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Macrophages exhibit multifunctional activity and play a central role in the response to infectious agents. It is commonly accepted that the plasticity of the response of macrophages depends on the type of stimuli. Here we re-evaluate whether the macrophage response is only dependent on the stimulus. We analyzed the transcriptomic profile of monocyte-derived macrophages (MDMs) that were activated with several pathogens and multiple in vitro-stimulations. The transcriptomic data were normalized using matched-pair analysis. Further analysis showed a clustering association with (i) specific signatures of the infectious agent and its strategy as well as (ii) a preponderance of MDM overall responses related to individuals. Currently, the null hypothesis H 0 is that the innate MDM response is globally associated with the pathogen. Our results reveal that the global innate MDM response is intrinsically and predominantly associated with the individual. Thus, the hypothesis is supported or negated depending on the transcriptomic analytical level., Author Summary: In modern medicine, diagnosis is based on objective criteria. Scientists are focused on the common denominators indicative of an infection. Analytical studies are based on this oriented approach, which defines the null hypothesis H 0 : the host immune response depends on the stimulus. We observe that the macrophage response to a given pathogen represents <0.4% of the expressed transcripts. The events to which the remaining 99.6% of transcripts are associated remain unclear. We find that 10.3% of the genes modulated during the response to the stimulus are related to the individual. They represent the overall response of the host, which integrates two responses: one associated with the stimulus and the other associated with the individual., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this mini-review, we focus on different strategies to bring nanotools specifically to cancer cells. We discuss about a better targeting of tumor, combining the characteristics of tumor environment, the increase in nanoparticles life time, the biomarkers overexpressed on cancer cells and different physical methods for non invasive therapies. Here we detail the necessity of a synergy between passive and active targeting for an actual specificity of cancer cells.

Background: Many tools used to analyze microarrays in different conditions have been described. However, the integration of deregulated genes within coherent metabolic pathways is lacking. Currently no objective selection criterion based on biological functions exists to determine a threshold demonstrating that a gene is indeed differentially expressed., Methodology/principal Findings: To improve transcriptomic analysis of microarrays, we propose a new statistical approach that takes into account biological parameters. We present an iterative method to optimise the selection of differentially expressed genes in two experimental conditions. The stringency level of gene selection was associated simultaneously with the p-value of expression variation and the occurrence rate parameter associated with the percentage of donors whose transcriptomic profile is similar. Our method intertwines stringency level settings, biological data and a knowledge database to highlight molecular interactions using networks and pathways. Analysis performed during iterations helped us to select the optimal threshold required for the most pertinent selection of differentially expressed genes., Conclusions/significance: We have applied this approach to the well documented mechanism of human macrophage response to lipopolysaccharide stimulation. We thus verified that our method was able to determine with the highest degree of accuracy the best threshold for selecting genes that are truly differentially expressed.

The protein tyrosine phosphatase PTPL1/PTPN13, the activity of which is decreased through allelic loss, promoter methylation, or somatic mutations in some tumors, has been proposed as a tumor suppressor gene. Moreover, our recent clinical study identified PTPL1 expression level as an independent prognostic indicator of a favorable outcome for patients with breast cancer. However, how PTPL1 can affect tumor aggressiveness has not been characterized. Here, we first show that PTPL1 expression, assessed by immunohistochemistry, is decreased in breast cancer and metastasis specimens compared with nonmalignant tissues. Second, to evaluate whether PTPL1 plays a critical role in breast cancer progression, RNA interference experiments were performed in poorly tumorigenic MCF-7 breast cancer cells. PTPL1 inhibition drastically increased tumor growth in athymic mice and also enhanced several parameters associated with tumor progression, including cell proliferation on extracellular matrix components and cell invasion. Furthermore, the inhibition of Src kinase expression drastically blocked the effects of PTPL1 silencing on cell growth. In PTPL1 knockdown cells, the phosphorylation of Src on tyrosine 419 is increased, leading to the activation of its downstream substrates Fak and p130cas. Finally, substrate-trapping experiments revealed that Src tyrosine 419 is a direct target of the phosphatase. Thus, by identification of PTPL1 as the first phosphatase able to inhibit Src through direct dephosphorylation in intact cells, we presently describe a new mechanism by which PTPL1 inhibits breast tumor aggressiveness.

The cation-independent mannose 6-phosphate receptor (CI-M6PR) is essential for the endocytosis of proteins bearing the mannose 6-phosphate (M6P) recognition marker. This study described the synthesis of M6P and M6S analogs presenting greater affinity for CI-M6PR than their natural compounds. Moreover, the finding of their lack of cytotoxicity for human cells and of their increased stability in human serum supports the high potential of these isosteric derivatives in therapies requiring CI-M6PR targeting.

