Mycobacterium avium complex activates nuclear factor kappaB via induction of inflammatory cytokines

BACKGROUND: Ultrasonography of the knee is a non-invasive, readily available and low-cost tool for demonstrating peri-articular tissues. OBJECTIVE: To correlate clinical features with US findings in the detection, quantification and follow-up of inflammatory signs of the knee in children with pauci-articular juvenile rheumatoid arthritis (JRA). MATERIALS AND METHODS: US of both knees was performed in 49 patients on the same day as the clinical examination. All joints were classified into two groups by clinical criteria: group A (active disease) or group B (quiescent disease). Thirteen patients underwent one or more follow-up examinations. US was performed with a small-parts, 7.5-MHz, electronic linear probe by using a technique previously reported. Quantitative assessment of any effusion and synovial thickening was evaluated at the level of the suprapatellar bursa. Wilcoxon and Spearman tests were employed to compare US findings between the two groups and to correlate clinical and US findings within each group, respectively. RESULTS: US demonstrated significant increase of effusion and synovial thickening in group A joints. US enabled visualisation of clinically undetected popliteal cysts in three patients. Correlation between clinical and US findings was significant in group A and positive, though not significant, in group B. CONCLUSIONS: US seems to be a sensitive and reliable method for the assessment and monitoring of knee joint involvement in pauci-articular JRA.     

Lipopolysaccharide induction of tissue factor in THP-1 cells involves Jun protein phosphorylation and nuclear factor kappaB nuclear translocation

Many Caenorhabditis elegans genes exist in operons in which polycistronic precursors are processed by cleavage at the 3' ends of upstream genes and trans splicing 100 to 400 nucleotides away, at the 5' ends of downstream genes, to generate monocistronic messages. Of the two spliced leaders, SL1 is trans spliced to the 5' ends of upstream genes, whereas SL2 is reserved for downstream genes in operons. However, there are isolated examples of what appears to be a different sort of operon, in which trans splicing is exclusively to SL1 and there is no intercistronic region; the polyadenylation signal is only a few base pairs upstream of the trans-splice site. We have analyzed the processing of an operon of this type by inserting the central part of mes-6/cks-1 into an SL2-type operon. In this novel context, cks-1 is trans spliced only to SL1, and mes-6 3'-end formation occurs normally, demonstrating that this unique mode of processing is indeed intrinsic to this kind of operon, which we herein designate "SL1-type." An exceptionally long polypyrimidine tract found in the 3' untranslated regions of the three known SL1-type operons is shown to be required for the accumulation of both upstream and downstream mRNAs. Mutations of the trans-splice and poly(A) signals indicate that the two processes are independent and in competition, presumably due to their close proximity, raising the possibility that production of upstream and downstream mRNAs is mutually exclusive.     

Cloning and characterization of the ponA gene encoding penicillin-binding protein 1 from Neisseria gonorrhoeae and Neisseria meningitidis

The ponA gene encoding penicillin-binding protein 1 (PBP 1) from Neisseria gonorrhoeae was cloned by a reverse genetic approach. PBP 1 was purified from solubilized membranes of penicillin-susceptible strain FA19 by covalent ampicillin affinity chromatography and used to obtain an NH2-terminal amino acid sequence. A degenerate oligonucleotide based on this protein sequence and a highly degenerate oligonucleotide based on a conserved amino acid motif found in all class A high-molecular-mass PBPs were used to isolate the PBP 1 gene (ponA). The ponA gene encodes a protein containing all of the conserved sequence motifs found in class A PBPs, and expression of the gene in Escherichia coli resulted in the appearance of a new PBP that comigrated with PBP 1 purified from N. gonorrhoeae. A comparison of the gonococcal ponA gene to its homolog isolated from Neisseria meningitidis revealed a high degree of identity between the two gene products, with the greatest variability found at the carboxy terminus of the two deduced PBP 1 protein sequences.     

