Oxidative stress involvement in the molecular pathogenesis and progression of multiple sclerosis: a literature review.

Multiple sclerosis (MS) is an autoimmune debilitating disease of the central nervous system caused by a mosaic of interactions between genetic predisposition and environmental factors. The pathological hallmarks of MS are chronic inflammation, demyelination, and neurodegeneration. Oxidative stress, a state of imbalance between the production of reactive species and antioxidant defense mechanisms, is considered one of the key contributors in the pathophysiology of MS. This review is a comprehensive overview of the cellular and molecular mechanisms by which oxidant species contribute to the initiation and progression of MS including mitochondrial dysfunction, disruption of various signaling pathways, and autoimmune response activation. The detrimental effects of oxidative stress on neurons, oligodendrocytes, and astrocytes, as well as the role of oxidants in promoting and perpetuating inflammation, demyelination, and axonal damage, are discussed. Finally, this review also points out the therapeutic potential of various synthetic antioxidants that must be evaluated in clinical trials in patients with MS.

Chagas Disease and Leishmaniasis, Sympatric Zoonoses Present in Human from Rural Communities of Venezuela.

PURPOSE: Trypanosoma cruzi and Leishmania spp. coexist in several endemic areas, and there are few studies of Chagas disease and leishmaniasis coinfection worldwide; for this reason, the objective of this work was to determine the Chagas disease and leishmaniasis coinfection in several rural communities co-endemic for these diseases. METHODS: A total of 1107 human samples from six co-endemic rural communities of Cojedes state, Venezuela, were analyzed. Serum samples were evaluated by ELISA, indirect hemagglutination, and indirect immunofluorescence for Chagas disease diagnosis, and individuals were evaluated for leishmaniasis by leishmanin skin test (LST). Approximately, 30% of the individuals were also analyzed by PCR (blood clot samples) for T. cruzi and for Leishmania spp. RESULTS: The 14.7% of the individuals were positive to Trypanosoma cruzi infection by serology, and 25.8% were positive to Leishmania spp. current or past infection by LST. Among the group with PCR results, 7.8% were positive for T. cruzi, and 9.4% for Leishmania spp. The coinfection T. cruzi/Leishmania spp. was 6.5%. The T. cruzi DTUs of the positive blood clot samples were TcI, revealed using the molecular markers: (i) intergenic region of the miniexon, (ii) D7 divergent domain of the 24Sα rDNA, (iii) size-variable domain of the 18S rDNA, and (iv) hsp60-PCR-RFLP (EcoRV). The Leishmania species identified were L. (Leishmania) mexicana and L. (Viannia) braziliensis. CONCLUSION: A high prevalence was found for T. cruzi and Leishmania spp. single and coinfections in almost all communities studied, being these results of relevance for the implementation of control programs in co-endemic areas.

Clostridium perfringens phospholipase C, an archetypal bacterial virulence factor, induces the formation of extracellular traps by human neutrophils.

Neutrophil extracellular traps (NETs) are networks of DNA and various microbicidal proteins released to kill invading microorganisms and prevent their dissemination. However, a NETs excess is detrimental to the host and involved in the pathogenesis of various inflammatory and immunothrombotic diseases. Clostridium perfringens is a widely distributed pathogen associated with several animal and human diseases, that produces many exotoxins, including the phospholipase C (CpPLC), the main virulence factor in gas gangrene. During this disease, CpPLC generates the formation of neutrophil/platelet aggregates within the vasculature, favoring an anaerobic environment for C. perfringens growth. This work demonstrates that CpPLC induces NETosis in human neutrophils. Antibodies against CpPLC completely abrogate the NETosis-inducing activity of recombinant CpPLC and C. perfringens secretome. CpPLC induces suicidal NETosis through a mechanism that requires calcium release from inositol trisphosphate receptor (IP3) sensitive stores, activation of protein kinase C (PKC), and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathways, as well as the production of reactive oxygen species (ROS) by the metabolism of arachidonic acid. Proteomic analysis of the C. perfringens secretome identified 40 proteins, including a DNAse and two 5´-nucleotidases homologous to virulence factors that could be relevant in evading NETs. We suggested that in gas gangrene this pathogen benefits from having access to the metabolic resources of the tissue injured by a dysregulated intravascular NETosis and then escapes and spreads to deeper tissues. Understanding the role of NETs in gas gangrene could help develop novel therapeutic strategies to reduce mortality, improve muscle regeneration, and prevent deleterious patient outcomes.

