The lack of effectiveness of hyperbaric oxygenation as a treatment for Leishmania major in a mouse model.

We aimed to study the effectiveness of hyperbaric oxygen therapy (HOT) (100% oxygen at 2 ATA for 70 minutes each session for 20 consecutive days) on BALB/c male mice infected with Leishmania major. Fifty-one mice were assigned to six groups. Group 1 was treated with HOT from 1 day after the inoculation. In Groups 2-5, treatment began when the cutaneous lesions appeared. Group 2 received HOT only, Group 3 received topical therapy with Leshcutan only, Groups 4 and 5 received a combination of HOT and Leshcutan for 5 and 10 days respectively, and Group 6 did not receive any treatment (control group). When comparing the control group with Group 1, treatment with HOT in Group 1 did not significantly affect the time of the appearance of the lesions. In contrast, mice treated with Leshcutan demonstrated a significant difference in lesion size and spleen dimensions as compared to the rest of the mice (p<0.001). The results show that HOT treatment has no positive effect on the course of Leishmaniasis in a BALB/c mice model infected with Leishmania major. Further studies are needed with a mouse model closer to humans and with different HOT protocols.

Leukocyte extravasation and vascular permeability are each controlled in vivo by different tyrosine residues of VE-cadherin.

Tyrosine phosphorylation of the adhesion molecule VE-cadherin is assumed to affect endothelial junction integrity. However, it remains unclear whether tyrosine residues of VE-cadherin are required for the induction of vascular permeability and the regulation of leukocyte extravasation in vivo. We found here that knock-in mice expressing a Y685F mutant of VE-cadherin had impaired induction of vascular permeability, but those expressing a Y731F mutant did not. In contrast, mice expressing the Y731F VE-cadherin mutant showed decreased neutrophil-extravasation in cremaster tissue, but those expressing the Y685F mutant did not. Whereas inflammatory mediators induced the phosphorylation of Tyr685 in vivo, Tyr731 showed high baseline phosphorylation. Leukocytes triggered dephosphorylation of Tyr731 via the tyrosine phosphatase SHP-2, which allowed the adaptin AP-2 to bind and initiate endocytosis of VE-cadherin. Thus, Tyr685 and Tyr731 of VE-cadherin distinctly and selectively regulate the induction of vascular permeability or leukocyte extravasation.

High-throughput screening of a collection of known pharmacologically active small compounds for identification of Candida albicans biofilm inhibitors.

Candida albicans is the most common etiologic agent of systemic fungal infections with unacceptably high mortality rates. The existing arsenal of antifungal drugs is very limited and is particularly ineffective against C. albicans biofilms. To address the unmet need for novel antifungals, particularly those active against biofilms, we have screened a small molecule library consisting of 1,200 off-patent drugs already approved by the Food and Drug Administration (FDA), the Prestwick Chemical Library, to identify inhibitors of C. albicans biofilm formation. According to their pharmacological applications that are currently known, we classified these bioactive compounds as antifungal drugs, as antimicrobials/antiseptics, or as miscellaneous drugs, which we considered to be drugs with no previously characterized antifungal activity. Using a 96-well microtiter plate-based high-content screening assay, we identified 38 pharmacologically active agents that inhibit C. albicans biofilm formation. These drugs were subsequently tested for their potency and efficacy against preformed biofilms, and we identified three drugs with novel antifungal activity. Thus, repurposing FDA-approved drugs opens up a valuable new avenue for identification and potentially rapid development of antifungal agents, which are urgently needed.

Identification of specific pluripotent stem cell death--inducing small molecules by chemical screening.

