PI3Kγ within a nonhematopoietic cell type negatively regulates diet-induced thermogenesis and promotes obesity and insulin resistance.

Obesity is associated with a chronic low-grade inflammation, and specific antiinflammatory interventions may be beneficial for the treatment of type 2 diabetes and other obesity-related diseases. The lipid kinase PI3Kγ is a central proinflammatory signal transducer that plays a major role in leukocyte chemotaxis, mast cell degranulation, and endothelial cell activation. It was also reported that PI3Kγ activity within hematopoietic cells plays an important role in obesity-induced inflammation and insulin resistance. Here, we show that protection from insulin resistance, metabolic inflammation, and fatty liver in mice lacking functional PI3Kγ is largely consequent to their leaner phenotype. We also show that this phenotype is largely based on decreased fat gain, despite normal caloric intake, consequent to increased energy expenditure. Furthermore, our data show that PI3Kγ action on diet-induced obesity depends on PI3Kγ activity within a nonhematopoietic compartment, where it promotes energetic efficiency for fat mass gain. We also show that metabolic modulation by PI3Kγ depends on its lipid kinase activity and might involve kinase-independent signaling. Thus, PI3Kγ is an unexpected but promising drug target for the treatment of obesity and its complications.

Surveillance of HIV type 1 drug resistance among naive patients from Venezuela.

We have studied 65 HIV-1-infected untreated patients recruited in Caracas, Venezuela with TCD4 counts > or =350/microl. The reverse transcriptase and protease sequences of the virus were sequenced, aligned with reference HIV-1 group M strains, and analyzed for drug resistance mutations. Most of the viruses were subtype B genotype in both the protease and RT genomic regions. Five of the 62 virus isolates successfully amplified showed evidence of recombination between protease and RT, with their protease region being non-B while their RT region was derived from subtype B. Four strains were found bearing resistance mutations either to NRTIs, NNRTIs, or PIs. The prevalence of HIV-1 isolates bearing resistance mutations was therefore above the 5% threshold of WHO.

Predictors for the emergence of the 2 multi-nucleoside/nucleotide resistance mutations 69 insertion and Q151M and their impact on clinical outcome in the Swiss HIV cohort study.

The 69 insertion and Q151M mutations are multi-nucleoside/nucleotide resistance mutations (MNR). The prevalence among 4078 antiretroviral therapy (ART)-experienced individuals was <1.3%. Combined ART fully prevented MNR in subtype B infections. Case-control studies were performed to identify risk factors. Control subjects were patients with ≥ 3 thymidine-analogue mutations. The 69 insertion study (27 control subjects, 14 case patients) identified didanosine exposure as a risk (odds ratio, 5.0 per year; P = .019), whereas the Q151M study (which included 44 control subjects and 25 case patients) detected no associations. Following detection, individuals with Q151M tended to have lower suppression rates and higher mortality rates, relative to control subjects. Additional studies are needed to verify these findings in non-subtype B infections.

Development of a cross-European virtual sample repository and HIV resistance database

BACKGROUND: Several European HIV observational data bases have, over the last decade, accumulated a substantial number of resistance test results and developed large sample repositories, There is a need to link these efforts together, We here describe the development of such a novel tool that allows to bind these data bases together in a distributed fashion for which the control and data remains with the cohorts rather than classic data mergers.METHODS: As proof-of-concept we entered two basic queries into the tool: available resistance tests and available samples. We asked for patients still alive after 1998-01-01, and between 180 and 195 cm of height, and how many samples or resistance tests there would be available for these patients, The queries were uploaded with the tool to a central web server from which each participating cohort downloaded the queries with the tool and ran them against their database, The numbers gathered were then submitted back to the server and we could accumulate the number of available samples and resistance tests.RESULTS: We obtained the following results from the cohorts on available samples/resistance test: EuResist: not availableI11,194; EuroSIDA: 20,71611,992; ICONA: 3,751/500; Rega: 302/302; SHCS: 53,78311,485, In total, 78,552 samples and 15,473 resistance tests were available amongst these five cohorts. Once these data items have been identified, it is trivial to generate lists of relevant samples that would be usefuI for ultra deep sequencing in addition to the already available resistance tests, Saon the tool will include small analysis packages that allow each cohort to pull a report on their cohort profile and also survey emerging resistance trends in their own cohort,CONCLUSIONS: We plan on providing this tool to all cohorts within the Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN) and will provide the tool free of charge to others for any non-commercial use, The potential of this tool is to ease collaborations, that is, in projects requiring data to speed up identification of novel resistance mutations by increasing the number of observations across multiple cohorts instead of awaiting single cohorts or studies to reach the critical number needed to address such issues.

