Melan-A/MART-1-specific CD4 T cells in melanoma patients: identification of new epitopes and ex vivo visualization of specific T cells by MHC class II tetramers.

Over the past decade, many efforts have been made to identify MHC class II-restricted epitopes from different tumor-associated Ags. Melan-A/MART-1(26-35) parental or Melan-A/MART-1(26-35(A27L)) analog epitopes have been widely used in melanoma immunotherapy to induce and boost CTL responses, but only one Th epitope is currently known (Melan-A51-73, DRB1*0401 restricted). In this study, we describe two novel Melan-A/MART-1-derived sequences recognized by CD4 T cells from melanoma patients. These epitopes can be mimicked by peptides Melan-A27-40 presented by HLA-DRB1*0101 and HLA-DRB1*0102 and Melan-A25-36 presented by HLA-DQB1*0602 and HLA-DRB1*0301. CD4 T cell clones specific for these epitopes recognize Melan-A/MART-1+ tumor cells and Melan-A/MART-1-transduced EBV-B cells and recognition is reduced by inhibitors of the MHC class II presentation pathway. This suggests that the epitopes are naturally processed and presented by EBV-B cells and melanoma cells. Moreover, Melan-A-specific Abs could be detected in the serum of patients with measurable CD4 T cell responses specific for Melan-A/MART-1. Interestingly, even the short Melan-A/MART-1(26-35(A27L)) peptide was recognized by CD4 T cells from HLA-DQ6+ and HLA-DR3+ melanoma patients. Using Melan-A/MART-1(25-36)/DQ6 tetramers, we could detect Ag-specific CD4 T cells directly ex vivo in circulating lymphocytes of a melanoma patient. Together, these results provide the basis for monitoring of naturally occurring and vaccine-induced Melan-A/MART-1-specific CD4 T cell responses, allowing precise and ex vivo characterization of responding T cells.

A new drug delivery system inhibits uveitis in an animal model after cataract surgery.

Cataract surgery is a common ocular surgical procedure consisting in the implantation of an artificial intraocular lens (IOL) to replace the ageing, dystrophic or damaged natural one. The management of postoperative ocular inflammation is a major challenge especially in the context of pre-existing uveitis. The association of the implanted IOL with a drug delivery system (DDS) allows the prolonged intraocular release of anti-inflammatory agents after surgery. Thus IOL-DDS represents an "all in one" strategy that simultaneously addresses both cataract and inflammation issues. Polymeric DDS loaded with two model anti-inflammatory drugs (triamcinolone acetonide (TA) and cyclosporine A (CsA)) were manufactured in a novel way and tested regarding their efficiency for the management of intraocular inflammation during the 3 months following surgery. The study involved an experimentally induced uveitis in rabbits. Experimental results showed that medicated DDS efficiently reduced ocular inflammation (decrease of protein concentration in aqueous humour, inflammatory cells in aqueous humour and clinical score). Additionally, more than 60% of the loading dose remained in the DDS at the end of the experiment, suggesting that the system could potentially cover longer inflammatory episodes. Thus, IOL-DDS were demonstrated to inhibit intraocular inflammation for at least 3 months after cataract surgery, representing a potential novel approach to cataract surgery in eyes with pre-existing uveitis.

Molecular features of hepatosplenic T-cell lymphoma unravels potential novel therapeutic targets.