A potential application of embryonic and inducible pluripotent stem cells for the therapy of degenerative diseases involves pure somatic cells, free of tumorigenic undifferentiated embryonic and inducible pluripotent stem cells. In complex collections of chemicals with pharmacological potential we expect to find molecules able to induce specific pluripotent stem cell death, which could be used in some cell therapy settings to eliminate undifferentiated cells. Therefore, we have screened a chemical library of 1120 small chemicals to identify compounds that induce specifically apoptotic cell death in undifferentiated mouse embryonic stem cells (ESCs). Interestingly, three compounds currently used as clinically approved drugs, nortriptyline, benzethonium chloride and methylbenzethonium chloride, induced differential effects in cell viability in ESCs versus mouse embryonic fibroblasts (MEFs). Nortriptyline induced apoptotic cell death in MEFs but not in ESCs, whereas benzethonium and methylbenzethonium chloride showed the opposite effect. Nortriptyline, a tricyclic antidepressant, has also been described as a potent inhibitor of mitochondrial permeability transition, one of two major mechanisms involved in mitochondrial membrane permeabilization during apoptosis. Benzethonium chloride and methylbenzethonium chloride are quaternary ammonium salts used as antimicrobial agents with broad spectrum and have also been described as anticancer agents. A similar effect of benzethonium chloride was observed in human induced pluripotent stem cells (hiPSCs) when compared to both primary human skin fibroblasts and an established human fibroblast cell line. Human fibroblasts and hiPSCs were similarly resistant to nortriptyline, although with a different behavior. Our results indicate differential sensitivity of ESCs, hiPSCs and fibroblasts to certain chemical compounds, which might have important applications in the stem cell-based therapy by eliminating undifferentiated pluripotent stem cells from stem cell-derived somatic cells to prevent tumor formation after transplantation for therapy of degenerative diseases.

Magnetic "fishing" assay to screen small-molecule mixtures for modulators of protein-protein interactions.

Protein-protein interactions are an intricate part of biological pathways and have become important targets for drug discovery. Here we present a two-stage magnetic bead assay to functionally screen small-molecule mixtures for modulators of protein-based interactions, with simultaneous affinity-based isolation of active compounds and identification by mass spectrometry. Proteins of interest interact in solution prior to the addition of Ni(II)-functionalized magnetic beads to recover an intact protein-protein complex through affinity capture of a polyhistidine-tagged primary target ("protein-complex fishing"). Protein-complex fishing, utilizing His(6)-tagged calmodulin (CaM) as the primary (bait) protein and melittin (Mel) as the target, was used to screen a mass-encoded library of 1000 bioactive compounds (50 mixtures, 20 compounds each) and successfully identified three known antagonists, three naturally occurring phenolic compounds previously reported to disrupt CaM-activated phosphodiesterase activity, and two newly identified modulators of the CaM-Mel interaction, methylbenzethonium and pempidine tartrate. The ability to produce quantitative inhibition data is also shown through the development of dose-dependent response curves and the determination of inhibition constants (K(I)) for the novel compound methylbenzethonium (K(I) = 14-49 nM) and two known antagonists, calmidazolium (K(I) = 1.7-7.5 nM) and trifluoperazine (K(I) = 1.2-3.0 µM), with the latter two values being in close agreement with literature values.

Is paromomycin an effective and safe treatment against cutaneous leishmaniasis? A meta-analysis of 14 randomized controlled trials.

