The Colibactin Genotoxin Generates DNA Interstrand Cross-Links in Infected Cells.

Colibactins are hybrid polyketide-nonribosomal peptides produced by Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae harboring the pks genomic island. These genotoxic metabolites are produced by pks-encoded peptide-polyketide synthases as inactive prodrugs called precolibactins, which are then converted to colibactins by deacylation for DNA-damaging effects. Colibactins are bona fide virulence factors and are suspected of promoting colorectal carcinogenesis when produced by intestinal E. coli Natural active colibactins have not been isolated, and how they induce DNA damage in the eukaryotic host cell is poorly characterized. Here, we show that DNA strands are cross-linked covalently when exposed to enterobacteria producing colibactins. DNA cross-linking is abrogated in a clbP mutant unable to deacetylate precolibactins or by adding the colibactin self-resistance protein ClbS, confirming the involvement of the mature forms of colibactins. A similar DNA-damaging mechanism is observed in cellulo, where interstrand cross-links are detected in the genomic DNA of cultured human cells exposed to colibactin-producing bacteria. The intoxicated cells exhibit replication stress, activation of ataxia-telangiectasia and Rad3-related kinase (ATR), and recruitment of the DNA cross-link repair Fanconi anemia protein D2 (FANCD2) protein. In contrast, inhibition of ATR or knockdown of FANCD2 reduces the survival of cells exposed to colibactin-producing bacteria. These findings demonstrate that DNA interstrand cross-linking is the critical mechanism of colibactin-induced DNA damage in infected cells.IMPORTANCE Colorectal cancer is the third-most-common cause of cancer death. In addition to known risk factors such as high-fat diets and alcohol consumption, genotoxic intestinal Escherichia coli bacteria producing colibactin are proposed to play a role in colon cancer development. Here, by using transient infections with genotoxic E. coli, we showed that colibactins directly generate DNA cross-links in cellulo Such lesions are converted into double-strand breaks during the repair response. DNA cross-links, akin to those induced by metabolites of alcohol and high-fat diets and by widely used anticancer drugs, are both severely mutagenic and profoundly cytotoxic lesions. This finding of a direct induction of DNA cross-links by a bacterium should facilitate delineating the role of E. coli in colon cancer and engineering new anticancer agents.

The Enterobacterial Genotoxins: Cytolethal Distending Toxin and Colibactin.

While the DNA damage induced by ionizing radiation and by many chemical compounds and drugs is well characterized, the genotoxic insults inflicted by bacteria are only scarcely documented. However, accumulating evidence indicates that we are exposed to bacterial genotoxins. The prototypes of such bacterial genotoxins are the Cytolethal Distending Toxins (CDTs) produced by Escherichia coli and Salmonella enterica serovar Typhi. CDTs display the DNase structure fold and activity, and induce DNA strand breaks in the intoxicated host cell nuclei. E. coli and certain other Enterobacteriaceae species synthesize another genotoxin, colibactin. Colibactin is a secondary metabolite, a hybrid polyketide/nonribosomal peptide compound synthesized by a complex biosynthetic machinery. In this review, we summarize the current knowledge on CDT and colibactin produced by E. coli and/or Salmonella Typhi. We describe their prevalence, genetic determinants, modes of action, and impact in infectious diseases or gut colonization, and discuss the possible involvement of these genotoxigenic bacteria in cancer.

Escherichia coli†ClbS is a colibactin resistance protein.

The genomic pks island codes for the biosynthetic machinery that produces colibactin, a peptide-polyketide metabolite. Colibactin is a genotoxin that contributes to the virulence of extra-intestinal pathogenic Escherichia coli and promotes colorectal cancer. In this work, we examined whether the pks-encoded clbS gene of unknown function could participate in the self-protection of E. coli-producing colibactin. A clbS mutant was not impaired in the ability to inflict DNA damage in HeLa cells, but the bacteria activated the SOS response and ceased to replicate. This autotoxicity phenotype was markedly enhanced in a clbS uvrB double mutant inactivated for DNA repair by nucleotide excision but was suppressed in a clbS clbA double mutant unable to produce colibactin. In addition, ectopic expression of clbS protected infected HeLa cells from colibactin. Thus, ClbS is a resistance protein blocking the genotoxicity of colibactin both in the procaryotic and the eucaryotic cells.

