Clinical benefit of fulvestrant in postmenopausal women with advanced breast cancer and primary or acquired resistance to aromatase inhibitors: final results of phase II Swiss Group for Clinical Cancer Research Trial (SAKK 21/00).

BACKGROUND: The aim of this study was to evaluate the efficacy and tolerability of fulvestrant, an estrogen receptor antagonist, in postmenopausal women with hormone-responsive tumors progressing after aromatase inhibitor (AI) treatment. PATIENTS AND METHODS: This is a phase II, open, multicenter, noncomparative study. Two patient groups were prospectively considered: group A (n=70) with AI-responsive disease and group B (n=20) with AI-resistant disease. Fulvestrant 250 mg was administered as intramuscular injection every 28 (+/-3) days. RESULTS: All patients were pretreated with AI and 84% also with tamoxifen or toremifene; 67% had bone metastases and 45% liver metastases. Fulvestrant administration was well tolerated and yielded a clinical benefit (CB; defined as objective response or stable disease [SD] for >or=24 weeks) in 28% (90% confidence interval [CI] 19% to 39%) of patients in group A and 37% (90% CI 19% to 58%) of patients in group B. Median time to progression (TTP) was 3.6 (95% CI 3.0 to 4.8) months in group A and 3.4 (95% CI 2.5 to 6.7) months in group B. CONCLUSIONS: Overall, 30% of patients who had progressed following prior AI treatment gained CB with fulvestrant, thereby delaying indication to start chemotherapy. Prior response to an AI did not appear to be predictive for benefit with fulvestrant.

Role of the stress sigma factor RpoS in GacA/RsmA-controlled secondary metabolism and resistance to oxidative stress in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens biocontrol strain CHA0, the two-component system GacS/GacA positively controls the synthesis of extracellular products such as hydrogen cyanide, protease, and 2,4-diacetylphloroglucinol, by upregulating the transcription of small regulatory RNAs which relieve RsmA-mediated translational repression of target genes. The expression of the stress sigma factor sigmaS (RpoS) was controlled positively by GacA and negatively by RsmA. By comparison with the wild-type CHA0, both a gacS and an rpoS null mutant were more sensitive to H2O2 in stationary phase. Overexpression of rpoS or of rsmZ, encoding a small RNA antagonistic to RsmA, restored peroxide resistance to a gacS mutant. By contrast, the rpoS mutant showed a slight increase in the expression of the hcnA (HCN synthase subunit) gene and of the aprA (major exoprotease) gene, whereas overexpression of sigmaS strongly reduced the expression of these genes. These results suggest that in strain CHA0, regulation of exoproduct synthesis does not involve sigmaS as an intermediate in the Gac/Rsm signal transduction pathway whereas sigmaS participates in Gac/Rsm-mediated resistance to oxidative stress.

Resistance to oxidative stress is associated with metastasis in mucocutaneous leishmaniasis.

Mucocutaneous leishmaniasis (MCL) in South and Central America is characterized by the dissemination (metastasis) of Leishmania Viannia subgenus parasites from a cutaneous lesion to nasopharyngeal tissues. Little is known about the pathogenesis of MCL, especially with regard to the virulence of the parasites and the process of metastatic dissemination. We previously examined the functional relationship between cytoplasmic peroxiredoxin and metastatic phenotype using highly, infrequently, and nonmetastatic clones isolated from an L. (V.) guyanensis strain previously shown to be highly metastatic in golden hamsters. Distinct forms of cytoplasmic peroxiredoxin were identified and found to be associated with the metastatic phenotype. We report here that peroxidase activity in the presence of hydrogen peroxide and infectivity differs between metastatic and nonmetastatic L. (V.) guyanensis clones. After hydrogen peroxide treatment or heat shock, peroxiredoxin was detected preferentially as dimers in metastatic L. (V.) guyanensis clones and in L. (V.) panamensis strains from patients with MCL, compared with nonmetastatic parasites. These data provide evidence that resistance to the first microbicidal response of the host cell by Leishmania promastigotes is linked to peroxiredoxin conformation and may be relevant to intracellular survival and persistence, which are prerequisites for the development of metastatic disease.

Rapid detection of bacterial resistance to antibiotics using AFM cantilevers as nanomechanical sensors.