BACKGROUND: High cost, poor compliance, and systemic toxicity have limited the use of pentavalent antimony compounds (SbV), the treatment of choice for cutaneous leishmaniasis (CL). Paromomycin (PR) has been developed as an alternative to SbV, but existing data are conflicting. METHODOLOGY/PRINCIPAL FINDINGS: We searched PubMed, Scopus, and Cochrane Central Register of Controlled Trials, without language restriction, through August 2007, to identify randomized controlled trials that compared the efficacy or safety between PR and placebo or SbV. Primary outcome was clinical cure, defined as complete healing, disappearance, or reepithelialization of all lesions. Data were extracted independently by two investigators, and pooled using a random-effects model. Fourteen trials including 1,221 patients were included. In placebo-controlled trials, topical PR appeared to have therapeutic activity against the old world and new world CL, with increased local reactions, when used with methylbenzethonium chloride (MBCL) compared to when used alone (risk ratio [RR] for clinical cure, 2.58 versus 1.01: RR for local reactions, 1.60 versus 1.07). In SbV-controlled trials, the efficacy of topical PR was not significantly different from that of intralesional SbV in the old world CL (RR, 0.70; 95% confidence interval, 0.26-1.89), whereas topical PR was inferior to parenteral SbV in treating the new world CL (0.67; 0.54-0.82). No significant difference in efficacy was found between parenteral PR and parenteral SbV in the new world CL (0.88; 0.56-1.38). Systemic side effects were fewer with topical or parenteral PR than parenteral SbV. CONCLUSIONS/SIGNIFICANCE: Topical PR with MBCL could be a therapeutic alternative to SbV in selected cases of the old world CL. Development of new formulations with better efficacy and tolerability remains to be an area of future research.

Cutaneous leishmaniasis.

Cutaneous leishmaniasis is a widespread tropical infection caused by numerous different species of Leishmania protozoa that are transmitted by sandflies. Its clinical presentations are extremely diverse and dependent on a variety of parasite and host factors that are poorly understood. Diagnosis should aim to identify the exact species involved, but this requires laboratory investigations that are not widely available. No single ideal treatment has been identified, and those available are limited by variable success rates and toxicity. Clinical guidelines are needed to make better use of the investigations and treatments that do exist. Prevention is currently limited to bite prevention measures.

Leishmania major: in vitro and in vivo anti-leishmanial activity of paromomycin ointment (Leshcutan) combined with the immunomodulator Imiquimod.

Paromomycin at 25, 50 and 100 microg/ml, inhibited the growth of Leishmania major amastigotes by 34.5%, 61.2%, 74.9% and 85.4%, 89.9%, 95.7% on the 2nd and the 4th day of treatment in culture, respectively. Methylbenzethonium chloride at 0.1 and 0.5 microg/ml and Imiquimod at 5 and 10 microg/ml, administered separately, inhibited the parasite development by 39.5% and 65.2% and 31.5% and 47.7%, respectively. Imiquimod (5-10 microg/ml) combined with either paromomycin (25, 50 and 100 microg/ml) or methylbenzethonium chloride (0.1 and 0.5 microg/ml) showed an anti-leishmanial additive effect. A 10 day topical treatment, twice daily, with an ointment containing 15% paromomycin and 12% methylbenzethonium chloride (Leshcutan), either undiluted or diluted 1:5 in soft white paraffin combined with 5% Imiquimod cream (Aldara), was as effective as Leshcutan given alone. The present study suggests that a combination of Aldara and Leshcutan is as effective as Leshcutan given alone in the topical treatment of CL caused by L. major.

Aqueous micellar two-phase system composed of hyamine-type hydrophobically modified ethylene oxide and application for cytochrome P450 BM-3 separation.

The hydrophobically modified ethylene oxide polymer, HM-EO, was modified with an alkyl halide to prepare a hyamine-type HM-EO, named N-Me-HM-EO, which could be used for forming N-Me-HM-EO/buffer aqueous micellar two-phase system. The critical micelle concentration of N-Me-HM-EO solution and the phase diagrams of N-Me-HM-EO/buffer systems were determined. By using this novel aqueous micellar two-phase system, the separation of cytochrome P450 BM-3 from cell extract was explored. The partitioning behavior of P450 BM-3 in N-Me-HM-EO/buffer systems was measured. The influences of some factors such as total proteins concentration, pH, temperature and salt concentration, on the partitioning coefficients of P450 BM-3 were investigated. Since the micellar aggregates in the N-Me-HM-EO enriched phase were positively charged, it was possible to conduct the proteins with different charges to top or bottom phases by adjusting pH and salt concentration in the system. A separation scheme consisting of two consecutive aqueous two-phase extraction steps was proposed: the first extraction with N-Me-HM-EO/buffer system at pH 8.0, and the second extraction in the same system at pH 6.0. The recovery of P450 BM-3 was 73.3% with the purification factor of 2.5. The results indicated that the aqueous micellar two-phase system composed of hyamine modified polysoap has a promising application for selective separation of biomolecules depending on the enhanced electrostatic interactions between micelles and proteins.

