Therapeutic Efficacy of Cintredekin Besudotox (IL13-PE38QQR) in Murine Lung Fibrosis Is Unaffected by Immunity to Pseudomonas aeruginosa Exotoxin A

Background: We have previously explored a therapeutic strategy for specifically targeting the profibrotic activity of IL-13 during experimental pulmonary fibrosis using a fusion protein comprised of human IL-13 and a mutated form of Pseudomonas aeruginosa exotoxin A (IL13-PE) and observed that the intranasal delivery of IL13-PE reduced bleomycin-induced pulmonary fibrosis through its elimination of IL-13-responsive cells in the lung. The aim of the present study was to determine whether the presence of an immune response to P. aeruginosa and/or its exotoxin A (PE) would diminish the anti-fibrotic properties of IL13-PE. Methodology/Principal Findings: Fourteen days after P. aeruginosa infection, C57BL/6 mice were injected with bleomycin via the intratracheal route. Other groups of mice received 4 doses of saline or IL13-PE by either intranasal or intraperitoneal application, and were challenged i.t. with bleomycin 28 days later. At day 21 after bleomycin, all mice received either saline vehicle or IL13-PE by the intranasal route and histopatological analyses of whole lung samples were performed at day 28 after bleomycin. Intrapulmonary P. aeruginosa infection promoted a neutralizing IgG2A and IgA antibody response in BALF and serum. Surprisingly, histological analysis showed that a prior P. aeruginosa infection attenuated the development of bleomycin-induced pulmonary fibrosis, which was modestly further attenuated by the intranasal administration of IL13-PE. Although prior intranasal administration of IL13-PE failed to elicit an antibody response, the systemic administration of IL13-PE induced a strong neutralizing antibody response. However, the prior systemic sensitization of mice with IL13-PE did not inhibit the anti-fibrotic effect of IL13-PE in fibrotic mice. Conclusions: Thus, IL13-PE therapy in pulmonary fibrosis works regardless of the presence of a humoral immune response to Pseudomonas exotoxin A. Interestingly, a prior infection with P. aeruginosa markedly attenuated the pulmonary fibrotic response suggesting that the immune elicitation by this pathogen exerts anti-fibrotic effects.

The role of disulfide bridges in the 3-D structures of the antimicrobial peptides gomesin and protegrin-1: a molecular dynamics study

Some antimicrobial peptides have a broad spectrum of action against many different kinds of microorganisms. Gomesin and protegrin-1 are examples of such antimicrobial peptides, and they were studied by molecular dynamics in this research. Both have a beta-hairpin conformation stabilized by two disulfide bridges and are active against Gram-positive and Gram-negative bacteria, as well as fungi. In this study, the role of the disulfide bridge in the maintenance of the tertiary peptide structure of protegrin-1 and gomesin is analyzed by the structural characteristics of these peptides and two of their respective variants, gomy4 and proty4, in which the four cysteines are replaced by four tyrosine residues. The absence of disulfide bridges in gomy4 and proty4 is compensated by overall reinforcement of the original hydrogen bonds and extra attractive interactions between the aromatic rings of the tyrosine residues. The net effects on the variants with respect to the corresponding natural peptides are: i) maintenance of the original beta-hairpin conformation, with great structural similarities between the mutant and the corresponding natural peptide; ii) combination of positive F and. Ramachandran angles within the hairpin head region with a qualitative change to a combination of positive (F) and negative (.) angles, and iii) significant increase in structural flexibility. Experimental facts about the antimicrobial activity of the gomesin and protegrin-1 variants have also been established here, in the hope that the detailed data provided in the present study may be useful for understanding the mechanism of action of these peptides.