The widespread misuse of drugs has increased the number of multiresistant bacteria, and this means that tools that can rapidly detect and characterize bacterial response to antibiotics are much needed in the management of infections. Various techniques, such as the resazurin-reduction assays, the mycobacterial growth indicator tube or polymerase chain reaction-based methods, have been used to investigate bacterial metabolism and its response to drugs. However, many are relatively expensive or unable to distinguish between living and dead bacteria. Here we show that the fluctuations of highly sensitive atomic force microscope cantilevers can be used to detect low concentrations of bacteria, characterize their metabolism and quantitatively screen (within minutes) their response to antibiotics. We applied this methodology to Escherichia coli and Staphylococcus aureus, showing that live bacteria produced larger cantilever fluctuations than bacteria exposed to antibiotics. Our preliminary experiments suggest that the fluctuation is associated with bacterial metabolism.

A genome-wide association study of resistance to HIV infection in highly exposed uninfected individuals with hemophilia A.

Human genetic variation contributes to differences in susceptibility to HIV-1 infection. To search for novel host resistance factors, we performed a genome-wide association study (GWAS) in hemophilia patients highly exposed to potentially contaminated factor VIII infusions. Individuals with hemophilia A and a documented history of factor VIII infusions before the introduction of viral inactivation procedures (1979-1984) were recruited from 36 hemophilia treatment centers (HTCs), and their genome-wide genetic variants were compared with those from matched HIV-infected individuals. Homozygous carriers of known CCR5 resistance mutations were excluded. Single nucleotide polymorphisms (SNPs) and inferred copy number variants (CNVs) were tested using logistic regression. In addition, we performed a pathway enrichment analysis, a heritability analysis, and a search for epistatic interactions with CCR5 Δ32 heterozygosity. A total of 560 HIV-uninfected cases were recruited: 36 (6.4%) were homozygous for CCR5 Δ32 or m303. After quality control and SNP imputation, we tested 1 081 435 SNPs and 3686 CNVs for association with HIV-1 serostatus in 431 cases and 765 HIV-infected controls. No SNP or CNV reached genome-wide significance. The additional analyses did not reveal any strong genetic effect. Highly exposed, yet uninfected hemophiliacs form an ideal study group to investigate host resistance factors. Using a genome-wide approach, we did not detect any significant associations between SNPs and HIV-1 susceptibility, indicating that common genetic variants of major effect are unlikely to explain the observed resistance phenotype in this population.

