Potent synergism of the combination of fluconazole and cyclosporine in Candida albicans.

Several types of drugs currently used in clinical practice were screened in vitro for their potentiation of the antifungal effect of the fungistatic agent fluconazole (FLC) on Candida albicans. These drugs included inhibitors of multidrug efflux transporters, antimicrobial agents, antifungal agents, and membrane-active compounds with no antimicrobial activity, such as antiarrhythmic agents, proton pump inhibitors, and platelet aggregation inhibitors. Among the drugs tested in an agar disk diffusion assay, cyclosporine (Cy), which had no intrinsic antifungal activity, showed a potent antifungal effect in combination with FLC. In a checkerboard microtiter plate format, however, it was observed that the MIC of FLC, as classically defined by the NCCLS recommendations, was unchanged when FLC and Cy were combined. Nevertheless, if a different reading endpoint corresponding to the minimal fungicidal concentration needed to decrease viable counts by at least 3 logs in comparison to the growth control was chosen, the combination was synergistic (fractional inhibitory concentration index of <1). This endpoint fitted to the definition of MIC-0 (optically clear wells) and reflected the absence of the trailing effect, which is the result of a residual growth at FLC concentrations greater than the MIC. The MIC-0 values of FLC and Cy tested alone in C. albicans were >32 and >10 microg/ml, respectively, and decreased to 0.5 and 0.625 microg/ml when the two drugs were combined. The combination of 0.625 microg of Cy per ml with supra-MICs of FLC resulted in a potent antifungal effect in time-kill curve experiments. This effect was fungicidal or fungistatic, depending on the C. albicans strain used. Since the Cy concentration effective in vitro is achievable in vivo, the combination of this agent with FLC represents an attractive perspective for the development of new management strategies for candidiasis.

Tegumental proteins of Schistosoma mansoni: complex biomolecules and potent antigens

The passive transfer of monoclonal antibodies, direct vaccination and in vitro assays have all shown that antigens associated with the tegumental membranes of Schistosoma mansoni are capable of mediating protective immune responses against the parasite in animal models. Furthermore, the principal antigens are highly antigenic during natural infection in man and stimulate strong humoral and cellular responses although, at present, their role in mediating protective immune responses in man remains equivocal. This presentation will review the current state of knowledge of the structure and expression of the major antigenic tegumental proteins of the schistosome and will attempt to relate the relevance of their structural features to possible function both in terms of protective immunity and parasite's ability to survive within the definitive host. A focus will be recent advances that have been made in the identification of means of anchoring of the antigenic proteins to the tegumental membrane. In addition, the implications of the structural complexity of the tegumental proteins in terms of their possible utility in vaccination and diagnosis will be considered.

The NAD biosynthesis inhibitor APO866 has potent antitumor activity against hematologic malignancies.

APO866 inhibits nicotinamide phosphoribosyltransferase (NMPRTase), a key enzyme involved in nicotinamide adenine dinucleotide (NAD) biosynthesis from the natural precursor nicotinamide. Intracellular NAD is essential for cell survival, and NAD depletion resulting from APO866 treatment elicits tumor cell death. Here, we determine the in vitro and in vivo sensitivities of hematologic cancer cells to APO866 using a panel of cell lines (n = 45) and primary cells (n = 32). Most cancer cells (acute myeloid leukemia [AML], acute lymphoblastic leukemia [ALL], mantle cell lymphoma [MCL], chronic lymphocytic leukemia [CLL], and T-cell lymphoma), but not normal hematopoietic progenitor cells, were sensitive to low concentrations of APO866 as measured in cytotoxicity and clonogenic assays. Treatment with APO866 decreased intracellular NAD and adenosine triphosphate (ATP) at 24 hours and 48 to72 hours, respectively. The NAD depletion led to cell death. At 96 hours, APO866-mediated cell death occurred in a caspase-independent mode, and was associated with mitochondrial dysfunction and autophagy. Further, in vivo administration of APO866 as a single agent prevented and abrogated tumor growth in animal models of human AML, lymphoblastic lymphoma, and leukemia without significant toxicity to the animals. The results support the potential of APO866 for treating hematologic malignancies.

