Evaluation in usual practice of the bevacizumab-FOLFIRI combination for the first-line treatment of patients with unresectable metastatic colorectal cancer treated in 2006: focus on resected patients and oncogeriatrics: AVASTIN OUEST cohort of the Observatory of Cancer of the Brittany and Pays de la Loire Areas (Observatoire dédié au Cancer Bretagne / Pays de la Loire).

BACKGROUND: In 2006, bevacizumab, a targeted therapy agent was combined with FOLFIRI for the firstline treatment of patients with unresectable metastatic colorectal cancer. METHODS/RESULTS: A study on a homogenous series of 111 patients from the Brittany and Pays de la Loire areas who received bevacizumab-FOLFIRI as first-line treatment in 2006 showed the following results: 51 responses, 29 stabilisations, 21 progressions and 10 cases of toxicity prior to assessment. Median overall survival (OS) was 25.1 months and median progression-free survival was 10.2 months. Surgery secondary to treatment tripled median OS which reached 59.2 months in resected patients versus 18.8 months in unresected patients. Comparison of patients aged more or less than 70 years showed no differences in terms of benefits or risks. CONCLUSION: Bevacizumab-FOLFIRI could be administered as part of a routine care protocol to elderly patients previously evaluated by a geriatric assessment and validated by a multidisciplinary staff.

En 2006, bevacizumab-FOLFIRI représente la thérapie ciblée administrable dès la première ligne chez les patients porteurs d'un cancer colorectal métastatique non opérable. Une série homogène de 111 patients colligés en région Bretagne et Pays de la Loire ayant reçu du bevacizumab- FOLFIRI en première ligne en 2006 révèle les résultats suivants: 51 réponses, 29 stabilités, 21 progressions et 10 toxicités avant évaluation. La médiane de survie globale (OS) est de 25,1 mois et la médiane de survie sans progression (PFS) de 10,2 mois. Dans le cas d'une chirurgie secondaire, l'OS médian triple de 18,8 mois chez les patients non réséqués versus 59,2 mois ceux réséqués. En comparant les sujets âgés de plus et de moins de 70 ans, aucune différence n'a été mise en évidence en termes de bénéfice ou de risque. Bevacizumab-FOLFIRI pourrait être administré en pratique courante chez les personnes âgées sous couvert d'une évaluation gériatrique et d'une approche multidisciplinaire.

[Management of endocrine dysfunctions after allogeneic hematopoietic stem cell transplantation: a report of the SFGM-TC on adrenal insufficiency and osteoporosis]. / Suivi et prise en charge des troubles endocriniens en post-greffe de cellules souches hématopoïétiques: insuffisance corticotrope et ostéoporose.

In the attempt to harmonize clinical practices between different French transplantation centers, the French Society of Bone Marrow Transplantation and Cell Therapy (SFGM-TC) set up the third annual series of workshops which brought together practitioners from all member centers and took place in October 2012 in Lille. Here we report our results and recommendations regarding the management of short and long-term endocrine dysfunction following allogeneic stem cell transplantation. The key aim of this workshop was to give an overview on secondary adrenal insufficiency and osteoporosis post-transplant.

[Management of endocrine dysfunctions after allogeneic hematopoietic stem cell transplantation: a report of the SFGM-TC on dyslipidemia and thyroid disorders]. / Suivi et prise en charge des troubles endocriniens en post-greffe de cellules souches hématopoïétiques: dyslipidémie et thyropathie.

In the attempt to harmonize clinical practices between different French transplantation centers, the French Society of Bone Marrow Transplantation and Cell Therapy (SFGM-TC) set up the third annual series of workshops which brought together practitioners from all member centers and took place in October 2012 in Lille. Here we report our results and recommendations regarding the management of short and long-term endocrine dysfunction following allogeneic stem cell transplantation. The key aim of this workshop was to give an overview on dyslipidemia and thyroid disorders post-transplant.

[Management of endocrine dysfunctions after allogeneic hematopoietic stem cell transplantation: a report of the SFGM-TC on gonadal failure and fertility]. / Suivi et prise en charge des troubles endocriniens en post-greffe de cellules souches hématopoïétiques: insuffisance gonadique et fertilité

In the attempt to harmonize clinical practices between different French transplantation centers, the French Society of Bone Marrow Transplantation and Cell Therapy (SFGM-TC) set up the third annual series of workshops which brought together practitioners from all member centers and took place in October 2012 in Lille. Here we report our results and recommendations regarding the management of short and long-term endocrine dysfunction following allogeneic stem cell transplantation. The key aim of this workshop was to give an overview gonadal failure, fertility preservation and post-transplant.

