Influence of iron binding in the structural stability and cellular internalization of bovine lactoferrin.

Lactoferrin (Lf) is an iron-binding glycoprotein and a component of many external secretions with a wide diversity of functions. Structural studies are important to understand the mechanisms employed by Lf to exert so varied functions. Here, we used guanidine hydrochloride and high hydrostatic pressure to cause perturbations in the structure of bovine Lf (bLf) in apo and holo (unsaturated and iron-saturated, respectively) forms, and analyzed conformational changes by intrinsic and extrinsic fluorescence spectroscopy. Our results showed that the iron binding promotes changes on tertiary structure of bLf and increases its structural stability. In addition, we evaluated the effects of bLf structural change on the kinetics of bLf internalization in Vero cells by confocal fluorescence microscopy, and observed that the holo form was faster than the apo form. This finding may indicate that structural changes promoted by iron binding may play a key role in the intracellular traffic of bLf. Altogether, our data improve the comprehension of bLf stability and uptake, adding knowledge to its potential use as a biopharmaceutical.

Lactoferrin affects rhinovirus B-14 entry into H1-HeLa cells.

Lactoferrin is part of the innate immune system, with antiviral activity against numerous DNA and RNA viruses. Rhinoviruses, the leading cause of the common cold, are associated with exacerbation of respiratory illnesses such as asthma. Here, we explored the effect of bovine lactoferrin (BLf) on RV-B14 infectivity. Using different assays, we show that the effect of BLf is strongest during adhesion of the virus to the cell and entry. Tracking the internalisation of BLf and virus revealed a degree of colocalisation, although their interaction was only confirmed in vitro using empty viral particles, indicating a possible additional influence of BLf on other infection steps.

Cellular and Molecular Immunology Approaches for the Development of Immunotherapies against the New Coronavirus (SARS-CoV-2): Challenges to Near-Future Breakthroughs.

The severe acute respiratory syndrome caused by the new coronavirus (SARS-CoV-2), termed COVID-19, has been highlighted as the most important infectious disease of our time, without a vaccine and treatment available until this moment, with a big impact on health systems worldwide, and with high mortality rates associated with respiratory viral disease. The medical and scientific communities have also been confronted by an urgent need to better understand the mechanism of host-virus interaction aimed at developing therapies and vaccines. Since this viral disease can trigger a strong innate immune response, causing severe damage to the pulmonary tract, immunotherapies have also been explored as a means to verify the immunomodulatory effect and improve clinical outcomes, whilst the comprehensive COVID-19 immunology still remains under investigation. In this review, both cellular and molecular immunopathology as well as hemostatic disorders induced by SARS-CoV-2 are summarized. The immunotherapeutic approaches based on the most recent clinical and nonclinical studies, emphasizing their effects for the treatment of COVID-19, are also addressed. The information presented elucidates helpful insights aiming at filling the knowledge gaps around promising immunotherapies that attempt to control the dysfunction of host factors during the course of this infectious viral disease.

Impairing the function of MLCK, myosin Va or myosin Vb disrupts Rhinovirus B14 replication.

Together, the three human rhinovirus (RV) species are the most frequent cause of the common cold. Because of their high similarity with other viral species of the genus Enterovirus, within the large family Picornaviridae, studies on RV infectious activities often offer a less pathogenic model for more aggressive enteroviruses, e.g. poliovirus or EV71. Picornaviruses enter via receptor mediated endocytosis and replicate in the cytosol. Most of them depend on functional F-actin, Rab proteins, and probably motor proteins. To assess the latter, we evaluated the role of myosin light chain kinase (MLCK) and two myosin V isoforms (Va and Vb) in RV-B14 infection. We report that ML-9, a very specific MLCK inhibitor, dramatically reduced RV-B14 entry. We also demonstrate that RV-B14 infection in cells expressing dominant-negative forms of myosin Va and Vb was impaired after virus entry. Using immunofluorescent localization and immunoprecipitation, we show that myosin Va co-localized with RV-B14 exclusively after viral entry (15 min post infection) and that myosin Vb was present in the clusters of newly synthesized RNA in infected cells. These clusters, observed at 180 min post infection, are reminiscent of replication sites. Taken together, these results identify myosin light chain kinase, myosin Va and myosin Vb as new players in RV-B14 infection that participate directly or indirectly in different stages of the viral cycle.

Antimicrobial activity and synergism of lactoferrin and lysozyme against cariogenic microorganisms.