Differential gene expression in three pairs of Trypanosoma cruzi populations or clones susceptible or resistant to benznidazole (BZ) was investigated by differential display (DD) and representation of differential expression (RDE). GenBank searches of 14 genes selected by DD showed that four sequences corresponded to different hypothetical proteins and the others were very similar to T. cruzi genes encoding mucin (TcMUC), dihydrolipoamide dehydrogenase (TcLipDH), the hexose transporter (TcHT), or a ribosomal protein. Sequence analysis was performed on 34 clones obtained by RDE; approximately half of these clones encoded 14 different hypothetical proteins and the other half encoded proteins involved with stress response, antioxidant defence, metabolism, transporter proteins, surface proteins, ribosomal proteins and others. The mRNA levels of eight T. cruzi genes obtained by RDE and DD were analysed by northern blotting to confirm the differential expression of these sequences. For six of the eight genes, TcLipDH, TcHT, TcFeSOD-A (iron superoxide dismutase-A), TcHSP70, TcHSP100 (heat shock protein) and Tc52 (thiol-transferase), mRNA levels in the drug-resistant T. cruzi population were at least twice those in the susceptible population. Further analysis of TcHSP70 showed that although the levels of TcHSP70 mRNA were four-fold higher in T. cruzi BZ-resistant population, no corresponding increase was observed in the levels of TcHSP70 protein expression. The results suggest that TcHSP70 is not directly associated with the T. cruzi drug resistance phenotype.

The first proteomic analysis of Trypanosoma cruzi resistance to Benznidazole (BZ) is presented. The differential proteome of T. cruzi with selected in vivo resistance to Benznidazole (BZR and Clone27R), its susceptible pairs (BZS and Clone9S), and a pair from a population with Benznidazole- in vitro-induced resistance (17LER) and the susceptible pair 17WTS were analyzed by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS) for protein identification. Out of 137 spots analyzed through MS, 110 were identified as 56 distinct proteins. Out of the 56 distinct proteins, 36 were present in resistant, 9 in susceptible, and 11 in both phenotypes. Among the proteins identified in resistant samples, 5 were found in Cl 27R and in BZR (calpain-like cysteine peptidase, hypothetical protein conserved 26 kDa, putative peptidase, peroxiredoxin and tyrosine amino transferase) and 4 in Cl 27R and 17LER (cyclophilin A, glutamate dehydrogenase, iron superoxide dismutase and nucleoside diphosphate kinase). As for the proteins identified in Benznidazole-susceptible samples, PGF-2a was found in BZS and 17WTS. A functional category analysis showed that the proteins involved with transcription and protein destination were overexpressed for the Benznidazole-resistant phenotype. Thus, the present study provides large-scale, protein-related information for investigation of the mechanism of T. cruzi resistance to Benznidazole.

Various biochemical differences exist between mammalian tyrosine aminotransferase (TAT) and its analogue in Trypanosoma cruzi (TcTAT), the causative agent of Chagas disease. Moreover, TcTAT is over-expressed in strains of the parasite that are resistant to benznidazole (BZ), a drug currently used in chemotherapy. TAT has thus been indicated as a potential target for the development of new chemotherapeutic agents. In the present study, the TcTAT gene has been characterised in 14 BZ-resistant and susceptible strains and clones of T. cruzi. A unique transcript of 2.0kb and similar levels of TcTAT mRNA were observed in all parasite populations. TcTAT gene is organized in a tandem multicopy array and is located on 8 chromosomal bands that vary from 785-2500kb. No amplification of TcTAT was observed in the parasite genome. A 42kDa protein expressed by TcTAT was present in all T. cruzi samples. The results suggest that TcTAT is not directly associated with the T. cruzi drug resistance phenotype. However, it may act as a general secondary compensatory mechanism or stress response factor rather than as a key component of the specific primary resistance mechanism in T. cruzi.

Estrogen receptors (ERs) are overexpressed in human breast cancers (BCs) and associated with differentiated tumors and with a more favorable prognosis. Paradoxically, ERs mediate the mitogenic action of estrogens in human BC cells and the efficacy of antiestrogens in adjuvant therapy of primary tumors. The exact mechanism underlying the ER protection against cancer progression to metastasis remains to be investigated. Herein, we show that ERs decrease invasiveness of BC cells. Detailed studies revealed that the unliganded and the E2-activated ERs decrease cancer cell invasion in vitro through two distinct mechanisms. In the presence of ligand, ERalpha inhibits invasion through a mechanism requiring the functional ERalpha domains involved in the transcriptional activation of target genes. Moreover, using different approaches, we found that cell-cell contacts were markedly increased by 17beta-estradiol (E2) treatment and decreased by the pure antiestrogen, ICI182,780. This cell-cell adhesion was associated with an increase of the major intercellular junctions, desmosomes. Conversely, in the absence of ligand, ERalpha also inhibits invasion through a distinct mechanism involving protein-protein interaction with the region of the first zinc finger of ERalpha. The relationship of these data with clinical studies and their potential therapeutic consequences will be discussed.