Virulence factors produced by strains of Staphylococcus aureus isolated from urinary tract infections.

Staphylococcus aureus infections are widely prevalent in West Africa and are often associated with urinary tract infections (UTIs). Virulence factors from S. aureus have rarely been described for such infections. The purpose of the current study was to determine the prevalence of toxins and adhesion factors obtained from S. aureus isolated from presumed primary UTIs at the Cotonou University Hospital (CUH) in Benin as compared with the Strasbourg University Hospital (SUH) in France. Both ambulatory and hospitalised patients were included in the study. Sixty-five independent strains of S. aureus from CUH and 35 strains from SUH were obtained over a four-month period. Virulence factors were characterised by immunodetection or multiplex polymerase chain reaction, and meticillin susceptibility was recorded. Approximately 50% of all isolates produced at least one enterotoxin. No isolate from SUH produced Panton-Valentine leucocidin (PVL), whereas 21.5% of the S. aureus isolates from CUH produced PVL (P<0.01). Six of 14 (43%) PVL-positive isolates were meticillin-resistant. At SUH, the incidence of MRSA (57%) was significantly higher (P<0.01) than at CUH (14%). Genes encoding clumping factor B, and elastin and laminin binding proteins were detected in almost all isolates (80%), irrespective of the geographical origin. The results for elastin binding protein differed significantly from published data regarding isolates from other clinical origins. Staphylococcal toxins and adhesion factors may be important in the physiopathology of UTI.

[Antimalarial drug retinopathy]. / Rétinopathie sévère aux antipaludéens de synthèse.

INTRODUCTION: Antimalarial drugs are largely used for the treatment of various systemic diseases. They can cause toxic retinopathy, which can lead to blindness. OBSERVATION: We report the case of a 32-year-old male with a systemic lupus erythematosus treated with hydroxychloroquine 400mg per day and then chloroquine 300mg per day during 8 and 9years respectively. Eighteen months after his latest visual examination, the patient experienced bilateral vision loss. Fundus examination revealed a bull's eye maculopathy. Additional tests including multifocal electroretinogram showed severe bilateral functional impairment in the parafoveal area leading to diagnosis of severe toxic retinopathy induced by antimalarial drugs. DISCUSSION: In 2016, the American Academy of Ophthalmology revised the previous 2011 recommendations concerning early retinal toxicity screening strategy which should be first based on both automated 10-2 visual fields and spectral-domain optical coherence tomography (SD OCT). Multifocal electroretinogram can be more helpful for diagnostic confirmation rather than screening. Although these recommendations are essential, they are not well known in clinical practice.

Metformin reduces angiotensin-mediated intracellular production of reactive oxygen species in endothelial cells through the inhibition of protein kinase C.

Oxidative stress plays a major role in the pathogenesis and in the onset of macrovascular complications of diabetes. We previously reported that the antihyperglycaemic drug metformin was able to decrease significantly intracellular reactive oxygen species (ROS) production of bovine aortic endothelial cells (BAEC) activated by high levels of glucose and angiotensin II (ANG). The aim of the present study was to investigate whether the antioxidant effect of metformin on BAEC could be mediated through a modulation of protein kinase C (PKC) activity, which plays a key role in the pathophysiology of diabetes. The effects of metformin on intracellular ROS production, PKC translocation and activity were studied on endothelial cells stimulated by PMA (a direct PKC activator), ANG or high levels of glucose as pathophysiological stimuli of endothelial dysfunction in diabetes. We showed that metformin decreased ROS production on PMA-, ANG- and glucose-stimulated BAEC in a similar manner to that obtained by PKC specific inhibitors (calphostin C, chelerythrine) alone. On the other hand, metformin reduced both PKC membrane translocation and kinase activity in ANG-stimulated cells. In PMA-activated cells, metformin reduced membrane PKC activity but we did not observe any alteration of PKC membrane translocation. Finally, in vitro incubation with purified PKC indicated that metformin had no direct effect on PKC activity. Taken together, our results suggest that metformin exerted intracellular antioxidant properties by decreasing ROS production through the inhibition of PKC activity.

Donor interleukin-22 and host type I interferon signaling pathway participate in intestinal graft-versus-host disease via STAT1 activation and CXCL10.