The pathogenesis of hepatosplenic T-cell lymphoma (HSTL), a rare entity mostly derived from γδ T cells and usually with a fatal outcome, remains largely unknown. In this study, HSTL samples (7γδ and 2αβ) and the DERL2 HSTL cell line were subjected to combined gene-expression profiling and array-based comparative genomic hybridization. Compared with other T-cell lymphomas, HSTL had a distinct molecular signature irrespective of TCR cell lineage. Compared with peripheral T-cell lymphoma, not otherwise specified and normal γδ T cells, HSTL overexpressed genes encoding NK-cell-associated molecules, oncogenes (FOS and VAV3), the sphingosine-1-phosphatase receptor 5 involved in cell trafficking, and the tyrosine kinase SYK, whereas the tumor-suppressor gene AIM1 (absent in melanoma 1) was among the most down-expressed. We found highly methylated CpG islands of AIM1 in DERL2 cells, and decitabine treatment induced a significant increase in AIM1 transcripts. Syk was present in HSTL cells and DERL2 cells contained phosphorylated Syk and were sensitive to a Syk inhibitor in vitro. Genomic profiles confirmed recurrent isochromosome 7q (n = 6/9) without alterations at the SYK and AIM1 loci. Our results identify a distinct molecular signature for HSTL and highlight oncogenic pathways that offer rationale for exploring new therapeutic options such as Syk inhibitors and demethylating agents.

Molecular imaging in the development of a novel treatment paradigm for glioblastoma (GBM): an integrated multidisciplinary commentary.

Current therapeutic strategies against glioblastoma (GBM) have failed to prevent disease progression and recurrence effectively. The part played by molecular imaging (MI) in the development of novel therapies has gained increasing traction in recent years. For the first time, using expertise from an integrated multidisciplinary group of authors, herein we present a comprehensive evaluation of state-of-the-art GBM imaging and explore how advances facilitate the emergence of new treatment options. We propose a novel next-generation treatment paradigm based on the targeting of multiple hallmarks of cancer evolution that will heavily rely on MI.

Meta-analysis of 28,141 individuals identifies common variants within five new loci that influence uric acid concentrations.

Elevated serum uric acid levels cause gout and are a risk factor for cardiovascular disease and diabetes. To investigate the polygenetic basis of serum uric acid levels, we conducted a meta-analysis of genome-wide association scans from 14 studies totalling 28,141 participants of European descent, resulting in identification of 954 SNPs distributed across nine loci that exceeded the threshold of genome-wide significance, five of which are novel. Overall, the common variants associated with serum uric acid levels fall in the following nine regions: SLC2A9 (p = 5.2x10(-201)), ABCG2 (p = 3.1x10(-26)), SLC17A1 (p = 3.0x10(-14)), SLC22A11 (p = 6.7x10(-14)), SLC22A12 (p = 2.0x10(-9)), SLC16A9 (p = 1.1x10(-8)), GCKR (p = 1.4x10(-9)), LRRC16A (p = 8.5x10(-9)), and near PDZK1 (p = 2.7x10(-9)). Identified variants were analyzed for gender differences. We found that the minor allele for rs734553 in SLC2A9 has greater influence in lowering uric acid levels in women and the minor allele of rs2231142 in ABCG2 elevates uric acid levels more strongly in men compared to women. To further characterize the identified variants, we analyzed their association with a panel of metabolites. rs12356193 within SLC16A9 was associated with DL-carnitine (p = 4.0x10(-26)) and propionyl-L-carnitine (p = 5.0x10(-8)) concentrations, which in turn were associated with serum UA levels (p = 1.4x10(-57) and p = 8.1x10(-54), respectively), forming a triangle between SNP, metabolites, and UA levels. Taken together, these associations highlight additional pathways that are important in the regulation of serum uric acid levels and point toward novel potential targets for pharmacological intervention to prevent or treat hyperuricemia. In addition, these findings strongly support the hypothesis that transport proteins are key in regulating serum uric acid levels.

Design, synthesis, and biological and crystallographic evaluation of novel inhibitors of Plasmodium falciparum enoyl-ACP-reductase (PfFabI).

