Magnetic purification of biotinylated cDNA removes false priming and ensures strand-specificity of RT-PCR for enteroviral RNAs

The detection of replicative intermediate RNAs as markers of active replication of RNA viruses is an essential tool to investigate pathogenesis in acute viral infections, as well as in their long-term sequelae. In this regard, strand-specific PCR has been used widely to distinguish (-) and (+) enteroviral RNAs in pathogenesis studies of diseases such as dilated cardiomyopathy. It has been generally assumed that oligonucleotide-primed reverse transcription of a given RNA generates only the corresponding specific cDNA, thus assuring the specificity of a PCR product amplified from it. Nevertheless, such assumed strand-specificity is a fallacy, because falsely primed cDNAs can be produced by RNA reverse transcription in the absence of exogenously added primers, (cDNA(primer)(-)), and such falsely primed cDNAs are amplifiable by PCR in the same way as the correctly primed cDNAs. Using as a prototype the coxsackievirus B5 (CVB5), a (+) strand RNA virus, it was shown that cDNA(primer)(-) renders the differential detection of viral (-) and (+) RNAs by conventional PCR virtually impossible, due to gross non-specificity. Using in vitro transcribed CVB5 RNAs (+) and (-), it was shown that cDNA(primer)(-) could be removed effectively by magnetic physical separation of correctly primed biotinylated cDNA. Such strategy enabled truly strand-specific detection of RNA (-) and (+), not only for CVB5, but also for other non-polio enteroviruses. These findings indicate that previous conclusions supporting a role for the persistence of actively replicating enterovirus in the pathogenesis of chronic myocarditis should be regarded with strong skepticism and purification of correctly primed cDNA should be used for strand-specific PCR of viral RNA in order to obtain reliable information on this important subject. (C) 2009 Elsevier B.V. All rights reserved.

Chagas disease

Chagas disease is a chronic, systemic, parasitic infection caused by the protozoan Trypanosoma cruzi, and was discovered in 1909. The disease affects about 8 million people in Latin America, of whom 30-40% either have or will develop cardiomyopathy, digestive megasyndromes, or both. In the past three decades, the control and management of Chagas disease has undergone several improvements. Large-scale vector control programmes and screening of blood donors have reduced disease incidence and prevalence. Although more effective trypanocidal drugs are needed, treatment with benznidazole (or nifurtimox) is reasonably safe and effective, and is now recommended for a widened range of patients. Improved models for risk stratification are available, and certain guided treatments could halt or reverse disease progression. By contrast, some challenges remain: Chagas disease is becoming an emerging health problem in non-endemic areas because of growing population movements; early detection and treatment of asymptomatic individuals are underused; and the potential benefits of novel therapies (eg, implantable cardioverter defibrillators) need assessment in prospective randomised trials.

Nitric Oxide-Induced Vasorelaxation in Response to PnTx2-6 Toxin from Phoneutria nigriventer Spider in Rat Cavernosal Tissue

Introduction. Priapism is one of several symptoms observed in accidental bites by the spider Phoneutria nigriventer. The venom of this spider is comprised of many toxins, and the majority has been shown to affect excitable ion channels, mainly sodium (Na+) channels. It has been demonstrated that PnTx2-6, a peptide extracted from the venom of P. nigriventer, causes erection in anesthetized rats and mice. Aim. We investigated the mechanism by which PnTx2-6 evokes relaxation in rat corpus cavernosum. Main Outcome Measures. PnTx2-6 toxin potentiates nitric oxide (NO)-dependent cavernosal relaxation. Methods. Rat cavernosal strips were incubated with bretylium (3 x 10-5 M) and contracted with phenylephrine (PE; 10-5 M). Relaxation responses were evoked by electrical field stimulation (EFS) or sodium nitroprusside (SNP) before and after 4 minutes of incubation with PnTx2-6 (10-8 M). The effect of PnTx2-6 on relaxation induced by EFS was also tested in the presence of atropine (10-6 M), a muscarinic receptor antagonist, N-type Ca2+ channel blockers (omega-conotoxin GVIA, 10-6 M) and sildenafil (3 x 10-8 M). Technetium99m radiolabeled PnTx2-6 subcutaneous injection was administrated in the penis. Results. Whereas relaxation induced by SNP was not affected by PnTx2-6, EFS-induced relaxation was significantly potentiated by this toxin as well as PnTx2-6 plus SNP. This potentiating effect was further increased by sildenafil, not altered by atropine, however was completely blocked by the N-type Ca2+ channels. High concentrated levels of radiolabeled PnTx2-6 was specifically found in the cavernosum tissue, suggesting PnTx2-6 is an important toxin responsible for P. nigriventer spider accident-induced priapism. Conclusion. We show that PnTx2-6 slows Na+ channels inactivation in nitrergic neurons, allowing Ca2+ influx to facilitate NO/cGMP signalling, which promotes increased NO production. In addition, this relaxation effect is independent of phosphodiesterase enzyme type 5 inhibition. Our data displays PnTx2-6 as possible pharmacological tool to study alternative treatments for erectile dysfunction. Nunes KP, Cordeiro MN, Richardson M, Borges MN, Diniz SOF, Cardoso VN, Tostes R, De Lima ME, Webb RC, and Leite R. Nitric oxide-induced vasorelaxation in response to PnTx2-6 toxin from Phoneutria nigriventer spider in rat cavernosal tissue. J Sex Med 2010;7:3879-3888.