Bacterial phospholipases C with dual activity: phosphatidylcholinesterase and sphingomyelinase.

Bacterial phospholipases and sphingomyelinases are lipolytic esterases that are structurally and evolutionarily heterogeneous. These enzymes play crucial roles as virulence factors in several human and animal infectious diseases. Some bacterial phospholipases C (PLCs) have both phosphatidylcholinesterase and sphingomyelinase C activities. Among them, Listeria monocytogenes PlcB, Clostridium perfringens PLC, and Pseudomonas aeruginosa PlcH are the most deeply understood. In silico predictions of substrates docking with these three bacterial enzymes provide evidence that they interact with different substrates at the same active site. This review discusses structural aspects, substrate specificity, and the mechanism of action of those bacterial enzymes on target cells and animal infection models to shed light on their roles in pathogenesis.

High Efficacy of Therapeutic Equine Hyperimmune Antibodies Against SARS-CoV-2 Variants of Concern.

SARS-CoV-2 variants of concern show reduced neutralization by vaccine-induced and therapeutic monoclonal antibodies; therefore, treatment alternatives are needed. We tested therapeutic equine polyclonal antibodies (pAbs) that are being assessed in clinical trials in Costa Rica against five globally circulating variants of concern: alpha, beta, epsilon, gamma and delta, using plaque reduction neutralization assays. We show that equine pAbs efficiently neutralize the variants of concern, with inhibitory concentrations in the range of 0.146-1.078 µg/mL, which correspond to extremely low concentrations when compared to pAbs doses used in clinical trials. Equine pAbs are an effective, broad coverage, low-cost and a scalable COVID-19 treatment.

Biological models in multiple sclerosis.

Considering the etiology of multiple sclerosis (MS) is still unknown, experimental models resembling specific aspects of this immune-mediated demyelinating human disease have been developed to increase the understanding of processes related to pathogenesis, disease evolution, evaluation of therapeutic interventions, and demyelination and remyelination mechanisms. Based on the nature of the investigation, biological models may include in vitro, in vivo, and ex vivo assessments. Even though these approaches have disclosed valuable information, every disease animal model has limitations and can only replicate specific features of MS. In vitro and ex vivo models generally do not reflect what occurs in the organism, and in vivo animal models are more likely used; nevertheless, they are able to reproduce only certain stages of the disease. In vivo MS disease animal models in mammals include: experimental autoimmune encephalomyelitis, viral encephalomyelitis, and induced demyelination. This review examines and describes the most common biological disease animal models for the study of MS, their specific characteristics and limitations.

Deficient Skeletal Muscle Regeneration after Injury Induced by a Clostridium perfringens Strain Associated with Gas Gangrene.

Gas gangrene, or clostridial myonecrosis, is usually caused by Clostridium perfringens and may occur spontaneously in association with diabetes mellitus, peripheral vascular disease, or some malignancies but more often after contamination of a deep surgical or traumatic lesion. If not controlled, clostridial myonecrosis results in multiorgan failure, shock, and death, but very little is known about the muscle regeneration process that follows myonecrosis when the infection is controlled. In this study, we characterized the muscle regeneration process after myonecrosis caused in a murine experimental infection with a sublethal inoculum of C. perfringens vegetative cells. The results show that myonecrosis occurs concomitantly with significant vascular injury, which limits the migration of inflammatory cells. A significant increase in cytokines that promote inflammation explains the presence of an inflammatory infiltrate; however, impaired interferon gamma (IFN-γ) expression, a reduced number of M1 macrophages, deficient phagocytic activity, and a prolongation of the permanence of inflammatory cells lead to deficient muscle regeneration. The expression of transforming growth factor ß1 (TGF-ß1) agrees with the consequent accumulation of collagen in the muscle, i.e., fibrosis observed 30 days after infection. These results provide new information on the pathogenesis of gas gangrene caused by C. perfringens, shed light on the basis of the deficient muscle regenerative activity, and may open new perspectives for the development of novel therapies for patients suffering from this disease.

