Cytotoxic activity and degradation patterns of structural proteins by corneal isolates of Acanthamoeba spp.

BACKGROUND: Proteolytic enzymes secreted by trophozoites (amoebic secretome) are suggested as the main virulence factor involved in the severity of Acanthamoeba keratitis. The degradation profile of the main glycoprotein components of anterior and posterior portions of the cornea and the cytopathic effect of secretomes on endothelial cells by contact-independent mechanism were evaluated. METHODS: Trophozoites were isolated primarily from corneal tissue samples (n = 11) and extracellular proteins were collected from axenic cell culture supernatants. The molecular weights of proteolytic enzymes were estimated by zymography. Enzymatic cleavage of laminin and fibronectin substrates by amoebic secretome was investigated and cluster analysis was applied to the proteolysis profiles. Primary cultures of endothelial cells were used in both qualitative and quantitative assays of cytophatogenicity. RESULTS: Differential patterns of proteolysis were observed among the Acanthamoeba secretomes that were analysed. The uniformity of laminin degradation contrasted with the diversity of the proteolysis profiles observed in the fibronectin substrate. Acanthamoeba secretome extracted from four clinical isolates was shown to be toxic when in contact with the endothelial cell monolayer (p < 0.01). Induction of apoptosis and membrane permeability, at different percentual values, were suggested as the main mechanisms that could induce endothelial cell death when in contact with amoebic secretome. CONCLUSIONS: Our results provide evidence that virulence factors secreted by Acanthamoeba trophozoites can be related to an increased pathogenicity pattern by an independent contact-trophozoite mechanism, through induction of endothelial cell death by apoptosis at a higher percentage than providing the lack of cell viability by the membrane-associated pore-forming toxin activity.

The venom of the Lonomia caterpillar: an overview.

Contact with the Lonomia caterpillar causes numerous accidents, especially in Venezuela and the southern region of Brazil, where it is considered a public health problem. The Lonomia obliqua venom causes disseminated intravascular coagulation and a consumptive coagulopathy, which can lead to a hemorrhagic syndrome. The venom of Lonomia achelous also causes hemorrhage, but through increased fibrinolysis. In vivo and in vitro studies have shown that the venom of the Lonomia caterpillar contains several toxins with procoagulant, anticoagulant and antithrombotic activities. These toxins also affect the endothelium. The recent construction of cDNA libraries of the transcripts from L. obliqua bristles enables the use of biotechnological approaches to study the venom. This paper presents an overview of the biochemical and biological properties of Lonomia caterpillar venom, discussing aspects of human accidents, experimental envenomation, toxins and targets and future perspectives.

The Use of Botulinum Toxin to Treat Infantile Esotropia: A Systematic Review With Meta-Analysis.

Purpose: The purpose of this review was to examine the efficacy of botulinum toxin in the treatment of infantile esotropia and to evaluate the average response of BT and its complication rates. Methods: A research was performed in the Latin American and Caribbean Literature on Health Sciences (LILACS), MEDLINE, and Cochrane Central Register of Controlled Trial (CENTRAL). The database was searched between December 28, 2016 and January 30, 2017. The selection was restricted to articles published in English, Spanish, or Portuguese. There were no date restrictions in the search. Results: Nine studies were eligible for inclusion. The grouped success rate of BT treatment in infantile esotropia was 76% (95% confidence interval [CI]: 61%-89%). For the success rate, I2 of 94.25% was observed, indicating a high heterogeneity (P < 0.001). The complication rates were also analyzed. The grouped consecutive exotropia (XT) rate was 1% (95% CI: 0%-2%). The grouped ptosis rate was 27% (95% CI: 21%-33%). The grouped vertical deviation rate was 12% (95% CI: 4%-22%). The mean change of the deviation after BT injection was -30.7 (95% CI: -37.7, -23.8), demonstrating a significant improvement in alignment. Conclusions: Botulinum toxin injection into medial recti muscles reveals to be a safe procedure and a valuable alternative to strabismus surgery in congenital esotropia, especially in moderate deviations.

Therapeutic agents and biocides for ocular infections by free-living amoebae of Acanthamoeba genus.

Acanthamoeba keratitis is a sight-threatening infectious disease. Resistance of the cystic form of the protozoan to biocides and the potential toxicity of chemical compounds to corneal cells are the main concerns related to long-term treatment with the clinically available ophthalmic drugs. Currently, a limited number of recognized antimicrobial agents are available to treat ocular amoebic infections. Topical application of biguanide and diamidine antiseptic solutions is the first-line therapy. We consider the current challenges when treating Acanthamoeba keratitis and review the chemical properties, toxicities, and mechanisms of action of the available biocides. Antimicrobial therapy using anti-inflammatory drugs is controversial, and aspects related to this topic are discussed. Finally, we offer our perspective on potential improvement of the effectiveness and safety of therapeutic profiles, with the focus on the quality of life and the advancement of individualized medicine.