Malaria, a disease of worldwide significance, is responsible for over one million deaths annually. The liver-stage of Plasmodium's life cycle is the first, obligatory, but clinically silent step in malaria infection. The P. falciparum type II fatty acid biosynthesis pathway (PfFAS-II) has been found to be essential for complete liver-stage development and has been regarded as a potential antimalarial target for the development of drugs for malaria prophylaxis and liver-stage eradication. In this paper, new coumarin-based triclosan analogues are reported and their biological profile is explored in terms of inhibitory potency against enzymes of the PfFAS-II pathway. Among the tested compounds, 7 and 8 showed the highest inhibitory potency against Pf enoyl-ACP-reductase (PfFabI), followed by 15 and 3. Finally, we determined the crystal structures of compounds 7 and 11 in complex with PfFabI to identify their mode of binding and to confirm outcomes of docking simulations.

Novel Bioconjugates of Aminolevulinic Acid with Nucleosides

Esters and amino acid derivatives of 5-aminolevulinic acid (ALA) are efficient prodrugs for the production of protoporphyrin IX (PpIX), which has been used in photodynamic cancer therapy (PDT). The synthesis of novel bioconjugates combining ALA with adenosine and thymidine is reported. The novel bioconjugates have been made using a robust methodology. The new class of prodrugs contains one, two, or three ALA per molecule. Preliminary cell tests in human cancer cell lines indicate that the thymidine conjugate of ALA is an efficient prodrug for PDT.

Searching for Novel Cellular Targets of the Hepatitis C Virus NS3-4A Protease

Background: The hepatitis C virus (HCV) NS3-4A protease isnot only an essential component of the viral replication complexand a prime target for antiviral intervention but also a key playerin the persistence and pathogenesis of HCV. It cleaves andthereby inactivates two crucial adaptor proteins in viral RNAsensing and innate immunity (MAVS and TRIF) as well as aphosphatase involved in growth factor signaling (TC-PTP). Theaim of this ongoing study is to identify novel cellular targets ofthe NS3-4A protease.Methods: Cell lines inducibly expressing the NS3-4A proteasewere established using a tetracycline-regulated geneexpression system. Cells were analyzed in basal as well asinterferon-α-stimulated states. Two-dimensional difference gelelectrophoresis (2D-DIGE) and stable isotopic labeling usingamino acids in cell culture (SILAC) proteomics analysescoupled with mass spectrometry were employed to search forcellular substrates of NS3-4A.Results: A number of candidate cellular targets have beenidentified by these proteomics approaches. These are currentlybeing validated by different experimental techniques. In parallel,we are in the process of further defining the determinants forsubstrate specificity of the NS3-4A protease.Conclusions: The identification of novel cellular targets of theHCV NS3-4A protase should yield new insights into thepathogenesis of hepatitis C and may reveal novel targets forantiviral intervention.

New insights into the mechanisms of organ-specific breast cancer metastasis.

Despite the substantial advances obtained in the treatment of localized malignancies, metastatic disease still lacks effective treatment and remains the primary cause of cancer mortality, including in breast cancer. Thus, in order to improve the survival of cancer patients it is necessary to effectively improve prevention or treatment of metastasis. To achieve this goal, complementary strategies can be envisaged: the first one is the eradication of established metastases by adding novel modalities to current treatments, such as immunotherapy or targeted therapies. A second one is to prevent tumor cell dissemination to secondary organs by targeting specific steps governing the metastatic cascade and organ-specific tropism. A third one is to block the colonization of secondary organs and subsequent cancer cell growth by impinging on the ability of disseminated cancer cells to adapt to the novel microenvironment. To obtain optimal results it might be necessary to combine these strategies. The development of therapeutic approaches aimed at preventing dissemination and organ colonization requires a deeper understanding of the specific genetic events occurring in cancer cells and of the host responses that co-operate to promote metastasis formation. Recent developments in the field disclosed novel mechanisms of metastasis. In particular the crosstalk between disseminated cancer cells and the host microenvironment is emerging as a critical determinant of metastasis. The identification of tissue-specific signals involved in metastatic progression will open the way to new therapeutic strategies. Here, we will review recent progress in the field, with particular emphasis on the mechanisms of organ specific dissemination and colonization of breast cancer.

A novel optical intracellular imaging approach for potassium dynamics in astrocytes.