Cross-reactivity, antivenomics, and neutralization of toxic activities of Lachesis venoms by polyspecific and monospecific antivenoms.

BACKGROUND: Bothrops, Crotalus and Lachesis represent the most medically relevant genera of pitvipers in Central and South America. Similarity in venom phenotype and physiopathological profile of envenomings caused by the four nominal Lachesis species led us to hypothesize that an antivenom prepared against venom from any of them may exhibit paraspecificity against all the other congeneric taxa. METHODS: To assess this hypothesis, in this work we have applied antivenomics and immunochemical methods to investigate the immunoreactivity of three monovalent antivenoms and two polyvalent antivenoms towards the venoms from different geographic populations of three different Lachesis species. The ability of the antivenoms to neutralize the proteolytic, hemorrhagic, coagulant, and lethal activities of the seven Lachesis venoms was also investigated. RESULTS: A conspicuous pattern of immunorecognition and cross-neutralization for all effects was evident by the polyspecific antivenoms, indicating large immunoreactive epitope conservation across the genus during more than 10 million years since the Central and South American bushmasters diverged. CONCLUSIONS: Despite the broad geographic distribution of Lachesis, antivenoms against venoms of different species are effective in the neutralization of congeneric venoms not used in the immunization mixture, indicating that they can be used equivalently for the clinical treatment of any lachesic envenoming. GENERAL SIGNIFICANCE: This study demonstrates that antivenoms raised against venom of different Lachesis species are indistinctly effective in the neutralization of congeneric venoms not used in the immunization mixture, indicating that antivenoms against conspecific venoms may be used equivalently for the clinical treatment of envenomings caused by any bushmaster species.

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors.

Bacterial sphingomyelinases and phospholipases are a heterogeneous group of esterases which are usually surface associated or secreted by a wide variety of Gram-positive and Gram-negative bacteria. These enzymes hydrolyze sphingomyelin and glycerophospholipids, respectively, generating products identical to the ones produced by eukaryotic enzymes which play crucial roles in distinct physiological processes, including membrane dynamics, cellular signaling, migration, growth, and death. Several bacterial sphingomyelinases and phospholipases are essential for virulence of extracellular, facultative, or obligate intracellular pathogens, as these enzymes contribute to phagosomal escape or phagosomal maturation avoidance, favoring tissue colonization, infection establishment and progression, or immune response evasion. This work presents a classification proposal for bacterial sphingomyelinases and phospholipases that considers not only their enzymatic activities but also their structural aspects. An overview of the main physiopathological activities is provided for each enzyme type, as are examples in which inactivation of a sphingomyelinase- or a phospholipase-encoding gene impairs the virulence of a pathogen. The identification of sphingomyelinases and phospholipases important for bacterial pathogenesis and the development of inhibitors for these enzymes could generate candidate vaccines and therapeutic agents, which will diminish the impacts of the associated human and animal diseases.

Clostridium perfringens phospholipase C induced ROS production and cytotoxicity require PKC, MEK1 and NFκB activation.

Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the most toxic extracellular enzyme produced by this bacteria and is essential for virulence in gas gangrene. At lytic concentrations, CpPLC causes membrane disruption, whereas at sublytic concentrations this toxin causes oxidative stress and activates the MEK/ERK pathway, which contributes to its cytotoxic and myotoxic effects. In the present work, the role of PKC, ERK 1/2 and NFκB signalling pathways in ROS generation induced by CpPLC and their contribution to CpPLC-induced cytotoxicity was evaluated. The results demonstrate that CpPLC induces ROS production through PKC, MEK/ERK and NFκB pathways, the latter being activated by the MEK/ERK signalling cascade. Inhibition of either of these signalling pathways prevents CpPLC's cytotoxic effect. In addition, it was demonstrated that NFκB inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of CpPLC in mice. Understanding the role of these signalling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridialmyonecrosis.