The present study evaluated the antimicrobial in vitro effects of the salivary proteins lactoferrin and lysozyme on microorganisms involved in the carious process, obtaining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Streptococcus mutans (ATCC 25175) and Lactobacillus casei (ATCC 7469) were submitted to broth macrodilution of lysozyme at 80 mg/mL and lactoferrin at 200 mg/mL. The tubes were read in a spectrophotometer after they had been incubated at 37 °C for 18 h, in a carbon dioxide chamber, in order to read the MIC. A new subculture was carried on agar plates to obtain the MBC. The agar diffusion method was also tested, using BHI agar with 100 µL of the standardized microbial inocula. Filter-paper disks soaked in 10 µL of the solutions lactoferrin (200 µg/mL) and lysozyme (80 µg/mL) were placed on the agar surface. Inhibition halos were not observed on the plates, showing the absence of the antimicrobial effects of these proteins in this method. The bactericidal and bacteriostatic effects of lysozyme on L. casei were 50.3 mg/mL and 43.1 mg/mL respectively. The bactericidal and bacteriostatic effects on S. mutans were 68.5 mg/mL and 58.7 mg/mL. Lactoferrin did not induce any inhibitory effects on any microorganism, even in the concentration of 200 mg/mL. There was not a synergic antimicrobial effect of proteins, when they were tested together, even in the concentration of 42.8 mg/mL of lysozyme and 114 mg/mL of lactoferrin (the highest values evaluated). S. mutans and L. casei were only inhibited by lysozyme, not affected by lactoferrin and by the synergic use of both proteins.

Antimicrobial Activity and Synergism of Lactoferrin and Lysozyme Against Cariogenic Microorganisms

The present study evaluated the antimicrobial in vitro effects of the salivary proteins lactoferrin and lysozyme on microorganisms involved in the carious process, obtaining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Streptococcus mutans (ATCC 25175) and Lactobacillus casei (ATCC 7469) were submitted to broth macrodilution of lysozyme at 80 mg/mL and lactoferrin at 200 mg/mL. The tubes were read in a spectrophotometer after they had been incubated at 37 °C for 18 h, in a carbon dioxide chamber, in order to read the MIC. A new subculture was carried on agar plates to obtain the MBC. The agar diffusion method was also tested, using BHI agar with 100 µL of the standardized microbial inocula. Filter-paper disks soaked in 10 µL of the solutions lactoferrin (200 µg/mL) and lysozyme (80 µg/mL) were placed on the agar surface. Inhibition halos were not observed on the plates, showing the absence of the antimicrobial effects of these proteins in this method. The bactericidal and bacteriostatic effects of lysozyme on L. casei were 50.3 mg/mL and 43.1 mg/mL respectively. The bactericidal and bacteriostatic effects on S. mutans were 68.5 mg/mL and 58.7 mg/mL. Lactoferrin did not induce any inhibitory effects on any microorganism, even in the concentration of 200 mg/mL. There was not a synergic antimicrobial effect of proteins, when they were tested together, even in the concentration of 42.8 mg/mL of lysozyme and 114 mg/mL of lactoferrin (the highest values evaluated). S. mutans and L. casei were only inhibited by lysozyme, not affected by lactoferrin and by the synergic use of both proteins.

O presente estudo avaliou, in vitro, o efeito antimicrobiano das proteínas salivares lactoferrina e lisozima sobre micro-organismos envolvidos no processo carioso, obtendo suas concentrações inibitórias mínimas (CIM) e bactericidas mínimas (CBM). Cepas de Streptococcus mutans (ATCC 25175) e Lactobacillus casei (ATCC 7469) foram submetidas a macrodiluição em caldo das soluções de lisozima a 80 mg/mL e lactoferrina a 200 mg/mL. A leitura dos tubos foi realizada em espectrofotômetro, após a incubação a 37 °C por 18 h em estufa de CO2, para verificação da CIM. Uma nova subcultura foi semeada em placas de ágar para a obtenção da CBM. O método de difusão em ágar foi também testado utilizando-se placas de Petri com ágar BHI com 100 µL do inóculo microbiano padronizado. Discos de filtro de papel embebidos com 10 µL das soluções de lactoferrina (200 µg/mL) e lisozima (80 µg/mL) foram colocados sobre a superfície do ágar. Não foi observado halo de inibição nas placas, demonstrando ausência de efeito antimicrobiano das proteínas neste teste. Os efeitos bactericida e bacteriostático da lisozima sobre L. casei foram 50,3 mg/mL e 43,1 mg/mL respectivamente. Os efeitos bactericida e bacteriostático sobre S. mutans foram 68,5 mg/mL e 58,7 mg/mL. A lactoferrina não induziu nenhum efeito inibitório sobre nenhuma bactéria, mesmo na concentração de 200 mg/mL. Não houve efeito antimicrobiano sinérgico das proteínas, quando testadas conjuntamente, e mesmo até em concentrações de 42,8 mg/mL de lisozima e 114 mg/mL de lactoferrina (os maiores valores avaliados). S. mutans e L. casei foram inibidos somente pela lisozima, não sendo afetados pela lactoferrina e pelo uso sinérgico de ambas proteínas.