CHARACTERISATION OF ARABIDOPSIS THALIANA PERMEABLE CUTICLE MUTANTS SHOWING A STRONG RESISTANCE TO BOTRYTIS CINEREA

La cuticule des plantes, composée de cutine, un polyester lipidique complexe et de cires cuticulaires, couvre l'épiderme de la plupart des parties aériennes des plantes. Elle est constituée d'une barrière hydrophobique primaire qui minimise les pertes en eau et en soluté et protège l'organisme de différents stress environnementaux tels que les rayons UV, la dessiccation et l'infection par des pathogènes. Elle est aussi impliquée dans la délimitation des organes durant le développement. La cutine est un polyester qui, dans la plupart des espèces végétales, est principalement composé d'acides gras ω-hydroxylés composé de 16 à 18 carbones. Cependant, la cutine des feuilles d'Arabidopsis a une composition différente et est principalement constituée d'acides dicarboxyliques à 16-18 carbones. Les cires sont présentes dans le polyester de la cutine ou le recouvrent. Chez Arabidopsis, un nombre de mutants, tel que 1er, bdg, hth, att1, wbc11, et des plantes transgéniques avec différents changement dans la structure de la cuticule dans les feuilles et la tige, ont récemment été décrits et servent d'outils pour étudier la relation entre la structure et la fonction de la cuticule.7 mutants d'Arabidopsis ont été isolés par une méthode de coloration qui permet de détecter une augmentation dans la perméabilité cuticulaire. Ces mutants ont été appelés pec pour permeable cuticle.Pour la première partie de mon projet, j'ai principalement travaillé avec pec9/bre1 (permeable cuticle 9/botrytis resistance 1). PEC9/BRE1 a été identifié comme étant LACS2 (LONG CHAIN ACYL-CoA SYNTHETASE 2). Dans ce mutant, la cuticule n'est pas visible sous microscopie électronique et la quantité en acides gras omega- hydroxylés et en leurs dérivés est fortement réduite. Ces altérations conduisent à une plus grande perméabilité de la cuticule qui est mise en évidence par une plus grande sensibilité à la sécheresse et aux xénobiotiques et une coloration plus rapide par bleu de toluidine. Le mutant Iacs2 démontre aussi une grande capacité de résistance à l'infection du champignon nécrotrophique B. cinerea. Cette résistance est due à l'extrusion sur les feuilles d'un composé antifongique durant l'infection. Ce travail a été publié dans EMBO journal (Bessire et al., 2007, EMBO Journal).Mon second projet était principalement concentré sur pec1, un autre mutant isolé par le premier crible. La caractérisation de pec1 a révélé des phénotypes similaires à ceux de Iacs2, mais à chaque fois dans des proportions moindres : sensibilité accrue à la sécheresse et aux herbicides, plus grande perméabilité au bleu de toluidine et au calcofluor white, altération de la structure cuticulaire et résistance à B. cinerea à travers la même activité antifongique. PEC1 a été identifié comme étant AtPDR4. Ce gène code pour un transporteur ABC de la famille PDR ("Pleiotropic Drugs Resistance") qui sont des transporteurs ayants un large spectre de substrats. Le mutant se différencie de Iacs2, en cela que la composition en acides gras de la cuticule n'est pas autant altérée. C'est principalement le dihydroxypalmitate des fleurs dont la quantité est réduite. L'expression du gène marqué avec une GFP sous le contrôle du promoteur endogène a permis de localiser le transporteur au niveau de la membrane plasmique des cellules de l'épiderme, de manière polaire. En effet, la protéine est principalement dirigée vers l'extérieure de la plante, là où se trouve la cuticule, suggérant une implication d'AtPDR4 dans le transport de composants de la cuticule. Ce travail est actuellement soumis à Plant Cell.Une étude phylogénétique a aussi montré qu'AtPDR4 était très proche d'OsPDR6 du riz. Le mutant du riz a d'ailleurs montré des phénotypes de nanisme et de perméabilité similaire au mutant chez Arabidopsis.AbstractThe cuticle, consisting principally of cutin and cuticular waxes, is a hydrophobic layer of lipidic nature, which covers all aerial parts of plants and protects them from different abiotic and biotic stresses. Recently, the research in this area has given us a better understanding of the structure and the formation of the cuticle. The Arabidopsis mutants permeable cuticle 1 (peel) and botrytis resistance 1 (brel) were identified in two screens to identify permeable cuticles. The screens used the fluorescent dye calcofluor to measure permeability and also resistance to the fungal pathogen Botrytis. These mutants have highly permeable cuticle characteristics such as higher water loss, intake of chemicals through the cuticle, higher resistance to Botrytis cinerea infection, and organ fusion.BRE1 was cloned and found to be LACS2, a gene previously identified which is important in the formation and biosynthetic pathway of the cuticle. In brel, the amount of the major component of cutin in Arabidopsis leaves and stems, dicarboxylic acids, is five times lower than in the wild type. Moreover, the permeability of the cuticle allows the release of antifungal compounds at the leaf surface that inhibits the growth of two necrotrophic fungi: Botrytis cinerea and Sclerotinia sclerotiorum.PEC1 was identified as AtPDR4, a gene that codes for a plasma membrane transporter of the Pleiotropic Drug Resistance family, a sub-family of the ABC- transporters. AtPDR4 is strongly expressed in the epidermis of expanding tissues. In the epidermis it is located in a polar manner on the external plasma membrane, facing the cuticle. Analysis of the monomer composition of the cutin reveals that in this mutant the amount of hydroxy-acids and dihydroxy-palmitate is 2-3 times lower in flowers, in which organ these cutin monomers are the major components. Thus AtPDR4 is thought to function as a putative cutin monomer transporter.

Analysis of hepatitis C virus resistance to silibinin in vitro and in vivo points to a novel mechanism involving nonstructural protein 4B.

Intravenous silibinin (SIL) is an approved therapeutic that has recently been applied to patients with chronic hepatitis C, successfully clearing hepatitis C virus (HCV) infection in some patients even in monotherapy. Previous studies suggested multiple antiviral mechanisms of SIL; however, the dominant mode of action has not been determined. We first analyzed the impact of SIL on replication of subgenomic replicons from different HCV genotypes in vitro and found a strong inhibition of RNA replication for genotype 1a and genotype 1b. In contrast, RNA replication and infection of genotype 2a were minimally affected by SIL. To identify the viral target of SIL we analyzed resistance to SIL in vitro and in vivo. Selection for drug resistance in cell culture identified a mutation in HCV nonstructural protein (NS) 4B conferring partial resistance to SIL. This was corroborated by sequence analyses of HCV from a liver transplant recipient experiencing viral breakthrough under SIL monotherapy. Again, we identified distinct mutations affecting highly conserved amino acid residues within NS4B, which mediated phenotypic SIL resistance also in vitro. Analyses of chimeric viral genomes suggest that SIL might target an interaction between NS4B and NS3/4A. Ultrastructural studies revealed changes in the morphology of viral membrane alterations upon SIL treatment of a susceptible genotype 1b isolate, but not of a resistant NS4B mutant or genotype 2a, indicating that SIL might interfere with the formation of HCV replication sites. CONCLUSION: Mutations conferring partial resistance to SIL treatment in vivo and in cell culture argue for a mechanism involving NS4B. This novel mode of action renders SIL an attractive candidate for combination therapies with other directly acting antiviral drugs, particularly in difficult-to-treat patient cohorts.

