Genomic characterization of Moraxella bovis and Moraxella bovoculi Uruguayan strains isolated from calves with infectious bovine keratoconjunctivitis.

Infectious bovine keratoconjunctivitis (IBK) is an ocular disease that affects bovines and has significant economic and health effects worldwide. Gram negative bacteria Moraxella bovis and Moraxella bovoculi are its main etiological agents. Antimicrobial therapy against IBK is often difficult in beef and dairy herds and, although vaccines are commercially available, their efficacy is variable and dependent on local strains. The aim of this study was to analyze for the first time the genomes of Uruguayan clinical isolates of M. bovis and M. bovoculi. The genomes were de novo assembled and annotated; the genetic basis of fimbrial synthesis was analyzed and virulence factors were identified. A 94% coverage in the reference genomes of both species, and more than 80% similarity to the reference genomes were observed. The mechanism of fimbrial phase variation in M. bovis was detected, and the tfpQ orientation of these genes confirmed, in an inversion region of approximately 2.18kb. No phase variation was determined in the fimbrial gene of M. bovoculi. When virulence factors were compared between strains, it was observed that fimbrial genes have 36.2% sequence similarity. In contrast, the TonB-dependent lactoferrin/transferrin receptor exhibited the highest percentage of amino acid similarity (97.7%) between strains, followed by cytotoxins MbxA/MbvA and the ferric uptake regulator. The role of these virulence factors in the pathogenesis of IBK and their potential as vaccine components should be explored.

Avaliação da lactoferrina e da calprotectina como marcadores de inflamação intestinal na pancreatite crônica / Evaluating lactoferrin and calprotectin as markers of intestinal inflammation inchronic pancreatitis

ABSTRACT Background: The treatment of chronic pancreatitis does not consistently solve intestinal abnormalities, and despite the implementation of various therapeutic measures, patients often continue to experience persistent diarrhea. Therefore, it is imperative to recognize that diarrhea may stem from factors beyond pancreatic insufficiency, and intestinal inflammation emerges as a potential contributing factor. Objective: The aim of this study was to assess fecal lactoferrin and calprotectin levels as indicators of intestinal inflammation in patients with chronic pancreatitis experiencing persistent diarrhea. Methods: In this study, 23 male patients with chronic pancreatitis primarily attributed to alcohol consumption and presenting with diarrhea (classified as Bristol stool scale type 6 or 7), underwent a comprehensive evaluation of their clinical and nutritional status. Fecal lactoferrin and calprotectin levels were mea­sured utilizing immunoassay techniques. Results: The average age of the participants was 54.8 years, 43.5% had diabetes, and 73.9% were smokers. Despite receiving enzyme replacement therapy and refraining from alcohol for over 4 years, all participants exhibited persistent diarrhea, accompanied by elevated calprotectin and lactoferrin levels indicative of ongoing intestinal inflammation. Conclusion: The findings of this study underscore that intestinal inflammation, as evidenced by elevated fecal biomarkers calprotectin and lactoferrin, may contribute to explaining the persistence of diarrhea in patients with chronic pancreatitis.

RESUMO Contexto: O tratamento da pancreatite crônica não resolve de forma consistente as anomalias intestinais e, apesar da implementação de várias medidas terapêuticas, os pacientes muitas vezes continuam a apresentar diarreia persistente. Portanto, é imperativo reconhecer que a diarreia pode resultar de fatores além da insuficiência pancreática, e a inflamação intestinal surge como um potencial fator contribuinte. Objetivo: O objetivo deste estudo foi avaliar os níveis fecais de lactoferrina e calprotectina como indicadores de inflamação intestinal em pacientes com pancreatite crônica com diarreia persistente. Métodos: Neste estudo, 23 pacientes do sexo masculino com pancreatite crônica atribuída principalmente ao consumo de álcool e apresentando diarreia (classificada na escala de fezes de Bristol tipo 6 ou 7), foram submetidos a uma avaliação abrangente de seu estado clínico e nutricional. Os níveis fecais de lactoferrina e calprotectina foram medidos utilizando técnicas de imunoensaio. Resultados: A idade média dos participantes foi de 54,8 anos, 43,5% tinham diabetes e 73,9% eram fumantes. Apesar de receber terapia de reposição enzimática e abster-se de álcool por mais de 4 anos, todos os participantes apresentaram diarreia persistente, acompanhada por níveis elevados de calprotectina e lactoferrina, indicativos de inflamação intestinal contínua. Conclusão: Os achados deste estudo ressaltam que a inflamação intestinal, evidenciada pelos biomarcadores fecais elevados calprotectina e lactoferrina, pode contribuir para explicar a persistência da diarreia em pacientes com pancreatite crônica.