The cation-independent mannose 6-phosphate receptor is a multifunctional protein which binds at the cell surface to two distinct classes of ligands, the mannose 6-phosphate (M6P) bearing proteins and IGF-II. Its major function is to bind and transport M6P-enzymes to lysosomes, but it can also modulate the activity of a variety of extracellular M6P-glycoproteins (i.e., latent TGFbeta precursor, urokinase-type plasminogen activator receptor, Granzyme B, growth factors, Herpes virus). The purpose of this review is to highlight the synthesis and potential use of high affinity M6P analogues able to target this receptor. Several M6P analogues with phosphonate, carboxylate or malonate groups display a higher affinity and a stronger stability in human serum than M6P itself. These derivatives could be used to favour the delivery of specific therapeutic compounds to lysosomes, notably in enzyme replacement therapies of lysosomal diseases or in neoplastic drug targeting. In addition, their potential applications in preventing clinical disorders, which are associated with the activities of other M6P-proteins involved in wound healing, cell growth or viral infection, will be discussed.

Superoxide dismutase (SOD) removes excess superoxide radicals via dismutation to oxygen and hydrogen peroxide. In this work, we have characterized TcFeSOD-A gene from 25 Trypanosoma cruzi populations and clones susceptible, naturally resistant or with in vitro-induced (17 LER) or in vivo-selected resistance to benznidazole (BZR). In the 17 LER T. cruzi population, the levels of TcFeSOD-A mRNA were at least 3-fold higher than its drug-susceptible counterpart 17 WTS. The levels of TcFeSOD-A mRNA were similar among the other T. cruzi populations and clones regardless of the drug-resistance phenotype. We determined whether the increase in mRNA levels was due to gene amplification using Southern blot analysis of the T. cruzi populations and clones. We found that the number of TcFeSOD-A gene copies was similar for all samples tested, except for 17 LER that presented twice as many copies. The chromosomal location of the TcFeSOD-A gene and polymorphisms detected in nucleotide and amino acid sequences of TcFeSOD-A were associated with the zymodeme of the T. cruzi strain but not with drug-resistance phenotype. We observed a 23 kDa TcFeSOD-A polypeptide in all analysed T. cruzi strains. The level of this polypeptide was increased only in the 17 LER population. Specific enzyme activity analysis of TcFeSOD in the T. cruzi samples revealed a correlation between expression and activity. Our findings show an increased expression of the TcFeSOD-A enzyme in the T. cruzi population with in vitro-induced resistance to benznidazole.

Old yellow enzyme (OYE) is a NAD(P)H flavin oxidoreductase that in Trypanosoma cruzi (TcOYE) catalyzes prostaglandin PGF2alpha synthesis and reduction of some trypanocidal drugs. We performed DNA microarray analysis and it revealed that the levels of transcription of the TcOYE gene were six-fold lower in a T. cruzi population with in vitro-induced resistance to benznidazole (BZ) (17LER) than in the wild-type (17WTS). Further we investigated the TcOYE levels in 15 T. cruzi strains and clones that were either susceptible or naturally resistant to BZ and nifurtimox, or had in vivo-selected resistance to BZ. Northern blot and real-time RT-PCR analyses confirmed our finding that TcOYE transcription levels were lower in 17LER than in 17WTS. In contrast, we detected no differences in TcOYE transcription levels between other T. cruzi samples. All T. cruzi strains contained four copies of TcOYE gene, except 17LER that contained only one. A 42kDa TcOYE protein was detected in all T. cruzi strains tested. The expression of this protein was similar for all samples, with the exception of 17LER for which the protein was nearly seven-fold less expressed. The chromosomal location of the TcOYE gene and the polymorphisms detected in TcOYE nucleotide and amino acid sequences of the T. cruzi strains are associated with the zymodeme but not with drug-resistance phenotype. Our data show that one of the mechanisms conferring in vitro-induced BZ resistance to T. cruzi correlates with deletion of copies of the TcOYE gene. In contrast, the in vivo and natural resistance to BZ are mediated by different mechanisms.