Astrocytes fulfill a central role in regulating K+ and glutamate, both released by neurons into the extracellular space during activity. Glial glutamate uptake is a secondary active process that involves the influx of three Na+ ions and one proton and the efflux of one K+ ion. Thus, intracellular K+ concentration ([K+]i) is potentially influenced both by extracellular K+ concentration ([K+]o) fluctuations and glutamate transport in astrocytes. We evaluated the impact of these K+ ion movements on [K+]i in primary mouse astrocytes by microspectrofluorimetry. We established a new noninvasive and reliable approach to monitor and quantify [K+]i using the recently developed K+ sensitive fluorescent indicator Asante Potassium Green-1 (APG-1). An in situ calibration procedure enabled us to estimate the resting [K+]i at 133±1 mM. We first investigated the dependency of [K+]i levels on [K+]o. We found that [K+]i followed [K+]o changes nearly proportionally in the range 3-10 mM, which is consistent with previously reported microelectrode measurements of intracellular K+ concentration changes in astrocytes. We then found that glutamate superfusion caused a reversible drop of [K+]i that depended on the glutamate concentration with an apparent EC50 of 11.1±1.4 µM, corresponding to the affinity of astrocyte glutamate transporters. The amplitude of the [K+]i drop was found to be 2.3±0.1 mM for 200 µM glutamate applications. Overall, this study shows that the fluorescent K+ indicator APG-1 is a powerful new tool for addressing important questions regarding fine [K+]i regulation with excellent spatial resolution.

Nouvelles techniques de stimulation cérébrale: perspectives thérapeutiques en psychiatrie [Novel brain stimulation techniques: therapeutic perspectives in psychiatry]

Recent advances have allowed the development of new physical techniques in neurology and psychiatry, such as Transcranial Magnetic Stimulation (TMS), Vagus Nerve Stimulation (VNS), and Deep Brain Stimulation (DBS). These techniques are already recognized as therapeutic approaches in several late stage refractory neurological disorders (Parkinson's disease, tremor, epilepsy), and currently investigated in psychiatric conditions, refractory to medical treatment (obsessive-compulsive disorder, resistant major depression). In Paralell, these new techniques offer a new window to understand the neurobiology of human behavior.

Safety of a new algorithm for the management of childhood illness (Almanach) to improve quality of care and rational use of drugs

Introduction New evidence from randomized controlled and etiology of fever studies, the availability of reliable RDT for malaria, and novel technologies call for revision of the IMCI strategy. We developed a new algorithm based on (i) a systematic review of published studies assessing the safety and appropriateness of RDT and antibiotic prescription, (ii) results from a clinical and microbiological investigation of febrile children aged <5 years, (iii) international expert IMCI opinions. The aim of this study was to assess the safety of the new algorithm among patients in urban and rural areas of Tanzania.Materials and Methods The design was a controlled noninferiority study. Enrolled children aged 2-59 months with any illness were managed either by a study clinician using the new Almanach algorithm (two intervention health facilities), or clinicians using standard practice, including RDT (two control HF). At day 7 and day 14, all patients were reassessed. Patients who were ill in between or not cured at day 14 were followed until recovery or death. Primary outcome was rate of complications, secondary outcome rate of antibiotic prescriptions.Results 1062 children were recruited. Main diagnoses were URTI 26%, pneumonia 19% and gastroenteritis (9.4%). 98% (531/541) were cured at D14 in the Almanach arm and 99.6% (519/521) in controls. Rate of secondary hospitalization was 0.2% in each. One death occurred in controls. None of the complications was due to withdrawal of antibiotics or antimalarials at day 0. Rate of antibiotic use was 19% in the Almanach arm and 84% in controls.Conclusion Evidence suggests that the new algorithm, primarily aimed at the rational use of drugs, is as safe as standard practice and leads to a drastic reduction of antibiotic use. The Almanach is currently being tested for clinician adherence to proposed procedures when used on paper or a mobile phone

New drugs and combinations for malignant glioma.