Essential role of nuclear factor kappaB in the induction of eosinophilia in allergic airway inflammation

The molecular mechanisms that contribute to an eosinophil-rich airway inflammation in asthma are unclear. A predominantly T helper 2 (Th2)-type cell response has been documented in allergic asthma. Here we show that mice deficient in the p50 subunit of nuclear factor (NF)- kappaB are incapable of mounting eosinophilic airway inflammation compared with wild-type mice. This deficiency was not due to a block in T cell priming or proliferation in the p50(-/-) mice, nor was it due to a defect in the expression of the cell adhesion molecules VCAM-1 and ICAM-1 that are required for the extravasation of eosinophils into the airways. The major defects in the p50(-/-) mice were the lack of production of the Th2 cytokine interleukin 5 and the chemokine eotaxin, which are crucial for proliferation and for differentiation and recruitment, respectively, of eosinophils into the asthmatic airway. Additionally, the p50(-/-) mice were deficient in the production of the chemokines macrophage inflammatory protein (MIP)-1alpha and MIP-1beta that have been implicated in T cell recruitment to sites of inflammation. These results demonstrate a crucial role for NF-kappaB in vivo in the expression of important molecules that have been implicated in the pathogenesis of asthma.     

Expression of sialyltransferase is not required for interaction of Neisseria gonorrhoeae with human epithelial cells and human neutrophils

Sialyltransferase (Stase) in Neisseria gonorrhoeae organisms (gonococci [GC]) transfers sialic acid (N-acetylneuraminic acid [NANA]) from cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) mainly to the terminal galactose (Gal) residue in the Gal beta-1,4 N-acetylglucosamine (Gal-GlcNAc)-R lipooligosaccharide (LOS) structure. Sialylated GC resist killing by normal human serum, sometimes show reduced invasion of epithelial cells, and have reduced adhesion to and stimulation of human neutrophils. We questioned whether Stase itself modulates the interactions of GC with human epithelial cells and neutrophils in the absence of exogenous CMP-NANA. To that end, we treated strain F62 with ethyl methanesulfonate and grew approximately 175,000 colonies on CMP-NANA plates, and screened them with monoclonal antibody 1B2-1B7 (MAb 1B2). MAb 1B2 is specific for Gal-GlcNAc and reacts only with asialylated GC. We isolated 13 MAb 1B2-reactive mutants, including five null mutants, that had Stase activities ranging from barely detectable to fivefold less than that of wild-type (WT) F62. The LOS phenotype of Stase null mutants was identical to that of WT F62, yet the mutants could not sialylate their LOS when grown with CMP-NANA. The Stase null phenotype was rescuable to Stase+ by transformation with chromosomal DNA from WT F62. Stase null mutants remained serum sensitive even when grown with CMP-NANA. One Stase null mutant, ST94A, adhered to and invaded the human cervical epithelial cell line ME-180 at levels indistinguishable from that of WT F62 in the absence of CMP-NANA. In human neutrophil studies, ST94A stimulated the oxidative burst in and adhered to human neutrophils at levels similar to those of WT F62. ST94A and WT F62 were also phagocytically killed by neutrophils at similar levels. These results indicate that expression of Stase activity is not required for interaction of GC with human cells.     

Two differently regulated nuclear factor kappaB activation pathways triggered by the cytoplasmic tail of CD40

CD40 signaling modulates the immune response at least in part by activation of nuclear factor kappaB (NFkappaB). It has been shown that two distinct domains in the CD40 cytoplasmic tail (cyt), namely cyt-N and cyt-C, independently activate NFkappaB. Although four members of the tumor necrosis factor receptor-associated factor (TRAF) family, including TRAF2, TRAF3, TRAF5, and TRAF6, bind to the CD40 cyt, how each TRAF protein contributes to the NFkappaB activation by CD40 is not clear. Here we report that TRAF2, TRAF3, and TRAF5 bind cyt-C, whereas TRAF6 binds cyt-N. cyt-N is conserved poorly between human and mouse CD40, while cyt-C is highly conserved. However, single aa substitution of Glu-235 in cyt-N of human CD40 with Ala abolishes the binding of TRAF6 to cyt-N and NFkappaB activation by cyt-N. Conservation of this Glu between mouse and human CD40 strongly suggests that TRAF6 could link cyt-N to signals essential for CD40-mediated immune response. Furthermore, NFkappaB activation by cyt-C is inhibited by a kinase-negative form of NFkappaB-inducing kinase more efficiently than that by cyt-N, consistent with the result that NFkappaB activation by TRAF2 and TRAF5 is inhibited by a kinase-negative form of NFkappaB-inducing kinase more efficiently than that by TRAF6. These results indicate that NFkappaB activating signals emanating from cyt-N and cyt-C are mediated by the different members of the TRAF family and could be regulated in a distinct manner.     