Acute graft-versus-host disease (aGVHD) remains a major complication following allogeneic hematopoietic cell transplantation, limiting the success of this therapy. We previously reported that interleukin-22 (IL-22) participates to aGVHD development, but the underlying mechanisms of its contribution remain poorly understood. In this study, we analyzed the mechanism of the pathological function of IL-22 in intestinal aGVHD. Ex-vivo colon culture experiments indicated that IL-22 was able to induce Th1-like inflammation via signal transducer and activator of transcription factor-1 (STAT1) and CXCL10 induction in the presence of type I interferon (IFN). To evaluate a potential synergy between IL-22 and type I IFN in aGVHD, we transplanted recipient mice, either wild-type (WT) or type I IFN receptor deficient (IFNAR(-/-)), with bone marrow cells and WT or IL-22 deficient (IL-22(-/-)) T cells. We observed a decreased GVHD severity in IFNAR(-/-) recipient of IL-22(-/-) T cells, which was associated with a lower level of STAT1 activation and reduced CXCL10 expression in the large intestine. Finally, immunohistochemistry staining of STAT1 performed on gastrointestinal biopsies of 20 transplanted patients showed exacerbated STAT1 activation in gastrointestinal tissues of patients with aGVHD as compared with those without aGVHD. Thus, interfering with both IL-22 and type I IFN signaling may provide a novel approach to limit aGVHD.

Bacterial death by DNA gyrase poisoning.

DNA gyrase is an essential topoisomerase that is found in all bacteria and is the target of potent antibiotics, such as the quinolones. By creating DNA lesions and inducing the bacterial SOS response, these drugs are not only highly cytotoxic but also mutagenic. Discovery and analysis of natural molecules with anti-gyrase activities, such as the CcdB or microcin B17 proteins, hold promise for understanding further topoisomerase reactions and for the design of new antibiotics.

Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes.

In Escherichia coli, the miniF plasmid CcdB protein is responsible for cell death when its action is not prevented by polypeptide CcdA. We report the isolation, localization, sequencing and properties of a bacterial mutant resistant to the cytotoxic activity of the CcdB protein. This mutation is located in the gene encoding the A subunit of topoisomerase II and produces an Arg462----Cys substitution in the amino acid sequence of the GyrA polypeptide. Hence, the mutation was called gyrA462. We show that in the wild-type strain, the CcdB protein promotes plasmid linearization; in the gyrA462 strain, this double-stranded DNA cleavage is suppressed. This indicates that the CcdB protein is responsible for gyrase-mediated double-stranded DNA breakage. CcdB, in the absence of CcdA, induces the SOS pathway. SOS induction is a biological response to DNA-damaging agents. We show that the gyrA462 mutation suppresses this SOS activation, indicating that SOS induction is a consequence of DNA damages promoted by the CcdB protein on gyrase-DNA complexes. In addition, we observe that the CcdBS sensitive phenotype dominates over the resistant phenotype. This is better explained by the conversion, in gyrA+/gyrA462 merodiploid strains, of the wild-type gyrase into a DNA-damaging agent. These results strongly suggest that the CcdB protein, like quinolone antibiotics and a variety of antitumoral drugs, is a DNA topoisomerase II poison. This is the first proteinic poison-antipoison mechanism that has been found to act via the DNA topoisomerase II.

Mini-F E protein: the carboxy-terminal end is essential for E gene repression and mini-F copy number control.

Mini-F is a segment of the conjugative plasmid F consisting of two origins of replication flanked by regulatory regions, which ensure a normal control of replication and partitioning. Adjacent to the ori-2 origin is a complex coding region that consists of the E gene overlapped by three open reading frames with the coding potential for 9000 Mr polypeptides here designated 9 kd-1, 9 kd-2 and 9 kd-3. In this paper, we show that open reading frame 9 kd-3 is preceded by active promoter and Shine-Dalgarno sequences. The E coding region specifies: an initiator of replication, which acts at the ori-2 site; a function that negatively regulates the expression of the E gene; and a function involved in mini-F copy number control. To assign one of these functions to one of the overlapping coding sequence, we have isolated, characterized and sequenced mutations mapping in the E coding region. In this paper, we analyse two mutations (cop5 and pla25) that abolish the repression of the E gene. As these mutations affect the primary structure of protein E itself but not the 9 kd polypeptides, we conclude that protein E takes part in the negative regulation of its own synthesis. In addition, the localization of the cop5 and pla25 mutations indicates that the carboxy-terminal end of the E protein is involved in the autorepression function. The cop5 mutation causes an eightfold increase of the mini-F copy number. The pla25 mutation leads to the inability of the derived mini-F plasmid to give rise to plasmid-harbouring bacteria. The ways in which the cop5 and pla25 mutations may lead to such phenotypes are discussed in relation to the different functions mapping in the E coding sequence.