Deltamethrin-impregnated dog collars have a potent anti-feeding and insecticidal effect on Lutzomyia longipalpis and Lutzomyia migonei

Deltamethrin-impregnated PVC dog collars were tested to assess if they were effective in protecting dogs from sand fly bites of Lutzomyia longipalpis and Lu. migonei. A protective effect against Old World species Phlebotomus perniciosus was demonstrated before. Four dogs wearing deltamethrin collars and three dogs wearing untreated collars (not impregnated with deltamethrin) were kept in separate kennels for over eight months in a village on the outskirts of Fortaleza in Ceará, Brazil. Periodically, a dog from each group was sedated, placed in a net cage for 2 h in which 150 female sand flies had been released 10-15 min before. Lu. longipalpis were used 4, 8, 12, 16, 22, 27, and 35 weeks after the attachment of the collars. Lu. migonei were used 3, 7, 11, 15, 22, 26, and 36 weeks after attachment. During 35 weeks, only 4.1% (81 of 2,022) Lu. longipalpis recovered from the nets with the deltamethrin collared dogs were engorged, an anti-feeding effect of 96%. Mortality initially was over 90% and at 35 weeks was 35% with half of the sand flies dying in the first 2 h. In contrast, 83% of the 2,094 Lu. longipalpis recovered from the nets containing the untreated collared dogs were engorged and the mortality ranged from zero to 18.8% on one occasion with 1.1% dying in the first 2 h. Similar findings were found with Lu. migonei: of 2,034 sand flies recovered over this period, only 70 were engorged, an anti-feeding effect of 96.5%, and mortality ranged from 91% initially to 46% at 36 weeks. In contrast, engorgement of controls ranged from 91 to71% and a mortality ranged from 3.5 to 29.8%. These studies show that deltamethrin impregnated collars can protect dogs against Brazilian sand flies for up to eight months. Thus, they should be useful in a program to control human and canine visceral leishmaniasis.

Comparative study and identification of potent eukaryotic transcriptional repressors in gene switch systems.

In mammalian cells, proper gene regulation is achieved by the complex interplay of transcription factors that activate or repress gene expression by binding to the regulatory regions of target promoters. While transcriptional activators have been extensively characterised and classified into functional groups, relatively little is known about the comparative strength and cell type-specificity of transcriptional repressors. Here, we have compared the ability of a series of eukaryotic repression domains to silence basal and activated transcription. A series of the most potent repression domains was further tested in the context of a gene therapy gene-switch system in various cell types. The results indicate that the analysed repression domains exert varying silencing activities in different promoter contexts. Furthermore, their potential for gene silencing varies also depending on the cellular context. When multimerised within one chimeric repressor protein, particular combinations of repressor domains were found to display synergistic repressing effects and efficient repression in a panel of cell lines. This approach thus allowed the identification of transcriptional repressors that are both potent and versatile in terms of cellular specificity as a basis for gene switch systems.

Selective PDE4 inhibitors as potent anti-inflammatory drugs for the treatment of airway diseases

Phosphodiesterases (PDEs) are responsible for the breakdown of intracellular cyclic nucleotides, from which PDE4 are the major cyclic AMP metabolizing isoenzymes found in inflammatory and immune cells. This generated greatest interest on PDE4 as a potential target to treat lung inflammatory diseases. For example, cigarette smoke-induced neutrophilia in BAL was dose and time dependently reduced by cilomilast. Beside the undesired side effects associated with the first generation of PDE4 inhibitors, the second generation of selective inhibitors such as cilomilast and roflumilast showed clinical efficacy in asthma and chronic obstrutive pulmonary diseases trials, thus re-enhancing the interest on these classes of compounds. However, the ability of PDE4 inhibitors to prevent or modulate the airway remodelling remains relatively unexplored. We demonstrated that selective PDE4 inhibitor RP 73-401 reduced matrix metalloproteinase (MMP)-9 activity and TGF-beta1 release during LPS-induced lung injury in mice and that CI-1044 inhibited the production of MMP-1 and MMP-2 from human lung fibroblasts stimulated by pro-inflammatory cytokines. Since inflammatory diseases of the bronchial airways are associated with destruction of normal tissue structure, our data suggest a therapeutic benefit for PDE4 inhibitors in tissue remodelling associated with chronic lung diseases.

Mitochondrial reactive oxygen species generation triggers inflammatory response and tissue injury associated with hepatic ischemia-reperfusion: Therapeutic potential of mitochondrially targeted antioxidants.