Association and direct activation of signal transducer and activator of transcription1alpha by platelet-derived growth factor receptor

The binding parameters of [3H]nociceptin were examined in membrane preparations of rat heart and compared with those of [3H]dynorphin A-(1-13) ([3H]Dyn A-(1-13)). Scatchard analysis of [3H]nociceptin binding revealed the presence of two distinct sites: a high affinity (Kd: 583 nM) low capacity (Bmax: 132 pmol/mg protein) site and a low affinity (Kd: 10,316 nM) high capacity (1552 pmol/mg protein) site. Dyn A and related peptides were potent competitors of the binding to the high affinity site with the following rank order of potency: alpha-neo-endorphin > Dyn A-(2-13) = Dyn A-(3-13) > Dyn A-(5-13) > Dyn A-(1-13) > Dyn A > Dyn B > Dyn A-(6-10) > Dyn A-(1-8). Nociceptin was 6.7 times less potent than Dyn A with a Ki of 4.8 microM as compared with 0.72 microM for Dyn A. The order of potency of the various peptides in inhibiting [3H]nociceptin binding correlated well (r = 0.93) with their ability to complete with the binding of [3H]Dyn A-(1-13) (Dumont and Lemaire, 1993). In addition, the high affinity [3H]nociceptin and non-opioid [3H]Dyn A-(1-13) sites were both sensitive to NaCl (120 mM) and the phospholipase C (PLC) inhibitors, U-73122 and neomycin (100 microM). The binding activities were less affected by the weak PLC inhibitor, U-73343, and no effect was observed with the non-hydrolysable GTP analogs. Gpp(NH)p and GTP-gamma-S. Nociceptin (1-50 microM) was also shown to inhibit the uptake of [3H]noradrenaline ([3H]NA) by cardiac synaptosomal preparations. In spontaneously hypertensive rats (SHR), the potency of nociceptin in inhibiting [3H]NA uptake was increased by 1.6-fold as compared with Wistar Kyoto (WKY) control rats and such effect was accompanied by comparable increased levels of cardiac ORL1 mRNA and [3H]nociceptin high affinity sites. These changes correlated well with the previously observed increased levels of non-opioid cardiac [3H]Dyn A-(1-13) sites in SHR (1.3 times as compared with WKY) and increased potency of Dyn A-(1-13) in inhibiting [3H]NA uptake by cardiac synaptosomes in SHR (2.2-fold as compared with WKY) (Dumont and Lemaire, 1995). The results demonstrate that in rat heart the characteristics of the high affinity, low capacity [3H]nociceptin binding site are similar to those of the non-opioid Dyn binding site. The stimulation of this site by nociceptin, Dyn A or related peptides is more likely to produce a modulation of PLC activity and [3H]NA uptake and may participate to the pathophysiology of hypertension.     

Synergistic upregulation of metalloproteinase-9 by growth factors and inflammatory cytokines: an absolute requirement for transcription factor NF-kappa B

