A unique role for p53 in the regulation of M2 macrophage polarization.

P53 is critically important in preventing oncogenesis but its role in inflammation in general and in the function of inflammatory macrophages in particular is not clear. Here, we show that bone marrow-derived macrophages exhibit endogenous p53 activity, which is increased when macrophages are polarized to the M2 (alternatively activated macrophage) subtype. This leads to reduced expression of M2 genes. Nutlin-3a, which destabilizes the p53/MDM2 (mouse double minute 2 homolog) complex, promotes p53 activation and further downregulates M2 gene expression. In contrast, increased expression of M2 genes was apparent in M2-polarized macrophages from p53-deficient and p53 mutant mice. Furthermore, we show, in mice, that p53 also regulates M2 polarization in peritoneal macrophages from interleukin-4-challenged animals and that nutlin-3a retards the development of tolerance to Escherichia coli lipopolysaccharide. P53 acts via transcriptional repression of expression of c-Myc (v-myc avian myelocytomatosis viral oncogene homolog) gene by directly associating with its promoter. These data establish a role for the p53/MDM2/c-MYC axis as a physiological 'brake' to the M2 polarization process. This work reveals a hitherto unknown role for p53 in macrophages, provides further insight into the complexities of macrophage plasticity and raises the possibility that p53-activating drugs, many of which are currently being trialled clinically, may have unforeseen effects on macrophage function.

No association in frequency of KIR receptors in patients with rheumatoid arthritis from Northern Ireland.

The frequency of inhibiting and activating killer cell immunoglobulin-like receptors was similar in 331 patients with rheumatoid arthritis and in 354 controls. Patients and controls came from a relatively homogeneous Caucasian population from Northern Ireland, thus limiting population stratification. Furthermore, no differences were found when the patients were sub-divided according to gender or shared epitope and when the presence of the human leucocyte antigen ligand was taken into account.

A randomised controlled trial of intra-articular corticosteroid injection of the carpometacarpal joint of the thumb in osteoarthritis.

OBJECTIVE: To investigate the efficacy of corticosteroid injections into the carpometacarpal joint of the thumb (CMCJ) in patients with osteoarthritis. DESIGN: A double blind, randomised controlled trial using 40 hospital referred patients with CMCJ osteoarthritis who received intra-articular injections of 5 mg triamcinolone hexacetonide (0.25 ml) or sterile 0.9% saline (0.25 ml). Injections were given under imaging control. MAIN OUTCOME MEASURES: The primary outcome was improvement in a pain visual analogue score (VAS) of 20% at 24 weeks. In addition patients were assessed at 4, 12, and 24 weeks for joint stiffness, joint tenderness, and physician and patient global assessments. Hand radiographs were evaluated for the degree of CMC joint space narrowing and marginal osteophytes according to the OARSI atlas. RESULTS: Baseline clinical variables were not significantly different between the two treatment groups. There was no improvement in the VAS of pain at 24 weeks. At each assessment point there was no significant difference between the steroid and placebo groups in median values for joint stiffness, joint tenderness, or patient and physician global assessments. Non-parametric analysis of each group individually revealed statistically significant improvements in patient and physician global assessments at weeks 4, 12, and 24 in the placebo group and at weeks 4 and 12 in the steroid group. CONCLUSIONS: No clinical benefit was gained from intra-articular steroid injection to the CMCJ in moderate to severe osteoarthritis compared with placebo injection.

A randomised study of two training programmes for general practitioners in the techniques of shoulder injection.

OBJECTIVES: To evaluate the impact of two different modes of shoulder injection training on the level of confidence and number of injections performed by general practitioners (GPs) METHODS: Demographic details, and information on referrals for shoulder problems, shoulder joint injection activity, and confidence in the six months before training were obtained for 40 GP principals at baseline. Standardised training in the techniques of shoulder joint injection using rubber mannequins was given to all GPs. Twenty of these GPs were randomly allocated to receive additional training on patients in hospital joint injection clinics. Six months after both forms of training the shoulder injection and referral activities of all GPs were reassessed. RESULTS: Both training groups had comparable demographic characteristics and baseline clinical activity. GPs who had additional training with patients reported a marked increase in their level of confidence in performing shoulder injections and the number performed. The number of shoulder referrals did not differ between the groups CONCLUSION: Training on patients in addition to conventional training on mannequins increased GPs' shoulder injection activity and their level of confidence. Hospital injection clinics may provide a suitable setting in which to train GPs interested in developing their shoulder joint injection skills.

Can diagnostic triage by general practitioners or rheumatology nurses improve the positive predictive value of referrals to early arthritis clinics?