A siderophore biosynthesis gene cluster from the fish pathogen Photobacterium damselae subsp. piscicida is structurally and functionally related to the Yersinia high-pathogenicity island.

Photobacterium damselae subsp. piscicida, the causative agent of fish pasteurellosis, produces a siderophore which is distinct from that produced by P. damselae subsp. damselae. Using suppression subtractive hybridization, a subsp. piscicida-specific DNA region of 35 kb was identified in strain DI21, and 11 genes were defined: dahP, araC1, araC2, frpA, irp8, irp2, irp1, irp3, irp4, irp9 and irp5. The sequence of the predicted proteins encoded by these genes showed significant similarity with the proteins responsible for the synthesis and transport of the siderophore yersiniabactin, encoded within the Yersinia high-pathogenicity island (HPI). Southern hybridization demonstrated that this gene cluster is exclusive to some European subsp. piscicida isolates. Database searches revealed that a similar gene cluster is present in Photobacterium profundum SS9 and Vibrio cholerae RC385. An irp1 gene (encoding a putative non-ribosomal peptide synthetase) insertional mutant (CS31) was impaired for growth under iron-limiting conditions and unable to produce siderophores, and showed an approximately 100-fold decrease in degree of virulence for fish. The subsp. piscicida DI21 strain, but not CS31, promoted the growth of a Yersinia enterocolitica irp1 mutant. Furthermore, a yersiniabactin-producing Y. enterocolitica strain as well as purified yersiniabactin were able to cross-feed strains DI21 and CS31, suggesting that the subsp. piscicida siderophore might be functionally and structurally related to yersiniabactin. The differential occurrence among P. damselae strains, and the low sequence similarity to siderophore synthesis genes described in other members of the Vibrionaceae, suggest that this genetic system might have been acquired by horizontal transfer in P. damselae subsp. piscicida, and might have a common evolutionary origin with the Yersinia HPI.

Comparison of the effectiveness of two topical paromomycin treatments versus meglumine antimoniate for New World cutaneous leishmaniasis.

The randomized, controlled study compared the therapeutic efficacy and safety of two paromomycin-containing topical preparations with the gold treatment standard, meglumine antimoniate, and with each other in 120 Ecuadorian patients with ulcerated lesions. The two paromomycin treatment comparisons were double-blinded. Group 1 (n = 14) received 15% paromomycin plus 12% methylbenzonium chloride (PR-MBCL) dissolved in a soft white paraffin base, applied twice daily for 30 days. Group 2 (n = 40) was also treated for 30 days with 15% paromomycin plus 10% urea (PR-U) dissolved in the same paraffin base. Group 3 (n = 40) received 20mg/kg/day of IM meglumine antimoniate (MA) for 10 days as per Ecuadorian Ministry of Public Health recommendations at the time of the study. The 10-day treatment was completed by 90% of the MA group compared to 72.5% of the PR-MBCL (X2 = 4.0, P = 0.045) and 75% of the PM-U (X2 = 3.1, P > 0.05) groups whose treatment regime lasted 20 days longer than the MA treatment. Post-treatment lesion burning, redness, inflammation, and soreness were more common in the two paromomycin groups compared to MA group (P < 0.05). The frequency of treatment-related side effects in the two paromomycin groups was similar. Six weeks after the start of treatment, 80.6% of MA subjects were clinically cured compared to 48.3% in the PR-MBCL (X2 = 6.1, P = 0.014) and 40% in the PM-U groups (X2 = 12.6, P = 0.002). By 12 weeks, the proportion of clinically cured subjects in the MA (91.7%) compared to PM-MBCL (79.3%) or PM-U (70%) groups was not significantly different (P > 0.05). MA-treated subjects clinically cured by 12 weeks had a faster mean healing time (29.5 +/- 12.2 days) compared to those in the PM-MBCL (versus 43.1 +/- 14.4 days, t = -3.7, P = 0.001) or PR-U groups (43.5 +/- 17 days; t = -3.2, P = 0.002). During the 48-week post-treatment follow-up period, infection reactivation was observed in 15.2% of the MA subjects compared to 17.4% in the PM-MBCL and 10.5% PM-U of subjects diagnosed as clinically healed by 12 weeks (P > 0.05). The results suggest that although the time required for the clinical healing of ulcerated lesions takes longer, topical paromomycin may be an acceptable therapeutic alternative in endemic areas where meglumine antimoniate is not available, is too costly or medically contraindicated.