A new chemotherapy agent and a method for local delivery of carmustine have recently been approved for the treatment of malignant glioma. However, the increase in survival remains modest at best with only a very select patients currently benefiting truly of these treatments. Combination regimen of different alkylating agents or prior O6-alkyltransferase depletion by O6-benzylguanine or continuous temozolomide administration schedules have shown some indication for increased activity. There is preclinical rational for combining temozolomide with radiotherapy and the initial results of a phase II clinical trial were promising. Several new cytotoxic agents are currently in clinical trials in patients with recurrent glioma. More importantly, targeted therapy and antiangiogenic agents have entered the clinical development phase also for patients with glioblastoma and anaplastic astrocytoma. The optimal timing of administration of non-cytotoxic substances and their integration into the currently available treatments remains a challenge. Novel study designs and identification of surrogate markers are necessary in order to make rapid and clinically meaningful progress. This review summarises the currently available evidence of activity of the recently approved drugs against malignant glioma and mentions also agents which have failed to demonstrate a significant antitumour activity. Study endpoints are critically discussed. Combination regimens with other agents and radiation therapy are reviewed. The rational for using antiangiogenic drugs in selected ongoing trials is discussed.

Genetic studies of body mass index yield new insights for obesity biology

Obesity is heritable and predisposes to many diseases. To understand the genetic basis of obesity better, here we conduct a genome-wide association study and Metabochip meta-analysis of body mass index (BMI), a measure commonly used to define obesity and assess adiposity, in up to 339,224 individuals. This analysis identifies 97 BMI-associated loci (P < 5 × 10(-8)), 56 of which are novel. Five loci demonstrate clear evidence of several independent association signals, and many loci have significant effects on other metabolic phenotypes. The 97 loci account for ∼2.7% of BMI variation, and genome-wide estimates suggest that common variation accounts for >20% of BMI variation. Pathway analyses provide strong support for a role of the central nervous system in obesity susceptibility and implicate new genes and pathways, including those related to synaptic function, glutamate signalling, insulin secretion/action, energy metabolism, lipid biology and adipogenesis.

In vitro characterization of PlySK1249, a novel phage lysin, and assessment of its antibacterial activity in a mouse model of Streptococcus agalactiae bacteremia.

Beta-hemolytic Streptococcus agalactiae is the leading cause of bacteremia and invasive infections. These diseases are treated with β-lactams or macrolides, but the emergence of less susceptible and even fully resistant strains is a cause for concern. New bacteriophage lysins could be promising alternatives against such organisms. They hydrolyze the bacterial peptidoglycan at the end of the phage cycle, in order to release the phage progeny. By using a bioinformatic approach to screen several beta-hemolytic streptococci, a gene coding for a lysin was identified on a prophage carried by Streptococcus dysgalactiae subsp. equisimilis SK1249. The gene product, named PlySK1249, harbored an original three-domain structure with a central cell wall-binding domain surrounded by an N-terminal amidase and a C-terminal CHAP domain. Purified PlySK1249 was highly lytic and bactericidal for S. dysgalactiae (2-log10 CFU/ml decrease within 15 min). Moreover, it also efficiently killed S. agalactiae (1.5-log10 CFU/ml decrease within 15 min) but not several streptococcal commensal species. We further investigated the activity of PlySK1249 in a mouse model of S. agalactiae bacteremia. Eighty percent of the animals (n = 10) challenged intraperitoneally with 10(6) CFU of S. agalactiae died within 72 h, whereas repeated injections of PlySK1249 (45 mg/kg 3 times within 24 h) significantly protected the mice (P < 0.01). Thus, PlySK1249, which was isolated from S. dysgalactiae, demonstrated high cross-lytic activity against S. agalactiae both in vitro and in vivo. These encouraging results indicated that PlySK1249 might represent a good candidate to be developed as a new enzybiotic for the treatment of systemic S. agalactiae infections.