Efficacy of benfluorex in combination with sulfonylurea in type 2 diabetic patients: an 18 to 34-week, open-label, extension period.

AIM: The aim of this trial was to obtain further data on the efficacy and safety of benfluorex as an add-on therapy in type 2 diabetic patients insufficiently controlled by sulfonylurea monotherapy who had a limitation for the use of metformin during a 4-month extension period following a 4-month double-blind trial. METHODS: Patients who completed the 18-week double-blind period entered the 16-week extension period. Patients in the benfluorex group during the double-blind period continued benfluorex 450 mg/day (B-B group), whilst patients in the placebo group switched to benfluorex 450 mg/day (P-B group). The main efficacy criterion was HbA(1c), analyzed as the change from week 18 (W18) to the end of treatment using a two-sided Student paired t-test. Secondary criteria were fasting plasma glucose (FPG), insulin resistance and lipids. RESULTS: Between W18 and the end of treatment, HbA(1c) decreased in the P-B group from 8.53+/-1.37% to 7.49+/-1.04% (P<0.001) and remained stable in the B-B group from 7.52+/-1.07% to 7.53+/-1.14% (NS). In the P-B group, parameters of glycemic control showed improvements from W18 to week 34 (W34) which were similar to those observed from baseline to W18 in the B-B group. Overall, the target HbA(1c) (

High expression of the POU factor Brn3a in aggressive neuroendocrine tumors.

A new family of POU transcription factors called Brn plays a role in development of the brain and some neuroendocrine structure. Because a member of this family, Brn3a, is present in the ACTH-producing mouse pituitary tumor AtT-20, binds to POMC promoter, and stimulates its activity, we studied its human homolog in ACTH-secreting or nonsecreting tumors of pituitary and bronchial origins. A specific and quantitative reverse transcription-PCR assay was developed to assess Brn3a transcripts in tumor ribonucleic acid. Brn3a transcript levels were invariably low (< 5 x 10(-6) arbitrary units) in four GH-, two PRL-, three gonadotropin-, and seven of eight ACTH-producing pituitary adenomas. A single highly invasive ACTH-secreting pituitary adenoma in a patient who ultimately died with liver metastases, and the mouse corticotroph tumor cell line AtT-20 had high Brn3a transcripts levels at 3 x 10(-5) and 4 x 10(-4) arbitrary units, respectively. Five typical bronchial carcinoids had barely detectable levels (< 5 x 10(-6) arbitrary units), whereas seven of eight small cell carcinomas of the lung (SCCLs) had extremely high levels (between 10(-3)-10(-1) arbitrary units); six of seven atypical bronchial carcinoids had intermediate values, between 10(-6) and 5 x 10(-3) arbitrary units. Although nine bronchial tumors produced POMC, there was no association between Brn3a levels and POMC gene expression; the two tumors with the highest POMC messenger ribonucleic acid contents were two bronchial carcinoids with barely detectable Brn3a levels. A gel mobility shift assay was performed with a rat CRH promoter probe that binds Brn3a; extracts of the POMC-producing human SCCL line DMS-79, which contained high levels of Brn3a transcripts, generated the same specific complex as did AtT-20 cell extracts. These data show that Brn3a gene expression in neuroendocrine tumors is not correlated with POMC gene expression; rather, it is strikingly elevated in the highly aggressive tumors, independently of their POMC status and their pituitary or nonpituitary origin.

Analysis of promoter and enhancer cell type specificities and the regulation of immunoglobulin gene expression.

