Nanoscale structural mapping as a measure of maturation in the murine frontal cortex.

Atomic force microscopy (AFM) is emerging as an innovative tool to phenotype the brain. This study demonstrates the utility of AFM to determine nanomechanical and nanostructural features of the murine dorsolateral frontal cortex from weaning to adulthood. We found an increase in tissue stiffness of the primary somatosensory cortex with age, along with an increased cortical mechanical heterogeneity. To characterize the features potentially responsible for this heterogeneity, we applied AFM scan mode to directly image the topography of thin sections of the primary somatosensory cortical layers II/III, IV and V/VI. Topographical mapping of the cortical layers at successive ages showed progressive smoothing of the surface. Topographical images were also compared with histochemically derived morphological information, which demonstrated the deposition of perineuronal nets, important extracellular components and markers of maturity. Our work demonstrates that high-resolution AFM images can be used to determine the nanostructural properties of cortical maturation, well beyond embryonic and postnatal development. Furthermore, it may offer a new method for brain phenotyping and screening to uncover topographical changes in early stages of neurodegenerative diseases.

The protective effects of zinc in experimental gentamicin induced acute renal failure in rats.

This study investigates the effects of zinc in acute kidney injury induced by gentamicin (Ge). We used Wistar male rats distributed in 4 groups of 12 animals each, treated intraperitoneally as follows: Group I (Control) treated with distilled water; Group II (Ge) with experimental induced acute renal failure with Ge; Group III (Ge + Zn) administration of ZnCl2 in animals with experimental induced renal failure with Ge, Group IV (Zn) treated with ZnCl2 as positive control. We measured serum levels of urea, creatinine, total antioxidant status, superoxide dismutase, glutathione peroxidase and urinary proteins before the nephrotoxicity induction (baseline) and 3, 7 and 10 days after Ge administration. The renal histopathological analysis was also done. The results showed an increase of urea and creatinine values in Ge + Zn group after 7 days compared to baseline, but less accentuated than those in Ge group. Zn supplementation was associated with an increase of the total antioxidant status in Ge + Zn group compared to Ge group (P < 0.01). It was also revealed a significant reduction of proteinuria in Ge + Zn group compared to Ge group (P < 0.001). The histopathological investigation highlighted the tubular necrosis affecting more than 90% of proximal tubules in Ge group. In Ge + Zn group it was observed a milder degree of tubular necrosis (influencing less than 25% of proximal tubules), a moderate inflammation and the presence of tubular regeneration. In conclusion, Zn administration proved a to have a protective role in experimental gentamicin-induced acute renal failure.

Analysis of Arrestin Recruitment to Chemokine Receptors by Bioluminescence Resonance Energy Transfer.

Chemokine receptors recruit the multifunctional scaffolding protein beta arrestin in response to binding of their chemokine ligands. Given that arrestin recruitment represents a signaling axis that is in part independent from G-protein signaling, it has become a hallmark of G protein-coupled receptor functional selectivity. Therefore, quantification of arrestin recruitment has become a requirement for the delineation of chemokine and drug candidate activity along different signaling axes. Bioluminescence resonance energy transfer (BRET) techniques provide methodology for such quantification that can reveal differences between nonredundant chemokines binding the same receptor, and that can be upscaled for high-throughput testing. We here provide protocols for the careful setup of BRET-based arrestin recruitment assays, and examples for the application of such systems in dose-response or time-course experiments. Suggestions are given for troubleshooting, optimizing test systems, and the interpretation of results obtained with BRET-based assays, which indeed yield an intricate blend of quantitative and qualitative information.

Mapping HA-tagged protein at the surface of living cells by atomic force microscopy.

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the ß2-adrenergic receptor (ß2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of ß2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process.

Treatment of high risk medulloblastomas in children above the age of 3 years: a SFOP study.