Internalization of Clostridium perfringens α-toxin leads to ERK activation and is involved on its cytotoxic effect.

Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, plays a key role in the pathogenesis of gas gangrene. CpPLC may lead to cell lysis at concentrations that cause extensive degradation of plasma membrane phospholipids. However, at sublytic concentrations it induces cytotoxicity without inducing evident membrane damage. The results of this work demonstrate that CpPLC becomes internalized in cells by a dynamin-dependent mechanism and in a time progressive process: first, CpPLC colocalizes with caveolin both at the plasma membrane and in vesicles, and later it colocalizes with early and late endosomes and lysosomes. Lysosomal damage in the target cells is evident 9 h after CpPLC exposure. Our previous work demonstrated that CpPLCinduces ERK1/2 activation, which is involved in its cytotoxic effect. In this work we found that cholesterol sequestration, dynamin inhibition, as well as inhibition of actin polymerization, prevent CpPLC internalization and ERK1/2 activation, involving endocytosis in the signalling events required for CpPLC cytotoxic effect at sublytic concentrations. These results provide new insights about the mode of action of this bacterial phospholipase C, previously considered to act only locally on cell membrane.

Snake venomics of Lachesis muta rhombeata and genus-wide antivenomics assessment of the paraspecific immunoreactivity of two antivenoms evidence the high compositional and immunological conservation across Lachesis.

We report the proteomic analysis of the Atlantic bushmaster, Lachesis muta rhombeata, from Brazil. Along with previous characterization of the venom proteomes of L. stenophrys (Costa Rica), L. melanocephala (Costa Rica), L. acrochorda (Colombia), and L. muta muta (Bolivia), the present study provides the first overview of the composition and distribution of venom proteins across this wide-ranging genus, and highlights the remarkable similar compositional and pharmacological profiles across Lachesis venoms. The paraspecificity of two antivenoms, produced at Instituto Vital Brazil (Brazil) and Instituto Clodomiro Picado (Costa Rica) using different conspecific taxa in the immunization mixtures, was assessed using genus-wide comparative antivenomics. This study confirms that the proteomic similarity among Lachesis sp. venoms is mirrored in their high immunological conservation across the genus. The clinical and therapeutic consequences of genus-wide venomics and antivenomics investigations of Lachesis venoms are discussed. BIOLOGICAL SIGNIFICANCE: The proteomics characterization of L. m. rhombeata venom completes the overview of Lachesis venom proteomes and confirms the remarkable toxin profile conservation across the five clades of this wide-ranging genus. Genus-wide antivenomics showed that two antivenoms, produced against L. stenophrys or L. m. rhombeata, exhibit paraspecificity towards all other congeneric venoms. Our venomics study shows that, despite the broad geographic distribution of the genus, monospecific antivenoms may achieve clinical coverage for any Lachesis sp. envenoming.

Snake venomics across genus Lachesis. Ontogenetic changes in the venom composition of Lachesis stenophrys and comparative proteomics of the venoms of adult Lachesis melanocephala and Lachesis acrochorda.