BACKGROUND: OG-VI is a solution composed of 30 mmol/l inosine, 30 mmol/l sodium 5'-guanylate, 30 mmol/l cytidine, 22.5 mmol/l uridine and 7.5 mmol/l thymidine; it limits myocardial stunning in dogs. We examined whether adenosine A1 receptors were involved in the mechanism of action of OG-VI. METHODS: Dogs anesthetized with pentobarbital were subjected to 20 min of left anterior descending coronary artery ligation followed by 30 min of reperfusion. Saline, OG-VI in several doses, adenosine or inosine was infused at 0.1 ml/kg/min, starting 30 min before the ischemia. In some experiments, 1 or 3 mg/kg 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective adenosine A1 receptor antagonist, was injected intravenously 15 min before the start of the OG-VI infusion. The percentage myocardial segment shortening (%SS) was measured by sonomicrometry. The tissue concentration of ATP was measured in the 30-min-reperfused hearts. RESULTS: In the saline group, %SS that had been decreased by ischemia returned toward pre-ischemic values after reperfusion, although the metabolic recovery was incomplete, with a low concentration of ATP. The %SS was almost completely restored by 12 and 1.2 mumol/kg/min OG-VI, but 0.4 mumol/kg/min was less effective. Administration of adenosine or inosine did not modify the changes in %SS during ischemia/reperfusion. Pretreatment with DPCPX worsened the recovery of %SS during reperfusion after ischemia in both the saline and the OG-VI groups. Infusion of DPCPX (3 mg/kg) with saline caused the animals to die shortly after the onset of ischemia. However, the enhancement of %SS recovery during OG-VI reperfusion was observed in the presence of DPCPX. CONCLUSION: OG-VI improves the recovery of %SS during reperfusion after brief ischemia in a dose-dependent manner. This effect is not brought about by stimulation of adenosine A1 receptors.     

T cell activation through the CD43 molecule leads to Vav tyrosine phosphorylation and mitogen-activated protein kinase pathway activation

CD43, the most abundant membrane protein of T lymphocytes, is able to initiate signals that lead to Ca2+ mobilization and interleukin-2 production, yet the molecular events involved in signal transduction pathway of the CD43 molecule are only beginning to be understood. We have shown recently that cross-linking CD43 on the cell surface of human T lymphocytes with the anti-CD43 monoclonal antibody L10 leads to CD43-Fyn kinase interactions and to Fyn phosphorylation on tyrosine residues. This interaction seems to be mediated by the SH3 domain of Fyn and a proline-rich sequence located in the cytoplasmic domain of CD43. Here we show that CD43-specific activation of human T lymphocytes induced tyrosine phosphorylation of the adaptor protein Shc and of the guanine exchange factor Vav, as well as the formation of a macromolecular complex that comprises Shc, GRB2, and Vav. CD43 ligation resulted in enhanced formation of Vav.SLP-76 complexes and in the activation and nuclear translocation of ERK2. Cross-linking of the CD43 molecule in 3T3-CD43(+) cells induced luciferase activity from a construct under the control of the Fos serum responsive element. Altogether, these data suggest that the mitogen-activated protein kinase pathway is involved in CD43-dependent interleukin-2 gene expression.     

Monocyte chemotactic protein 1 regulates oral tolerance induction by inhibition of T helper cell 1-related cytokines

Intracerebroventricular (i.c.v.) choline (50-150 microg) increased blood pressure and decreased heart rate in spinal cord transected, hypotensive rats. Choline administered intraperitoneally (60 mg/kg), also, increased blood pressure, but to a lesser extent. The pressor response to i.c.v. choline was associated with an increase in plasma vasopressin. Mecamylamine pretreatment (50 microg; i.c.v.) blocked the pressor, bradycardic and vasopressin responses to choline (150 microg). Atropine pretreatment (10 microg; i.c.v.) abolished the bradycardia but failed to alter pressor and vasopressin responses. Hemicholinium-3 [HC-3 (20 microg; i.c.v.)] pretreatment attenuated both bradycardia and pressor responses to choline. The vasopressin V1 receptor antagonist, (beta-mercapto-beta,beta-cyclopenta-methylenepropionyl1, O-Me-Tyr2, Arg8)-vasopressin (10 microg/kg) administered intravenously 5 min after choline abolished the pressor response and attenuated the bradycardia-induced by choline. These data show that choline restores hypotension effectively by activating central nicotinic receptors via presynaptic mechanisms, in spinal shock. Choline-induced bradycardia is mediated by central nicotinic and muscarinic receptors. Increase in plasma vasopressin is involved in cardiovascular effects of choline.