Integrin-dependent homotypic adhesion of neutrophils. Arachidonic acid activates Raf-1/Mek/Erk via a 5-lipoxygenase- dependent pathway.

AA stimulates integrin-dependent neutrophil adhesion, a critical early step in acute inflammation. However, neither the signaling pathway(s) of AA-stimulated adhesion, nor whether AA acts directly or through the generation of active metabolites, has been elucidated. Previously, we have observed a tight association between neutrophil Erk activation and homotypic adhesion in response to chemoattractants acting through G protein-linked receptors. We now report a similar association between homotypic adhesion and Erk activation in response to AA. Erk activation was cyclooxygenase independent and required AA metabolism to 5(S)- hydroperoxyeicosatetraenoic acid (5-HpETE) via 5-lipoxygenase, but not the further lipoxygenase-dependent metabolism of 5-HpETE to leukotrienes. AA stimulation of Erk was accompanied by Raf-1 activation and was sensitive to inhibitors of Raf-1 and Mek. Whereas activation of Erk by AA was pertussis toxin sensitive, [3H]-AA binding to neutrophils was not saturable, suggesting that an AA metabolite activates a G protein. Consistent with this hypothesis, Erk activation by 5(S)-hydroxyeicosatetraenoic acid (5-HETE; lipoxygenase-independent metabolite of 5-HpETE) was also pertussis toxin sensitive. These data suggest that a 5-lipoxygenase metabolite of AA, e.g., 5-HETE, is released from AA-treated cells to engage a plasma membrane-associated, pertussis toxin-sensitive, G protein-linked receptor, leading to activation of Erk and adhesion via the Raf-1/Mek signal transduction pathway.     

Activation-dependent carboxyl methylation of neutrophil G-protein gamma subunit.

The gamma subunits of heterotrimeric guanine nucleotide-binding regulatory (G) proteins (G gamma) are post-translationally processed at their C termini by prenylation, proteolysis, and carboxyl methylation. Whereas prenylation of G gamma is required for membrane association of G proteins, the role of carboxyl methylation is unknown. Here we show that human neutrophils express G gamma 2 but not G gamma 3 or G gamma 7 and that carboxyl methylation of G gamma 2 is associated with signal transduction. In a reconstituted cell-free system, neutrophil G gamma 2 was labeled by the methyl donor S-[methyl-3H]adenosyl-L-methionine. Carboxyl methylation was confirmed by alkaline hydrolysis and quantitation of volatile [3H]methanol. Neutrophil G gamma 2 methylation was stimulated by activation of G protein with guanosine 5'-[beta, gamma-thio]triphosphate. We estimate that after 1 hr of G-protein activation at least 6% of the total pool of G gamma 2 was carboxyl-methylated. The inflammatory agonist fMet-Leu-Phe stimulated guanosine 5'-[beta,gamma-thio]triphosphate-dependent carboxyl methylation. Methylation of G gamma 2 was inhibited by the carboxyl methyltransferase inhibitor N-acetyl-S-trans,trans-farnesylcysteine at concentrations that affected signal transduction in neutrophils. These results demonstrate that activation of neutrophil Gi is associated with alpha-carboxyl methyl esterification of G gamma 2 and suggest that carboxyl methylation of G gamma may play a role in signal transduction.     

Adenosine A2A receptors in diffuse dermal fibrosis: pathogenic role in human dermal fibroblasts and in a murine model of scleroderma

OBJECTIVE: Adenosine regulates inflammation and tissue repair, and adenosine A2A receptors promote wound healing by stimulating collagen matrix production. We therefore examined whether adenosine A2A receptors contribute to the pathogenesis of dermal fibrosis. METHODS: Collagen production by primary human dermal fibroblasts was analyzed by real-time polymerase chain reaction, 14C-proline incorporation, and Sircol assay. Intracellular signaling for dermal collagen production was investigated using inhibitors of MEK-1 and by demonstration of ERK phosphorylation. In vivo effects were studied in a bleomycin-induced dermal fibrosis model using adenosine A2A receptor-deficient wild-type littermate mice, C57BL/6 mice, and mice treated with adenosine A2A receptor antagonist. Morphometric features and levels of hydroxyproline were determined as measures of dermal fibrosis. RESULTS: Adenosine A2A receptor occupancy promoted collagen production by primary human dermal fibroblasts, which was blocked by adenosine A2A, but not A1 or A2B, receptor antagonism. Adenosine A2A receptor ligation stimulated ERK phosphorylation, and A2A receptor-mediated collagen production by dermal fibroblasts was blocked by MEK-1 inhibitors. Adenosine A2A receptor-deficient and A2A receptor antagonist-treated mice were protected from developing bleomycin-induced dermal fibrosis. CONCLUSION: These results demonstrate that adenosine A2A receptors play an active role in the pathogenesis of dermal fibrosis and suggest a novel therapeutic target in the treatment and prevention of dermal fibrosis in diseases such as scleroderma.     

