whereas urinary methylhistamine and serum orosomucoid levels did not differ from another.No significant side effects were recorded with the use of loratadine.Conclusion: This pilot study shows that low-dose HIreceptor antagonist treatment as adjunctive therapy in UC exhibits a clear steroid-sparing effect.In comparison with steroids and 5ASA, HI-receptor blockage appears to mediate additional anti-inflammatory effects in UC.The beneficial effect of 10 mg loratadine/day in UC treated with conventional drugs needs one to two months treatment and may be further augmented in future trials, when using higher daily antihistamine doses.

NF-kappa B plays a critical role in the transcriptional regulation of proinflammatory gene expression in various cells. Cytokine-mediated activation of NF-kappa B requires activation of various kinases, which ultimately leads to the phosphorylation and degradation of I kappa B, the NF-kappa B cytoplasmic inhibitor. The food derivative curcumin has been shown to inhibit NF-kappa B activity in some cell types. In this report we investigate the mechanism of action of curcumin on cytokine-induced proinflammatory gene expression using intestinal epithelial cells (IEC). Curcumin inhibited IL-1 beta-mediated ICAM-1 and IL-8 gene expression in IEC-6, HT-29, and Caco-2 cells. Cytokine-induced NF-kappa B DNA binding activity, RelA nuclear translocation, I kappa B alpha degradation, I kappa B serine 32 phosphorylation, and I kappa B kinase (IKK) activity were blocked by curcumin treatment. Wound-induced p38 phosphorylation was not inhibited by curcumin treatment. In addition, mitogen-activated protein kinase/ERK kinase kinase-1-induced IL-8 gene expression and 12-O-tetraphorbol 12-myristate 13-acetate-responsive element-driven luciferase expression were inhibited by curcumin. However, I kappa B alpha degradation induced by ectopically expressed NF-kappa B-inducing kinase or IKK was not inhibited by curcumin treatment. Therefore, curcumin blocks a signal upstream of NF-kappa B-inducing kinase and IKK. We conclude that curcumin potently inhibits cytokine-mediated NF-kappa B activation by blocking a signal leading to IKK activity.

In neutrophils of a chronic granulomatous disease (CGD) patient with a lack of p67phox the mRNA for p67phox was present in normal amount and size. This mRNA was reverse transcribed, and the coding region was analyzed by single-strand conformation polymorphism analysis. Direct DNA sequencing allowed the identification of a A479-to-T and A481-to-G substitution in exon 5 of the p67phox gene resulting in a double nonconservative amino acid change160Lys-to-Glu and161Asp-to-Val (D160V-K161E). This defect was found in the genomic DNA of this patient in heterozygous state and does not correspond to those previously found in other cases of CGD lacking the p67phox.