Avaliação de fatores de risco e antígeno prostático específico no rastreamento de câncer de próstata / Evaluation of risk factors and prostate-specific antigen in prostate cancerscreening

Introdução: no Brasil, o câncer de maior incidência nos homens é o câncer de próstata (CaP), com 6,9% de mortalidade. Atualmente, discute-se a aplicabilidade do antígeno prostático específico (PSA) em políticas de rastreamento para CaP e os riscos associados ao sobrediagnóstico. Objetivo: correlacionar a dosagem do PSA com fatores de risco, história clínica e a presença de neoplasia prostática. Metodologia: estudo descritivo transversal que analisou, comparativamente, dados clínico-epidemiológicos e níveis séricos de PSA de 200 pacientes. Valores de PSA foram estratificados em três categorias (<2,5, 2,5­10,0 e >10 ng/ml). Resultados: os fatores de risco analisados foram relacionados significativamente com o aumento do PSA e neoplasia prostática. A prevalência de CaP (11%) e hiperplasia prostática (61%) foi observada nos pacientes com maior dosagem de PSA, enquanto 1% dos pacientes apresentou CaP sem alteração do PSA e 4% tiveram CaP com 2,5­10,0 ng/ml de PSA. Maiores níveis séricos do biomarcador foram relacionados a diabetes (70%), hipertensão (77%), uso crônico de medicações (60%) e ausência de exames periódicos (58%). O grupo com PSA >10 ng/ml teve média de idade maior que o primeiro (p = 0,002) e o segundo grupos (p = 0,027). Conclusão: a prevalência de hiperplasia prostática benigna associada à alteração do PSA, e o elevado risco de exames falso-positivos evidenciam a preocupação com o sobrediagnóstico. No contexto dos dados clinico-epidemiológicos avaliados, a possibilidade de resultados falso-positivos e falso-negativos associados à dosagem do PSA deve ser considerada, ressaltando a importância de adoção de exames complementares para rastreio do CaP.

Introduction: in Brazil, the cancer with the highest incidence in men is prostate cancer (PCa), with 6.9% mortality. Currently, the applicability of prostate specific antigen (PSA) in screening policies for PCa and the risks associated with overdiagnosis are discussed. Objective: to correlate the PSA level with risk factors, clinical history and the presence of prostatic neoplasm. Methods: a cross-sectional descriptive study that analyzed, comparatively, clinical-epidemiological data and serum PSA levels of 200 patients. PSA values were stratified into three categories (<2.5, 2.5­10.0 and> 10 ng / ml). Results: the risk factors analyzed were significantly related to the increase in PSA and prostatic neoplasm. The prevalence of PCa (11%) and prostatic hyperplasia (61%) was observed in patients with higher levels of PSA, while 1% of patients had PCa without PSA changes and 4% had PCa with 2.5­10.0 ng/ml PSA. Increased serum levels of the biomarker were related to diabetes (70%), hypertension (77%), chronic use of medications (60%) and periodic exams (58%). The group with PSA> 10 ng/ml had a mean age greater than the first (p = 0.002) and the second group (p = 0.027). Conclusion: the prevalence of benign prostatic hyperplasia associated with PSA change and an increased risk of false-positive tests show a concern with overdiagnosis. In the context of clinical-epidemiological data, the possibility of false-positive and false-negative results associated with the PSA measurement have to be considered, highlighting the importance of complementary tests for PCa screening.

Toxicity of Intracameral Injection of Fourth-Generation Fluoroquinolones on the Corneal Endothelium.

PURPOSE: The aim of this study was to compare the cellular susceptibility patterns and morphologic changes in the corneal endothelium associated with the use of fourth-generation fluoroquinolones. METHOD: Endothelial susceptibility was assessed through intracameral injection of besifloxacin, gatifloxacin, and moxifloxacin. Human umbilical vein endothelial cells (HUVECs) were used as the standard cellular lineage to assess the quantitative toxicity of each antibiotic solution. Qualitative changes in the morphologic character of the corneal structure and the endothelial layer were generated using a combination of ex vivo and in vivo assays. Experimental assays were conducted in triplicate, and the results were statistically analyzed. RESULTS: At 1 hour of exposure, all HUVECs exposed to antibiotics showed viability above 85%, after 3 hours of exposure to besifloxacin, gatifloxacin, and moxifloxacin, the percentages of viable cells were 68.3 ± 4.0 (P < 0.001), 90.7 ± 4.2 (P < 0.05), and 93.3 ± 1.5 (P > 0.05), respectively. All fluoroquinolones tested showed toxicity to HUVECs, resulting in significant (P < 0.001) loss of cellular viability after 24 hours of drug exposure. Giant endothelial cells were observed in animals treated with the 3 fluoroquinolones in contrast to the absence of these abnormal cells in the untreated group. Early cellular detachment was seen in the endothelial layer after exposure to gatifloxacin and moxifloxacin. CONCLUSIONS: We concluded that injection of fourth-generation fluoroquinolones in the aqueous humor did not adversely affect the corneal endothelium. However, these results suggested that prophylactic intracameral injection of besifloxacin, gatifloxacin, or moxifloxacin, if needed, should be administered as a last therapeutic resource in clinical practice, with careful and constant monitoring of corneal endothelium.