Total synthesis and preliminary biological evaluation of cis-solamin isomers.

An efficient total synthesis of cis-solamin (1) has been achieved in 21% overall yield and with a longest linear sequence of just 11 steps from aldehyde 8. A key feature of the approach was the use of asymmetric permanganate-promoted oxidative cyclization to introduce four of the five required stereocenters in a single step. The use of robust and chemoselective methodology meant that the use of protecting groups could be avoided during the assembly of cis-solamin (1) from the three fragments 23, 6, and 4. The methodology was also applied to the synthesis of three further cis-solamin isomers 2, ent-1, and ent-2. Cytotoxicity and hemolytic properties of cis-solamin isomers and synthetic intermediates are reported.

Randomized, controlled, double-blind trial of topical treatment of cutaneous leishmaniasis with paromomycin plus methylbenzethonium chloride ointment in Guatemala.

A double-blind, randomized trial was undertaken in Guatemala to determine the therapeutic efficacy of an ointment for the treatment of cutaneous leishmaniasis that contained 15% paromomycin and 12% methylbenzethonium chloride and that was applied twice a day for 20 days. The treatment group included 35 patients, and the placebo group included 33 patients. The initial clinical response rate (13 weeks after completing the treatment) was 91.4% in the treatment group and 39.4% in the placebo group. The final clinical response rate at the 12-month follow-up examination was 85.7% (31 of 35) in the treatment group and 39.4% (13 of 33) in the placebo group (P < or = 0.001). In general, the treatment was well tolerated and was never interrupted because of adverse effects. The number of adverse effects reported in the placebo group was lower than in the treatment group (16 events versus 30 events). All adverse effects reported by patients disappeared within 1 week of completing the treatment. Our findings show that the combination of paromomycin with methylbenzethonium chloride for 20 days is a good alternative for antimonial treatments of cutaneous leishmaniasis in Guatemala.

Successful topical treatment of murine cutaneous leishmaniasis with a combination of paromomycin (Aminosidine) and gentamicin.

Cutaneous leishmaniasis is presently treated with 20 days of parenteral therapy with a frequently toxic drug (antimony). Topical formulations of paromomycin (15%) plus methylbenzethonium chloride (MBCL, 12%) or plus urea (10%) in soft white paraffin have been tested for Old and New World disease in humans. We compared the efficacy of a new topical formulation, WR 279,396 (paromomycin [15%] plus gentamicin [0.5%]) to the clinical formulations in the treatment of cutaneous disease in BALB/c mice. Sixty-day-old lesions were treated twice a day for 10 days, and the response to therapy was determined over a further 70 days. For ulcers due to Leishmania major or to Leishmania mexicana, 100% of lesions in the WR 279,396 group healed by day 20 after therapy and did not relapse by day 70; 83% of lesions healed without relapse in the paromomycin-MBCL group. In the paromomycin-urea group, 100% of L. major lesions healed by day 30 but 30% relapsed. For ulcers due to Leishmania panamensis or Leishmania amazonensis, all lesions treated with WR 279,396 healed and did not relapse; < 50% of lesions treated with paromomycin-MBCL healed by day 30, and all lesions relapsed by day 70. In addition to being active, WR 279,396 was not toxic in this model and appears to have a cosmetic effect (promoting hair growth, healing, and limiting the size of the scar).

