A Novel Screening System for Claudin Binder Using Baculoviral Display

Recent progress in cell biology has provided new insight into the claudin (CL) family of integral membrane proteins, which contains more than 20 members, as a target for pharmaceutical therapy. Few ligands for CL have been identified because it is difficult to prepare CL in an intact form. In the present study, we developed a method to screen for CL binders by using the budded baculovirus (BV) display system. CL4-displaying BV interacted with a CL4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), but it did not interact with C-CPE that was mutated in its CL4-binding region. C-CPE did not interact with BV and CL1-displaying BV. We used CL4-displaying BV to select CL4-binding phage in a mixture of a scFv-phage and C-CPE-phage. The percentage of C-CPE-phage in the phage mixture increased from 16.7% before selection to 92% after selection, indicating that CL-displaying BV may be useful for the selection of CL binders. We prepared a C-CPE phage library by mutating the functional amino acids. We screened the library for CL4 binders by affinity to CL4-displaying BV, and we found that the novel CL4 binders modulated the tight-junction barrier. These findings indicate that the CL-displaying BV system may be a promising method to produce a novel CL binder and modulator

Diet supplementation for 5 weeks with polyphenol-rich cereals improves several functions and the redox state of mouse leucocytes

BACKGROUND: Cereals naturally contain a great variety of polyphenols, which exert a wide range of physiological effects both in vitro and in vivo. Many of their protective effects, including an improvement of the function and redox state of immune cells in unhealthy or aged subjects come from their properties as powerful antioxidant compounds. However, whether cereal-based dietary supplementation positively affects the immune function and cellular redox state of healthy subjects remains unclear. AIM OF THE STUDY: To investigate the effects of supplementation (20% wt/wt) for 5 weeks with four different cereal fractions on healthy mice. METHODS: Several parameters of function and redox state of peritoneal leukocytes were measured. The cereals, named B (wheat germ), C (buckwheat flour), D (fine rice bran) and E (wheat middlings) contained different amounts of gallic acid, p-hydroxybenzoic acid, vanillic acid, sinapic acid, p-coumaric acid, ferulic acid, quercetin, catechin, rutin and oryzanol as major polyphenols. RESULTS: In general, all cereal fractions caused an improvement of the leukocyte parameters studied such as chemotaxis capacity, microbicidal activity, lymphoproliferative response to mitogens, interleukin-2 (IL-2) and tumor necrosis factor (TNFα) release, as well as oxidized glutathione (GSSG), GSSG/GSH ratio, catalase (CAT) activity and lipid oxidative damage. We observed similar effects among the cereal fractions. CONCLUSIONS: The results suggest that some of these effects may due, at least partially, to the antioxidant activity of the polyphenols naturally present in cereals. Since an appropriate function of the leukocytes has been proposed as marker of the health state, a short-term intake of cereals seems to be sufficient to exert a benefit in the health of the general population. However, further studies are needed to assess the optimal doses and to find out which active polyphenols are able to mediate the observed physiological effects before recommending their regular consumption

Active case detection, treatment of falciparum malaria with combined chloroquine and sulphadoxine/pyrimethamine and vivax malaria with chloroquine and molecular markers of anti-malarial resistance in the Republic of Vanuatu

