Mutation of the DRY motif reveals different structural requirements for the CC chemokine receptor 5-mediated signaling and receptor endocytosis

CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor that governs migration of leukocytes and serves as a coreceptor for the R5 tropic strains of human immunodeficiency virus (HIV). CCR5-mediated signaling in response to CC chemokines relies on G protein activation. Desensitization, which rapidly turns off G protein-dependent signaling, involves phosphorylation of CCR5 that promotes interaction of the receptor with beta-arrestins for endocytosis. Whether coupling to G proteins, desensitization, and endocytosis of CCR5 require the same structural determinants remains a matter of investigation. Here, we show that CCR5 displayed agonist-independent coupling to G proteins. This constitutive activity of the receptor was abrogated by TAK779 (N,N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbonyl]amino]benzyl]tetrahydro-2H-pyran-4-aminium chloride), a nonpeptidic CCR5 ligand that inhibits HIV infection and was found to depend on the integrity of the Asp-Arg-Tyr (DRY) motif. Changing Arg-126 by the neutral residue Asn (R126N-CCR5 mutant) abolished CCR5-mediated activation of G proteins, either constitutively or in response to agonists. In contrast, R126N-CCR5 not only retained agonist-promoted phosphorylation and beta-arrestin-dependent endocytosis but also displayed a higher basal phosphorylation than wild-type CCR5. Expression of beta-arrestin in R126N-CCR5-expressing cells resulted in receptor down-regulation, thereby suggesting that R126N-CCR5 spontaneously interacts with beta-arrestins. However, although expression of beta-arrestin favored wild-type CCR5-mediated chemotaxis, it failed to promote migration of cells expressing R126N-CCR5. Overall, these data indicate that structural requirements for CCR5-mediated activation of G proteins, albeit not involved in receptor desensitization and internalization, are needed for beta-arrestin-mediated chemotaxis. These results have implications for how distinct biological responses of CCR5 might rely on a different set of receptor conformations.     

Meeting report: summary of IARC monographs on formaldehyde, 2-butoxyethanol, and 1-tert-butoxy-2-propanol

An international, interdisciplinary working group of expert scientists met in June 2004 to develop IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans (IARC Monographs) on formaldehyde, 2-butoxyethanol, and 1-tert-butoxy-2-propanol. Each IARC Monograph includes a critical review of the pertinent scientific literature and an evaluation of an agent's potential to cause cancer in humans. After a thorough discussion of the epidemiologic, experimental, and other relevant data, the working group concluded that formaldehyde is carcinogenic to humans, based on sufficient evidence in humans and in experimental animals. In the epidemiologic studies, there was sufficient evidence that formaldehyde causes nasopharyngeal cancer, "strong but not sufficient" evidence of leukemia, and limited evidence of sinonasal cancer. The working group also concluded that 2-butoxyethanol and 1-tert-butoxy-2-propanol are not classifiable as to their carcinogenicity to humans, each having limited evidence in experimental animals and inadequate evidence in humans. These three evaluations and the supporting data will be published as Volume 88 of the IARC Monographs.     

Accuracy of the Sauvegrain method in determining skeletal age during puberty

BACKGROUND: The method of Sauvegrain et al. for the assessment of skeletal age from radiographs of the elbow is useful during the two years of the pubertal growth spurt. The purpose of this study was to determine the accuracy of the method and its value in pediatric orthopaedics. METHODS: The Sauvegrain method uses four anatomical landmarks of the elbow: the lateral condyle, trochlea, olecranon apophysis, and proximal radial epiphysis. It is based on a 27-point scoring system. The scores for these structures are summed, and a total score is determined. A graph is then used to determine the skeletal age. The method was evaluated by three independent observers who used it to assess skeletal age on anteroposterior and lateral radiographs of the left elbow of sixty boys and sixty girls and compared the results with assessments made with use of the Greulich and Pyle atlas on posteroanterior radiographs of the left hand and wrist. Skeletal age determinations were performed twice by each observer at a four-week interval. RESULTS: The skeletal age determination from radiographs of the elbow was more precise because a clear semiannual age determination was possible. On the basis of the rating by the observers, the Sauvegrain method presented excellent interobserver correlation (r = 0.93) and excellent reproducibility (r = 0.96). The correlation between the methods of Sauvegrain et al. and Greulich and Pyle was good (r = 0.85). Nevertheless, certain elbow growth centers showed an intermediate developmental morphology, which failed to correspond to the score described by Sauvegrain et al. This led to errors in the interpretation of data. We suggest an intermediate score for these cases, and we modified the original graph to make it more accurate. CONCLUSIONS: The modified method of Sauvegrain et al. is simple, reliable, and reproducible, and it complements the Greulich and Pyle atlas. In clinical practice, maturity can best be evaluated by associating skeletal age, annual growth rate, and secondary sexual characteristics. Therefore, this method is useful when major decisions such as the timing of epiphysiodesis or spinal arthrodesis are necessary during puberty.     