We have analysed the properties of IgH promoter (VH) and enhancer (Ig) regions which were used to drive the expression of the chloramphenicol acetyl transferase (CAT) gene (cat) in recombinant plasmids. We observe little synergistic effect between the VH promoter and Ig enhancer on cat gene expression in our constructs. Replacing the VH promoter by the thymidine kinase (TK) promoter does not affect the enhancer-mediated B-cell-specific expression of the cat gene. However, replacement of the VH promoter by the mouse renin gene promoter, which is not normally expressed in B cells, completely abolishes cat gene expression in cells of this lineage. When the Ig enhancer is replaced by the SV40 enhancer (SV), CAT activity is restricted to B cells. The VH promoter is as efficient as the TK promoter in a preB cell line. Extending the size of the VH promoter fragment to include sequences between 126 to 639 bp upstream from the transcription start point results in an eight-fold decrease in CAT activity. In this situation, the tissue specificity of the promoter cat fusion is maintained. Among the various combinations tested here, the association of the TK promoter and the Ig enhancer expresses the cat gene most efficiently. The implications of these observations are discussed.

A mouse renin promoter containing the conserved decanucleotide element binds the same B-cell factors as an authentic immunoglobulin heavy chain promoter.

A mouse renin-1 gene promoter fragment, normally inactive in B-cells, becomes a potent promoter in these cells after insertion of the highly conserved decanucleotide (dc/cd sequence) of immunoglobulin heavy and light chain promoters [(1987) EMBO J. 6, 1685-1690]. We observe retarded complexes of the same electrophoretic mobility when the cd-containing renin promoter fragment or an authentic immunoglobulin heavy chain promoter fragment is incubated with a nuclear extract from myeloma cells, suggesting that the renin promoter is activated due to its acquired ability to bind a B-cell-specific positive factor. No retarded complexes are observed with the original renin promoter fragment thus questioning the presence of a repressor as an explanation for its lack of activity in B-cells.

Functional analysis of the human pro-opiomelanocortin promoter in the small cell lung carcinoma cell line DMS-79.

DMS-79 is a human cell line derived from a small cell lung carcinoma (SCLC), which expresses the pro-opiomelanocortin (POMC) gene. We took it as a model in which to study the mechanism of POMC gene expression in these tumors: precursor processing is altered and gene expression is essentially unresponsive to glucocorticoids. POMC gene structure appeared normal by Southern blot analysis, indicating that gene rearrangement was not responsible for its expression in DMS-79. Indeed, using transient expression of human POMC-luciferase fusion genes in DMS-79, we showed that (1) the normal human POMC promoter was functional in DMS-79, and (2) the same proximal promoter region (-417; + 21) produced the full transcriptional activity in DMS-79 and in the mouse pituitary cell line AtT-20. Progressive 5' deletion analysis revealed differences between AtT-20 and DMS-79: region (-611; -376) was active in AtT-20 and not in DMS-79, whereas region (-95; -161) was active in both cell lines and (-376; -417) was only active in DMS-79. The latter partially overlaps a motif homologous to the DE-2 rat element which confers the tissue-specific expression of POMC in AtT-20 cells; however, this motif had no effect in DMS-79. These data suggest that POMC gene transcription is achieved through a different set of transacting factors in DMS-79 and AtT-20. Altogether, our results provide evidence that DMS-79 is a valid model of tumors responsible for the ectopic ACTH syndrome and that the mechanism of POMC gene expression in these SCLC cells is different from that in pituitary cells.

[Pheochromocytoma, first manifestation of Von Hippel-Lindau disease: a possibility to be considered]. / Phéochromocytome, première manifestation de la maladie de von Hippel-Lindau: une éventualité à connaître.

