Glucans: A Therapeutic Alternative for Sepsis Treatment.

Sepsis treatment is a challenging condition due to its complexity, which involves host inflammatory responses to a severe and potentially fatal infection, associated with organ dysfunction. The aim of this study was to analyze the scientific literature on the immunomodulatory effects of glucans in a murine model of systemic infection induced by cecal ligation and puncture. This study comprises an integrative literature review based on systematic steps, with searches carried out in the PubMed, ScienceDirect, Scopus, Web of Science, and Embase databases. In most studies, the main type of glucan investigated was ß-glucan, at 50 mg/kg, and a reduction of inflammatory responses was identified, minimizing the occurrence of tissue damage leading to increased animal survival. Based on the data obtained and discussed in this review, glucans represent a promising biotechnological alternative to modulate the immune response and could potentially be used in the clinical management of septic individuals.

Exploring the therapeutic potential of an antinociceptive and anti-inflammatory peptide from wasp venom.

Animal venoms are rich sources of neuroactive compounds, including anti-inflammatory, antiepileptic, and antinociceptive molecules. Our study identified a protonectin peptide from the wasp Parachartergus fraternus' venom using mass spectrometry and cDNA library construction. Using this peptide as a template, we designed a new peptide, protonectin-F, which exhibited higher antinociceptive activity and less motor impairment compared to protonectin. In drug interaction experiments with naloxone and AM251, Protonectin-F's activity was decreased by opioid and cannabinoid antagonism, two critical antinociception pathways. Further experiments revealed that this effect is most likely not induced by direct action on receptors but by activation of the descending pain control pathway. We noted that protonectin-F induced less tolerance in mice after repeated administration than morphine. Protonectin-F was also able to decrease TNF-α production in vitro and modulate the inflammatory response, which can further contribute to its antinociceptive activity. These findings suggest that protonectin-F may be a potential molecule for developing drugs to treat pain disorders with fewer adverse effects. Our results reinforce the biotechnological importance of animal venom for developing new molecules of clinical interest.

An Experimental Model of Chromoblastomycosis Caused by Fonsecaea sp. Species.

The experimental rodent models for the fungal disease are a handy tool for understanding host-fungus interactions. To Fonsecaea sp., one of the causative agents of chromoblastomycosis, there is an extra challenge because the animals preferably used show a spontaneous cure; so until now, there is no model to reproduce the long-term disease similar to human chronic disease. In this chapter, we described an experimental model using rats and mice with a subcutaneous route, with the checkpoints of acute-like and chronic-like lesion analysis comparable with human lesions, the fungal burden, and the lymphocytes investigation.

Anti-CEA tagged iron nanoparticles for targeting triple-negative breast cancer.

Systemic therapy is generally required for breast cancer. However, treatment toxicity and side effects are a concern, especially for triple-negative breast cancer (TNBC), a subtype that usually develops resistance to chemotherapy. To overcome this issue, new nanoformulations capable of targeting cancer cells have been developed and alternative biomarkers have been explored as target molecules for TNBC management. In this study, we performed anin vivoassay in a murine orthotopic TNBC model to evaluate the targeting ability of anti-carcinoembryonic antigen (anti-CEA) loaded nanoparticles (labelled MFCEA), which had been previously synthetized by our research group. 4T1 cells were injected in the mammary gland of balb-c mice, and tumors were evaluated for CEA expression by immunohistochemistry. Tumor-bearing mice received targeted (MFCEA) and non-targeted (MF) nanoparticles intraperitoneally. Tumors were removed 1, 4, 15 and 24 h after treatment, and Prussian blue iron staining was performed. Our results showed, as far as we know for the first time, that 4T1 induced tumors are CEA positive, and this opens up new prospects for treating TNBC. Furthermore, MFCEA nanoparticles were able to target malignant tissue and were retained in the tumor for longer than MF nanoparticles. The retention property of MFCEA, together with the absence of toxicity observed in the MTT assay, make these nanoparticles a promising device for management of CEA positive tumors and perhaps for TNBC. Nevertheless, further studies must be carried out to improve their performance and ensure safety for clinical studies.

Genomics and Virulence of Fonsecaea pugnacius, Agent of Disseminated Chromoblastomycosis.