AIM: Improvement of EFS of children older than 3 years with high risk medulloblastoma. METHODS: Between 1993 and 1999, 115 patients (3-18 years, mean 8 years) with high risk medulloblastoma were included. After surgery treatment consisted of chemotherapy ('8in1' and etoposide/carboplatin) before and after craniospinal radiotherapy. RESULTS: Patients were staged using Chang-criteria (PF residue only, M1 and M2/M3) by local investigator as well as by central review panel (82.4% concordance). Chemotherapy was well tolerated without major delays in radiotherapy. With a mean follow up of 81 months (9-119), 5-year EFS was 49.8% and OS 60.1%. In detail according to subgroups EFS was 68.8% for PF residue only, 58.8% for M1 disease and 43.1% for M2/M3. CONCLUSION: M1 patients are legitimate high risk patients. Survival rates are still very low for high risk medulloblastoma patients and future trials should therefore focus on more intensive (chemotherapy/radiotherapy) treatment.

Standard-risk medulloblastoma treated by adjuvant chemotherapy followed by reduced-dose craniospinal radiation therapy: a French Society of Pediatric Oncology Study.

OBJECTIVE: The primary objective of this study was to decrease the late effects of prophylactic radiation without reducing survival in standard-risk childhood medulloblastoma. PATIENTS AND METHODS: Inclusion criteria were as follows: children between the ages of 3 and 18 years with total or subtotal tumor resection, no metastasis, and negative postoperative lumbar puncture CSF cytology. Two courses of eight drugs in 1 day followed by two courses of etoposide plus carboplatin (500 and 800 mg/m(2) per course, respectively) were administered after surgery. Radiation therapy had to begin 90 days after surgery. Delivered doses were 55 Gy to the posterior fossa and 25 Gy to the brain and spinal canal. RESULTS: Between November 1991 and June 1998, 136 patients (median age, 8 years; median follow-up, 6.5 years) were included. The overall survival rate and 5-year recurrence-free survival rate were 73.8% +/- 7.6% and 64.8% +/- 8.1%, respectively. Radiologic review showed that 4% of patients were wrongly included. Review of radiotherapy technical files demonstrated a correlation between the presence of a major protocol deviation and treatment failure. The 5-year recurrence-free survival rate of patients included in this study with all optimal quality controls of histology, radiology, and radiotherapy was 71.8% +/- 10.5%. In terms of sequelae, 31% of patients required growth hormone replacement therapy and 25% required special schooling. CONCLUSION: Reduced-dose craniospinal radiation therapy can be proposed in standard-risk medulloblastoma provided staging and radiation therapy are performed under optimal conditions.

[Medical and economic evaluation of organ transplantation in France. The example of liver transplantation]. / L'évaluation médico-économique des greffes d'organe en France. L'exemple de la transplantation hépatique.

BACKGROUND: Organ transplantation is an expensive and risky medical procedure. Estimating the cost is difficult because the care is complex and involves many actors. We present a methodological framework for the economic evaluation of organ transplantation in France which include a detailed collection of all the direct costs and the simultaneous analysis of health status. It was applied to evaluate the cost of liver transplantation in France. METHODS: All consecutive adults transplanted or placed on the waiting list of liver transplantation in the Department of Surgery of Cochin Hospital, Paris, between 1994 and 1996 were included. All resource use was measured during one year: staff wages, pharmacy and blood, laboratory and radiology, supplies, overhead hospital services. Mean quality of life was estimated by the survival weighted by the Karnofsky index. RESULTS: Transplantation: 38 patients were included. The first year mean cost after transplantation was 561,000FF (included rehospitalizations cost of 120,000FF). Care outside the hospital induced 10% of the total cost. Mean quality of life was 63% (from 0% to 93%) and increased with time, whereas cost decreased. Waiting list: 26 of 33 patients on waiting list were transplanted. The first year mean cost was 95,000FF (included 32,000FF for first evaluation) and increased with time. 44% of cost was supported by another hospital than the transplanting one. Sickness allowance added 20% to the cost. The mean quality of life was 56% during the first year. CONCLUSIONS: This complete approach of organ transplantation cost respected the medical procedure over the time. Detailed costs take into account the care outside the hospital. This method can be used in other countries and generalised to all surgical or medical procedure as heavy as the organ transplantation.