Von Hippel-Lindau (VHL) disorder is an autosomal dominant disease characterized by the almost constant development of hemangioblastomas in the central nervous system (cerebellum, spinal cord and retina). In addition, various types of tumors including renal cell carcinomas, pancreatic cysts and pheochromocytomas are frequently observed in VHL gene carriers. Linkage of the VHL locus to the RAF-1 oncogene on the short arm of chromosome 3 (3p25-26) has been recently reported. Pheochromocytoma is of particular interest because of the risk of inaugural malignant hypertensive crisis but especially because of a great degree of interfamily variability (from 0 to 92% of affected members in previously reported large kindreds). We have studied a French series of 25 pheochromocytoma (11 males, 14 females) in VHL affected patients. Twenty pheochromocytoma (80%) occurred in a familial context, whereas 5 (20%) were consistent with "apparent sporadic cases". The mean age at pheochromocytoma diagnosis was 27 years (5-55 years). Bilateral tumours have been documented in 13 cases (52%). The prevalence of pheochromocytoma revealing VHL was 14 out 25 (56%). In these cases, VHL diagnosis was considered up to 25 years later. In 6 cases (2 deceased) pheochromocytoma was the only manifestation of VHL. Thus, search for VHL must be systematic in the presence of pheochromocytoma, in the interest of the patients themselves and of potential at-risk family members (prevention of hypertensive crisis linked to latent tumours). Basic check-up (neurological and somatic examination, ophthalmoscopy, familial inquiry) may be completed with cerebral CT scan or MRI and abdominal ultrasonography followed, if positive or doubtful, by abdominal MRI or selective angiography.

Population PKPD modelling of the long-term hypoglycaemic effect of gliclazide given as a once-a-day modified release (MR) formulation.

AIMS: To study the relationship between the pharmacokinetics (PK) of gliclazide and its long-term pharmacodynamic (PD) effect in a large population of Type 2 diabetic patients and to identify factors predicting intersubject variability. METHODS: A PKPD database of 634 Type 2 diabetic patients with a total of 5,258 fasting plasma glucose (FPG) samples was built up from the data collected during the clinical development of a modified release formulation of gliclazide (gliclazide MR). The PKPD analysis used a nonlinear mixed effect modelling approach. A mixture model was used to identify patients with a FPG response to treatment. In patients identified as responders, the decrease in FPG was related to gliclazide exposure (AUC) by an Emax relationship. An effect compartment was used to describe the link between PK and PD. A linear disease-progression model was used to assess the glycaemic deterioration observable over several months of treatment. Simulations were performed to evaluate the predictive performance of the PKPD model and to illustrate the time course of the antidiabetic effect of gliclazide MR. RESULTS: Disease state was found to be the main explanatory factor for intersubject variability in response to gliclazide. The percentage of responders to gliclazide, used as monotherapy, increased inversely to the number of classes of antidiabetic agents received prior to entry in the studies. In responders, the initial dose (30 mg) of the gliclazide MR dosing regimen induced half of the maximum hypoglycaemic effect. The equilibration half-life between the PK and PD steady states was 3 weeks (intersubject variability of 84%). The rate of disease progression was 0.84 mmol l(-1) year(-1) (intersubject variability 143%). The PKPD model adequately predicted the FPG profiles of 234 patients who received the current formulation of gliclazide. Simulation of a 1-year parallel dose ranging clinical trial illustrated the influence of dose, time and type of previous antidiabetic treatment on the percentage of patients with clinically significant improvement of blood glucose control. CONCLUSIONS: This population PKPD analysis has characterized the relationship between the exposure to gliclazide and its long-term hypoglycaemic effect, and has established that the intersubject variability in response is mostly related to disease state. These results underline the clinical interest of quickly increasing the dose of gliclazide MR according to the response to treatment in order to achieve effective blood glucose control.

Loss of catalase activity in Tn1545-induced mutants does not reduce growth of Listeria monocytogenes in vivo.

Two catalase-negative mutants of Listeria monocytogenes were obtained by chromosomal insertions of the conjugative transposon Tn1545. The loss of catalase activity did not reduce the level of virulence of these mutants in mice. Indeed, both mutants were capable of growing in host tissues at the same rate as the parental catalase-positive strain. These results favor the view that catalase does not play a critical role in the resistance of L. monocytogenes to macrophage killing.

Isolation of DNA-protein complexes based on streptavidin and biotin interaction.