Among agents of chromoblastomycosis, Fonsecaea pugnacius presents a unique type of infection because of its secondary neurotropic dissemination from a chronic cutaneous case in an immunocompetent patient. Neurotropism occurs with remarkable frequency in the fungal family Herpotrichiellaceae, possibly associated with the ability of some species to metabolize aromatic hydrocarbons. In an attempt to understand this new disease pattern, were conducted genomic analysis of Fonsecaea pugnacius (CBS 139214) performed with de novo assembly, gene prediction, annotation and mitochondrial genome assembly, supplemented with animal infection models performed with Tenebrio molitor in Mus musculus lineages BALB/c and C57BL/6. The genome draft of 34.8 Mb was assembled with a total of 12,217 protein-coding genes. Several proteins, enzymes and metabolic pathways related to extremotolerance and virulence were recognized. The enzyme profiles of black fungi involved in chromoblastomycosis and brain infection were analyzed with the Carbohydrate-Active Enzymes (CAZY) and peptidases database (MEROPS). The capacity of the fungus to survive inside Tenebrio molitor animal model was confirmed by histopathological analysis and by presence of melanin and hyphae in host tissue. Although F. pugnacius was isolated from brain in a murine model following intraperitoneal infection, cytokine levels were not statistically significant, indicating a profile of an opportunistic agent. A dual ecological ability can be concluded from presence of metabolic pathways for nutrient scavenging and extremotolerance, combined with a capacity to infect human hosts.

Fenbendazole Controls In Vitro Growth, Virulence Potential, and Animal Infection in the Cryptococcus Model.

The human diseases caused by the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are associated with high indices of mortality and toxic and/or cost-prohibitive therapeutic protocols. The need for affordable antifungals to combat cryptococcal disease is unquestionable. Previous studies suggested benzimidazoles as promising anticryptococcal agents combining low cost and high antifungal efficacy, but their therapeutic potential has not been demonstrated so far. In this study, we investigated the antifungal potential of fenbendazole, the most effective anticryptococcal benzimidazole. Fenbendazole was inhibitory against 17 different isolates of C. neoformans and C. gattii at a low concentration. The mechanism of anticryptococcal activity of fenbendazole involved microtubule disorganization, as previously described for human parasites. In combination with fenbendazole, the concentrations of the standard antifungal amphotericin B required to control cryptococcal growth were lower than those required when this antifungal was used alone. Fenbendazole was not toxic to mammalian cells. During macrophage infection, the anticryptococcal effects of fenbendazole included inhibition of intracellular proliferation rates and reduced phagocytic escape through vomocytosis. Fenbendazole deeply affected the cryptococcal capsule. In a mouse model of cryptococcosis, the efficacy of fenbendazole to control animal mortality was similar to that observed for amphotericin B. These results indicate that fenbendazole is a promising candidate for the future development of an efficient and affordable therapeutic tool to combat cryptococcosis.

HDAC genes play distinct and redundant roles in Cryptococcus neoformans virulence.

The human fungal pathogen Cryptococcus neoformans undergoes many phenotypic changes to promote its survival in specific ecological niches and inside the host. To explore the role of chromatin remodeling on the expression of virulence-related traits, we identified and deleted seven genes encoding predicted class I/II histone deacetylases (HDACs) in the C. neoformans genome. These studies demonstrated that individual HDACs control non-identical but overlapping cellular processes associated with virulence, including thermotolerance, capsule formation, melanin synthesis, protease activity and cell wall integrity. We also determined the HDAC genes necessary for C. neoformans survival during in vitro macrophage infection and in animal models of cryptococcosis. Our results identified the HDA1 HDAC gene as a central mediator controlling several cellular processes, including mating and virulence. Finally, a global gene expression profile comparing the hda1Δ mutant versus wild-type revealed altered transcription of specific genes associated with the most prominent virulence attributes in this fungal pathogen. This study directly correlates the effects of Class I/II HDAC-mediated chromatin remodeling on the marked phenotypic plasticity and virulence potential of this microorganism. Furthermore, our results provide insights into regulatory mechanisms involved in virulence gene expression that are likely shared with other microbial pathogens.

Comparative Genomics of Sibling Species of Fonsecaea Associated with Human Chromoblastomycosis.