Cost-effectiveness of monolayers and human papillomavirus testing compared to that of conventional Papanicolaou smears for cervical cancer screening: protocol of the study of the French Society of Clinical Cytology.

The French Society of Clinical Cytology is conducting a study to compare the cost-effectiveness of monolayers and human papillomavirus (HPV) testing with that of conventional Papanicolaou (Pap) smears for cervical cancer screening. The protocol of this study is presented. It includes 3,000 women who will be evaluated by the three methods (conventional Pap smears, or monolayers with or without HPV testing) and by the reference method: colposcopy followed, in cases with abnormalities, by cervical biopsy. Efficacy or performance of the methods will be compared on the basis of sensitivity. Cost comparisons and cost-effectiveness modeling will be based on the costs associated with methods themselves and also the costs of "false positives." This will require specific collection of data concerning the costs of the three methods, as these costs have not previously been accurately documented. Patient recruiting and data collection started in September 1999 and will be complete in June 2000. The first results are expected to be available in spring 2001.

Mutation of Asn-391 within the conserved NPXXY motif of the cholecystokinin B receptor abolishes Gq protein activation without affecting its association with the receptor.

Among the most conserved regions in the G-protein-coupled receptors is the (N/D)PX(2-3)Y motif of the seventh transmembrane domain (X represents any amino acid). The mutation of the Asn/Asp residue of this motif in different G-protein-coupled receptors was shown to affect the activation of either adenylyl cyclase or phospholipase C. We have mutated the Asn residue (Asn-391) of the NPXXY motif in the CCKBR to Ala and determined the effects of the mutation on binding, signaling, and G-proteins coupling after expression of the mutated receptor in COS cells. The mutated receptor displayed similar expression levels and high affinity CCK binding compared with the wild type CCKBR. However, unlike the wild type CCKBR, the mutated receptor was completely unable to mediate activation of either phospholipase C and protein kinase C-dependent and -independent mitogen-activated protein kinase pathways, indicating an essential role of Asn-391 in CCKBR signaling. Coimmunoprecipitation experiments allowed us to show that the inactive mutant retains an intact capacity to form stable complexes with G(q)alpha subunits in response to CCK. These results indicate that the formation of high affinity CCK-receptor-G(q) protein complexes is not sufficient to activate G(q) and suggest that Asn-391 is specifically involved in G(q) proteins activation.

Evidence for a direct interaction between the penultimate aspartic acid of cholecystokinin and histidine 207, located in the second extracellular loop of the cholecystokinin B receptor.

Recently, we reported that the mutation of His(207) to Phe located in the second extracellular loop of the cholecystokinin B receptor strongly affected cholecystokinin (CCK) binding (Silvente-Poirot, S., Escrieut, C., and Wank, S. A. (1998) Mol. Pharmacol. 54, 364-371). To characterize the functional group in CCK that interacts with His(207), we first substituted His(207) to Ala. This mutation decreased the affinity and the potency of CCK to produce total inositol phosphates 302-fold and 456-fold without affecting the expression of the mutant receptor. The screening of L-alanine-modified CCK peptides to bind and activate the wild type and mutant receptors allowed the identification of the interaction of the C-terminal Asp(8) of CCK with His(207). The H207A-CCKBR mutant, unlike the wild type receptor, was insensitive to substitution of Asp(8) of CCK to other amino acid residues. This interaction was further confirmed by mutating His(207) to Asp. The affinity of CCK for the H207D-CCKBR mutant was 100-fold lower than for the H207A-CCKBR mutant, consistent with an electrostatic repulsion between the negative charges of the two interacting aspartic acids. Peptides with neutral amino acids in position eight of CCK reversed this effect and displayed a gain of affinity for the H207D mutant compared with CCK. To date, this is the first report concerning the identification of a direct contact point between the CCKB receptor and CCK.