The Plk1 inhibitor BI 2536 in patients with refractory or relapsed non-Hodgkin lymphoma: a phase I, open-label, single dose-escalation study.

Polo-like kinase 1 (Plk1) is expressed during mitosis and overexpressed in multiple cancers, including non-Hodgkin lymphoma (NHL). This phase I study determined the maximum tolerated dose (MTD) of BI 2536, a Plk1 inhibitor, as a 1 h infusion once every 3 weeks in post-transplant relapsed (n = 17) and transplant-naive (n = 24) patients with relapsed/refractory NHL. Median treatment cycles were 2 and 1.5, respectively. MTD was 175 mg for both populations; dose-limiting toxicities were grade 4 thrombocytopenia and neutropenia. Most treatment-related adverse events were grade 1/2; drug-related grade 3/4 events included thrombocytopenia and neutropenia. Four patients achieved responses (three complete and one partial at doses ≥ 150 mg, all post-transplant relapsed patients) for an overall response rate of 9.8%. BI 2536 exhibited multi-compartmental pharmacokinetics with a high volume of distribution. The activity and safety of BI 2536 in this pretreated patient population support Plk inhibitors as a therapeutic strategy in oncology.

Time course of improvement of different symptom clusters in patients with major depression and pain treated with duloxetine or placebo.

OBJECTIVE: This post hoc analysis assessed improvements in a broad range of psychopathological dimensions and in interference of pain with functioning as well as the time course of these improvements in patients with major depressive disorder (MDD) and pain treated with duloxetine versus placebo. RESEARCH DESIGN AND METHODS: Data were derived from an 8-week, double-blind, placebo-controlled study in adult outpatients with MDD and non-specific physical pain. Mean times between improvement in Brief Pain Inventory (BPI) pain severity and interference of pain with functioning, depression severity, and dimensions of the Symptom Checklist-90 Revised (SCL-90-R) subscales were evaluated by responder analysis. RESULTS: For all SCL-90-R subscores, a higher percentage of duloxetine-treated patients reached responder status (50% improvement) as compared to placebo, of these anger/hostility and interpersonal sensitivity had the highest response rates. In the duloxetine-treated group, response for anger/hostility, phobic anxiety, psychoticism, and most items assessing interference of pain with functioning was reached earlier than response for pain severity. The times to response for Montgomery-Asberg Depression Rating Scale (MADRS) and for pain severity were similar. In the placebo-treated group, times to response for depression, anxiety, and MADRS were longer than response for pain severity. CONCLUSIONS: Duloxetine, and to a lesser degree placebo, not only improved depressive symptomatology and pain severity but also a much broader range of psychopathological symptoms. Time courses of improvements were different for duloxetine and placebo, in that depression and interference of pain with functioning improved earlier than pain severity in duloxetine-treated patients but not in placebo-treated patients. These results suggest that time to response is a valuable means of characterizing treatment effects. LIMITATIONS: Pain was only assessed as a symptom and no further clinical diagnosis for pain syndromes were performed. CLINICAL TRIAL REGISTRY ID: www.clinicaltrial.gov - NCT00191919.

[Modeling of antiretroviral response from taxonomy of CD4 cells count trajectories in profound immunodeficiency setting]. / Modélisation de la réponse antirétrovirale en méta-trajectoires de taux de CD4 en situation d'immunodépression profonde.

Modeling of CD4 cells counts response was performed through a Non-Hierarchical-descendant process with profoundly immunocompromised symptomatic patients under nevirapine or efavirenz-based antiretroviral regimen in Abidjan. Similar CD4 cells count trajectories have been modelled in meta-trajectories linked to patients' classes. Global immunological response is similar between "nevirapine group" and "efavirenz group" but the model showed an internal variation of this response in each group. In the both groups, some variables presented a significant variation between classes: average CD4, CD4 Nadir, CD4 peak and average gain of CD4. In "nevirapine group", these following parameters vary significantly between classes: mean weight, mean haemoglobin count and mean increase in haemoglobin count and sex. It's also important to note that, all meta-trajectories began with distinctive categories of baseline CD4 cells counts. Other explanatory factors must be sought because the characteristics we have chosen to describe patients'classes, are not exhaustive.

Escherichia coli induces DNA damage in vivo and triggers genomic instability in mammalian cells.

