Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake

The evaluation of new antimalarial agents using older methods of monitoring sensitivity to antimalarial drugs are laborious and poorly suited to discriminate stage-specific activity. We used flow cytometry to study the effect of established antimalarial compounds, cysteine protease inhibitors, and a quinolone against asexual stages of Plasmodium falciparum. Cultured P. falciparum parasites were treated for 48 h with different drug concentrations and the parasitemia was determined by flow cytometry methods after DNA staining with propidium iodide. P. falciparum erythrocytic life cycle stages were readily distinguished by flow cytometry. Activities of established and new antimalarial compounds measured by flow cytometry were equivalent to results obtained with microscopy and metabolite uptake assays. The antimalarial activity of all compounds was higher against P. falciparum trophozoite stages. Advantages of flow cytometry analysis over traditional assays included higher throughput for data collection, insight into the stage-specificity of antimalarial activity avoiding use of radioactive isotopes.

Effects of immunosuppressive drugs on T helper cells subsets and potential to promote tolerance in a stringent experimental transplantation model.

To achieve the goal of sustained donor-specifi c transplantation (Tx) tolerance, research efforts are now focusing on therapies based on specifi c cell subsets with regulatory properties. We and others have previously highlighted the therapeutic potential of naturally occurring CD4+CD25+Foxp3+ regulatory T cells (nTreg) in promoting long-term graft acceptance. Using more stringent experimental Tx models, we were however confronted to limitations. Indeed, while the transfer of antigenspecifi c nTreg promoted long-term MHC-mismatched skin allograft acceptance in lymphopenic mice in the absence of any immunosuppressive drug, allograft survival was only slightly prolonged when nTreg were transferred alone into non-lymphopenic mice. This suggested that in more stringent conditions, adjuvant therapies may be needed to effectively control alloreactive T cells (Teff). Whether and how the expansion of the Treg pool could be best combined with current immunosuppressive regimens in clinical settings remains to be defi ned. In this study, we have used in vitro assays and an in vivo skin Tx model to investigate the effects of various immunosuppressive drugs on the survival, proliferation and effector function of Teff and nTreg in response to alloantigens. Teff proliferation was inhibited in a dose-dependent manner by rapamycin and cyclosporine A, while anti-CD154 mAb only marginally affected Teff survival, proliferation and effector fucntion in vitro. Rapamycin promoted apoptosis of Teff as compared to nTreg that were more resistant in the presence of IL-2. In vivo, the transfer and/or expansion of Treg could be advantageously combined with rapamycin and anti-CD154 mAb treatment to signifi cantly prolong MHC-mismatched skin allografts survival in non-lymphopenic recipients. Taken together our data indicate that immunosuppressive drugs differentially target T-cell subsets and that some regimens could promote Treg expansion while controlling the Teff pool in response to alloantigens.

Engineering, conjugation, and immunogenicity assessment of Escherichia coli O121 O antigen for its potential use as a typhoid vaccine component.

State-of-the-art production technologies for conjugate vaccines are complex, multi-step processes. An alternative approach to produce glycoconjugates is based on the bacterial N-linked protein glycosylation system first described in Campylobacter jejuni. The C. jejuni N-glycosylation system has been successfully transferred into Escherichia coli, enabling in vivo production of customized recombinant glycoproteins. However, some antigenic bacterial cell surface polysaccharides, like the Vi antigen of Salmonella enterica serovar Typhi, have not been reported to be accessible to the bacterial oligosaccharyltransferase PglB, hence hamper development of novel conjugate vaccines against typhoid fever. In this report, Vi-like polysaccharide structures that can be transferred by PglB were evaluated as typhoid vaccine components. A polysaccharide fulfilling these requirements was found in Escherichia coli serovar O121. Inactivation of the E. coli O121 O antigen cluster encoded gene wbqG resulted in expression of O polysaccharides reactive with antibodies raised against the Vi antigen. The structure of the recombinantly expressed mutant O polysaccharide was elucidated using a novel HPLC and mass spectrometry based method for purified undecaprenyl pyrophosphate (Und-PP) linked glycans, and the presence of epitopes also found in the Vi antigen was confirmed. The mutant O antigen structure was transferred to acceptor proteins using the bacterial N-glycosylation system, and immunogenicity of the resulting conjugates was evaluated in mice. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with E. coli O121 LPS. One animal developed a significant rise in serum immunoglobulin anti-Vi titer upon immunization.