Eumelanin- and pheomelanin-based colour advertise resistance to oxidative stress in opposite ways.

The control mechanisms and information content of melanin-based colourations are still debated among evolutionary biologists. Recent hypotheses contend that molecules involved in melanogenesis alter other physiological processes, thereby generating covariation between melanin-based colouration and other phenotypic attributes. Interestingly, several molecules such as agouti and glutathione that trigger the production of reddish-brown pheomelanin have an inhibitory effect on the production of black/grey eumelanin, whereas other hormones, such as melanocortins, have the opposite effect. We therefore propose the hypothesis that phenotypic traits positively correlated with the degree of eumelanin-based colouration may be negatively correlated with the degree of pheomelanin-based colouration, or vice versa. Given the role played by the melanocortin system and glutathione on melanogenesis and resistance to oxidative stress, we examined the prediction that resistance to oxidative stress is positively correlated with the degree of black colouration but negatively with the degree of reddish colouration. Using the barn owl (Tyto alba) as a model organism, we swapped eggs between randomly chosen nests to allocate genotypes randomly among environments and then we measured resistance to oxidative stress using the KRL assay in nestlings raised by foster parents. As predicted, the degree of black and reddish pigmentations was positively and negatively correlated, respectively, with resistance to oxidative stress. Our results reveal that eumelanin- and pheomelanin-based colourations can be redundant signals of resistance to oxidative stress.

A stroma-related gene signature predicts resistance to neoadjuvant chemotherapy in breast cancer.

To better understand the relationship between tumor-host interactions and the efficacy of chemotherapy, we have developed an analytical approach to quantify several biological processes observed in gene expression data sets. We tested the approach on tumor biopsies from individuals with estrogen receptor-negative breast cancer treated with chemotherapy. We report that increased stromal gene expression predicts resistance to preoperative chemotherapy with 5-fluorouracil, epirubicin and cyclophosphamide (FEC) in subjects in the EORTC 10994/BIG 00-01 trial. The predictive value of the stromal signature was successfully validated in two independent cohorts of subjects who received chemotherapy but not in an untreated control group, indicating that the signature is predictive rather than prognostic. The genes in the signature are expressed in reactive stroma, according to reanalysis of data from microdissected breast tumor samples. These findings identify a previously undescribed resistance mechanism to FEC treatment and suggest that antistromal agents may offer new ways to overcome resistance to chemotherapy.

Skeletal muscle triglycerides, diacylglycerols, and ceramides in insulin resistance: another paradox in endurance-trained athletes?