<p>Abstract</p> <p>Background</p> <p>Chloroquine-resistant <it>Plasmodium falciparum </it>was first described in the Republic of Vanuatu in the early 1980s. In 1991, the Vanuatu Ministry of Health instituted new treatment guidelines for uncomplicated <it>P. falciparum </it>infection consisting of chloroquine/sulphadoxine-pyrimethamine combination therapy. Chloroquine remains the recommended treatment for <it>Plasmodium vivax</it>.</p> <p>Methods</p> <p>In 2005, cross-sectional blood surveys at 45 sites on Malo Island were conducted and 4,060 adults and children screened for malaria. Of those screened, 203 volunteer study subjects without malaria at the time of screening were followed for 13 weeks to observe peak seasonal incidence of infection. Another 54 subjects with malaria were followed over a 28-day period to determine efficacy of anti-malarial therapy; chloroquine alone for <it>P. vivax </it>and chloroquine/sulphadoxine-pyrimethamine for <it>P. falciparum </it>infections.</p> <p>Results</p> <p>The overall prevalence of parasitaemia by mass blood screening was 6%, equally divided between <it>P. falciparum </it>and <it>P. vivax</it>. Twenty percent and 23% of participants with patent <it>P. vivax </it>and <it>P. falciparum </it>parasitaemia, respectively, were febrile at the time of screening. In the incidence study cohort, after 2,303 person-weeks of follow-up, the incidence density of malaria was 1.3 cases per person-year with <it>P. vivax </it>predominating. Among individuals participating in the clinical trial, the 28-day chloroquine <it>P. vivax </it>cure rate was 100%. The 28-day chloroquine/sulphadoxine-pyrimethamine <it>P. falciparum </it>cure rate was 97%. The single treatment failure, confirmed by <it>merozoite surface protein-2 </it>genotyping, was classified as a day 28 late parasitological treatment failure. All <it>P. falciparum </it>isolates carried the Thr-76 <it>pfcrt </it>mutant allele and the double Asn-108 + Arg-59 <it>dhfr </it>mutant alleles. <it>Dhps </it>mutant alleles were not detected in the study sample.</p> <p>Conclusion</p> <p>Peak seasonal malaria prevalence on Malo Island reached hypoendemic levels during the study observation period. The only <it>in vivo </it>malaria drug efficacy trial thus far published from the Republic of Vanuatu showed chloroquine/sulphadoxine-pyrimethamine combination therapy for <it>P. falciparum </it>and chloroquine alone for <it>P. vivax </it>to be highly efficacious. Although the chloroquine-resistant <it>pfcrt </it>allele was present in all <it>P. falciparum </it>isolates, mutant alleles in the <it>dhfr </it>and <it>dhps </it>genes do not yet occur to the extent required to confer sulphadoxine-pyrimethamine resistance in this population.</p

CD4-Specific Designed Ankyrin Repeat Proteins Are Novel Potent HIV Entry Inhibitors with Unique Characteristics

Here, we describe the generation of a novel type of HIV entry inhibitor using the recently developed Designed Ankyrin Repeat Protein (DARPin) technology. DARPin proteins specific for human CD4 were selected from a DARPin DNA library using ribosome display. Selected pool members interacted specifically with CD4 and competed with gp120 for binding to CD4. DARPin proteins derived in the initial selection series inhibited HIV in a dose-dependent manner, but showed a relatively high variability in their capacity to block replication of patient isolates on primary CD4 T cells. In consequence, a second series of CD4-specific DARPins with improved affinity for CD4 was generated. These 2nd series DARPins potently inhibit infection of genetically divergent (subtype B and C) HIV isolates in the low nanomolar range, independent of coreceptor usage. Importantly, the actions of the CD4 binding DARPins were highly specific: no effect on cell viability or activation, CD4 memory cell function, or interference with CD4-independent virus entry was observed. These novel CD4 targeting molecules described here combine the unique characteristics of DARPins—high physical stability, specificity and low production costs—with the capacity to potently block HIV entry, rendering them promising candidates for microbicide development

Toxicological aspects of the use of phenolic compounds in disease prevention

The consumption of a diet low in fat and enhanced by fruits and vegetables, especially rich in phenolic compounds, may reduce risks of many civilization diseases. The use of traditional medicines, mainly derived from plant sources, has become an attractive segment in the management of many lifestyle diseases. Concerning the application of dietary supplements (based on phenolic compounds) in common practice, the ongoing debate over possible adverse effects of certain nutrients and dosage levels is of great importance. Since dietary supplements are not classified as drugs, their potential toxicities and interactions have not been thoroughly evaluated. First, this review will introduce phenolic compounds as natural substances beneficial for human health. Second, the potential dual mode of action of flavonoids will be outlined. Third, potential deleterious impacts of phenolic compounds utilization will be discussed: pro-oxidant and estrogenic activities, cancerogenic potential, cytotoxic effects, apoptosis induction and flavonoid-drug interaction. Finally, future trends within the research field will be indicated