Comparison of copper-67- and iodine-125-labeled anti-CEA monoclonal antibody biodistribution in patients with colorectal tumors.

Copper-67 has comparable beta-particle emissions to that of 131I, but it displays more favorable gamma emission characteristics for application in radioimmunotherapy (RIT). This study investigates the potential of 67Cu-labeled monoclonal antibody (MAb) 35 for RIT of colorectal carcinoma. METHODS: Biokinetics of simultaneously injected 67Cu- and 125I-labeled MAb35 were studied in six patients scheduled for surgery of primary colorectal cancer. RESULTS: Whole-body clearance (T 1/2) of 67Cu, estimated from sequential anterior and posterior whole-body scans and corrected for decay of 67Cu, was 41 hr. Serum clearance of 67Cu was faster (27.41 hr) than that of 125I (38.33 hr). Mean tumor uptake of the 67Cu-labeled compound (0.0133% ID/g) exceeded that of 125I (0.0095% ID/g), and tumor-to-blood ratios were higher for 67Cu than for 125I, with averages of 6.07 and 2.41, respectively. The average 67Cu/125I ratio was 1.9 for tumor uptake, 0.7 for blood and 2.6 for tumor-to-blood ratios. Nonspecific liver uptake of 67Cu as calculated from whole-body scans was high in four patients, up to 25% of residual whole-body activity at 48 hr, but did not increase with time. We also observed some nonspecific bowel activity, as well as moderate to high uptake in benign polyps. CONCLUSION: Copper-67-labeled MAb35 is more favorable than its radioiodine-labeled counterpart for RIT of colorectal carcinoma due to higher tumor-to-blood ratios, but the problem of nonspecific liver and bowel uptake must first be overcome. The absolute accumulation of activity in tumor remains low, however, so the probability of cure with this compound alone is questionable. The use of 67Cu as one component of a multimodality adjuvant treatment seems to remain the most appropriate application for RIT.

Lutzomyia longipalpis in Clorinda, Formosa province, an area of potential visceral leishmaniasis transmission in Argentina

Phlebotomine captures were performed during 2004 in Clorinda, Argentina. Clorinda is located across the branches of the Paraguay river in front of Asunción city, Paraguay. Reports of canine and human visceral leishmaniasis in Asunción have been increasing since 1997, however neither leishmaniasis cases nor sand flies were ever recorded from Clorinda. Light traps were located in migration paths (bridges, port), and peridomestic environments of Clorinda and surrounding localities. Lutzomyia longipalpis was found in Clorinda and Puerto Pilcomayo, first report in a potential visceral leishmaniasis transmission area for Argentina. Active surveillance is required immediately in the localities involved and the surrounding area.

Bacterial and fungal colonization of burn wounds

A prospective study of fungal and bacterial flora of burn wounds was carried out from February 2004 to February 2005 at the Burns Unit of Hospital Regional da Asa Norte, Brasília, Brazil. During the period of the study, 203 patients were treated at the Burns Unit. Wound swab cultures were assessed at weekly intervals for four weeks. Three hundred and fifty four sampling procedures (surface swabs) were performed from the burn wounds. The study revealed that bacterial colonization reached 86.6% within the first week. Although the gram-negative organisms, as a group, were more predominant, Staphylococcus aureus (28.4%) was the most prevalent organism in the first week. It was however surpassed by Pseudomonas aeruginosa form third week onwards. For S. aureus and P. aeruginosa vancomycin and polymyxin were found to be the most effective drugs. Most of the isolates showed high level resistance to antimicrobial agents. Fungi were found to colonize the burn wound late during the second week postburn, with a peak incidence during the third and fourth weeks. Species identification of fungi revealed that Candida tropicalis was the most predominant, followed by Candida parapsilosis. It is crucial for every burn institution to determine the specific pattern of burn wound microbial colonization, the time-related changes in the dominant flora, and the antimicrobial sensitivity profiles. This would enable early treatment of imminent septic episodes with proper empirical systemic antibiotics, without waiting for culture results, thus improving the overall infection-related morbidity and mortality.