Purification, circular dichroism analysis, crystallization and preliminary X-ray diffraction analysis of the F plasmid CcdB killer protein.

Large crystals of the Escherichia coli F plasmid CcdB killer protein were grown from solutions containing 32% ammonium sulphate. The crystals belong to space group P4(2)2(1)2 with a = b = 104.52 A and c = 88.45 A or P2(1)2(1)2(1) with a = 77.62 A, b = 93.28 A and c = 141.44 A. Both crystal forms diffract to 2.6 A resolution. Structure determination by multiple isomorphous replacement is under way.

The interaction of the F plasmid killer protein, CcdB, with DNA gyrase: induction of DNA cleavage and blocking of transcription.

We have studied the interaction of the F plasmid killer protein CcdB with its intracellular target DNA gyrase. We confirm that CcdB can induce DNA cleavage by gyrase and show that this cleavage reaction requires ATP hydrolysis when the substrate is linear DNA, but is independent of hydrolysis when negatively supercoiled DNA is used. The 64 kDa domain of the gyrase A protein, which can catalyse DNA cleavage in the presence of the B protein and quinolone drugs, is unable to cleave DNA in the presence of CcdB unless the C-terminal 33 kDa domain of the gyrase A protein is also present. CcdB-induced DNA cleavage by gyrase requires a minimum length of DNA (> approximately 160 bp), whereas in the presence of quinolone drugs gyrase can cleave much shorter DNA molecules. We show that CcdB, like quinolones, can form a complex with gyrase which can block transcription by RNA polymerase. A model for the interaction of CcdB with gyrase involving the trapping of a post-strand-passage intermediate is suggested. We conclude that CcdB can stabilise a cleavage complex between DNA gyrase and DNA in a manner distinct from quinolones but, like the quinolone-induced cleavage complex, the CcdB-stabilised complex can also form a barrier to the passage of polymerases.

The F plasmid CcdB protein induces efficient ATP-dependent DNA cleavage by gyrase.

DNA topoisomerases perform essential roles in DNA replication, gene transcription, and chromosome segregation. Recently, we identified a new type of topoisomerase II poison: the CcdB protein of plasmid F. When its action is not prevented by CcdA protein, the CcdB protein is a potent cytotoxin. In this paper, using purified CcdB, CcdA and gyrase, we show that CcdB protein efficiently traps gyrase in a cleavable complex. The CcdA protein not only prevents the gyrase poisoning activity of CcdB but also reverses its effect on gyrase. The mechanism by which the CcdB protein induces DNA strand breakage is closely related to the action of quinolone antibiotics. However, the ATP dependence of the CcdB cleavage process differentiates the CcdB mechanism from quinolone-dependent reactions because the quinolone antibiotics stimulate efficient DNA breakage, whether or not ATP is present. We previously showed that bacteria resistant to quinolone antibiotics are sensitive to CcdB and vice versa. Elucidation of the mechanism of action of CcdB protein may permit the design of drugs targeting gyrase so as to take advantage of this new poisoning mechanism.

Crystal structure of CcdB, a topoisomerase poison from E. coli.

The crystal structure of CcdB, a protein that poisons Escherichia coli gyrase, was determined in three crystal forms. The protein consists of a five-stranded antiparallel beta-pleated sheet followed by a C-terminal alpha-helix. In one of the loops of the sheet, a second small three-stranded antiparallel beta-sheet is inserted that sticks out of the molecule as a wing. This wing contains the LysC proteolytic cleavage site that is protected by CcdA and, therefore, forms a likely CcdA recognition site. A dimer is formed by sheet extension and by extensive hydrophobic contacts involving three of the five methionine residues and the C terminus of the alpha-helix. The surface of the dimer on the side of the alpha-helix is overall negatively charged, while the opposite side as well as the wing sheet is dominated by positive charges. We propose that the CcdB dimer binds into the central hole of the 59 kDa N-terminal fragment of GyrA, after disruption of the head dimer interface of GyrA.

Positive-selection vectors using the F plasmid ccdB killer gene.