An evaluation of the expression profiles of salivary proteins lactoferrin and lysozyme and their association with caries experience and activity / Avaliação do padrão de expressão das proteínas salivares lactoferrina e lisozima e sua associação com experiência e atividade de cárie

Purpose: Saliva contains both specific and non-specific protective factors of the immune system, such as antimicrobial proteins, which can inhibit the adhesion and viability of cariogenic microorganisms. The association between caries experience/activity and the electrophoretic profiles of salivary proteins lactoferrin and lysozyme was evaluated. Methods: Eighty 12-year-old students from public schools in Londrina, PR, Brazil, were selected and divided into two groups: Group A - with decayed teeth and Group B - with caries-free teeth. The parent/guardian of each child signed a consent form and filled out a questionnaire regarding the oral and systemic health of his/her child. A clinical examination to diagnose the presence or absence of dental caries, by means of the DMFT index, was conducted. A total of 1 mL of saliva was collected for protein analysis using a polyacrylamide gel electrophoresis (SDS-PAGE). Results: A total of 58.8% of the children were caries-inactive; in contrast, 63.3% showed caries experience. There was a slight association between lysozyme concentrations and DMFT. Lactoferrin was positively correlated with both DMFT and restored teeth. Conclusion: The quantification of lactoferrin and lysozyme enabled an assessment of possible associations with caries status, thus improving the understanding of the biological and etiological aspects of caries.

Objetivo: A saliva contém fatores de defesa adquiridos e não adquiridos, como proteínas antimicrobianas capazes de inibir a aderência e a viabilidade dos microrganismos cariogênicos. Avaliou-se a associação entre a experiência/atividade de cárie e o perfil eletroforético das proteínas salivares lactoferrina e lisozima.Metodologia: Oitenta escolares aos 12 anos de idade da Rede Estadual de Ensino de Londrina, PR, Brasil, foram divididos em dois grupos: Grupo A – com cárie e Grupo B – sem cárie. Os responsáveis legais assinaram um termo de consentimento informado, e responderam um questionário sobre a saúde bucal e sistêmica das crianças. Foi realizado exame clínico, para diagnosticar a presença ou ausência de cárie através do Índice CPO-D e coletado 1 mLde saliva para análise das proteínas por meio da eletroforese em gel de poliacrilamida (SDS-PAGE).Resultados: Observou-se que 58,8% das crianças eram cárie-inativas, apesar de 63,3% possuírem experiência de cárie. Houve uma tendência de associação entre a concentração de lisozima com o CPO-D. A proteína lactoferrina correlacionou-se positivamente com CPO-D e dentes restaurados.Conclusão: A quantificação destas proteínas permitiu observar possível associação com a cárie, favorecendo uma melhor compreensão do aspecto biológico e etiológico da doença.

Conformational selection, dynamic restriction and the hydrophobic effect coupled to stabilization of the BIR3 domain of the human X-linked inhibitor of apoptosis protein by the tetrapeptide AVPI.

The XIAP-BIR3 domain blocks a substantial portion of the apoptosis pathway and is an attractive target for novel anticancer agents. The tetrapeptide AVPI, from the protein Smac/DIABLO, binds to the XIAP-BIR3 domain, allowing the cancer cells to die. Here we characterize the binding parameters of AVPI to XIAP-BIR3 and analyze its effects on the thermodynamic stability of this domain. XIAP-BIR3 was exceptionally stable against physical and chemical treatments and became even more stable by interaction with AVPI. Nuclear magnetic resonance experiments demonstrated that conformational selection is taking place upon AVPI interaction with XIAP-BIR3. Molecular dynamics simulations corroborate that the flexibility of XIAP-BIR3 is significantly reduced. The positive binding entropy associated with a loss of conformational entropy involved in the binding indicates that hydrophobic interactions play an important role in the interaction and domain stabilization. The mechanism of XIAP-BIR3 stabilization and its implications for drug affinity optimization are discussed.