Mitochondrial reactive oxygen species generation has been implicated in the pathophysiology of ischemia-reperfusion (I/R) injury; however, its exact role and its spatial-temporal relationship with inflammation are elusive. Herein we explore the spatial-temporal relationship of oxidative/nitrative stress and inflammatory response during the course of hepatic I/R and the possible therapeutic potential of mitochondrial-targeted antioxidants, using a mouse model of segmental hepatic ischemia-reperfusion injury. Hepatic I/R was characterized by early (at 2h of reperfusion) mitochondrial injury, decreased complex I activity, increased oxidant generation in the liver or liver mitochondria, and profound hepatocellular injury/dysfunction with acute proinflammatory response (TNF-α, MIP-1α/CCL3, MIP-2/CXCL2) without inflammatory cell infiltration, followed by marked neutrophil infiltration and a more pronounced secondary wave of oxidative/nitrative stress in the liver (starting from 6h of reperfusion and peaking at 24h). Mitochondrially targeted antioxidants, MitoQ or Mito-CP, dose-dependently attenuated I/R-induced liver dysfunction, the early and delayed oxidative and nitrative stress response (HNE/carbonyl adducts, malondialdehyde, 8-OHdG, and 3-nitrotyrosine formation), and mitochondrial and histopathological injury/dysfunction, as well as delayed inflammatory cell infiltration and cell death. Mitochondrially generated oxidants play a central role in triggering the deleterious cascade of events associated with hepatic I/R, which may be targeted by novel antioxidants for therapeutic advantage.

Limits to potent antiretroviral therapy.

The availability of potent combination antiretroviral therapy (ART), also known as highly active antiretroviral therapy or HAART has changed the prognosis of HIV infection. However, the benefits have to be seen in the context of deficiencies of current therapy: failure to eradicate the virus, the slow and potentially incomplete recovery of the immune system, the high prevalence of resistance, and the potential for long-term toxicity. Treatment strategies need to take into account these limits to better target those HIV-infected patients who could benefit the most from antiretroviral therapy.

A potent trypanocidal component from the fungus Lentinus strigosus inhibits trypanothione reductase and modulates PBMC proliferation

The fungus Lentinus strigosus (Pegler 1983) (Polyporaceae, basidiomycete) was selected in a screen for inhibitory activity on Trypanosoma cruzi trypanothione reductase (TR). The crude extract of L. strigosus was able to completely inhibit TR at 20 µg/ml. Two triquinane sesquiterpenoids (dihydrohypnophilin and hypnophilin), in addition to two panepoxydol derivatives (neopanepoxydol and panepoxydone), were isolated using a bioassay-guided fractionation protocol. Hypnophilin and panepoxydone displayed IC50 values of 0.8 and 38.9 µM in the TR assay, respectively, while the other two compounds were inactive. The activity of hypnophilin was confirmed in a secondary assay with the intracellular amastigote forms of T. cruzi, in which it presented an IC50 value of 2.5 µ M. Quantitative flow cytometry experiments demonstrated that hypnophilin at 4 µM also reduced the proliferation of human peripheral blood monocluear cells (PBMC) stimulated with phytohemaglutinin, without any apparent interference on the viability of lymphocytes and monocytes. As the host immune response plays a pivotal role in the adverse events triggered by antigen release during treatment with trypanocidal drugs, the ability of hypnophilin to kill the intracellular forms of T. cruzi while modulating human PBMC proliferation suggests that this terpenoid may be a promising prototype for the development of new chemotherapeutical agents for Chagas disease.

Targeting aquaporin function: potent inhibition of aquaglyceroporin-3 by a gold-based compound.

Aquaporins (AQPs) are membrane channels that conduct water and small solutes such as glycerol and are involved in many physiological functions. Aquaporin-based modulator drugs are predicted to be of broad potential utility in the treatment of several diseases. Until today few AQP inhibitors have been described as suitable candidates for clinical development. Here we report on the potent inhibition of AQP3 channels by gold(III) complexes screened on human red blood cells (hRBC) and AQP3-transfected PC12 cells by a stopped-flow method. Among the various metal compounds tested, Auphen is the most active on AQP3 (IC(50) = 0.8±0.08 µM in hRBC). Interestingly, the compound poorly affects the water permeability of AQP1. The mechanism of gold inhibition is related to the ability of Au(III) to interact with sulphydryls groups of proteins such as the thiolates of cysteine residues. Additional DFT and modeling studies on possible gold compound/AQP adducts provide a tentative description of the system at a molecular level. The mapping of the periplasmic surface of an homology model of human AQP3 evidenced the thiol group of Cys40 as a likely candidate for binding to gold(III) complexes. Moreover, the investigation of non-covalent binding of Au complexes by docking approaches revealed their preferential binding to AQP3 with respect to AQP1. The high selectivity and low concentration dependent inhibitory effect of Auphen (in the nanomolar range) together with its high water solubility makes the compound a suitable drug lead for future in vivo studies. These results may present novel metal-based scaffolds for AQP drug development.