OBJECTIVE-Chronic exercise and obesity both increase intra-myocellular triglycerides (IMTGs) despite having opposing effects on insulin sensitivity. We hypothesized that chronically exercise-trained muscle would be characterized by lower skeletal muscle diacylglycerols (DAGs) and ceramides despite higher IMTGs and would account for its higher insulin sensitivity. We also hypothesized that the expression of key skeletal muscle proteins involved in lipid droplet hydrolysis, DAG formation, and fatty-acid partitioning and oxidation would be associated with the lipotoxic phenotype.RESEARCH DESIGN AND METHODS-A total of 14 normal-weight, endurance-trained athletes (NWA group) and 7 normal-weight sedentary (NWS group) and 21 obese sedentary (OBS group) volunteers were studied. Insulin sensitivity was assessed by glucose clamps. IMTGs, DAGs, ceramides, and protein expression were measured in muscle biopsies.RESULTS-DAG content in the NWA group was approximately twofold higher than in the OBS group and similar to 50% higher than in the NWS group, corresponding to higher insulin sensitivity. While certain DAG moieties clearly were associated with better insulin sensitivity, other species were not. Ceramide content was higher in insulin-resistant obese muscle. The expression of OXPAT/perilipin-5, adipose triglyceride lipase, and stearoyl-CoA desaturase protein was higher in the NWA group, corresponding to a higher mitochondrial content, proportion of type 1 myocytes, DAGs, and insulin sensitivity.CONCLUSIONS-Total myocellular DAGs were markedly higher in highly trained athletes, corresponding with higher insulin sensitivity, and suggest a more complex role for DAGs in insulin action. Our data also provide additional evidence in humans linking ceramides to insulin resistance. Finally, this study provides novel evidence supporting a role for specific skeletal muscle proteins involved in intramyocellular lipids, mitochondrial oxidative capacity, and insulin resistance. Diabetes 60:2588-2597, 2011

Identification of Multiple Mechanisms of Resistance to Vemurafenib in a Patient with BRAFV600E-Mutated Cutaneous Melanoma Successfully Rechallenged after Progression.

PURPOSE: To investigate the mechanism(s) of resistance to the RAF-inhibitor vemurafenib, we conducted a comprehensive analysis of the genetic alterations occurring in metastatic lesions from a patient with a BRAF(V600E)-mutant cutaneous melanoma who, after a first response, underwent subsequent rechallenge with this drug. EXPERIMENTAL DESIGN: We obtained blood and tissue samples from a patient diagnosed with a BRAF(V600E)-mutant cutaneous melanoma that was treated with vemurafenib and achieved a near-complete response. At progression, he received additional lines of chemo/immunotherapy and was successfully rechallenged with vemurafenib. Exome and RNA sequencing were conducted on a pretreatment tumor and two subcutaneous resistant metastases, one that was present at baseline and previously responded to vemurafenib (PV1) and one that occurred de novo after reintroduction of the drug (PV2). A culture established from PV1 was also analyzed. RESULTS: We identified two NRAS-activating somatic mutations, Q61R and Q61K, affecting two main subpopulations in the metastasis PV1 and a BRAF alternative splicing, involving exons 4-10, in the metastasis PV2. These alterations, known to confer resistance to RAF inhibitors, were tumor-specific, mutually exclusive, and were not detected in pretreatment tumor samples. In addition, the oncogenic PIK3CA(H1047R) mutation was detected in a subpopulation of PV1, but this mutation did not seem to play a major role in vemurafenib resistance in this metastasis. CONCLUSIONS: This work describes the coexistence within the same patient of different molecular mechanisms of resistance to vemurafenib affecting different metastatic sites. These findings have direct implications for the clinical management of BRAF-mutant melanoma. Clin Cancer Res; 19(20); 5749-57. ©2013 AACR.

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.

Can resistance to trastuzumab be reversed by endocrine therapy? Combination of trastuzumab and letrozole after resistance to sequential trastuzumab and aromatase inhibitor monotherapies in patients with ER-positive, HER-2 positive, advanced breast cancer: a proof-of-concept trial (SAKK 23/03)