CC-chemokine receptors: a potential therapeutic target for Trypanosoma cruzi-elicited myocarditis

The comprehension of the pathogenesis of Trypanosoma cruzi-elicited myocarditis is crucial to delineate new therapeutic strategies aiming to ameliorate the inflammation that leads to heart dysfunction, without hampering parasite control. The augmented expression of CCL5/RANTES and CCL3/MIP-1alpha, and their receptor CCR5, in the heart of T. cruzi-infected mice suggests a role for CC-chemokines and their receptors in the pathogenesis of T. cruzi-elicited myocarditis. Herein, we discuss our recent results using a CC-chemokine receptor inhibitor (Met-RANTES), showing the participation of CC-chemokines in T. cruzi infection and unraveling CC-chemokine receptors as an attractive therapeutic target for further evaluation in Chagas disease.

A Novel Pulse-Chase SILAC Strategy Measures Changes in Protein Decay and Synthesis Rates Induced by Perturbation of Proteostasis with an Hsp90 Inhibitor.

Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc) variant of SILAC (stable isotope labeling by amino acids in cell culture). pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 "clients". We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants kd and synthesis rates Vs for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538.

Arylfurans as potential Trypanosoma cruzi trypanothione reductase inhibitors

The natural lignans veraguensin and grandisin have been reported to be active against Trypanosoma cruzi bloodstream forms. Aiming at the total synthesis of these and related compounds, we prepared three 2-arylfurans and eight 2,5-diarylfurans. They were evaluated for their potential as T. cruzi trypanothione reductase (TR) inhibitors as well against the parasite's intracellular (amastigote) and bloodstream (trypomastigote) forms. Compound 12 was the most effective against TR with an IC50 of 48.5 µM while 7 and 14 were active against amastigotes, inhibiting the parasite development by 60% at 20 µg/ml (59 and 90 µM, respectively). On the other hand, none of the compounds was significantly active against the parasite bloodstream forms even at 250 µg/ml (0.6-1.5 mM).

A one-year monitoring of nicotine use in sport: frontier between potential performance enhancement and addiction issues

Tobacco consumption is a global epidemic responsible for a vast burden of disease. With pharmacological properties sought-after by consumers and responsible for addiction issues, nicotine is the main reason of this phenomenon. Accordingly, smokeless tobacco products are of growing popularity in sport owing to potential performance enhancing properties and absence of adverse effects on the respiratory system. Nevertheless, nicotine does not appear on the 2011 World Anti-Doping Agency (WADA) Prohibited List or Monitoring Program by lack of a comprehensive large-scale prevalence survey. Thus, this work describes a one-year monitoring study on urine specimens from professional athletes of different disciplines covering 2010 and 2011. A method for the detection and quantification of nicotine, its major metabolites (cotinine, trans-3-hydroxycotinine, nicotine-N′-oxide and cotinine-N-oxide) and minor tobacco alkaloids (anabasine, anatabine and nornicotine) was developed, relying on ultra-high pressure liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-TQ-MS/MS). A simple and fast dilute-and-shoot sample treatment was performed, followed by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) operated in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. After method validation, assessing the prevalence of nicotine consumption in sport involved analysis of 2185 urine samples, accounting for 43 different sports. Concentrations distribution of major nicotine metabolites, minor nicotine metabolites and tobacco alkaloids ranged from 10 (LLOQ) to 32,223, 6670 and 538 ng/mL, respectively. Compounds of interest were detected in trace levels in 23.0% of urine specimens, with concentration levels corresponding to an exposure within the last three days for 18.3% of samples. Likewise, hypothesizing conservative concentration limits for active nicotine consumption prior and/or during sport practice (50 ng/mL for nicotine, cotinine and trans-3-hydroxycotinine and 25 ng/mL for nicotine-N′-oxide, cotinine-N-oxide, anabasine, anatabine and nornicotine) revealed a prevalence of 15.3% amongst athletes. While this number may appear lower than the worldwide smoking prevalence of around 25%, focusing the study on selected sports highlighted more alarming findings. Indeed, active nicotine consumption in ice hockey, skiing, biathlon, bobsleigh, skating, football, basketball, volleyball, rugby, American football, wrestling and gymnastics was found to range between 19.0 and 55.6%. Therefore, considering the adverse effects of smoking on the respiratory tract and numerous health threats detrimental to sport practice at top level, likelihood of smokeless tobacco consumption for performance enhancement is greatly supported.