The Thermal Effects of Therapeutic Lasers with 810 and 904 nm Wavelengths on Human Skin

Objective: To investigate the effect of therapeutic infrared class 3B laser irradiation on skin temperature in healthy participants of differing skin color, age, and gender. Background: Little is known about the potential thermal effects of Low Level Laser Therapy (LLLT) irradiation on human skin. Methods: Skin temperature was measured in 40 healthy volunteers with a thermographic camera at laser irradiated and control (non-irradiated) areas on the skin. Six irradiation doses (2-12 J) were delivered from a 200mW, 810nm laser and a 60mW, 904nm laser, respectively. Results: Thermal effects of therapeutic LLLT using doses recommended in the World Association for Laser Therapy (WALT) guidelines were insignificant; below 1.5 degrees C in light, medium, and dark skin. When higher irradiation doses were used, the 60mW, 904 nm laser produced significantly (p < 0.01) higher temperatures in dark skin (5.7, SD +/- 1.8 degrees C at 12 J) than in light skin, although no participants requested termination of LLLT. However, irradiation with a 200mW, 810nm laser induced three to six times more heat in dark skin than in the other skin color groups. Eight of 13 participants with dark skin asked for LLLT to be stopped because of uncomfortable heating. The maximal increase in skin temperature was 22.3 degrees C. Conclusions: The thermal effects of LLLT at doses recommended by WALT-guidelines for musculoskeletal and inflammatory conditions are negligible (< 1.5 degrees C) in light, medium, and dark skin. However, higher LLLT doses delivered with a strong 3B laser (200mW) are capable of increasing skin temperature significantly and these photothermal effects may exceed the thermal pain threshold for humans with dark skin color.

Anti-Plasmodium Activity of Angiotensin II and Related Synthetic Peptides

Plasmodium species are the causative agents of malaria, the most devastating insect-borne parasite of human populations. Finding and developing new drugs for malaria treatment and prevention is the goal of much research. Angiotensins I and II (ang I and ang II) and six synthetic related peptides designated Vaniceres 1-6 (VC1-VC6) were assayed in vivo and in vitro for their effects on the development of the avian parasite, Plasmodium gallinaceum. Ang II and VC5 injected into the thoraces of the insects reduced mean intensities of infection in the mosquito salivary glands by 88% and 76%, respectively. Although the mechanism(s) of action is not completely understood, we have demonstrated that these peptides disrupt selectively the P. gallinaceum cell membrane. Additionally, incubation in vitro of sporozoites with VC5 reduced the infectivity of the parasites to their vertebrate host. VC5 has no observable agonist effects on vertebrates, and this makes it a promising drug for malaria prevention and chemotherapy.

Evaluation of therapeutic ultrasound equipments performance

The therapeutic ultrasound (US) is one of the resources mostly used by physiotherapists; however the use of uncalibrated equipments results in inefficient or even harmful therapies to the patient. In this direction, the objective of this study was to evaluate the performance and the procedures of utilization and maintenance of US in use in clinics and Physical-therapy offices. A questionnaire with questions related to the procedures applied in service during the use of therapeutic ultrasound was applied to physiotherapists. The performance of 31 equipments of 6 different brands and 13 different models was evaluated according to the IEC 61689 norm. The parameters measured were: acoustic power; effective radiating area (AER); non-uniformity ratio of the beam (RBN); maximum effective intensity; acoustic frequency of operation, modulation factor and wave form on pulsate mode. As for the questionnaires, it was evident that the professionals are not concerned about the calibration of the equipment. The results demonstrated that only 32.3% of the equipments were in accordance with the norms for the variables power and effective radiation area. The frequency analysis indicated that 20% of the 3 MHz transducers and 12.5% of the 1 MHz contemplated the norms. In the pulsate mode, 12.7% presented relation rest/duration inside allowed limits. A great variation of the ultrasonic field was observed on the obtained images, which presented beams not centered, sometimes with bifurcation of its apex. The results allow concluding that, although used in therapeutic sessions with the population, none of the equipments presents all the analyzed variables inside technical norms. (C) 2010 Elsevier B. V. All rights reserved.