Introduction: Trastuzumab (T) is a cornerstone in the treatment of patients with HER2-overexpressing advanced breast cancer and development of resistance to T is a major therapeutic problem. HER-2 is part of a highly interactive signaling network that may impair efficacy of endocrine therapy. A sequential treatment design was chosen in this trial to ensure complete resistance to single agent therapy before receiving both a non-steroidal aromatase inhibitor (AI) and T. Any kind of clinical activity with combined treatment of AI and T after progression of single agent treatments could indicate restoration of sensitivity as a consequence of cross-talking and networking between both pathways. Methods: Key eligibility criteria included postmenopausal patients (pts.) with advanced, measurable, HER-2 positive (assessed by FISH, ratio (≥2)), HR positive disease and progression on prior treatment with a non-steroidal AI, e.g. letrozole or anastrozole, either in an adjuvant or advanced setting. Pts. received standard dose T monotherapy either weekly or three-weekly in step 1 and upon disease progression, continued T in combination with letrozole in step 2. The primary endpoint was clinical benefit response (CBR: CR, PR or SD for at least 24 weeks (+/- 1 week) according to RECIST) in step 2. Results: Thirteen pts. were enrolled in five centers in Switzerland. In step 1, six pts. (46%) achieved CBR. Median time to progression (TTP) was 161 days (Range: 50 - 627). Based on data collected until the end of May 2010, CBR was observed in seven out of the eleven evaluable pts. (64%) in step 2, including one pt. with partial response. Four of the seven pts. within step 2 that achieved CBR also had CBR in step 1. Seven out of eleven pts. have documented tumor progression during step 2 treatment. Median TTP for all eleven pts. was 184 days (range 61 - 471). Mean time on study treatment (TTP in step 1 plus TTP in step 2) for pts. reaching step 2 was 380 days (range 174 - 864). Adverse events were generally mild. Conclusion: Results of this proof-of-principle trial suggest that complete resistance to both AI and T can be overcome in a proportion of pts. by combined treatment of AI and T, as all pts. served as their own control. Our results appear promising for a new treatment strategy which offers a chemotherapy-free and well-tolerated option for at least a subset of the pts. with HR positive, HER-2 positive breast cancer. Further trials will need to corroborate this finding.

Prévention et prise en charge de la résistance antivirale dans l'hépatite B chronique [A primer on the management of antiviral resistance in chronic hepatitis B]

Treatment options for chronic hepatitis B have significantly expanded over the last decade. Six nucleoside or nucleotide analogs (NA) with activity against the hepatitis B virus are currently available. Prolonged NA treatment is required in many cases to maintain viral suppression, with an inherent risk of the development of antiviral resistance. The purpose of this concise review is to provide an introduction to the prevention, diagnosis and management of antiviral resistance in chronic hepatitis B.

Sputum containing zinc enhances carbapenem resistance, biofilm formation and virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa chronic lung infections are the leading cause of mortality in cystic fibrosis patients, a serious problem which is notably due to the numerous P. aeruginosa virulence factors, to its ability to form biofilms and to resist the effects of most antibiotics. Production of virulence factors and biofilm formation by P. aeruginosa is highly coordinated through complex regulatory systems. We recently found that CzcRS, the zinc and cadmium-specific two-component system is not only involved in metal resistance, but also in virulence and carbapenem antibiotic resistance in P. aeruginosa. Interestingly, zinc has been shown to be enriched in the lung secretions of cystic fibrosis patients. In this study, we investigated whether zinc might favor P. aeruginosa pathogenicity using an artificial sputum medium to mimic the cystic fibrosis lung environment. Our results show that zinc supplementation triggers a dual P. aeruginosa response: (i) it exacerbates pathogenicity by a CzcRS two-component system-dependent mechanism and (ii) it stimulates biofilm formation by a CzcRS-independent mechanism. Furthermore, P. aeruginosa cells embedded in these biofilms exhibited increased resistance to carbapenems. We identified a novel Zn-sensitive regulatory circuit controlling the expression of the OprD porin and modifying the carbapenem resistance profile. Altogether our data demonstrated that zinc levels in the sputum of cystic fibrosis patients might aggravate P. aeruginosa infection. Targeting zinc levels in sputum would be a valuable strategy to curb the increasing burden of P. aeruginosa infections in cystic fibrosis patients.

Persistent Candida albicans colonization and molecular mechanisms of azole resistance in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) patients.