We describe a method for the purification of proteins binding to specific DNA sites based on the strong interaction between streptavidin and biotin. We tested the efficiency of this method using the Escherichia coli lactose operon operator-repressor system. dUTP coupled to biotin is incorporated into a DNA fragment containing the lactose operator. A crude E. coli extract is first incubated with the biotinylated fragment and the reaction mixture is filtered on a streptavidin-agarose column. Proteins retained on the column are either eluted alone by high salt or isopropyl beta-D-thiogalactoside, or as a complex with the DNA site by enzymatic digestion of the DNA. We thus obtained a 3400-fold enrichment of the repressor complexed to the operator in one step. The method is simple and makes use of commercially available reagents. The large concentration of biotin-binding sites of the streptavidin-agarose matrix (0.1 mumol/ml packed gel) provides a very high capacity for the concentration and purification of large amounts of proteins. The advantage of this method for the detection and purification of other DNA-binding proteins is discussed.

Liver collagen mRNA and serum amino-terminal peptide of type III procollagen (PIIINP) levels in patients with alcoholic liver disease.

We have investigated the alpha 1 (I), alpha 2 (I) and alpha 1 (III) liver collagen mRNA levels in 38 patients with alcoholic liver disease. The patients were divided into 3 groups according to the severity of their liver disease. Liver collagen mRNA levels were estimated by densitometric analysis after hybridization with the corresponding cDNA. Serum amino-terminal peptide of type III procollagen (PIIINP) was determined by radioimmunoassay in 30 patients. The results indicated that there was no increase but rather a decrease in the liver alpha 1 (I), alpha 2 (I) and alpha 1 (III) collagen mRNA in patients with the most severe liver lesions as compared to those with minimal changes. This decrease was significant for alpha 2 (I) and alpha 1 (III) cDNA probes. In contrast, serum PIIINP levels showed a positive correlation with the severity of the disease. Thus this study indicates that collagen accumulation in the liver as well as elevation of the serum PIIINP during the development of alcoholic liver disease probably reflects posttranscriptional events in collagen synthesis.

Clonal analysis of human adrenocortical carcinomas and secreting adenomas.

OBJECTIVES: Adrenocortical tumours in man are characterized mainly on biochemical, anatomical and histological grounds which establish their secretory pattern and, with some uncertainty, their benign or malignant nature. To study further these tumours and eventually to shed some light on their pathogenesis, we determined their clonal composition. METHODS: Clonal composition was determined by X-chromosome inactivation analysis on tumour and leucocyte DNA using three markers: M27 beta, phospho-glycero-kinase (PGK) and hypoxanthine-phosphoribosyl transferase (HPRT) with 88, 33 and 27% heterozygosity rates respectively. PATIENTS: Clonal analysis was performed on 25 tumours from 19 heterozygous female patients: four had a carcinoma, 14 had a single secreting adenoma, and one had autonomous bilateral macronodular hyperplasia with Cushing's syndrome (seven adenomas examined). RESULTS: The malignant tumours had patterns indicative of monoclonality. The single adenomas displayed contrasting results with patterns indicative of monoclonality in eight cases, and patterns indicative of polyclonality in six cases; monoclonal adenomas were larger and had a higher prevalence of nuclear pleomorphism than the apparently polyclonal adenomas. In the patient with bilateral macronodular hyperplasia, different clonal patterns were present in different adenomas: whereas a clear monoclonal pattern was observed in the three adenomas of the right gland, in which the active X-allele was not always the same, in two interpretable adenomas of the left gland, a moderately skewed pattern suggested a partial monoclonal component. CONCLUSIONS: These data show that adrenocortical carcinomas are monoclonal and suggest that adenomas may arise from a single cell or from more than one cell under the putative action of local growth factors. In adenomas, which until now had appeared homogeneous, this genetic heterogeneity may reflect different pathophysiological mechanisms or it may represent different stages of a common multistep process exceptionally occurring in a single patient with bilateral macronodular hyperplasia.