We report the proteomic analysis of ontogenetic changes in venom composition of the Central American bushmaster, Lachesis stenophrys, and the characterization of the venom proteomes of two congeneric pitvipers, Lachesis melanocephala (black-headed bushmaster) and Lachesis acrochorda (Chochoan bushmaster). Along with the previous characterization of the venom proteome of Lachesis muta muta (from Bolivia), our present outcome enables a comparative overview of the composition and distribution of the toxic proteins across genus Lachesis. Comparative venomics revealed the close kinship of Central American L. stenophrys and L. melanocephala and support the elevation of L. acrochorda to species status. Major ontogenetic changes in the toxin composition of L. stenophrys venom involves quantitative changes in the concentration of vasoactive peptides and serine proteinases, which steadily decrease from birth to adulthood, and age-dependent de novo biosynthesis of Gal-lectin and snake venom metalloproteinases (SVMPs). The net result is a shift from a bradykinin-potentiating and C-type natriuretic peptide (BPP/C-NP)-rich and serine proteinase-rich venom in newborns and 2-years-old juveniles to a (PI>PIII) SVMP-rich venom in adults. Notwithstanding minor qualitative and quantitative differences, the venom arsenals of L. melanocephala and L. acrochorda are broadly similar between themselves and also closely mirror those of adult L. stenophrys and L. muta venoms. The high conservation of the overall composition of Central and South American bushmaster venoms provides the ground for rationalizing the "Lachesis syndrome", characterized by vagal syntomatology, sensorial disorders, hematologic, and cardiovascular manifestations, documented in envenomings by different species of this wide-ranging genus. This finding let us predict that monospecific Lachesic antivenoms may exhibit paraspecificity against all congeneric species.

Reactive oxygen species and the MEK/ERK pathway are involved in the toxicity of clostridium perfringens α-toxin, a prototype bacterial phospholipase C.

Clostridium perfringens, the most broadly distributed pathogen in nature, produces a prototype phospholipase C, also called α-toxin, which plays a key role in the pathogenesis of gas gangrene. α-Toxin causes plasma membrane disruption at high concentrations, but the role of intracellular mediators in its toxicity at low concentrations is unknown. This work demonstrates that α-toxin causes oxidative stress and activates the MEK/ERK pathway in cultured cells and furthermore provides compelling evidence that O(2)(-.), hydrogen peroxide, and the OH(.) radical are involved in its cytotoxic and myotoxic effects. The data show that antioxidants and MEK1 inhibitors reduce the cytotoxic and myotoxic effects of α-toxin and demonstrate that edaravone, a clinically used hydroxyl radical trap, reduces the myonecrosis and the mortality caused by an experimental infection with C. perfringens in a murine model of gas gangrene. This knowledge provides new insights for the development of novel therapies to reduce tissue damage during clostridial myonecrosis.

Household social determinants of ascariasis and trichuriasis in North Central Venezuela.

Ascariasis and trichuriasis are highly prevalent parasitoses in tropical countries caused by Ascaris lumbricoides and Trichuris trichiura, respectively. Their prevalence is associated with poor sanitation and hygiene, among other biological and social factors. In this study, the associations of household social determinants with these soil-transmitted helminthiases in North Central Venezuela was assessed in the context of a National Study on Human Growth and Development (SENACREDH). This was a probabilistic, stratified, cross-sectional survey with a sample of 3388 individuals that weighted represents 4 675 433 inhabitants from the region. The prevalence of ascariasis and trichuriasis was 3.73% and 1.13%, respectively. In the univariate and multivariate analyses, these prevalences were significantly higher in those living in vulnerable houses [adjusted OR (AOR) = 1.479, 95% CI 1.428-1.532; and AOR = 10.519, 95% CI 9.971-11.097, respectively], houses located in rural areas (AOR = 2.067, 95% CI 2.035-2.101; and AOR = 1.918, 95% CI 1.868-1.970, respectively) and houses with a soil floor (AOR = 5.027, 95% CI 4.895-5.162; and AOR = 5.190, 95% CI 4.944-5.448, respectively), among other factors. People living in rural households with inappropriate building materials, insufficient basic public services such as water and waste disposal, and surrounding disease-prone environments are at higher risk of acquiring A. lumbricoides and T. trichiura infections.

Unexpected wide substrate specificity of C. perfringens α-toxin phospholipase C.

Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the main virulence factor for gas gangrene in humans. The lipase activity serves the bacterium to generate lipid signals in the host eukaryotic cell, and ultimately to degrade the host cell membranes. Several previous reports indicated that CpPLC was specific for phosphatidylcholine and sphingomyelin. Molecular docking studies described in this paper predict favorable interactions of the CpPLC active site with other phospholipids, e.g. phosphatidylethanolamine, phosphatidylinositol and, to a lesser extent, phosphatidylglycerol. On the basis of these predictions, we have performed experimental studies showing α-toxin to degrade all the phospholipids mentioned above. The molecular docking data also provide an explanation for the observed lower activity of CpPCL on sphingomyelin as compared to the glycerophospholipids.

Profiling the venom gland transcriptomes of Costa Rican snakes by 454 pyrosequencing.

BACKGROUND: A long term research goal of venomics, of applied importance for improving current antivenom therapy, but also for drug discovery, is to understand the pharmacological potential of venoms. Individually or combined, proteomic and transcriptomic studies have demonstrated their feasibility to explore in depth the molecular diversity of venoms. In the absence of genome sequence, transcriptomes represent also valuable searchable databases for proteomic projects. RESULTS: The venom gland transcriptomes of 8 Costa Rican taxa from 5 genera (Crotalus, Bothrops, Atropoides, Cerrophidion, and Bothriechis) of pitvipers were investigated using high-throughput 454 pyrosequencing. 100,394 out of 330,010 masked reads produced significant hits in the available databases. 5.165,220 nucleotides (8.27%) were masked by RepeatMasker, the vast majority of which corresponding to class I (retroelements) and class II (DNA transposons) mobile elements. BLAST hits included 79,991 matches to entries of the taxonomic suborder Serpentes, of which 62,433 displayed similarity to documented venom proteins. Strong discrepancies between the transcriptome-computed and the proteome-gathered toxin compositions were obvious at first sight. Although the reasons underlaying this discrepancy are elusive, since no clear trend within or between species is apparent, the data indicate that individual mRNA species may be translationally controlled in a species-dependent manner. The minimum number of genes from each toxin family transcribed into the venom gland transcriptome of each species was calculated from multiple alignments of reads matched to a full-length reference sequence of each toxin family. Reads encoding ORF regions of Kazal-type inhibitor-like proteins were uniquely found in Bothriechis schlegelii and B. lateralis transcriptomes, suggesting a genus-specific recruitment event during the early-Middle Miocene. A transcriptome-based cladogram supports the large divergence between A. mexicanus and A. picadoi, and a closer kinship between A. mexicanus and C. godmani. CONCLUSIONS: Our comparative next-generation sequencing (NGS) analysis reveals taxon-specific trends governing the formulation of the venom arsenal. Knowledge of the venom proteome provides hints on the translation efficiency of toxin-coding transcripts, contributing thereby to a more accurate interpretation of the transcriptome. The application of NGS to the analysis of snake venom transcriptomes, may represent the tool for opening the door to systems venomics.

Effects of bilayer composition and physical properties on the phospholipase C and sphingomyelinase activities of Clostridium perfringens α-toxin.

α-Toxin, a major determinant of Clostridium perfringens toxicity, exhibits both phospholipase C and sphingomyelinase activities. Our studies with large unilamellar vesicles containing a variety of lipid mixtures reveal that both lipase activities are enhanced by cholesterol and by lipids with an intrinsic negative curvature, e.g. phosphatidylethanolamine. Conversely lysophospholipids, that possess a positive intrinsic curvature, inhibit the α-toxin lipase activities. Phospholipids with a net negative charge do not exert any major effect on the lipase activities, and the same lack of effect is seen with the lysosomal lipid bis (monoacylglycero) phosphate. Ganglioside GT1b has a clear inhibitory effect, while the monosialic ganglioside GM3 is virtually ineffectual even when incorporated at 6mol % in the vesicles. The length of the lag periods appears to be inversely related to the maximum (post-lag) enzyme activities. Moreover, and particularly in the presence of cholesterol, lag times increase with pH. Both lipase activities are sensitive to vesicle size, but in opposite ways: while phospholipase C is higher with larger vesicles, sphingomyelinase activity is lower. The combination of our results with previous structural studies suggests that α-toxin lipase activities have distinct, but partially overlapping and interacting active sites.