Hypomorphic mutation in the site-1 protease Mbtps1 endows resistance to persistent viral infection in a cell-specific manner.

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), which naturally persists in rodents, represents a model for HIV, HBV, and HCV. Cleavage of the viral glycoprotein precursor by membrane-bound transcription factor peptidase, site 1 (Mbtps1 or site-1 protease), is crucial for the life cycle of arenaviruses and therefore represents a potential target for therapy. Recently, we reported a viable hypomorphic allele of Mbtps1 (woodrat) encoding a protease with diminished enzymatic activity. Using the woodrat allele, we examine the role of Mbtps1 during persistent LCMV infection. Surprisingly, Mbtps1 inhibition limits persistent but not acute viral infection and is associated with an organ/cell type-specific decrease in viral titers. Analysis of bone marrow-derived dendritic cells from woodrat mice supports their specific role in resolving persistent viral infection. These results support in vivo targeting of Mbtps1 in the treatment of arenavirus infections and demonstrate a critical role for dendritic cells in persistent viral infections.

In vivo expansion of T reg cells with IL-2-mAb complexes: induction of resistance to EAE and long-term acceptance of islet allografts without immunosuppression.

Via a transcription factor, Foxp3, immunoregulatory CD4(+)CD25(+) T cells (T reg cells) play an important role in suppressing the function of other T cells. Adoptively transferring high numbers of T reg cells can reduce the intensity of the immune response, thereby providing an attractive prospect for inducing tolerance. Extending our previous findings, we describe an in vivo approach for inducing rapid expansion of T reg cells by injecting mice with interleukin (IL)-2 mixed with a particular IL-2 monoclonal antibody (mAb). Injection of these IL-2-IL-2 mAb complexes for a short period of 3 d induces a marked (>10-fold) increase in T reg cell numbers in many organs, including the liver and gut as well as the spleen and lymph nodes, and a modest increase in the thymus. The expanded T reg cells survive for 1-2 wk and are highly activated and display superior suppressive function. Pretreating with the IL-2-IL-2 mAb complexes renders the mice resistant to induction of experimental autoimmune encephalomyelitis; combined with rapamycin, the complexes can also be used to treat ongoing disease. In addition, pretreating mice with the complexes induces tolerance to fully major histocompatibility complex-incompatible pancreatic islets in the absence of immunosuppression. Tolerance is robust and the majority of grafts are accepted indefinitely. The approach described for T reg cell expansion has clinical potential for treating autoimmune disease and promoting organ transplantation.

Combination of trastuzumab and letrozole after resistance to sequential trastuzumab and aromatase inhibitor monotherapies in patients with estrogen receptor-positive, HER-2-positive advanced breast cancer: a proof-of-concept trial (SAKK 23/03).

A sequential treatment design was chosen in this trial to ensure complete resistance to single-agent non-steroidal aromatase inhibitor (AI) and trastuzumab both given as monotherapy before receiving the combination of a non-steroidal AI and trastuzumab. Key eligibility criteria included postmenopausal patients with advanced, measurable, human epidermal growth factor receptor-2 (HER-2)-positive disease (assessed by FISH, ratio (≥2)), hormone receptor (HR)-positive disease, and progression on prior treatment with a non-steroidal AI, e.g. letrozole or anastrozole, either in the adjuvant or in the advanced setting. Patients received standard dose trastuzumab monotherapy in step 1 and upon disease progression continued trastuzumab in combination with letrozole in step 2. The primary endpoint was clinical benefit rate (CBR) in step 2. Totally, 13 patients were enrolled. In step 1, six patients (46%) achieved CBR. Median time to progression (TTP) was 161 days (95% confidence interval (CI): 82-281). In step 2, CBR was observed in eight out of the 11 evaluable patients (73%), including one patient with partial response. Median TTP for all the 11 patients was 188 days (95% CI: 77-not reached). Results of this proof-of-concept trial suggest that complete resistance to both AI and trastuzumab can be overcome in a proportion of patients by combined treatment of AI and trastuzumab, as all patients served as their own control. Our results appear promising for a new treatment strategy that offers a chemotherapy-free option for at least a subset of patients with HR-positive, HER-2-positive breast cancer over a clinically relevant time period.

