Cytokine upregulation of proteinase-activated-receptors 2 and 4 expression mediated by p38 MAP kinase and inhibitory kappa B kinase beta in human endothelial cells

BACKGROUND AND PURPOSE: Up-regulation of proteinase-activated receptor-2 (PAR2) is a factor in a number of disease states and we have therefore examined the signalling pathways involved in the expression of the receptor. EXPERIMENTAL APPROACH: We investigated the effects of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), trypsin and the PAR2 activating peptide, 2-furoyl(2f)-LIGKV-OH on both mRNA and functional expression of PAR2 in human umbilical vein endothelial cells (HUVECs). The effect of specific chemical inhibitors and dominant negative adenovirus constructs of the mitogen-activated protein kinase (MAPK) cascade and the nuclear factor kappa B (NF-kappaB) signalling pathway was assessed. Methods included semi-quantitative and quantitative RT-PCR, [(3)H]inositol phosphate (IP) accumulation and Ca(2+)-dependent fluorescence. KEY RESULTS: The above agonists induced both mRNA and functional expression of PAR2; PAR4 mRNA, but not that for PAR1 or PAR-3, also increased following TNFalpha treatment. Inhibition of p38 MAP kinase reduced PAR2 and PAR4 expression, whilst inhibition of MEK1/ERK/JNK was without effect. A similar dependency upon p38 MAP kinase was observed for the expression of PAR4. TNFalpha -induced enhancement of PAR2 stimulated [(3)H]-inositol phosphate accumulation (IP) and Ca(2+) signalling was abolished following SB203580 pre-treatment. Infection with adenovirus encoding dominant-negative IKKbeta (Ad.IKKbeta(+/-)) and to a lesser extent dominant-negative IKKalpha (Ad.IKKalpha(+/-)), substantially reduced both control and IL-1beta- induced expression of both PAR2 and PAR4 mRNA and enhancement of PAR2-stimulated IP accumulation and Ca(2+) mobilisation. CONCLUSIONS AND IMPLICATIONS: These data reveal for the first time the signalling events involved in the upregulation of both PAR2 and PAR4 during pro-inflammatory challenge.     

Highly homologous Mycobacterium tuberculosis chaperonin 60 proteins with differential CD14 dependencies stimulate cytokine production by human monocytes through cooperative activation of p38 and ERK1/2 mitogen-activated protein kinases

Tuberculosis is a chronic inflammatory and destructive disease caused by infection with Mycobacterium tuberculosis. We have previously shown that the mycobacterial chaperonin (Cpn)60.1 and 60.2 proteins stimulate human monocytes to secrete pro-inflammatory cytokines. Identification of the cellular mechanisms that contribute to the chronic inflammation characterised by myobacterial infection is therefore of potential therapeutic benefit. In the present study we have investigated the role of the extracellular signal-regulated (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) families in Cpn60-induced cytokine synthesis, and have compared the effects of the bacterial proteins with those of lipopolysaccharide (LPS). Exposure to Cpn60.1, Cpn60.2 or LPS enhanced ERK1/2 activation with increases in phosphorylation evident between 10 and 30 min and maximal after 60-90 min stimulation. Phosphorylation of ERK1/2 in Cpn60-stimulated monocytes was maintained whereas ERK1/2 was rapidly dephosphorylated in LPS-stimulated cells. Exposure to the chaperonins also caused rapid activation of p38(mapk) with kinetics of phosphorylation comparable to those observed in response to LPS. Selective inhibitors of p38(mapk) (SB203580) or of MEK1/2, the direct upstream activator of ERK1/2 (PD98059), reduced the synthesis of IL-1beta, TNFalpha, IL-6 and IL-8 induced by either the chaperonins or LPS. Experiments in which cells were exposed to a combination of both inhibitors led to a nearly complete abrogation of agonist-induced cytokine synthesis. These results show that the p38(mapk) and ERK1/2 signalling pathways are important regulators of the cellular response to mycobacterial chaperonins and that these pathways cooperate to regulate pro-inflammatory cytokine production by human monocytes.     

Epidemiology of frequent attenders: a 3-year historic cohort study comparing attendance, morbidity and prescriptions of one-year and persistent frequent attenders

Background  

General Practitioners spend a disproportionate amount of time on frequent attenders. So far, trials on the effect of interventions on frequent attenders have shown negative results. However, these trials were conducted in short-term frequent attenders. It would be more reasonable to target intervention at persistent frequent attenders. Typical characteristics of persistent frequent attenders, as opposed to 1-year frequent attenders and non-frequent attenders, may generate hypotheses regarding modifiable factors on which new randomized trials may be designed.     