Inhibition of Snake Venoms and Phospholipases A(2) by Extracts from Native and Genetically Modified Eclipta alba: Isolation of Active Coumestans

We genetically modified Eclipta alba using Agrobacterium rhizogenes LBA 9402, with the aim of producing secondary metabolites with pharmacological properties against phospholipase A(2) and the myotoxic activities of snake venom. Extracts from in natura aerial parts and roots, both native and genetically modified (in vitro), were prepared and analysed by high-performance liquid chromatography. In natura materials showed the coumestan wedelolactone at higher concentration in the aerial parts, while demethylwedelolactone appeared at higher concentration in roots. Among the modified roots, clone 19 showed higher concentrations of these coumestans. Our results show that the in natura extracts of plants collected from Botucatu and Ribeirao Preto were efficient in inhibiting snake venom phospholipase A(2) activity. Regarding in vitro material, the best effect against Crotalus durissus terrificus venom was that of clone 19. Clone 19 and isolated coumestans (wedelolactone and demethylwedelolactone) inhibited the myotoxic activity induced by basic phospholipases A(2) isolated from the venoms of Crotalus durissus terrificus (CB) and Bothrops jararacussu (BthTX-I and II). The search for antivenom is justified by the need of finding active principles that are more efficient in neutralizing snake venoms and also as an attempt to complement serum therapy.

Disruption of sarcolemmal dystrophin and beta-dystroglycan may be a potential mechanism for myocardial dysfunction in severe sepsis

Evidence from our laboratory has shown alterations in myocardial structure in severe sepsis/septic shock. The morphological alterations are heralded by sarcolemmal damage, characterized by increased plasma membrane permeability caused by oxidative damage to lipids and proteins. The critical importance of the dystrophin-glycoprotein complex (DGC) in maintaining sarcolemmal stability led us to hypothesize that loss of dystrophin and associated glycoproteins could be involved in early increased sarcolemmal permeability in experimentally induced septic cardiomyopathy. Male C57Bl/6 mice were subjected to sham operation and moderate (MSI) or severe (SSI) septic injury induced by cecal ligation and puncture (CLP). Using western blot and immunofluorescence, a downregulation of dystrophin and beta-dystroglycan expression in both severe and moderate injury could be observed in septic hearts. The immunofluorescent and protein amount expressions of laminin-alpha 2 were similar in SSI and sham-operated hearts. Consonantly, the evaluation of plasma membrane permeability by intracellular albumin staining provided evidence of severe injury of the sarcolemma in SSI hearts, whereas antioxidant treatment significantly attenuated the loss of sarcolemmal dystrophin expression and the increased membrane permeability. This study offers novel and mechanistic data to clarify subcellular events in the pathogenesis of cardiac dysfunction in severe sepsis. The main finding was that severe sepsis leads to a marked reduction in membrane localization of dystrophin and beta-dystroglycan in septic cardiomyocytes, a process that may constitute a structural basis of sepsis-induced cardiac depression. In addition, increased sarcolemmal permeability suggests functional impairment of the DGC complex in cardiac myofibers. In vivo observation that antioxidant treatment significantly abrogated the loss of dystrophin expression and plasma membrane increased permeability supports the hypothesis that oxidative damage may mediate the loss of dystrophin and beta-dystroglycan in septic mice. These abnormal parameters emerge as therapeutic targets and their modulation may provide beneficial effects on future cardiovascular outcomes and mortality in sepsis. Laboratory Investigation (2010) 90, 531-542; doi: 10.1038/labinvest.2010.3; published online 8 February 2010