Topical paromomycin/methylbenzethonium chloride plus parenteral meglumine antimonate as treatment for American cutaneous leishmaniasis: controlled study.

We determined the efficacy of the combination of the topical formulation 15% paromomycin sulfate/12% methylbenzethonium chloride (MBCL) and a short course (7 days) of parenteral meglumine antimonate (pentavalent antimony [Sb]) as treatment of American cutaneous leishmaniasis in Colombian patients. Patients were randomly assigned in unequal allocation (2:1:1:1) to group 1 (topical paromomycin/MBCL plus injectable Sb for 7 days), group 2 (topical placebo plus injectable Sb for 7 days), group 3 (topical paromomycin/MBCL plus injectable Sb for 3 days), and group 4 (injectable Sb for 20 days). Cure was defined as complete reepithelialization of all lesions without relapse. Cure rates among groups were as follows: 58% (34 of 59), group 1; 53% (16 of 30), group 2; 20% (6 of 30), group 3; and 84% (26 of 31), group 4. Seventy-one percent of the organisms identified to the species level were Leishmania braziliensis panamensis. We conclude that 10 days of therapy with paromomycin/MBCL does not augment the response of cutaneous leishmaniasis (predominately due to L. braziliensis panamensis) to a short course of treatment with meglumine antimonate.

Cellulose materials modified by antiseptics and their antimicrobial properties.

Adsorption properties of dressing cellulose materials with respect to surfactant antiseptics were studied. These antiseptics are a complex of the copolymer of N-vinylpyrrolidone and crotonic acid with dimethylbenzylalkylammonium chloride (a synthetic polymer with a wide spectrum of antimicrobial effect) and its low molecular weight analogue (dimethylbenzylalkylammonium chloride). It was established that cellulose materials reversibly adsorb mentioned surfactant antiseptics depending on their concentration in the initial solutions. Maximum release of surfactant antiseptics is achieved at solutions at pH = 7.0. Microbiological tests of cellulose materials modified by antiseptics have shown that they exhibit antimicrobial activity. These results can be used in medical practice in clinics for imparting antimicrobial properties to dressing materials.

Measurements of fatty acid and carbohydrate metabolism in the isolated working rat heart.

The isolated working rat heart is a useful experimental model which allows contractile function to be measured in hearts perfused at physiologically relevant workloads. To maintain these high workloads the heart is required to generate a tremendous amount of energy. In vivo this energy is derived primarily from the oxidation of fatty acids. In many experimental situations it is desirable to perfuse the isolated working heart in the presence of physiologically relevant concentrations of fatty acids. This is particularly important when studying energy metabolism in the heart, or in determining how fatty acids alter the outcome of myocardial ischemic injury [1, 2]. The other major source of energy for the heart is derived from the oxidation of carbohydrates (glucose and lactate), with a smaller amount of ATP also being derived from glycolysis. Two byproducts of both fatty acid and carbohydrate metabolism are H2O and CO2. By labeling the glucose, lactate, or fatty acids in the perfusate with 3H or 14C the experimenter can quantitatively collect either 3H2O or 14CO2 produced by the heart. By using radioisotopes that are labeled at specific hydrogen or carbon molecules on the various energy substrates, and by knowing the specific activity of the radiolabeled substrate used, it is possible to determine the actual rate of flux through these individual pathways. This paper will describe the experimental protocols for directly measuring fatty acid and carbohydrate metabolism in isolated working rat hearts.