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.

Soil fungal communities of grasslands are environmentally structured at a regional scale in the Alps.

Studying patterns of species distributions along elevation gradients is frequently used to identify the primary factors that determine the distribution, diversity and assembly of species. However, despite their crucial role in ecosystem functioning, our understanding of the distribution of below-ground fungi is still limited, calling for more comprehensive studies of fungal biogeography along environmental gradients at various scales (from regional to global). Here, we investigated the richness of taxa of soil fungi and their phylogenetic diversity across a wide range of grassland types along a 2800 m elevation gradient at a large number of sites (213), stratified across a region of the Western Swiss Alps (700 km(2)). We used 454 pyrosequencing to obtain fungal sequences that were clustered into operational taxonomic units (OTUs). The OTU diversity-area relationship revealed uneven distribution of fungal taxa across the study area (i.e. not all taxa are everywhere) and fine-scale spatial clustering. Fungal richness and phylogenetic diversity were found to be higher in lower temperatures and higher moisture conditions. Climatic and soil characteristics as well as plant community composition were related to OTU alpha, beta and phylogenetic diversity, with distinct fungal lineages suggesting distinct ecological tolerances. Soil fungi, thus, show lineage-specific biogeographic patterns, even at a regional scale, and follow environmental determinism, mediated by interactions with plants.

Schistosoma mansoni major egg antigen Smp40: molecular modeling and potential immunoreactivity for anti-pathology vaccine development

The pathogenesis of Schistosoma mansoni infection is largely determined by host T-cell mediated immune responses such as the granulomatous response to tissue deposited eggs and subsequent fibrosis. The major egg antigens have a valuable role in desensitizing the CD4+ Th cells that mediate granuloma formation, which may prevent or ameliorate clinical signs of schistosomiasis.S. mansoni major egg antigen Smp40 was expressed and completely purified. It was found that the expressed Smp40 reacts specifically with anti-Smp40 monoclonal antibody in Western blotting. Three-dimensional structure was elucidated based on the similarity of Smp40 with the small heat shock protein coded in the protein database as 1SHS as a template in the molecular modeling. It was figured out that the C-terminal of the Smp40 protein (residues 130 onward) contains two alpha crystallin domains. The fold consists of eight beta strands sandwiched in two sheets forming Greek key. The purified Smp40 was used for in vitro stimulation of peripheral blood mononuclear cells from patients infected with S. mansoni using phytohemagglutinin mitogen as a positive control. The obtained results showed that there is no statistical difference in interferon-g, interleukin (IL)-4 and IL-13 levels obtained with Smp40 stimulation compared with the control group (P > 0.05 for each). On the other hand, there were significant differences after Smp40 stimulation in IL-5 (P = 0.006) and IL-10 levels (P < 0.001) compared with the control group. Gaining the knowledge by reviewing the literature, it was found that the overall pattern of cytokine profile obtained with Smp40 stimulation is reported to be associated with reduced collagen deposition, decreased fibrosis, and granuloma formation inhibition. This may reflect its future prospect as a leading anti-pathology schistosomal vaccine candidate.

Dynamics of feeding and defecation in Triatoma vitticeps (Stal, 1859) (Hemiptera, Reduviidae, Triatominae) and its potential in the transmission of Trypanosoma cruzi

Adults of Triatoma vitticeps infected by flagellates similar to Trypanosoma cruzi are frequently captured by the inhabitants of rural areas in the Brazilian state of Espírito Santo. The dynamics of feeding and defecation were observed in three groups of adult triatomines, consisting of sylvatic T. vitticeps and laboratory-reared specimens of this species and T. infestans. Triatomines were observed from the moment they were presented with an immobilized chicken as a bloodmeal source until 240 min after feeding had ended. Mean times between the end of feeding and defecation for T. infestans, wild T. vitticeps and laboratory-reared specimens of the latter species were 1.2, 21.1, and 64 min respectively. All T. infestans defecated within 10 min of feeding, while only 29.9 of wild and 52.8% laboratory-reared specimens of T. vitticeps did so within this period. These results may explain the low efficiency of T. vitticeps in T. cruzi transmission to man. The shorter time between feeding and defecation in laboratory-reared T. vitticeps may indicate a change in behaviour of this species as a result of adaptation to an artificial environment.