Isoproterenol induces primary loss of dystrophin in rat hearts: correlation with myocardial injury

The mechanism of isoproterenol-induced myocardial damage is unknown, but a mismatch of oxygen supply vs. demand following coronary hypotension and myocardial hyperactivity is the best explanation for the complex morphological alterations observed. Severe alterations in the structural integrity of the sarcolemma of cardiomyocytes have been demonstrated to be caused by isoproterenol. Taking into account that the sarcolemmal integrity is stabilized by the dystrophin-glycoprotein complex (DGC) that connects actin and laminin in contractile machinery and extracellular matrix and by integrins, this study tests the hypothesis that isoproterenol affects sarcolemmal stability through changes in the DGC and integrins. We found different sensitivity of the DGC and integrin to isoproterenol subcutaneous administration. Immunofluorescent staining revealed that dystrophin is the most sensitive among the structures connecting the actin in the cardiomyocyte cytoskeleton and the extracellular matrix. The sarcomeric actin dissolution occurred after the reduction or loss of dystrophin. Subsequently, after lysis of myofilaments, gamma-sarcoglycan, beta-dystroglycan, beta 1-integrin, and laminin alpha-2 expressions were reduced followed by their breakdown, as epiphenomena of the myocytolytic process. In conclusion, administration of isoproterenol to rats results in primary loss of dystrophin, the most sensitive among the structural proteins that form the DGC that connects the extracellular matrix and the cytoskeleton in cardiomyocyte. These changes, related to ischaemic injury, explain the severe alterations in the structural integrity of the sarcolemma of cardiomyocytes and hence severe and irreversible injury induced by isoproterenol.

Insights on calcium-independent phospholipid membrane damage by Lys49-PLA(2) using tryptophan scanning mutagenesis of bothropstoxin-I from Bothrops jararacussu

Bothropstoxin-I (BthTx-I) is a homodimerie Lys49-PLA(2) from the venom of the snake Bothrops jararacussu, which lacks hydrolytic activity against phospholipid substrates, yet permeabilizes membranes by a Ca2+- independent mechanism. The interaction of the BthTx-I with model membranes has been studied by intrinsic tryptophan fluorescence emission (ITFE) spectroscopy. Nine separate mutants have been created each with a unique tryptophan residue located at a different position in the interfacial recognition site (IRS) of the protein. The rapid and efficient Ca2+-independent membrane damage against unilamellar liposomes composed of DPPC/DMPA in a 9:1 molar ratio was unaffected by these substitutions. Binding studies revealed low protein affinity for these liposomes and no changes were observed in the ITFE properties. In contrast, the binding of all mutants to DPPC/DMPA liposomes in a 1:1 molar ratio was stronger, and was correlated with altered ITFE properties. The blue-shifted emission spectra and increased emission intensity of mutants at positions 31, 67 and 115-117 in the interface recognition surface of the protein suggest these regions are partially inserted into the membrane. These results are consistent with a model for the Ca2+-independent membrane damaging mechanism that involves a transient interaction of the protein with the outer phospholipid leaflet of the target membrane. (C) 2007 Elsevier Masson SAS. All rights reserved.

The involvement of CD4(+)CD25(+) T cells in the acute phase of Trypanosoma cruzi infection

The infection with Trypanosoma cruzi leads to a vigorous and apparently uncontrolled inflammatory response in the heart. Although the parasites trigger specific immune response, the infection is not completely cleared out, a phenomenon that in other parasitic infections has been attributed to CD4(+)CD25(+) T cells (Tregs). Then, we examined the role of natural Tregs and its signaling through CD25 and GITR in the resistance against infection with T. cruzi. Mice were treated with mAb against CD25 and GITR and the parasitemia, mortality and heart pathology analyzed. First, we demonstrated that CD4(+)CD25(+)GITR(+)Foxp3(+) T cells migrate to the heart of infected mice. The treatment with anti-CD25 or anti-GITR resulted in increased mortality of these infected animals. Moreover, the treatment with anti-GITR enhanced the myocarditis, with increased migration of CD4(+), CD8(+), and CCR5(+) leukocytes, TNF-alpha production, and tissue parasitism, although it did not change the systemic nitric oxide synthesis. These data showed a limited role for CD25 signaling in controlling the inflammatory response during this protozoan infection. Also, the data suggested that signaling through GITR is determinant to control of the heart inflammation, parasite replication, and host resistance against the infection. (C) 2008 Elsevier Masson SAS. All rights reserved.