Synthetic peptides as an antigenic base in an ELISA for laboratory diagnosts of schistosomiasis mansoni

A pool of five synthetic peptides was used as an antigenic base in an ELISA (ELISA-Pp) for laboratory diagnosis of Schistosoma mansoni. Serum samples were obtained from individuals with acute (n = 23) and chronic (n = 30) schistosomiasis, with other parasitoses (n = 39) or without parasitic infections (n = 100). ELISA-Pp was compared with other immunoenzymatic methods for detection of IgM (IgM-ELISA) or IgG (IgG-ELISA) as well as an immunofluorescence test for detection of IgM antibodies (IgM-IFT). The sensitivity and specificity of ELISA-Pp was 86.8% and 94.2% when tested on the schistosomiasis group and the non-schistosomiasis group, respectively. Comparison of ELISA-Pp with other serological methods resulted in K concordance indices varying from 0.59 to 0.75. Evaluation of anti-peptide IgG antibodies showed higher Levels in patients with acute compared with chronic schistosomiasis (P = 0.001). ELISA-Pp showed satisfactory sensitivity and high specificity and may constitute a potentially useful method for laboratory diagnosis of schistosomiasis mansoni. (c) 2007 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Micromethod for Quantification of Carbamazepine, Phenobartital and Phenytoin in Human Plasma by HPLC-UV Detection for Therapeutic Drug Monitoring Application

A simple, rapid, selective and sensitive analytical method by HPLC with UV detection was developed for the quantification of carbamazepine, phenobarbital and phenytoin in only 0.2 mL of plasma. A C18 column (150 x 3.9 mm, 4 micra) using a binary mobile phase consisting of water and acetonitrile (70:30, v/v) at a flow rate of 0.5 mL/min were proposed. Validation of the analytical method showed a good linearity (0.3 to 20.0 mg/L for CBZ, 0.9 to 60.0 mg/L for PB and 0.6 to 40.0 mg/L for PHT), high sensitivity (LOQ: 0.3, 0.9 and 0.6 mg/L respectively). The method was applied for drug monitoring of antiepileptic drugs (AED) in 27 patients with epilepsy under polytherapy.

Classical and alternative components of the mitochondrial respiratory chain in pathogenic fungi as potential therapeutic targets

The frequency of opportunistic fungal infection has increased drastically, mainly in patients who are immunocompromised due to organ transplant, leukemia or HIV infection. In spite of this, only a few classes of drugs with a limited array of targets, are available for antifungal therapy. Therefore, more specific and less toxic drugs with new molecular targets is desirable for the treatment of fungal infections. In this context, searching for differences between mitochondrial mammalian hosts and fungi in the classical and alternative components of the mitochondrial respiratory chain may provide new potential therapeutic targets for this purpose.

Nitric oxide donor trans-[RuCl([15]aneN(4))NO]2+as a possible therapeutic approach for Chagas` disease

Background and purpose: Benznidazole (Bz) is the therapy currently available for clinical treatment of Chagas` disease. However, many strains of Trypanosoma cruzi parasites are naturally resistant. Nitric oxide (NO) produced by activated macrophages is crucial to the intracellular killing of parasites. Here, we investigate the in vitro and in vivo activities against T. cruzi, of the NO donor, trans-[RuCl([15]aneN(4))NO]2+. Experimental approach: Trans-[RuCl([15]aneN(4))NO]2+ was incubated with a partially drug-resistant T. cruzi Y strain and the anti-proliferative (epimastigote form) and trypanocidal activities (trypomastigote and amastigote) evaluated. Mice were treated during the acute phase of Chagas` disease. The anti-T. cruzi activity was evaluated by parasitaemia, survival rate, cardiac parasitism, myocarditis and the curative rate. Key results: Trans-[RuCl([15]aneN(4))NO]2+ was 10- and 100-fold more active than Bz against amastigotes and trypomastigotes respectively. Further, trans-[RuCl([15]aneN(4))NO]2+ (0.1 mM) induced 100% of trypanocidal activity (trypomastigotes forms) in vitro. Trans-[RuCl([15]aneN(4))NO]2+ induced permanent suppression of parasitaemia and 100% survival in a murine model of acute Chagas` disease. When the drugs were given alone, parasitological cures were confirmed in only 30 and 40% of the animals treated with the NO donor (3.33 mu mol center dot kg-1 center dot day-1) and Bz (385 mu mol center dot kg-1 center dot day-1), respectively, but when given together, 80% of the animals were parasitologically cured. The cured animals showed an absence of myocarditis and a normalisation of cytokine production in the sera. In addition, no in vitro toxicity was observed at the tested doses. Conclusions and implications: These findings indicate that trans-[RuCl([15]aneN(4))NO]2+ is a promising lead compound for the treatment of human Chagas` disease. This article is commented on by Machado et al., pp. 258-259 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00662.x and to view a related paper in this issue by Silva et al. visit http://dx.doi.org/10.1111/j.1476-5381.2010.00524.x.