OBJECTIVES: To determine whether diagnostic triage by general practitioners (GPs) or rheumatology nurses (RNs) can improve the positive predictive value of referrals to early arthritis clinics (EACs). METHODS: Four GPs and two RNs were trained in the assessment of early inflammatory arthritis (IA) by four visits to an EAC supervised by hospital rheumatologists. Patients referred to one of three EACs were recruited for study and assessed independently by a GP, an RN and one of six rheumatologists. Each assessor was asked to record their clinical findings and whether they considered the patient to have IA. Each was then asked to judge the appropriateness of the referral according to predetermined guidelines. The rheumatologists had been shown previously to have a satisfactory level of agreement in the assessment of IA. RESULTS: Ninety-six patients were approached and all consented to take part in the study. In 49 cases (51%), the rheumatologist judged that the patient had IA and that the referral was appropriate. The assessments of GPs and RNs were compared with those of the rheumatologists. Levels of agreement were measured using the kappa value, where 1.0 represents total unanimity. The kappa value was 0.77 for the GPs when compared with the rheumatologists and 0.79 for the RNs. Significant stiffness in the morning or after rest and objective joint swelling were the most important clinical features enabling the GPs and RNs to discriminate between IA and non-IA conditions. CONCLUSION: Diagnostic triage by GPs or RNs improved the positive predictive value of referrals to an EAC with a degree of accuracy approaching that of a group of experienced rheumatologists.

Active site labeling of the gentamicin resistance enzyme AAC(6')-APH(2") by the lipid kinase inhibitor wortmannin.

BACKGROUND: Aminoglycoside antibiotic resistance is largely the result of the production of enzymes that covalently modify the drugs including kinases (APHs) with structural and functional similarity to protein and lipid kinases. One of the most important aminoglycoside resistance enzymes is AAC(6')-APH(2"), a bifunctional enzyme with both aminoglycoside acetyltransferase and kinase activities. Knowledge of enzyme active site structure is important in deciphering the molecular mechanism of antibiotic resistance and here we explored active site labeling techniques to study AAC(6')-APH(2") structure and function. RESULTS: AAC(6')-APH(2") was irreversibly inactivated by wortmannin, a potent phosphatidylinositol 3-kinase inhibitor, through the covalent modification of a conserved lysine in the ATP binding pocket. 5'-[p-(Fluorosulfonyl)benzoyl]adenosine, an electrophilic ATP analogue and known inactivator of other APH enzymes such as APH(3')-IIIa, did not inactivate AAC(6')-APH(2"), and reciprocally, wortmannin did not inactivate APH(3')-IIIa. CONCLUSIONS: These distinct active site label sensitivities point to important differences in aminoglycoside kinase active site structures and suggest that design of broad range, ATP binding site-directed inhibitors against APHs will be difficult. Nonetheless, given the sensitivity of APH enzymes to both protein and lipid kinase inhibitors, potent lead inhibitors of this important resistance enzyme are likely to be found among the libraries of compounds directed against other pharmacologically important kinases.

Vgb from Staphylococcus aureus inactivates streptogramin B antibiotics by an elimination mechanism not hydrolysis.

The streptogramin antibiotics were identified almost 50 years ago but have only recently found clinical use as a consequence of the increase in multidrug-resistant bacteria. Despite the fact that these antibiotics have historically not found intense clinical use, resistance to streptogramins exists. Streptogramins consist of a mixture of two components: cyclic polyunsaturated macrolactones (group A) and cyclic hexadepsipeptides (group B). The latter are cyclized through an ester bond between the hydroxyl group of an N-terminal threonine and the C-terminal carboxyl. Resistance to the B streptogramins can occur through the production of enzymes such as Vgb from Staphylococcus aureus. This enzyme had been assumed to be a lactonase that inactivates the cyclic antibiotic by linearization through hydrolytic cleavage of the ester bond. We have expressed recombinant Vgb in quantity and, using a combination of mass spectrometry, NMR, and synthesis of model depsipeptides, show unequivocally that streptogramin B inactivation does not involve hydrolysis of the ester bond. Rather, the hexadepsipeptide is linearized through an elimination reaction across the ester bond generating an N-terminal dehydrobutyrine group. Therefore, Vgb is not a hydrolase but a lyase. We also have explored the activity of Vgb orthologues present in the chromosomes of various bacteria including Bordetella pertussis and Streptomyces coelicolor and have determined that these enzymes also show streptogramin B inactivation through an elimination mechanism indistinguishable to that used by Vgb. These results demonstrate that Vgb is a member of a large group of streptogramin B lyases that are present not only in resistant clinical isolates but also in the chromosomes of many bacteria. There is therefore a significant reservoir of streptogramin resistance enzymes in the environment, which has the potential to impact the long-term utility of these antibiotics. This research establishing the molecular mechanism of streptogramin resistance therefore has the potential to be exploited in the discovery of inhibitory compounds that could rescue antibiotic activity even in the presence of resistance elements.