Escherichia coli is a normal inhabitant of the human gut. However, E. coli strains of phylogenetic group B2 harbor a genomic island called "pks" that codes for the production of a polyketide-peptide genotoxin, Colibactin. Here we report that in vivo infection with E. coli harboring the pks island, but not with a pks isogenic mutant, induced the formation of phosphorylated H2AX foci in mouse enterocytes. We show that a single, short exposure of cultured mammalian epithelial cells to live pks(+) E. coli at low infectious doses induced a transient DNA damage response followed by cell division with signs of incomplete DNA repair, leading to anaphase bridges and chromosome aberrations. Micronuclei, aneuploidy, ring chromosomes, and anaphase bridges persisted in dividing cells up to 21 d after infection, indicating occurrence of breakage-fusion-bridge cycles and chromosomal instability. Exposed cells exhibited a significant increase in gene mutation frequency and anchorage-independent colony formation, demonstrating the infection mutagenic and transforming potential. Therefore, colon colonization with these E. coli strains harboring the pks island could contribute to the development of sporadic colorectal cancer.

Patient-assessed versus physician-assessed disease severity and outcome in patients with nonspecific pain associated with major depressive disorder.

OBJECTIVES: This post hoc analysis compared how patients and physicians estimate disease severity and global improvement during 8 weeks of treatment for major depressive disorder (MDD) with associated nonspecific pain. In addition, predictors of pain and depression were identified. METHOD: Data were derived from a double-blind, placebo-controlled, multicenter, European study (conducted from May 2005 to May 2006) in adult outpatients with MDD (DSM-IV criteria) and moderate pain not attributable to a diagnosed organic pain syndrome (Brief Pain Inventory-Short Form [BPI-SF] average pain score ≥ 3). Patients were randomly assigned to duloxetine 60 mg/day or placebo and treated for 8 weeks. Physicians were asked to rate severity of depression by using the Montgomery-Asberg Depression Rating Scale (MADRS) and the Clinical Global Impressions-Severity of Illness (CGI-S) and CGI-Improvement (CGI-I) scales. Patients were asked to assess pain using the BPI-SF, psychological symptomatology (9 domains including depression) with the Symptom Checklist-90-Revised (SCL-90-R), and overall improvement with the Patient Global Impression of Improvement (PGI-I). Multivariate linear regressions were performed as post hoc analyses to identify predictors of disease assessment at baseline and at the end of the study using a last-observation-carried-forward approach. RESULTS: All SCL-90-R domains improved during the 8 weeks of treatment. At baseline, the MADRS was associated only with the SCL-90-R obsessive-compulsive score, while the SCL-90-R depression score was associated with the BPI-SF average pain score and with many SCL-90-R subscores. The global impression of improvement was rated higher by the physicians than by the patients. At the end of the study, CGI-I was significantly associated with a decrease in depression severity (MADRS; p < .0001), younger age (p = .0005), and a decrease of the SCL-90-R interpersonal sensitivity score (p = .0359), but not with BPI-SF average pain. In contrast, patient-rated PGI-I was significantly associated with the SCL-90-R depressive domain (p < .0001), BPI-SF average pain (p = .0003), and the SCL-90-R anxiety domain (p = .0041) scores. CONCLUSION: In patients with MDD associated with at least moderate nonspecific pain, physicians consider mainly the change in depressive symptoms as measured by MADRS in their CGI-I ratings, while patients also consider pain, depression, and anxiety in their PGI-I ratings. When treating depression and assessing treatment outcome, a broad spectrum of symptoms needs to be monitored. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00191919.

Efficacy and safety of duloxetine 60 mg once daily in the treatment of pain in patients with major depressive disorder and at least moderate pain of unknown etiology: a randomized controlled trial.