Antinociceptive and anti-inflammatory properties of 7-hydroxycoumarin in experimental animal models: potential therapeutic for the control of inflammatory chronic pain

Objectives In the present study we investigated the anti nociceptive, anti-inflammatory and antipyretic effects of 7-hydroxycoumarin (7-HC) in animal models. Methods The effects of oral 7-HC were tested against acetic acid-induced writhing, formalin test, tail flick test, complete Freund`s adjuvant (CFA)-induced hypemociception, carrageenan-induced paw oedema, lipopolysaccharide-induced fever and the rota rod test. Key findings 7-HC (3-60 mg/kg) produced a dose-related antinociception against acetic acid-induced writhing in mice and in the formalin test. In contrast, treatment with 7-HC did not prevent thermal nociception in the tail flick test. A single treatment with 7-HC, 60 mg/kg, produced a long-lasting antinociceptive effect against CFA-induced hypernociception, a chronic inflammatory pain stimulus. Notably, at 60 mg/kg per day over 4 days the administration of 7-HC produced a continuous antinociceptive effect against CFA-induced hypernociception. 7-HC (30-120 mg/kg) produced anti-inflammatory and antipyretic effects against carrageenan-induced inflammation and lipopolysaccharide-induced fever, respectively. Moreover, 7-HC was found to be safe with respect to ulcer induction. In the rota rod test, 7-HC-treated mice did not show any motor performance alterations. Conclusions The prolonged antinociceptive and anti-inflammatory effects of 7-HC, in association with its low ulcerogenic activity, indicate that this molecule might be a good candidate for development of new drugs for the control of chronic inflammatory pain and fever.

Enhanced skin penetration of P20 phosphopeptide using protein transduction domains

Protein transduction domains (PTDs) were recently demonstrated to increase the penetration of the model peptide P20 when the PTD and P20 were covalently attached. Here, we evaluated whether non-covalently linked PTDs were capable of increasing the skin penetration of P20. Two different PTDs were studied: YARA and WLR. Porcine ear skin mounted in a Franz diffusion cell was used to assess the penetration of P20 in the stratum corneum (SC) and viable skin (VS); VS consists of dermis and epidermis without SC. The transdermal delivery of P20 was also assessed. At 1 mM, YARA promoted a 2.33-fold increase in the retention of P20 in the SC but did not significantly increase the amount of P20 that reached VS. WLR significantly increased (2.88-fold) the penetration of P20 in VS. Compared to the non-attached form, the covalently linked WLR fragment was two times more effective in promoting the penetration of P20 into VS. None of the PTDs promoted transdermal delivery of P20 at 4 h post-application. It was concluded that selected non-covalently linked PTDs can be used as a penetration enhancer, but greater skin penetration efficiency can be achieved by covalently binding the PTD to the therapeutic agent. (c) 2007 Elsevier B.V. All rights reserved.