Avaluació de vitamines i antioxidants en aliments vegetals mitjançant tècniques HPLC-DAD i UPLC-MS/MS

El programa experimental s’ha portat a terme dins el marc de les activitats del projecte TRUEFOOD, finançat per la UE per als anys 2007-2010. L’objectiu principal d’aquesta activitat ha estat l’avaluació dels continguts en àcid ascòrbic (vitamina C), polifenols totals, àcids fenòlics i flavonoides en mostres de tomàquet i enciam, produïts sota diferents condicions de camp (producció ecològica i convencional). Per aconseguir els resultats s’han utilitzat mètodes analítics basats en tècniques de cromatografia líquida d’alta eficàcia (HPLC) i d’ultra-alta eficàcia (UHPLC) acoblades a sistemes de detecció de diode array (DAD) i espectrometria de masses (MSn). Per a l’àcid ascòrbic, s’ha desenvolupat un mètode ràpid que ha permès la determinació d’aquest compost en diferents matrius vegetals amb el mínim pretractament de les mostres, utilitzant una fase estacionària HILIC (Fluorinated Stationary Phase). Els mètodes desenvolupats d’anàlisi de compostos fenòlics han permès realitzar les anàlisi de forma ràpida, a fi de processar el màxim nombre de mostres per a obtenir resultats representatius. S’ha realitzat una completa caracterització dels extractes de tomàquet i enciam, ampliant el coneixement descrit en la bibliografia sobre la seva composició fenòlica. En el cas de l’enciam, s’ha identificat quatre compostos fenòlics que mai abans han estat descrits i quantificats en aquesta hortalissa. La definició, amb precisió, dels continguts en vitamina C i compostos fenòlics en les mostres analitzades ha permès comparar els efectes de diferents tècniques de cultiu sobre les característiques nutricionals dels vegetals objecte de l’estudi. Els mètodes d’anàlisi desenvolupats i els resultats derivats del projecte seran publicats properament en revistes científiques de reconegut prestigi.

Modulation of TCR avidity of primary HIV-1-specific CD8 T-cell responses by early and potent antiviral therapy responses

Background: CD8 T-cells play a critical role in antiviral immunity. However, mechanisms of virus control and immune correlates of protection are still not fully understood. Among other factors, TCR avidity (antigen sensitivity) is thought to play a critical role. Whereas there is a large consensus that high TCR avidity T-cell responses are correlated to higher efficacy against cancer and acute viral infections, it may be not the case in chronic persistent viral infections. Methods: TCR avidity (measured by the effect concentration 50% [EC50]) of HIV-1-specific CD8 T-cell responses directed against optimal epitopes was investigated in different cohorts of HIV-1- infected subjects (n¼114) including early acute and chronic (progressive and non-progressive) HIV-1-infection. Overall, TCR avidity was investigated in 245 HIV-1-specific CD8 T-cell responses. The relationships between TCR avidity, T-cell differentiation and functional profile including cytokine secretion, proliferation and cytotoxic potential (determined by polychromatic flow cytometry) were analyzed. Results: HIV-1-specific CD8 T-cell responses from patients with acute infection had significantly lower TCR avidity as compared to patients with chronic (progressive or non-progressive) HIVinfection (P¼0.03 and 0.003, respectively). These differences remained significant when the analyses were restricted to common epitopes (same epitopes restricted by the same class I HLA). Interestingly, some patients treated during acute infection underwent spontaneous treatment interruption. Re-exposure to high viral load induced two major effects: a) the increase in TCR avidity of pre-existing high avidity (EC50<0.01) T-cell responses (P<0.02) and b) the generation of new T-cell responses with higher TCR avidity as compared to the average pre-existing T-cell responses. Conclusion: These results suggest that high TCR avidity T-cell responses are selected during the course of HIV-1 infection and that one of the potential driving mechanisms is continuous exposure to HIV-1 antigens. These results advance our understanding of the relationship between TCR avidity and Ag exposure of antiviral memory CD8 T-cells.

Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide.

We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.