Effect of the carcinogen N-nitrosomethylurea on the tRNA methylase activity of cells cultured in vitro

A comparison has been made between the activity of enzymes responsible for methylation of tRNA in a permanent line of adult Chinese hamster ovary fibroblasts maintained in in vitro culture, and that of cells derived from this line after exposure in vitro to the alkylating carcinogen N-nitrosomethylurea. A significantly elevated activity has been observed in several clones of cells which had received a single acute dose of the nitrosamide. A series of chronic treatments in which the single acute dose was given over a period of 8 weeks illustrated the cumulative effect of the fractionated doses on the methylase enzymes, and confirmed the repeatability of findings after a single acute dose. Evidence has been obtained from morphological criteria that a correlation appears to exist between the extent of morphological conversion within the cell population and the activity of the enzymes. Kinetic evidence suggests that this alteration in activity is not related to changes in growth rate alone. These results are discussed in relation to the concept that methylation of tRNA may relate to the degree of maturation and differentiation in a particular cell population.     

Periodontal disease and the oral microbiota in new‐onset rheumatoid arthritis

Objective

To profile the abundance and diversity of subgingival oral microbiota in patients with never‐treated, new‐onset rheumatoid arthritis (RA).

Methods

Periodontal disease (PD) status, clinical activity, and sociodemographic factors were determined in patients with new‐onset RA, patients with chronic RA, and healthy subjects. Multiplexed‐454 pyrosequencing was used to compare the composition of subgingival microbiota and establish correlations between the presence/abundance of bacteria and disease phenotypes. Anti–Porphyromonas gingivalis antibody testing was performed to assess prior exposure to the bacterial pathogen P gingivalis.

Results

The more advanced forms of periodontitis were already present at disease onset in patients with new‐onset RA. The subgingival microbiota observed in patients with new‐onset RA was distinct from that found in healthy controls. In most cases, however, these microbial differences could be attributed to the severity of PD and were not inherent to RA. The presence and abundance of P gingivalis were also directly associated with the severity of PD and were not unique to RA. The presence of P gingivalis was not correlated with anti–citrullinated protein antibody (ACPA) titers. Overall exposure to P gingivalis was similar between patients with new‐onset RA and controls, observed in 78% of patients and 83% of controls. The presence and abundance of Anaeroglobus geminatus correlated with the presence of ACPAs/rheumatoid factor. Prevotella and Leptotrichia species were the only characteristic taxa observed in patients with new‐onset RA irrespective of PD status.

Conclusion

Patients with new‐onset RA exhibited a high prevalence of PD at disease onset, despite their young age and paucity of smoking history. The subgingival microbiota profile in patients with new‐onset RA was similar to that in patients with chronic RA and healthy subjects whose PD was of comparable severity. Although colonization with P gingivalis correlated with the severity of PD, overall exposure to P gingivalis was similar among the groups. The role of A geminatus and Prevotella/Leptotrichia species in this process merits further study.

     

Sacral nerve stimulation for faecal incontinence

Faecal incontinence is a common problem. Conservative measures are effective in a significant proportion of patients. Failure of conservative management has until recently meant recourse to surgical intervention. Surgical treatment is often associated with disappointing results. Recently, sacral nerve stimulation (SNS) has been developed as a minimally invasive, effective technique for idiopathic and acquired faecal incontinence. The technique uses chronic low-level electrical stimulation of the sacral nerves, or neuromodulation, to produce a clinically beneficial effect on the distal colon and rectum, the pelvic floor and the anal sphincter complex. SNS is a 2-stage procedure: a diagnostic stage - temporary percutaneous nerve evaluation (PNE), and a therapeutic stage - permanent SNS. The predictive value of PNE is high, and the surgical trauma and morbidity of both procedures extremely low. The technique has been adapted from its original application in urinary dysfunction. It is almost impossible to produce level 1 evidence for this type of intervention; however, the results are superior to other interventions. Patient selection criteria are evolving, but there is a growing body of evidence that supports its use as first-line treatment for faecal incontinence in patients where conservative measures have failed.     

Atmospheric entry simulations of Mars lander bioload--experiments in support of Beagle 2

Simulations of the temperature and vacuum effects of Martian atmospheric entry upon Bacillus atrophaeus (formerly Bacillus subtilis var niger; 8058; NCIMB) endospores were carried out inside a purpose-built vacuum chamber. The work formed part of the study in support of planetary protection for the Beagle 2 Mars lander and investigated to what extent the outer surface of the lander's back heat shield would be sterilised during Mars atmospheric entry. The spores were heated to peak temperatures up to 300 degrees C over 30 s under vacuum conditions (10(-3) mbar). There was no effect on spore viability until peak temperatures reached 180-200 degrees C (12-15 s of heat exposure). Spore viability then fell rapidly with increasing temperature. Once peak temperatures exceeded 300 degrees C, no further spore viability was detected. The average heating rate was rapid (10 degrees C s(-1)); thus spores were exposed to peak temperatures for less than a second. These data inform on the process of determining bioburden reduction and control steps necessary for external surfaces of spacecraft which are non-sterile at launch, as well as providing new information about the ability of a model resistant organism to survive rapid, short-duration heating.