Isolation and Characterization of a Natriuretic Peptide from Crotalus oreganus abyssus (Grand Canyon Rattlesnake) and its Effects on Systemic Blood Pressure and Nitrite Levels

Studies on the therapeutic potential of venom peptides have significantly advanced the development of new peptide drugs. A good example is captopril, a synthetic peptide drug, which acts as an anti-hypertensive and potentiating bradykinin, inhibiting the angiotensin-converting enzyme, whose precursor was isolated from the venom of Bothrops jararacussu. The natriuretic peptide (NPs) family comprises three members, ANP (atrial natriuretic peptide), BNP (B-type natriuretic peptide) and CNP (C-type natriuretic peptide), and has an important role in blood pressure regulation and electrolyte homeostasis. In this study, we describe, for the first time, the isolation and characterization of a novel natriuretic-like peptide (Coa_NP), isolated from Crotalus Oreganus abyssus venom. The peptide has 32 amino acids and its complete sequence is SKRLSNGCFGLKLDRIGAMSGLGCWRLINESK. The Coa_NP has an average molecular mass of 3510.98 Da and its amino acid sequence presents the loop region that is characteristic of natriuretic peptides (17 amino acids, NP domain consensus; CFGXXXDRIXXXSGLGC). Coa_NP is a natriuretic peptide of the ANP/BNP-like family, since the carboxy terminal region of CNP has its own NP domain. The functional experiments showed that Coa_NP produced biological effects similar to those of the other natriuretic peptides: (1) a dose-dependent decrease in mean arterial pressure; (2) significant increases in plasma nitrite levels, and (3) vasorelaxation in thoracic aortic rings that were pre-contracted with phenylephrine. The structural and biological aspects confirm Coa_NP as a natriuretic peptide isolated from snake venom, thus expanding the diversification of venom components.

Effect of a Pool of Peptides Isolated from Crotalus durissus terrificus (South American Rattlesnake) Venom on Glucose Levels of Mice Fed on a High-Fat Diet

This study investigated the effect of a pool of peptides, isolated from venom of Crotalus durissus terrificus (South American rattlesnake) on glucose concentration in C57BL/6 mice fed on a high-fat diet for 6 weeks. The pool of peptides (molecular mass around of 10 kDa) was obtained using a MidJet apparatus with a cartridge of 10 KDa. The peptide pool was injected intraperitoneally in mice in a single dose (0.5 mg/animal) or multiple doses (0.2 mg/dose). After predetermined times (30, 60, 90 and 120 min) post injections, venous blood samples were collected for enzymatic measurement of serum glucose using a commercial glucose kit (glucose oxidase method). High-fat fed mice showed an increase in blood glucose concentration, in comparison with mice fed on the chow diet. Thirty minutes after a single dose of the peptide pool, high-fat fed animals showed a significant decrease (similar to 47%) in glycemia. However, the glucose level increased again at 60 and 120 min. Conversely, after multiple injections of the pool of peptides administered every 30 min, the blood glucose concentration in the high-fat mice was significantly decreased (similar to 37%) and remained at low levels until 120 min. These results suggest that the tested pool of peptides from Crotalus durissus terrificus contained a peptide (or peptides) with a beneficial role on glucose-lowering action of high-fat fed mice.

Wolbachia neither induces nor suppresses transcripts encoding antimicrobial peptides

Wolbachia are intracellular maternally inherited microorganisms that are associated with reproductive abnormalities such as cytoplasmic incompatibility (CI), feminization and parthenogenesis in the various arthropod species they infect. Surveys indicate that these bacteria infect more than 16% of all insect species as well as isopods, mites and nematodes, making Wolbachia one of the most ubiquitous parasites yet described. However, nothing is known about the interactions of this bacterium with the host's immune system. We studied the expression of inducible antimicrobial markers in the adults of two Wolbachia infected insect species, Drosophila simulans and Aedes albopictus. The lack of available immune markers in the mosquito species led us to clone part of the defensin gene from this species, which was found to be very similar to the other mosquito defensins cloned from Anopheles gambiae and Aedes aegypti. Comparisons of the expression pattern of the antibacterial markers between Wolbachia-infected and cured lines, and also between bacteria-challenged and unchallenged adults indicated that Wolbachia does not either constitutively induce or suppress the transcription of these antibacterial genes. In addition, no difference in the transcription of these genes was found between double and single Wolbachia-infected strains or between strains in which Wolbachia has different tissue tropisms.