Peroxisomal and microsomal lipid pathways associated with resistance to hepatic steatosis and reduced pro-inflammatory state.

Accumulation of fat in the liver increases the risk to develop fibrosis and cirrhosis and is associated with development of the metabolic syndrome. Here, to identify genes or gene pathways that may underlie the genetic susceptibility to fat accumulation in liver, we studied A/J and C57Bl/6 mice that are resistant and sensitive to diet-induced hepatosteatosis and obesity, respectively. We performed comparative transcriptomic and lipidomic analysis of the livers of both strains of mice fed a high fat diet for 2, 10, and 30 days. We found that resistance to steatosis in A/J mice was associated with the following: (i) a coordinated up-regulation of 10 genes controlling peroxisome biogenesis and β-oxidation; (ii) an increased expression of the elongase Elovl5 and desaturases Fads1 and Fads2. In agreement with these observations, peroxisomal β-oxidation was increased in livers of A/J mice, and lipidomic analysis showed increased concentrations of long chain fatty acid-containing triglycerides, arachidonic acid-containing lysophosphatidylcholine, and 2-arachidonylglycerol, a cannabinoid receptor agonist. We found that the anti-inflammatory CB2 receptor was the main hepatic cannabinoid receptor, which was highly expressed in Kupffer cells. We further found that A/J mice had a lower pro-inflammatory state as determined by lower plasma levels and IL-1β and granulocyte-CSF and reduced hepatic expression of their mRNAs, which were found only in Kupffer cells. This suggests that increased 2-arachidonylglycerol production may limit Kupffer cell activity. Collectively, our data suggest that genetic variations in the expression of peroxisomal β-oxidation genes and of genes controlling the production of an anti-inflammatory lipid may underlie the differential susceptibility to diet-induced hepatic steatosis and pro-inflammatory state.

Résistance aux diurétiques de l'anse en clinique [Resistance to loop diuretics in clinical practice]

Loop diuretics belong to the most common medications used in ambulatory and hospitalized patients, especially in situation of hypervolemia and chronic renal failure. Prolonged used of these agents in particular medical conditions can lead to attenuation of their diuretic effect, commonly known as "resistance" to diuretics. This article intends to review the main mechanisms of resistance to loop diuretic and the ways to counteract them in clinical practice.

Resistance to nucleoside analog reverse transcriptase inhibitors mediated by human immunodeficiency virus type 1 p6 protein.

Resistance of human immunodeficiency virus type 1 (HIV-1) to antiretroviral agents results from target gene mutation within the pol gene, which encodes the viral protease, reverse transcriptase (RT), and integrase. We speculated that mutations in genes other that the drug target could lead to drug resistance. For this purpose, the p1-p6(gag)-p6(pol) region of HIV-1, placed immediately upstream of pol, was analyzed. This region has the potential to alter Pol through frameshift regulation (p1), through improved packaging of viral enzymes (p6(Gag)), or by changes in activation of the viral protease (p6(Pol)). Duplication of the proline-rich p6(Gag) PTAP motif, necessary for late viral cycle activities, was identified in plasma virus from 47 of 222 (21.2%) patients treated with nucleoside analog RT inhibitor (NRTI) antiretroviral therapy but was identified very rarely from drug-naïve individuals. Molecular clones carrying a 3-amino-acid duplication, APPAPP (transframe duplication SPTSPT in p6(Pol)), displayed a delay in protein maturation; however, they packaged a 34% excess of RT and exhibited a marked competitive growth advantage in the presence of NRTIs. This phenotype is reminiscent of the inoculum effect described in bacteriology, where a larger input, or a greater infectivity of an organism with a wild-type antimicrobial target, leads to escape from drug pressure and a higher MIC in vitro. Though the mechanism by which the PTAP region participates in viral maturation is not known, duplication of this proline-rich motif could improve assembly and packaging at membrane locations, resulting in the observed phenotype of increased infectivity and drug resistance.