Fonsecaea and Cladophialophora are genera of black yeast-like fungi harboring agents of a mutilating implantation disease in humans, along with strictly environmental species. The current hypothesis suggests that those species reside in somewhat adverse microhabitats, and pathogenic siblings share virulence factors enabling survival in mammal tissue after coincidental inoculation driven by pathogenic adaptation. A comparative genomic analysis of environmental and pathogenic siblings of Fonsecaea and Cladophialophora was undertaken, including de novo assembly of F. erecta from plant material. The genome size of Fonsecaea species varied between 33.39 and 35.23 Mb, and the core genomes of those species comprises almost 70% of the genes. Expansions of protein domains such as glyoxalases and peptidases suggested ability for pathogenicity in clinical agents, while the use of nitrogen and degradation of phenolic compounds was enriched in environmental species. The similarity of carbohydrate-active vs. protein-degrading enzymes associated with the occurrence of virulence factors suggested a general tolerance to extreme conditions, which might explain the opportunistic tendency of Fonsecaea sibling species. Virulence was tested in the Galleria mellonella model and immunological assays were performed in order to support this hypothesis. Larvae infected by environmental F. erecta had a lower survival. Fungal macrophage murine co-culture showed that F. erecta induced high levels of TNF-α contributing to macrophage activation that could increase the ability to control intracellular fungal growth although hyphal death were not observed, suggesting a higher level of extremotolerance of environmental species.

DNAhsp65 Vaccine as Therapy against Paracoccidioidomycosis.

The conventional treatment for fungal diseases usually shows long periods of therapy and the high frequency of relapses and sequels. New strategies of the treatment are necessary. We have shown that the Mycobacterium leprae HSP65 gene can be successfully used as therapy against murine Paracoccidioidomycosis (PCM). Here, we described the methodology of DNAhsp65 immunotherapy in mice infected with the dimorphic fungus Paracoccidioides brasiliensis, one of PCM agent, evaluating cytokines levels, fungal burden, and lung injury. Our results provide a new prospective on the immunotherapy of mycosis.

Evaluation of the antimicrobial activity of the mastoparan Polybia-MPII isolated from venom of the social wasp Pseudopolybia vespiceps testacea (Vespidae, Hymenoptera).

Mastoparans, a class of peptides found in wasp venom, have significant effects following a sting as well as useful applications in clinical practice. Among these is their potential use in the control of micro-organisms that cause infectious diseases with a significant impact on society. Thus, the present study describes the isolation and identification of a mastoparan peptide from the venom of the social wasp Pseudopolybia vespiceps and evaluated its antimicrobial profile against bacteria (Staphylococcus aureus and Mycobacterium abscessus subsp. massiliense), fungi (Candida albicans and Cryptococcus neoformans) and in vivo S. aureus infection. The membrane pore-forming ability was also assessed. The mastoparan reduced in vitro and ex vivo mycobacterial growth by 80% at 12.5 µM in infected peritoneal macrophages but did not affect the shape of bacterial cells at the dose tested (6.25 µM). The peptide also showed potent action against S. aureus in vitro (EC50 and EC90 values of 1.83 µM and 2.90 µM, respectively) and reduced the in vivo bacterial load after 6 days of topical treatment (5 mg/kg). Antifungal activity was significant, with EC50 and EC90 values of 12.9 µM and 15.3 µM, respectively, for C. albicans, and 11 µM and 22.70 µM, respectively, for C. neoformans. Peptides are currently attracting interest for their potential in the design of antimicrobial drugs, particularly due to the difficulty of micro-organisms in developing resistance to them. In this respect, Polybia-MPII proved to be highly effective, with a lower haemolysis rate compared with peptides of the same family.

Activity of Scorpion Venom-Derived Antifungal Peptides against Planktonic Cells of Candida spp. and Cryptococcus neoformans and Candida albicans Biofilms.