Plasmids pKIL18/19 are positive-selection cloning vectors containing an active cytotoxic ccdB gene under the control of the lacP promoter. They are derivatives of high-copy-number pUC18/19 plasmids in which the ccdB killer gene has been fused in phase downstream from the lacP MCS18 and MCS19 multiple cloning sites. When an Escherichia coli wild-type gyrA+ strain is transformed by such vectors, the ccdB gene product blocks bacterial growth. However, if ccdB is inactivated by insertion of a foreign DNA fragment, this recombinant plasmid no longer interferes with host viability. The positive selection of recombinant clones is highly efficient and bench manipulations are simplified to the utmost: E. coli transformants are plated on rich medium and only cells containing recombinant plasmids give rise to colonies. The CcdB protein is a potent poison of gyrase and the gyrA462 mutation confers total resistance to CcdB [Bernard and Couturier, J. Mol. Biol. 226 (1992) 735-745]. Therefore, pKIL18/19 vectors can be amplified and prepared in large quantities in a gyrA462 host. Like pUC vectors, pKIL vectors are designed for general cloning/sequencing procedures.

Direct selection cloning vectors adapted to the genetic analysis of gram-negative bacteria and their plasmids.

A range of specific and unusual biological pathways are found in Gram-negative bacteria. It is possible to express the genes involved in these processes in Escherichia coli, however, some genes prove lethal when cloned into high copy number vectors in common usage. Conversely, various genetic functions remain silent in E. coli and require to be transferred into their original host for expression and subsequent analysis. To facilitate the cloning and the characterisation of bacterial genes, we have constructed CcdB 'positive-selection' vectors that possess one or more of the following properties: (i) low or medium copy number; (ii) narrow or broad replication host range; (iii) conjugational mobilisation. In this communication, we illustrate the use of these new cloning tools and analyse the CcdB toxicity in different bacterial species.

In vitro study of the cytotoxicity of isolated oxidized lipid low-density lipoproteins fractions in human endothelial cells: relationship with the glutathione status and cell morphology.

Toxic effects of oxidized lipid compounds contained in oxidized LDL to endothelial cells are involved in the pathogenesis of atherosclerosis. Glutathione (GSH) plays an important role in the redox status of the cell and in the protective effect against oxidant injuries. However, little is known about the respective effect of these different oxidized lipid compounds toward cytotoxicity and GSH status of the cell. In this report, we isolated by high-performance liquid chromatography oxidized lipid compounds from low-density lipoproteins (LDL) oxidized by copper and we examined their effects on cultured endothelial cells. Cytotoxicity and GSH status were determined after incubation of endothelial cells with crude LDL or isolated lipid fractions derived from cholesterol, phospholipids, or cholesteryl esters. Their effects on cell morphology were also assessed. Oxidized lipids coming from cholesteryl esters (hydroperoxides or short-chain polar derivatives) induced a slight but significant GSH depletion without inducing cytotoxicity. The same species coming from phospholipids induced a more pronounced GSH depletion and a cytotoxic effect which is only present for the more polar compounds (short-chain polar derivatives) and corresponding to a total GSH depletion. In contrast, fractions containing oxysterols had a larger cytotoxic effect than their effect on GSH depletion suggesting that their cytotoxic effects are mediated by a GSH-independent pathway. All together, these data suggest that LDL-associated oxidized lipids present in copper-oxidized LDL exert cytotoxicity by an additional or synergistic effect on GSH depletion, but also by another mechanism independent of the redox status of the cell.

Short- and long-term vitamin A treatment in children with cholestasis.

Newborns have limited reserve supplies of vitamin A. Infants with chronic cholestasis are in a precarious nutritional state because of their limited ability to build these stores even though the vitamin is present in their diet. In this study, we investigated liver concentrations of vitamin A in 30 children with extrahepatic biliary atresia. We demonstrate that correction of the deficiency occurs after intramuscular administration of a water-miscible solution of retinyl palmitate (100,000 IU, or 30 mg retinol equivalent). Furthermore, we evaluated the effect of vitamin A injections on liver and blood concentrations in nine children with chronic cholestasis over a 1-y period. We conclude this treatment is efficient and is well tolerated.

Influence of two cell harvesting methods on intracellular ATP and amino acid concentrations in human fibroblast cultures.

The intracellular ATP and amino acid concentrations were determined in human fibroblast cultures reaching confluence. The values obtained were very different, depending on the cell harvesting method: trypsinization or scraping. Trypsinization appeared to be the better method for measuring the ATP concentrations (21.25 +/- 0.96 nmol per mg cell protein), this level being much lower with scraping. On the contrary, scraping was the most appropriate method for amino acid measurement. This work underlines the importance of harvesting methods for metabolic studies in human cell cultures.