Antibothropic action of Casearia sylvestris Sw. (flacourtiaceae) extracts

Casearia sylvestris Sw., popularly known in Brazil as `guacatonga`, has been used as antitumor, antiseptic, antiulcer, local anaesthetic and healer in folk medicine. Snakebite envenomation by Bothrops jararacussu (Bjssu) constitutes a relevant public health hazard capable of inducing serious local damage in victims. This study examined the pharmacological action of apolar and polar C sylvestris leaf extracts in reverting the neuromuscular blockade and myonecrosis, which is induced by Bjssu venom and its major toxin bothropstoxin-I on the mouse phrenic nerve-diaphragm preparations. The polar methanol extract (ME) was by far the most efficacious. ME not only prevented myonecrosis and abolished the blockade, but also increased ACh release. Such facilitation in neuromuscular transmission was observed with ME alone, but was accentuated in preparations incubated with ME plus venom or toxin. This established synergy opens an interesting point of investigation because the venom or toxin in contact with ME changes from a blocking to a facilitating effect. It is suggested that rutin, known to have potent antioxidant properties, and one of the components present in the ME, could have a role in the observed effects. Since commercial rutin did not reproduce the ME effects, it is likely that a rutin-containing phytocomplex is neutralizing the bothropic envenoming effects. Copyright (C) 2008 John Wiley & Sons, Ltd.

Expressed sequence tags (ESTs) from the salivary glands of the tick Amblyomma cajennense (Acari : Ixodidae)

The neotropical tick Amblyomma cajennense is a significant pest to domestic animals, the most frequently human-biting tick in South America and the main vector of Brazilian spotted fever (caused by Rickettsia rickettsii), a deadly human disease. The purpose of this study is to characterize the adult A. cajennense salivary gland transcriptome by expressed sequence tags (ESTs). We report the analysis of 1754 clones obtained from a cDNA library, which reveal mainly transcripts related to proteins involved in the hemostatic processes, especially proteases and their inhibitors. Remarkably, five types of possible serine protease inhibitors were found, including a molecule with a distinguished structure that contains repeats of the active motif of hirudin inhibitors. Besides, other components that may be active over the host immune system or acting as defensins against infecting microorganisms were also described, including a molecule similar to insect venom allergens. The conjunction of components from this transcriptome suggests a diverse strategy of A. cajennense tick during feeding, but emphasized in the coagulation system. (c) 2008 Published by Elsevier Ltd.

Enhanced programmed death 1 (PD-1) and PD-1 ligand (PD-L1) expression in patients with actinic cheilitis and oral squamous cell carcinoma

PD-1 and PD-L1 can be involved in tumor escape, and little is known about the role of these molecules in oral tumors or pre-malignant lesions. In the present study, we investigated the expression of PD-1 and PD-L1 in the blood and lesion samples of patients with actinic cheilitis (AC) and oral squamous cell carcinoma (OSCC). Our results showed that lymphocytes from peripheral blood and tissue samples exhibited high expression of PD-1 in both groups analyzed. Patients with AC presented higher percentage as well as the absolute numbers of CD4(+)PD-1(+) and CD8(+)PD-1(+) lymphocytes in peripheral blood mononuclear cells (PBMC) than healthy individuals, while patients with OSCC presented an increased frequency of CD8(+)PD1(+) in PBMC when compared with controls. On the other hand, increased frequency of CD4(+) and CD8(+) T cells expressing PD-1(+) accumulate in samples from OSCC, and the expression of PD-L1 was intense in OSCC and moderate in AC lesion sites. Lower levels of IFN-gamma and higher levels of TGF-beta were detected in OSCC samples. Our data demonstrate that PD-1 and PD-L1 molecules are present in blood and samples of AC and OSCC patients. Further studies are required to understand the significance of PD-1 and PD-L1 in oral tumors microenvironment.