The incidence of fungal infections has been increasing in the last decades, while the number of available antifungal classes remains the same. The natural and acquired resistance of some fungal species to available therapies, associated with the high toxicity of these drugs on the present scenario and makes an imperative of the search for new, more efficient and less toxic therapeutic choices. Antimicrobial peptides (AMPs) are a potential class of antimicrobial drugs consisting of evolutionarily conserved multifunctional molecules with both microbicidal and immunomodulatory properties being part of the innate immune response of diverse organisms. In this study, we evaluated 11 scorpion-venom derived non-disulfide-bridged peptides against Cryptococcus neoformans and Candida spp., which are important human pathogens. Seven of them, including two novel molecules, showed activity against both genera with minimum inhibitory concentration values ranging from 3.12 to 200 µM and an analogous activity against Candida albicans biofilms. Most of the peptides presented low hemolytic and cytotoxic activity against mammalian cells. Modifications in the primary peptide sequence, as revealed by in silico and circular dichroism analyses of the most promising peptides, underscored the importance of cationicity for their antimicrobial activity as well as the amphipathicity of these molecules and their tendency to form alpha helices. This is the first report of scorpion-derived AMPs against C. neoformans and our results underline the potential of scorpion venom as a source of antimicrobials. Further characterization of their mechanism of action, followed by molecular optimization to decrease their cytotoxicity and increase antimicrobial activity, is needed to fully clarify their real potential as antifungals.

Draft Genome Sequence of Fonsecaea monophora Strain CBS 269.37, an Agent of Human Chromoblastomycosis.

The black yeast Fonsecaea monophora is one of the main etiologic agents of chromoblastomycosis in humans. Its pathogenicity profile is more invasive than that of related Fonsecaea species, causing brain infection in addition to (sub)cutaneous infections.

Clavanin A improves outcome of complications from different bacterial infections.

The rapid increase in the incidence of multidrug-resistant infections today has led to enormous interest in antimicrobial peptides (AMPs) as suitable compounds for developing unusual antibiotics. In this study, clavanin A, an antimicrobial peptide previously isolated from the marine tunicate Styela clava, was selected as a purposeful molecule that could be used in controlling infection and further synthesized. Clavanin A was in vitro evaluated against Staphylococcus aureus and Escherichia coli as well as toward L929 mouse fibroblasts and skin primary cells (SPCs). Moreover, this peptide was challenged here in an in vivo wound and sepsis model, and the immune response was also analyzed. Despite displaying clear in vitro antimicrobial activity toward Gram-positive and -negative bacteria, clavanin A showed no cytotoxic activities against mammalian cells, and in acute toxicity tests, no adverse reaction was observed at any of the concentrations. Moreover, clavanin A significantly reduced the S. aureus CFU in an experimental wound model. This peptide also reduced the mortality of mice infected with E. coli and S. aureus by 80% compared with that of control animals (treated with phosphate-buffered saline [PBS]): these data suggest that clavanin A prevents the start of sepsis and thereby reduces mortality. These data suggest that clavanin A is an AMP that could improve the development of novel peptide-based strategies for the treatment of wound and sepsis infections.

Expression of metalloproteinases and interleukins on anastomoses in septic rats.

BACKGROUND: Anastomotic dehiscence is the most severe complication of colorectal surgery and its incidence increases in the presence of infection. It has been reported that immune factors or the activity of matrix metalloproteinases (MMP) may mediate the loss of anastomotic strength in the first postoperative days. In this study, we investigated the effects of abdominal sepsis on the MMP and interleukin (IL) gene expression in left colonic anastomoses in rats. MATERIALS AND METHODS: Forty rats were divided into two groups of 20 animals according to the presence (group S) or absence (group N) of sepsis induction by cecal ligation and perforation during left colonic anastomosis. Each group was divided into subgroups for euthanasia on the third (N3 and S3) or seventh (N7 and S7) postoperative day (POD). A colonic segment containing anastomosis was removed for analysis of the expression of MMP1a, MMP8, MMP13, IL1ß, IL6, IL10, TNFα, and IFNγ genes. RESULTS: The anastomoses with abdominal sepsis showed increased MMP1a gene expression and decreased MMP8 gene expression both on the third and seventh POD. There was no change in the expression of MMP13. There was an increase in the expression of IL10 only on the third POD and a negative modulation of IL1ß, IFNγ, and IL6 genes on both periods. The TNFα gene expression was negatively modulated on the third POD and became not modulated on the seventh POD. CONCLUSION: Abdominal sepsis induced a specific inflammatory pattern with increased MMP1a and IL10 gene expression and negative modulation of MMP8, IL1ß, IFNγ, and TNFα gene expression in left colonic anastomoses in rats.