Costs and outcomes of liver transplantation in adults: a prospective, 1-year, follow-up study. GRETHECO study group.

BACKGROUND: Orthotopic liver transplantation (OLT) is widely used to treat patients with end-stage liver disease. However, data on the cost of the procedure are fragmentary. We evaluated the costs, as calculated from resource use, and outcomes of OLT in adults, from registration on the transplant waiting list to the end of the 1st-year of follow-up after the transplant. METHODS: Two parallel cohort studies were conducted from 1994 to 95. All patients ages 18 years and older, on the waiting list (n=33) according to national criteria or having undergone transplants (n=38) were followed for 1 year or until either the transplant (waiting list cohort) or death (waiting list and transplantation cohorts). RESULTS: Eighty percent of the patients undergoing transplants were alive after 1 year, and no patient died while on the waiting list. However, the estimated cost of the procedure was high: more than 55,000 pound silver for the 1st year after OLT, to be added to 5,500 pound silver for evaluation and further costs motivated by the planned transplant during an average 6.5 months on the waiting list. Age over 40 and a baseline Child-Pugh score of 10 and over were predictive of high costs. The proportion of costs associated with immunosuppressive therapy and rejection were very high. CONCLUSIONS: This medical and economic cohort study suggests that OLT is still expensive; the study identifies sources of extra cost that could be limited either by improved selection of patients or, in the future, by technological advances in immunosuppressive therapy that help avoid medical complications. It also suggests the situation is precarious, with outcomes and costs being very sensitive to variation in graft availability.

Homologous regulation of the alpha2C-adrenoceptor subtype in human hepatocarcinoma, HepG2.

1. Previous studies of the regulation of the alpha2C-adrenoceptor in OK and in transfected cells have led to discrepant conclusions. In the present work, we examined the homologous regulation of the human alpha2C-adrenoceptor in the hepatocarcinoma cell-line, HepG2; a model which expresses this subtype spontaneously. 2. Short-period treatment of the cells with UK14304 provoked neither a diminution of the potency of the alpha2-agonist to inhibit forskolin-induced cyclic AMP-accumulation nor a change in the degree of receptor coupling to G-proteins. 3. Long-period exposure to UK14304 resulted in a large reduction of [3H]MK912 binding sites (55% decrease). The action of UK14304 was dose-dependent (EC50 = 190 +/- 45 nM), rapid (t1/2 = 4.2 h) and reversible. Receptor down-regulation was also observed with clonidine or (-)adrenaline (38 and 36% decrease, respectively) and was blocked by the addition of alpha2-antagonists. 4. Conversely to that observed with alpha2-agonists, treatment of the cells with RX821002 or yohimbine alone, but not with phentolamine, promoted a significant increase of the receptor expression. 5. The observed alterations of receptor density are not the reflection of changes at the alpha2C4 mRNA level. Estimation of the receptor protein turnover and measurement of its half-life demonstrated that down-regulation by alpha2-agonists and up-regulation by alpha2-antagonists, with inverse-agonist efficacy, are respectively the consequence of increased and decreased rate of receptor degradation. 6. In conclusion, our data show that alpha2C-adrenoceptor does not undergo desensitization but is down-regulated in HepG2. The lack of desensitization agrees with previous results obtained in cells transfected with the alpha2C4 gene, but not with observations made in OK cells. Inversely, down-regulation fits with results obtained in OK but not in transfected cells. The reasons for these discrepancies are discussed. Our results also demonstrated that certain alpha2-antagonists behave as inverse agonist on the HepG2 model and thus provide for the first time evidence of inverse efficacy of antagonists on a cellular model expressing physiological level of a wild-type alpha2-adrenoceptor.

Cross-cultural adaptation of a health status classification system in children with cancer. First results of the French adaptation of the Health Utilities Index Marks 2 and 3.