Regulation of Trypanosoma cruzi-Induced Myocarditis by Programmed Death Cell Receptor

Trypanosoma cruzi infection causes intense myocarditis, leading to cardiomyopathy and severe cardiac dysfunction. Protective adaptive immunity depends on balanced signaling through a T cell receptor and coreceptors expressed on the T cell surface. Such coreceptors can trigger stimulatory or inhibitory signals after binding to their ligands in antigen-presenting cells (APC). T. cruzi modulates the expression of coreceptors in lymphocytes after infection. Deregulated inflammation may be due to unbalanced expression of these molecules. Programmed death cell receptor 1 (PD-1) is a negative T cell coreceptor that has been associated with T cell anergy or exhaustion and persistent intracellular infections. We aimed to study the role of PD-1 during T. cruzi-induced acute myocarditis in mice. Cytometry assays showed that PD-1 and its ligands are strongly upregulated in lymphocytes and APC in response to T. cruzi infection in vivo and in vitro. Lymphocytes infiltrating the myocardium exhibited high levels of expression of these molecules. An increased cardiac inflammatory response was found in mice treated with blocking antibodies against PD-1, PD-L1, and to a lesser extent, PD-L2, compared to that found in mice treated with rat IgG. Similar results in PD-1(-/-) mice were obtained. Moreover, the PD-1 blockade/deficiency led to reduced parasitemia and tissue parasitism but increased mortality. These results suggest the participation of a PD-1 signaling pathway in the control of acute myocarditis induced by T. cruzi and provide additional insight into the regulatory mechanisms in the pathogenesis of Chagas` disease.

Species and regional variations in the effectiveness of antivenom against the in Vitro neurotoxicity of death adder (Acanthophis) venoms

Although viperlike in appearance and habit, death adders belong to the Elapidae family of snakes. Systemic envenomation represents a serious medical problem with antivenom, which is raised against Acanthophis antarcticus venom, representing the primary treatment. This study focused on the major Acanthophis variants from Australia and islands in the Indo-Pacific region. Venoms were profiled using liquid chromatography-mass spectrometry, and analyzed for in vitro neurotoxicity (0.3-10 mug/ml), as well as the effectiveness of antivenom. (1-5 units/ml; 10 min prior to the addition of 10 mug/ml venom). The following death adder venoms were examined: A. antarcticus (from separate populations in New South Wales, Queensland, South Australia, and Western Australia), A. hawkei, A. praelongus, A. pyrrhus, A. rugosus, A. wellsi, and venom from an unnamed species from the Indonesian island of Seram. All venoms abolished indirect twitches of the chick isolated biventer cervicis nerve-muscle preparation in a dose-dependent manner. In addition, all venoms blocked responses to exogenous acetylcholine (1 m-M) and carbachol (20 muM), but not KCl (40 mM), suggesting postsynaptic neurotoxicity. Death adder antivenom (1 unit/ml) prevented the neurotoxic effects of A. pyrrhus, A. praelongus, and A. hawkei venoms, although it was markedly less effective against venoms from A. antarcticus (NSW, SA, WA), A. rugosus, A. wellsi, and A. sp. Scram. However, at 5 units/ml, antivenom was effective against all venoms tested. Death adder venoms, including those from A. antarcticus geographic variants, differed not only in their venom composition but also in their neurotoxic activity and susceptibility to antivenom. For the first time toxicological aspects of A. hawkei, A. wellsi, A. rugosus, and A. sp. Seram venoms were studied. (C) 2001 Academic Press.

Alpha-conotoxins: Nicotinic acetylcholine receptor antagonists as pharmacological tools and potential drug leads

Alpha-Conotoxins are small disulfide rich peptides from the venoms of marine cone snails. They target specific nicotinic acetylcholine receptor (nAChR) subtypes with high affinity and potency and are therefore valuable as neurophamacological probes and potential drug leads. This article gives a general overview of the chemical and biological features of alpha -conotoxins, including their pharmacology, binding interactions and structure. A detailed analysis of recently reported three-dimensional structures from members of different subfamilies of the alpha -conotoxins, including those with 3/5, 4/3, 4/6 and 4.7 spacings of their two intracysteine loops is given. The structures are generally well defined and represent useful frameworks for the display of amino acid residues to target molecules.