Mutation of a single amino acid in the ligand-binding domain (LBD) of the human androgen receptor (hAR) can induce functional abnormalities in androgen binding, stabilization of active conformation, or interaction with coactivators. The Gly708Ala and Gly708Val substitutions are associated with partial and complete androgen insensitivity syndromes, respectively. In this work, we introduced Ala, Val, and aromatic Phe mutations at position 708 on helix H3 of the hAR-LBD and tested the functional and structural consequences on hAR activity in the presence of steroidal or nonsteroidal agonists and antagonists. The residues involved in the specific recognition of these androgen ligands were identified and analyzed in the light of in vitro biological experiments and the 3D hAR-LBD structure. Our study demonstrated that the Gly708Ala mutation influenced the agonist versus antagonist activity of the ligands and confirmed the crucial role of this residue within the ligand-binding pocket (LBP) in the modulation of androgen agonists. The Gly708Ala mutation transformed the antiandrogen cyproterone acetate (CPA), a partial agonist, into a pure antiandrogen, and the pure nonsteroidal antiandrogen hydroxyflutamide in a partial agonist. From the docking studies, we suggest that CPA acts on AR through the novel mechanism called "passive antagonism".

To gain further information concerning the regulation by androgen of AR mRNA expression in cultured genital skin fibroblasts (GSF), we first developed a quantitative reverse transcription-competitive polymerase chain reaction (RT-PCR). This method used an ethidium bromide stain analysis of the PCR products for the accurate quantitation of low levels of human androgen receptor (hAR) mRNA in GSF. To control for variations due to sample preparation, and to minimize the disparity of the reverse transcriptase efficiency between samples after the RT procedure, we produced an initial PCR for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and then adjusted the amount of cDNA to that of this housekeeping gene. Competitive PCR for hAR was then immediately performed on normalized cDNA with a competitor DNA that exhibited a 13 bp deletion as compared to the 163 bp for the target fragment, and the PCR products were easily separated by 3.5% agarose gel electrophoresis. This quantitation procedure involved no additional steps, such as enzymatic cleavage of the PCR products, nor the use of radioactivity. In GSF from individuals, we found that the normal amount of AR mRNA was 5.6 attomoles/microg RNA, (+/-1.0, s.e.m.) with an intra- and an inter-assay of 8.4 and 14.7%, respectively. We observed a biphasic pattern of AR mRNA expression in normal human GSF in the presence of physiological concentration of androgen. Quantitative RT-PCR of AR mRNA may be useful for studying AR mRNA expression in experimental or clinical conditions.

Serotonin (5-HT) analysis by high pressure liquid chromatography with electrochemical detection (HPLC-ED) has been designed to perform micro assays below the picogram range. We demonstrated that the oxidation potential of 5-HT was tightly linked to the pH of the buffer used as eluent phase. The electrochemistry of 5-HT in pH 4.00 acetate buffer and in pH 6.80 phosphate buffer allowed quantification down to 2 x 10(-18) g. The oxidization current was linked to the 5-HT amount added. The electrochemical response of 5-HT was different for low amounts in the 10(-18) g range and for amounts above the picogram range. Two curves were obtained according to the electrochemical response of 5-HT: the first one was for the 10(-18) g range and the second one was for the picogram range and above. It thus appeared between concentration ranges that assays are not be feasible. This method has been used to analyse 20 microns frozen slices of rat spinal cord. These data open new insights into scarce quantification of 5-HT without the use of radio-elements.

Several hereditary characteristics are found in Mongoloid populations (Asian and Amerindian) with a much higher frequency than in other populations. These are the mongoloid facial features, the presence of alcohol dehydrogenase beta 2, the presence of inactive mitochondrial aldehyde dehydrogenase 2, and the high rate of urinary excretion of beta aminoisobutyric acid. Analyses carried out in France and Switzerland have been made to research a possible correlation between these diverse characteristics. This correlation does not seem to exist. With regard to the excretion of beta aminoisobutyric acid, the hypothetical role of mercury in dental fillings has been checked in Caucasoid subjects. If this role is not completely excluded, it is far from being statistically significant.

In vitro benznidazole resistance was induced in cloned T. cruzi epimastigotes using a continuous drug pressure protocol. Stocks were selected according to their previous genetic characterization by multilocus enzyme electrophoresis. All the resistant clones were able to grow in long term cultivation in the presence of at least 50 microM benznidazole, which is the drug plasma level during chemotherapy in man. The highest level of resistance achieved was 220 microM. After differentiation of the epimastigote into the amastigote forms, the drug-resistance level was not affected. In both, the resistant epimastigotes and the resistant amastigotes, growth curves exhibited a lower growth rate than the sensitive counterparts without affecting the viability of the parasites. These data could be significant in basic research, to study the drug-resistance phenotype on relevant chemoresistant clones of T. cruzi, and to follow this phenotype after in vivo cycles.