Our objective was to adapt and validate the Health Utilities Index Mark 2 (HUI 2) and HUI 3 health status classification systems self-report questionnaire in a population of children with cancer, a group of 42 children already included in a multi-centre database designed by the Group on Brain Tumors in Children of the French Society for Pediatric Oncology. Children were recruited during a routine consultation. Most of them had completed treatment. The version of the questionnaire for French adults was adapted linguistically for children. Open-ended queries by children about the comprehensiveness of the questions and very low non-response rates showed a good acceptability of the questionnaire. The main psychometric properties of the HUI 2 and HUI 3 classification systems were assessed in 3 groups of raters (child, parent, physician): construct validity was tested against the rating of the child's health state on a Likert scale and through comparison with clinical data, and internal consistency was determined through multi-trait analysis. Weighted and unweighted kappa values were used to measure the inter-rater agreement between the child's, parent's and physician's assessment of the child's health state. The convergent validity was satisfactory, with better results when the physician's assessment was used. The most affected attributes were the expected ones (i.e., cognition, pain and emotion). Disagreement was observed between the 3 raters, more often in the same direction: taking the child's assessment as the reference, the parents tended to under-estimate the health status while physicians tended to over-estimate it.

Social and economic aspects of osteoporosis.

The first part of this article draws together descriptive studies on the direct medical costs of osteoporosis. The second part is devoted to economic evaluation of 1) osteoporosis treatment on the basis of clinical criteria and 2) preventive strategies, some of them including bone mineral density screening. These analyses point out the importance of taking into account quality of life and compliance issues. Research into dependency of the elderly has made it possible to obtain knowledge of the long-term economic consequences of osteoporosis. Development of research into dependency and its causes leads to the question as to whether the treatment and prevention of osteoporosis should be regulated in an isolated manner or whether it should be part of a larger health care policy with respect to aging.

Characterization of humoral immune responses induced by immunization with plasmid DNA expressing HIV-1 Nef accessory protein.

Mice immunized with plasmid DNA encoding Nef accessory protein of human immunodeficiency virus type 1 developed high levels of anti-Nef antibodies which were maintained for at least 16 months. These antibodies produced in response to Nef-expressing plasmid DNA did not recognize the linear peptides except the long C-terminal peptide for three of the ten sera. With anti-Nef antibodies produced in mice immunized with the protein Nef without any adjuvant, the same restraint epitope binding was found. On the contrary, anti-Nef antibodies from mice immunized with the protein in Freund's adjuvant showed a broader epitope reactivity pattern. Interestingly, the analysis of immunoglobulin isotype profiles of antibodies generated by the different protocols of immunization showed that plasmid DNA immunization induced predominantly IgG2a, whereas immunization with Nef protein, with or without adjuvant, yielded a preponderance of IgG1 antibodies.

French cross-cultural adaptation of the Health Utilities Indexes Mark 2 (HUI2) and 3 (HUI3) classification systems. Clinical and Economic Working Groups.

The McMaster Health Utilities Indexes Mark 2 (HUI2) and 3 (HUI3) are multiattribute health classification systems, for which multiattribute preference functions have been developed in Canada. They provide a comprehensive instrument for use in economic evaluations and population health survey studies. This paper reports on the first results on the adaptation of the HUI2 and HUI3 systems cross-culturally and the assessment of the validity and reliability of the French self-report questionnaire in different patient populations. The cross-cultural adaptation included translation, backtranslations, an expert consensus meeting and pre-test with a few patients and healthy subjects in order to produce a conceptually equivalent French version of the 15 question self-report questionnaire and the HUI2 and HUI3 classification systems. Different groups of patients attending specialized clinics (n = 709) completed the questionnaire and another generic questionnaire (the Sickness Impact Profile (SIP)) for validity assessment. Physicians and patients were also asked for a global subjective assessment of the patient's health status. The French questionnaire was well received by the patients. The criterion and convergent validities of both classification systems (correlations with the patients' and physicians' assessments and with the responses to the SIP questionnaire) were satisfactory. The internal consistency was acceptable too (Cronbach's alpha = 0.81), as was the 3 day test-retest reproducibility. These first results authorize careful use of the 15 question self-report questionnaire in French. An assessment of the multiattribute preference function for the HUI3 system in France will be the study's next objective.