Antitumor effect and toxicity of free rhodium (II) citrate and rhodium (II) citrate-loaded maghemite nanoparticles in mice bearing breast cancer.

BACKGROUND: Magnetic fluids containing superparamagnetic iron oxide nanoparticles represent an attractive platform as nanocarriers in chemotherapy. Recently, we developed a formulation of maghemite nanoparticles coated with rhodium (II) citrate, which resulted in in vitro cytotoxicity enhanced up to 4.6 times when compared to free rhodium (II) citrate formulation on breast carcinoma cells. In this work, we evaluate the antitumor activity and toxicity induced by these formulations in Balb/c mice bearing orthotopic 4T1 breast carcinoma. METHODS: Mice were evaluated with regard to the treatments' toxicity through analyses of hemogram, serum levels of alanine aminotransferase, iron, and creatinine; DNA fragmentation and cell cycle of bone marrow cells; and liver, kidney and lung histology. In addition, the antitumor activity of rhodium (II) citrate and maghemite nanoparticles coated with rhodium (II) citrate was verified by tumor volume reduction, histology and immunohistochemistry. RESULTS: Regarding the treatments' toxicity, no experimental groups had alterations in levels of serum ALT or creatinine, and this suggestion was corroborated by the histopathologic examination of liver and kidney of mice. Moreover, DNA fragmentation frequency of bone marrow cells was lower than 15% in all experimental groups. On the other hand, the complexes rhodium (II) citrate-functionalized maghemite and free rhodium (II) citrate led to a marked growth inhibition of tumor and decrease in CD31 and Ki-67 staining. CONCLUSIONS: In summary, we demonstrated that both rhodium (II) citrate and maghemite nanoparticles coated with rhodium (II) citrate formulations exhibited antitumor effects against 4T1 metastatic breast cancer cell line following intratumoral administration. This antitumor effect was followed by inhibition of both cell proliferation and microvascularization and by tumor tissue injury characterized as necrosis and fibrosis. Remarkably, this is the first published report demonstrating the therapeutic efficacy of maghemite nanoparticles coated with rhodium (II) citrate. This treatment prolonged the survival period of treated mice without inducing apparent systemic toxicity, which strengthens its use for future breast cancer therapeutic applications.

Predicting antimicrobial peptides from eukaryotic genomes: in silico strategies to develop antibiotics.

A remarkable and intriguing challenge for the modern medicine consists in the development of alternative therapies to avoid the problem of microbial resistance. The cationic antimicrobial peptides present a promise to be used to develop more efficient drugs applied to human health. The in silico analysis of genomic databases is a strategy utilized to predict peptides of therapeutic interest. Once the main antimicrobial peptides' physical-chemical properties are already known, the correlation of those features to search on these databases is a tool to shorten identifying new antibiotics. This study reports the identification of antimicrobial peptides by theoretical analyses by scanning the Paracoccidioides brasiliensis transcriptome and the human genome databases. The identified sequences were synthesized and investigated for hemocompatibility and also antimicrobial activity. Two peptides presented antifungal activity against Candida albicans. Furthermore, three peptides exhibited antibacterial effects against Staphylococcus aureus and Escherichia coli; finally one of them presented high potential to kill both pathogens with superior activity in comparison to chloramphenicol. None of them showed toxicity to mammalian cells. In silico structural analyses were performed in order to better understand function-structure relation, clearly demonstrating the necessity of cationic peptide surfaces and the exposition of hydrophobic amino acid residues. In summary, our results suggest that the use of computational programs in order to identify and evaluate antimicrobial peptides from genomic databases is a remarkable tool that could be used to abbreviate the search of peptides with biotechnological potential from natural resources.

Murine dendritic cells transcriptional modulation upon Paracoccidioides brasiliensis infection.