Involvement of PKCα and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of μ-opioid receptors in mature brain neurons

Background and purpose:

The ability of an agonist to induce desensitization of the µ-opioid receptor (MOR) depends upon the agonist used. Furthermore, previous data suggest that the intracellular mechanisms underlying desensitization may be agonist-specific. We investigated the mechanisms underlying MOR desensitization, in adult mammalian neurons, caused by morphine (a partial agonist in this system) and DAMGO (a high-efficacy agonist).

Experimental approach:

MOR function was measured in locus coeruleus neurons, by using whole-cell patch-clamp electrophysiology, in rat and mouse brain slices (both wild-type and protein kinase C (PKC)α knockout mice). Specific isoforms of PKC were inhibited by using inhibitors of the receptors for activated C-kinase (RACK), and in vivo viral-mediated gene-transfer was used to transfect neurons with dominant negative mutants (DNMs) of specific G-protein-coupled receptor kinases (GRKs).

Key results:

Morphine-induced desensitization was attenuated by using RACK inhibitors that inhibit PKCα, but not by other isoform-specific inhibitors. Further, the PKC component of morphine-induced desensitization was absent in locus coeruleus neurons from PKCα knockout mice. The PKC-enhanced morphine-induced desensitization was not affected by over-expression of a GRK2 dominant negative mutant (GRK2 DNM). In contrast, DAMGO-induced MOR desensitization was independent of PKC activity but was reduced by over-expression of the GRK2 DNM but not by that of a GRK6 DNM.

Conclusions and implications:

In mature mammalian neurons, different MOR agonists can induce MOR desensitization by different mechanisms, morphine by a PKCα-mediated, heterologous mechanism and DAMGO by a GRK-mediated, homologous mechanism. These data represent functional selectivity at the level of receptor desensitization.     

Quantitative analysis of two isoflavones in <Emphasis Type="Italic">Pueraria lobata</Emphasis> flowers from eleven Chinese provinces using high performance liquid chromatography

Background  

Pueraria lobata flower (Gehua) is a medicinal herb to treat intoxication, hepatic and gastrointestinal tract lesion induced by alcohol. This study aims to develop a new HPLC method for the determination of two major isoflavones in P. lobata flowers, namely tectoridin and 6"-O-xylosyl-tectoridin.     

Primary omental torsion in children

Objective : A retrospective review was conducted to establish the prevalence and clinical features of omental torsion or infarction as a cause of acute abdominal pain in childhood.
Methodology : The case records were analysed for all patients admitted with primary omental pathology to the Department of General Surgery, Royal Children's Hospital, Melbourne, between January 1975 and July 1994.
Results : From 1975 to 1994 (20 years) 13 children were admitted to our General Surgical Department with primary omental disease. There were nine males and four females under 16 years of age. The presenting complaint was abdominal pain with vomiting or diarrhoea. Four children had major medical conditions. Pre-operative diagnosis in all cases was acute appendicitis. Appendicectomy and omentectomy were performed without complication in all cases. Histology of the omentum demonstrated torsion, infarction or haemorrhage.
Conclusions : All children presented with features of acute appendicitis, a majority were male, and two out of the 13 patients were obese. The absence of any children under 4 years was consistent with the relative paucity of omental fat in younger children. We found no clear mechanism for primary omental torsion, although rotation around the right epiploic artery was observed.     

Cyclosporin as initial treatment for Crohn's disease

Childhood Crohn's disease may cause significant morbidity. T cell activation is considered to be central to Crohn's disease pathology, and as cyclosporin is a powerful inhibitor of T cell activation, and has been used in adult Crohn's disease with encouraging results, it may offer the prospect of remission if given early in the course of disease. Children with newly diagnosed Crohn's disease or those relapsing off treatment were therefore given cyclosporin or conventional treatment (enteral nutrition or corticosteroids) by random allocation. Evaluation was performed initially and at two months. Twenty four children were studied (10 on cyclosporin and 14 on conventional treatment; one child on cyclosporin withdrew). Significant clinical improvement occurred in the group on conventional treatment, but not in the cyclosporin group. Colonoscopic improvement was noted in 5/9 on cyclosporin and 8/14 on conventional treatment, but neither group produced a significant fall in median colonoscopic index. Histological improvement was seen in 7/8 on cyclosporin and 8/13 on conventional treatment, but cyclosporin was not significantly better. Cyclosporin produced improved clinical and histological appearance without matched improvement in blood disease indices. It was not better than conventional treatment, and simple oral administration is probably not suitable for newly diagnosed patients with Crohn's disease.