Objectives: Patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, APS-I) suffer from chronic candidosis caused mainly by Candida albicans, and repeated courses of azole antifungals have led to the development of resistance in the APECED patient population in Finland. The aim of our study was to address whether the patients are persistently colonized with the same or genetically closely related strains, whether epidemic strains are present and which molecular mechanisms account for azole resistance. Methods: Sets of C. albicans (n?=?19) isolates from nine APECED patients reported with decreased susceptibility to fluconazole isolated up to 9 years apart were included. The strains were typed by multilocus sequence typing. CDR1/2, MDR1 and ERG11 mRNA expression was analysed by northern blotting and Cdr1, Cdr2 and Mdr1 protein expression by western blotting, and TAC1 and ERG11 genes were sequenced. Results: All seven patients with multiple C. albicans isolates analysed were persistently colonized with the same or a genetically closely related strain for a mean of 5 years. All patients were colonized with different strains and no epidemic strains were found. The major molecular mechanisms behind the azole resistance were mutations in TAC1 contributing to overexpression of CDR1 and CDR2. Six new TAC1 mutations were found, one of which (N740S) is likely to be a gain-of-function mutation. Most isolates were found to have gained multiple TAC1 and ERG11 point mutations. Conclusions: Despite clinically successful treatment leading to relief of symptoms, colonization by C. albicans strains is persistent within APECED patients. Microevolution and point mutations occur within strains, leading to the development of azole-resistant isolates.

Investigations of a thermosensitive gel to temporarily occlude crural arteries in femoro-distal bypass surgery.

OBJECTIVES: Long occlusions in calcified crural arteries are a major cause of endovascular technical failure in patients with critical limb ischaemia. Therefore, distal bypasses are mainly performed in patients with heavily calcified arteries and with consequently delicate clamping. A new reverse thermosensitive polymer (RTP) is an alternative option to occlude target vessels. The aim of the study is to report our technical experience with RTP and to assess its safety and efficiency to temporarily occlude small calcified arteries during anastomosis time. METHODS: Between July 2010 and December 2011, we used RTP to occlude crural arteries in 20 consecutive patients with 20 venous distal bypasses. We recorded several operative parameters, such as volume of injected RTP, duration of occlusion and anastomotic time. Quality of occlusion was subjectively evaluated. Routine on-table angiography was performed to search for plug emboli. Primary patency, limb salvage and survival rates were reported at 6 months. RESULTS: In all patients, crural artery occlusion was achieved with the RTP without the use of an adjunct occlusion device. Mean volume of RTP used was 0.3 ml proximally and 0.25 ml distally. Mean duration of occlusion was 14.4 ± 4.5 min, while completion of the distal anastomosis lasted 13.4 ± 4.3 min. Quality of occlusion was judged as excellent in eight cases and good in 12 cases. Residual plugs were observed in two patients and removed with an embolectomy catheter, before we amended the technique for dissolution of RTP. At 6 months, primary patency rate was 75% but limb salvage rate was 87.5%. The 30-day mortality rate was 10%. CONCLUSIONS: This study shows that RTP is safe when properly dissolved and effective to occlude small calcified arteries for completion of distal anastomosis.

Inborn resistance of mice to myxoviruses: macrophages express phenotype in vitro.

A strain of avian influenza A virus was adapted to grow in mouse peritoneal macrophages in vitro. The adapted strain, called M-TUR, induced a marked cytopathic effect in macrophages from susceptible mice. Mice homozygous (A2G) or heterozygous (F1 hybrids between A2G and several susceptible strains) for the gene Mx, shown previously to induce a high level of resistance towards lethal challenge by a number of myxoviruses in vivo, yielded peritoneal macrophages which were not affected by M-TUR. Peritoneal macrophages could be classified as resistant or susceptible to M-TUR without sacrificing the cell donor. Backcrosses were arranged between (A2G X A/J)F1 and A/J mice. 64 backcross animals could be tested individually both for resistance of their macrophages in vitro after challenge with M-TUR, and for resistance of the whole animal in vivo after challenge with NWS (a neurotropic variant of human influenza A virus). Macrophages from 36 backcross mice were classified as susceptible, and all of these mice died after challenge. Macrophages from 28 mice were classified as resistant, and 26 mice survived challenge. We conclude that resistance of macrophages and resistance of the whole animal are two facets of the same phenomenon.