Limited information is available regarding the modulation of genes involved in the innate host response to Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis. Therefore, we sought to characterize, for the first time, the transcriptional profile of murine bone marrow-derived dendritic cells (DCs) at an early stage following their initial interaction with P. brasiliensis. DCs connect innate and adaptive immunity by recognizing invading pathogens and determining the type of effector T-cell that mediates an immune response. Gene expression profiles were analyzed using microarray and validated using real-time RT-PCR and protein secretion studies. A total of 299 genes were differentially expressed, many of which are involved in immunity, signal transduction, transcription and apoptosis. Genes encoding the cytokines IL-12 and TNF-α, along with the chemokines CCL22, CCL27 and CXCL10, were up-regulated, suggesting that P. brasiliensis induces a potent proinflammatory response in DCs. In contrast, pattern recognition receptor (PRR)-encoding genes, particularly those related to Toll-like receptors, were down-regulated or unchanged. This result prompted us to evaluate the expression profiles of dectin-1 and mannose receptor, two other important fungal PRRs that were not included in the microarray target cDNA sequences. Unlike the mannose receptor, the dectin-1 receptor gene was significantly induced, suggesting that this ß-glucan receptor participates in the recognition of P. brasiliensis. We also used a receptor inhibition assay to evaluate the roles of these receptors in coordinating the expression of several immune-related genes in DCs upon fungal exposure. Altogether, our results provide an initial characterization of early host responses to P. brasiliensis and a basis for better understanding the infectious process of this important neglected pathogen.

Polymorphism analysis of the CTLA-4 gene in paracoccidioidomycosis patients.

The CTLA-4 protein is expressed in activated T cells and plays an essential role in the immune response through its regulatory effect on T cell activation. Polymorphisms of the CTLA-4 gene have been correlated with autoimmune, neoplastic and infectious illnesses. This work aimed to verify possible associations between single nucleotide polymorphisms (SNPs) in CTLA-4, -318C/T in the promoter and +49A/G in exon 1 and paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis. For this purpose, 66 chronic form PCM patients and 76 healthy controls had their allele, genotype and haplotype frequencies determined. The genetic admixture structure of the patients and controls was evaluated to eliminate ancestral bias. The comparison of frequencies indicated no significant differences between patients and controls that could link the SNPs to PCM. Groups were admixture matched with no difference observed in population ancestry inference, indicating that the absence of association between CTLA-4 polymorphisms and PCM could not be attributed to ancestral bias. This study showed that there was no association between the CTLA-4 SNPs -318 and +49 and the resistance or susceptibility to PCM.

In vitro antifungal activity and toxicity of itraconazole in DMSA-PLGA nanoparticles.

Itraconazole (ITZ) is a drug used to treat various fungal infections and may cause side effects. The aim of this study was to develop and evaluate the in vitro activity of DMSA-PLGA nanoparticles loaded with ITZ against Paracoccidioides brasiliensis, as well as their cytotoxicity. Nanoparticles were prepared using the emulsification-evaporation technique and characterized by their encapsulation efficiency, morphology (TEM), size (Nanosight) and charge (zeta potential). Antifungal efficacy in P. brasiliensis was determined by minimal inhibition concentration (MIC), and cytotoxicity using MTT assay. ITZ was effectively incorporated in the PLGA-DMSA nanoparticles with a loading efficiency of 72.8 +/- 3.50%. The shape was round with a solid polymeric structure, and a size distribution of 174 +/- 86 nm (Average +/- SD). The particles were negatively charged. ITZ-NANO presented antifungal inhibition (MIC = 6.25 ug/mL) against P. brasiliensis and showed lower in vitro cytotoxicity than free drug (ITZ).

Polymorphism analysis of the CTLA-4 gene in paracoccidioidomycosis patients

The CTLA-4 protein is expressed in activated T cells and plays an essential role in the immune response through its regulatory effect on T cell activation. Polymorphisms of the CTLA-4 gene have been correlated with autoimmune, neoplastic and infectious illnesses. This work aimed to verify possible associations between single nucleotide polymorphisms (SNPs) in CTLA-4, -318C/T in the promoter and +49A/G in exon 1 and paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis. For this purpose, 66 chronic form PCM patients and 76 healthy controls had their allele, genotype and haplotype frequencies determined. The genetic admixture structure of the patients and controls was evaluated to eliminate ancestral bias. The comparison of frequencies indicated no significant differences between patients and controls that could link the SNPs to PCM. Groups were admixture matched with no difference observed in population ancestry inference, indicating that the absence of association between CTLA-4 polymorphisms and PCM could not be attributed to ancestral bias. This study showed that there was no association between the CTLA-4 SNPs -318 and +49 and the resistance or susceptibility to PCM.