[Conditions of use and access to care of sickle cell anemia patients. Analysis of the literature]. / Conditions d'usage et d'accès aux soins des drépanocytaires. Analyse de la littérature.

In order to identify the care access and life conditions and non-compliance risk factors of sickle-cell patients, we performed a literature survey on these subjects, and on other chronic pathologies and care systems used as references. The data showed a positive influence of good quality of life, social support and family environment on diabetes mellitus patients compliance, and the negative effect of poor socioeconomic status on adequate prenatal care. Concerning sickle-cell disease, there were few data on the non-compliance risk factors. However, the authors identified factors which influence the patient health status and showed the negative result of the disease on the quality of life of the patient and his family. A new study has been induced by these conclusions on the sickle-cell disease cohort of Guadeloupe reference center.

[Cost-effectiveness analysis of the treatment of chronic hepatitis C]. / Analyse coût-efficacité du traitement de l'hépatite chronique C.

OBJECTIVES: Chronic active hepatitis C is an important public health issue because of its prevalence, evolution, and overall cost. Treatment by recombinant alpha-interferon is both expensive and exacting and its effectiveness is limited. We report the results of a cost-effectiveness analysis of alpha-interferon treatment in patients with chronic active hepatitis C. METHODS: Direct medical costs of caring for patients with chronic active hepatitis C and its complications, based on treatment or no treatment, were assessed with retrospective data collected from the files of 137 hospital patients. Seventy-seven patients were treated with alpha-interferon between 1988 and 1994. The overall costs of caring for chronic active hepatitis C patients, without treatment or with alpha-interferon treatment (3 millions units three times a week) for 6 months (strategy A), 12 months (strategy B), or 12 months but discontinuing treatment when there was no response (strategy C), was reported and compared to the respective effectiveness of each. RESULTS: With an actualization rate of 5%, the real overall cost of caring for a chronic active hepatitis C patient was 143290 FF. Considering the contraindication rate (15%), the treatment acceptance (85%), the response rate to treatment (50%), and the prolonged response rate (25 to 30%), treating patients with strategy A induced a real overall cost of caring to 140731 FF to avoid 0.11 cases of cirrhosis, to 150277 FF to avoid 0.13 cases of cirrhosis with strategy B, and to 136947 FF to avoid 0.13 cases of cirrhosis with strategy C. CONCLUSION: Alpha-interferon treatment in patients with chronic active hepatitis C provides a long-term saving compared to cases which receive no treatment. Strategy C was the most cost-effective, inducing the reduction of both the number of cases of cirrhosis and the cost of care.

[Medical costs associated with the risk of maternal-fetal contamination by HIV. Results of a prospective survey]. / Coûts médicaux associés au risque de contamination maternofoetale par le VIH. Résultats d'une enquête prospective.

BACKGROUND: Assessment of economic implications of pediatric HIV infection allows to document efforts which are devoted by health care system to the care of children born to HIV infected women. This paper presents the results of the first analysis, in the French health care context, of direct medical costs related to HIV mother-to-child risk of transmission. MATERIAL AND METHODS: Statistical prospective analysis was performed on 853 files concerning 152 children followed in three medical centers. Because the medical consumptions data were linked to clinical status, average direct medical costs per medical contact could be defined for each center. RESULTS: Results were consistent with the hypothesis that more medical care is necessary when the clinical picture is uncertain (indeterminate status) or reflects health state deterioration. However, results are very different according to HIV clinical status and disparities may be so large that costs per clinical status hierarchy may be disrupted. Explicative factors for these variabilities are expressed in terms of patient recruitment, medical uncertainty management and resources disponibility.