OBJECTIVE: Experience of pain in major depressive disorder (MDD) can complicate diagnosis and impair treatment outcomes. This study evaluated the efficacy and safety of duloxetine in the treatment of patients with moderate pain associated with depression. METHOD: In this double-blind, placebo-controlled, 8-week study, conducted from May 2005 to May 2006, outpatients 18 years of age or older, presenting with major depressive disorder (DSM-IV criteria; Montgomery-Asberg Depression Rating Scale [MADRS] score >or= 20), moderate pain (Brief Pain Inventory-Short Form [BPI-SF] average pain score >or= 3), and Clinical Global Impressions-Severity of Illness scale (CGI-S) score >or= 4 were randomly assigned to either placebo (N = 165) or duloxetine 60 mg (N = 162) once daily. Primary outcome was change in item 5 score (average pain in the last 24 hours) of the BPI-SF from baseline. Secondary measures were MADRS total score, other BPI-SF items, CGI-S, CGI-Improvement scale, Patient Global Impressions-Improvement scale, Symptom Checklist-90-Revised, response and remission rates, safety, and tolerability. RESULTS: Duloxetine, compared with placebo, significantly reduced pain and improved depression with significant mean changes at endpoint in both BPI-SF average pain scores (-2.57 vs. -1.64, p < .001) and in MADRS total scores (-16.69 vs. -11.31, p < .001). Remission of MDD and response rates in pain and MDD were significantly (p or= 10%) in duloxetine-treated patients were nausea, hyperhidrosis, and dry mouth. CONCLUSION: These results support duloxetine's efficacy and tolerability in the treatment of pain and depression in patients with at least moderate pain associated with depression. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT00191919 (http://www.clinicaltrials.gov).

Meat and cancer: haemoglobin and haemin in a low-calcium diet promote colorectal carcinogenesis at the aberrant crypt stage in rats.

High intake of red meat, but not of white meat, is associated with an increased risk of colon cancer. However, red meat does not promote cancer in rodents. Haemin, added to low-calcium diets, increases colonic proliferation, and haemoglobin, added to high-fat diets, increases the colon tumour incidence in rats, an effect possibly due to peroxyl radicals. We thus speculated that haem might be the promoting agent in meat, and that prevention strategies could use calcium and antioxidants. These hypotheses were tested in rats at the aberrant crypt foci (ACF) stage at 100 days. F344 rats (n = 124) were given an injection of azoxymethane and were then randomized to 11 groups fed with low-calcium (20 micro mol/g) AIN76-based diets, containing 5% safflower oil. Haemin (0.25, 0.5 and 1.5 micro mol/g) or haemoglobin (1.5 and 3 micro mol haem/g) was added to five experimental diets, compared with a control diet without haem. Three other high-haemin diets (1.5 micro mol/g) were supplemented with calcium (250 micro mol/g), antioxidant butylated hydroxyanisole and rutin (0.05% each), and olive oil, which replaced safflower oil. Faecal water was assayed for lipid peroxidation by thiobarbituric acid reactive substances (TBARs) test, and for cytolytic activity. Haemin strikingly increased the ACF size, dose-dependently, from 2.6 to 11.4 crypts/ACF (all P < 0.001). The high-haemin diet also increased the number of ACF per colon (P < 0.001). Promotion was associated with increased faecal water TBARs and cytotoxicity. Calcium, olive oil and antioxidants each inhibited the haemin-induced ACF promotion, and normalized the faecal TBARs and cytotoxicity. The haemoglobin diets increased the number of ACF and faecal TBARs, but not the ACF size or the faecal cytotoxicity. In conclusion, dietary haemin is the most potent known ACF promoter. Haemoglobin is also a potent promoter of colorectal carcinogenesis. The results suggest that myoglobin in red meat could promote colon cancer. Diets high in calcium, or in oxidation-resistant fats, may prevent the possible cancer-promoting effect of red meat.

A Bayesian analysis of pharmacoeconomic data from a clinical trial on schizophrenia.

Pharmacoeconomic studies are performed in a higher frequency to assess the economic interest of new drugs. However, a standard methodology does not still completely exist. We present here the principles and results of a cost-effectiveness Bayesian analysis on data from 146 patients (interim analysis) collected during a clinical trial. This trial was originally planned to enrol 245 patients with predominantly negative schizophrenia symptoms and involved four treatment groups (a new treatment given at low dose and high dose, a comparator and a placebo). First, some prior distributions of the cost-effectiveness ratio were numerically deduced from the effectiveness parameter clinical priors (based on investigators' opinions and questionnaires before going to blind breaking) and from cost function priors. The costs taken into account were hospitalizations, sick leave days, treatments, visits to the doctor, laboratory exams and suicide attempts. The effectiveness parameter was the change from baseline on SANS (scale for the assessment of negative symptoms). Posterior distributions were elaborated for the cost-effectiveness ratio by combining the cost-effectiveness ratio priors and likelihood together using the Bayes theorem. Results lead to a conclusion in favour of the new treatment given at high dose.