Association of Aneurysmal and Occlusive Lesions in Behçet’s Disease

Arterial involvement in Behçets disease is rare, occurring in various locations with multiple clinical expressions. When Behçets disease is associated with large arteries, lesions are usually in the form of aneurysms or occlusions. The simultaneous occurrence of these two lesions is even more unusual. We present a case of Behçets disease in which arterial involvement included an iliac artery thrombosis and an asymptomatic aneurysm of the infrarenal abdominal aorta. Behçets disease must be considered in the diagnosis of any unexplained inflammatory arteriopathy. Surgery is indicated for the majority of aneurysms and severe symptoms. The postoperative follow-up is based on noninvasive radiologic examinations.     

Prognostic significance of wound infections following major head and neck cancer surgery: an open non-comparative prospective study

Objective We evaluated the incidence, risk factors and consequences of wound infection (WI) following major head and neck cancer surgery in an open non-comparative study.Patients and methods The study group, comprising 95 patients who underwent clean-contaminated procedures with opening of the upper aerodigestive tract for biopsy-proven squamous cell cancer, were studied over a 1-year period. Antibiotic prophylaxis was amoxicillin and clavulanic acid. More than 20 variables were prospectively recorded for each patient. The mean follow-up was 30 months.Main results The overall WI rate was 50.5% (48/95). Most pathogens isolated from samples were gram-negative rods. In univariate analysis, we found three risk factors for WI: alcohol consumption (P=0.07), a hypopharyngeal location (P=0.02) and laryngectomy stoma (P=0.01). WI were associated with postoperative fever (P=1.5×10^–11), postoperative antibiotic therapy (P=1.5×10^–5) and postoperative death (P=0.043). Patients without WI had a median postoperative hospital stay of 15 days compared with 29 days for those with WI (P<0.001). Healing of WI was achieved after a median time of 48 days. WI delayed postoperative radiation therapy in 21 out of 33 evaluable patients. But overall survival, and local and metastatic failures were similar with and without WI.Conclusions WI are associated with a heavy postoperative morbidity, but have no prognostic impact on cancer control.     

Population pharmacokinetics and Bayesian estimation of mycophenolic acid concentrations in stable renal transplant patients

BACKGROUND: Therapeutic drug monitoring of mycophenolic acid (MPA) may minimise the risk of acute rejection after transplantation. Area under the curve (AUC) rather than trough concentration-based monitoring is recommended and models for AUC estimation are needed. OBJECTIVES: To develop a population pharmacokinetic model suitable for Bayesian estimation of individual AUC in stable renal transplant patients. PATIENTS AND METHODS: The population pharmacokinetics of MPA were studied using nonlinear mixed effects modelling (NONMEM) in 60 patients (index group) receiving MPA on a twice-daily basis. Ten blood samples were collected at fixed timepoints from ten patients and four blood samples were collected at sparse timepoints from 50 patients. Bayesian estimation of individual AUC was made on the basis of three blood concentration measurements and covariates. The predictive performances of the Bayesian procedure were evaluated in an independent group of patients (test group) comprising ten subjects in whom ten blood samples were collected at fixed timepoints. RESULTS: A two-compartment model with zero-order absorption best fitted the data. Covariate analysis showed that bodyweight was positively correlated with oral clearance. However, the weak magnitude of the reduction in variability (from 34.8 to 28.2%) indicates that administration on a per kilogram basis would be of limited value in decreasing interindividual variability in MPA exposure. Bayesian estimation of pharmacokinetic parameters using samples drawn at 20 minutes and 1 and 3 hours enabled estimation of individual AUC with satisfactory accuracy (bias 7.7%, range of prediction errors 0.43-15.1%) and precision (root mean squared error 12.4%) as compared with the reference value obtained using the trapezoidal method. CONCLUSION: This paper reports for the first time population pharmacokinetic data for MPA in stable renal transplant patients, and shows that Bayesian estimation can allow accurate prediction of AUC with only three samples. This method provides a tool for therapeutic drug monitoring of MPA or for concentration-effect studies. Its application to MPA monitoring in the early period post-transplantation needs to be evaluated.     

Polyploidisation of metastatic colon carcinoma cells by microtubule and tubulin interacting drugs: effect on proteolytic activity and invasiveness

When SW620 colon cancer-derived metastatic cells were exposed to nanomolar concentrations of Taxol, colchicine or (Z)-3,5,4'-trimethoxystilbene (R3), huge aneuploid, polynuclear cells survived the treatment. These cells released considerable amounts of the matrix metalloproteinase matrilysin (MMP-7), and tissue-type plasminogen activator (tPA) into the surrounding culture medium. MMP-7, and other proteolytic enzymes were highly expressed by these cells. In spite of their enormous size, the polyploid cells exhibited a considerable migratory capacity, as was demonstrated by their migration through an artificial basement membrane. While colchicine and R3-treated cells showed an inverse relationship between drug concentration and invasiveness, treatment with Taxol increased the capacity of the SW620 cells to penetrate through the membrane. The invasive capacity was not correlated with the induction and release of proteolytic enzymes. The idea that expression and release of proteolytic enzymes is a fundamental prerequisite of tumour cell invasiveness is generally accepted. The ability of the cells to respond to chemotactic signalling, and the filamentous structures of the cells, together with several cell adhesion factors, which are the basis of cell migration, are prerequisites of invasiveness. These factors are presumably different in the aneuploid cells produced by Taxol, colchicine and R3, and await scrutiny.