Rational development of novel leads from animal secretion based on coagulation and cell targets: 1. In silico analysis to explore a peptide derivative as lipocalins' signature.

Animal venoms and secretions have been screened, in our research group, to discover, identify and isolate peptide molecules active in the mammalian haemostatic system. As result, this kind of research has provided a portfolio of promising drug candidates. These novel recombinant proteins have turned out to be multifunctional molecules, and are currently under different development phases. Lopap from bristles of the Lonomia obliqua moth caterpillar, for instance, is a prothrombin activator which belongs to the lipocalin family. It displays serine protease-like activity with procoagulant effect, and also induces cytokine secretion and antiapoptotic pathways in human cultured endothelial cells. Furthermore, a Lopap-derived peptide has showed to induce collagen synthesis in fibroblast culture and in animal dermis. Here, the molecular properties (steric, electronic, hydrophobic, geometric), which are strongly dependent on chemical structure, were investigated by applying chemometric and computational chemistry methods. It was considered different patterns of amino acid substitution related to the lipocalins' motif 2, which was recently shown to modulate cell survival. The calculated molecular properties were generally maintained in all investigated peptides extracted from three-dimensional structures of Protein Data Bank (1t0v, 1bbp, 1kxo, 2hzr, 1iiu, 1jyj, 1gka, 1s44, 3ebw) when compared to Lopap-derived peptide, specially the molecular shape and electronic density distribution, validating the lipocalin sequence signature previously reported. Indeed, those two properties are quite important for the molecular recognition process.

Antimicrobial action of biguanides on the viability of Acanthamoeba cysts and assessment of cell toxicity.

PURPOSE: To assess dose- and concentration-dependent rates of biguanides on the viability of Acanthamoeba cysts isolated from severe ulcerative keratitis, and to correlate cysticidal activites with cytotoxic profiles in corneal and endothelial cells. METHODS: Cysticidal activities of polyhexamethylene biguanide and chlorhexidine digluconate were evaluated in the Acanthamoeba castellanii strain and clinical isolates of Acanthamoeba spp obtained from two severe and recurrent cases of ulcerative keratitis. The molecular characterization of protozoa used in the experimental assays was performed by sequencing reactions of the 18S rDNA gene. Acanthamoeba cysts were exposed at different dosages and concentrations of both biguanides; the application of double-biguanides was also evaluated. Automated cell viability assessment of cysts was performed using the trypan blue dye exclusion method. Cytotoxicity assays of biguanides were conducted using primary cultures of endothelial cells alone or in coculture with Acanthamoeba cysts. Human corneal epithelial cells were used as a comparative pattern to assess the toxicity of biguanide compounds. Cell viability was measured using both quantitative and qualitative methods. Statistical analyses were applied to the data. RESULTS: The in vitro study showed that all dosages, concentrations, and combinations of biguanides tested had a cysticidal effect on Acanthamoeba spp strains tested compared with control cultures not exposed to any antimicrobials; the difference in response was statistically significant. The use of both biguanides in combination demonstrated the best cysticidal effect. The use of isolated biguanides was associated with greater cytotoxic effects than with biguanides used in combination. Chlorhexidine digluconate used alone tended to have greater cytotoxicity than polyhexamethylene biguanide. Furthermore, the double-biguanide application had a statistically significant decrease in the deleterious effect on endothelial cells at higher dosage and concentration. Quantitative and qualitative analyses demonstrated the toxic effect of biguanide compounds on the viability of corneal epithelial cells, under single or in combination usage. CONCLUSIONS: We demonstrated that the combined use of biguanides had greater cysticidal activity than individual drug application as well as a possible protective effect on endothelial cells. The biguanide compounds tested were able to induce corneal epithelial cell death in time and concentration-independent fashions. Findings support the hypothesis concerning the cysticidal effect and the differential patterns of toxicity expressed by polyhexamethylene biguanide and chlorhexidine digluconate on the endothelial and corneal cells.

A lipocalin-derived Peptide modulating fibroblasts and extracellular matrix proteins.

Lipocalin family members have been implicated in development, regeneration, and pathological processes, but their roles are unclear. Interestingly, these proteins are found abundant in the venom of the Lonomia obliqua caterpillar. Lipocalins are ß-barrel proteins, which have three conserved motifs in their amino acid sequence. One of these motifs was shown to be a sequence signature involved in cell modulation. The aim of this study is to investigate the effects of a synthetic peptide comprising the lipocalin sequence motif in fibroblasts. This peptide suppressed caspase 3 activity and upregulated Bcl-2 and Ki-67, but did not interfere with GPCR calcium mobilization. Fibroblast responses also involved increased expression of proinflammatory mediators. Increase of extracellular matrix proteins, such as collagen, fibronectin, and tenascin, was observed. Increase in collagen content was also observed in vivo. Results indicate that modulation effects displayed by lipocalins through this sequence motif involve cell survival, extracellular matrix remodeling, and cytokine signaling. Such effects can be related to the lipocalin roles in disease, development, and tissue repair.