In vitro prebiotic activity of rhLf and galactooligosaccharideson infant intestinal microbiota / Actividad prebiótica in vitro de lactoferrina y galactooligosácridossobre la microbiota intestinal infantil

Objective: human lactoferrin (Lf) and human milk oligosaccharides possess a wide range of functions. So, the present study focusses on the role of Lf and/or galactooligosaccharides (GOS) in the modulation of gut microbiota composition.Methods: recombinant human lactoferrin (rhLf) was added to the first infant formula (0.10, 0.15, 0.20 %) alone or in combination with GOS (1 %) in vessels of a small-scale batch culture fermentation model. Short-chain fatty acids (SCFAs), microbial population groups, and pH were monitored through fermentation for 24 hours. Results: insignificant changes were observed in pH values and acetic acid accumulated during fermentation. Propionic acid content has been insignificantly increased while butyric acid has been insignificantly decreased. Moreover, increments in all bacterial groups except for Bacteroides were observed through the fermentation process. Lactobacillus and Bifidobacterium showed an increase in relation to initial time over thefermentation process, demonstrating the prebiotic effect of lactoferrin and GOS. After 24 hours of fermentation, all tested ingredients showed significant similarities in Enterococcus for controls except for 0.20 % rhLf + 1 % GOS, which provoked a diminution of Enterococci growth. Conclusion: despite the importance of the batch culture fermentation technique in uncovering the prebiotic activity of food ingredients, it is not useful for detecting the prebiotic nature of Lf due to its nature as a protein. Thus, Lf maybe shows its prebiotic activity on the gut microbiota through other mechanisms.(AU)

Objetivo: la lactoferrina humana (Lf) y los oligosacáridos de leche materna presentan un amplio rango de funciones. El presente estudio se centra en el papel de la Lf y/o galactooligosácridos (GOS) en la modulación de la composición de la microbiota intestinal. Métodos: se añadió lactoferrina humana recombinante (rhLf) a fórmula infantil (0,10, 0,15, 0,20 %), sola o en combinación con GOS (1 %) en botes de fermentación colónica. A lo largo de 24 horas de fermentación, se monitorizaron ácidos grasos de cadena corta, grupos de poblaciones microbianas y pH. Resultados: se observaron pequeños cambios en valores de pH y cantidad de ácido acético durante la fermentación. El contenido de ácido propiónico aumentó ligeramente, mientras que el butírico sufrió un ligero descenso. Todos los grupos bacterianos estudiados incrementaron, excepto los Bacteroides, durante la fermentación. Lactobacillus and Bifidobacterium mostraron un incremento respecto al valor inicial, demostrando el efecto prebiótico de la lactoferrina y los GOS. A las 24 horas de fermentación, todos los ingredientes estudiados mostraron similitud al control en cuanto a Enterococcus, excepto para 0,20 % rhLf + 1 % GOS, donde disminuyó el crecimiento de los enterococos. Conclusión: a pesar de la importancia de los estudios de fermentación in vitro para descubrir potenciales ingredientes prebióticos, no fue útil en el caso de lactoferrina debido a su naturaleza proteica. Por tanto, la lactoferrina podría mostrar su actividad prebiótica en la microbiota intestinal a través de otros mecanismos.(AU)

In vitro prebiotic activity of rhLf and galactooligosaccharides on infant intestinal microbiota.

Introduction: Objective: human lactoferrin (Lf) and human milk oligosaccharides possess a wide range of functions. So, the present study focusses on the role of Lf and/or galactooligosaccharides (GOS) in the modulation of gut microbiota composition. Methods: recombinant human lactoferrin (rhLf) was added to the first infant formula (0.10, 0.15, 0.20 %) alone or in combination with GOS (1 %) in vessels of a small-scale batch culture fermentation model. Short-chain fatty acids (SCFAs), microbial population groups, and pH were monitored through fermentation for 24 hours. Results: insignificant changes were observed in pH values and acetic acid accumulated during fermentation. Propionic acid content has been insignificantly increased while butyric acid has been insignificantly decreased. Moreover, increments in all bacterial groups except for Bacteroides were observed through the fermentation process. Lactobacillus and Bifidobacterium showed an increase in relation to initial time over the fermentation process, demonstrating the prebiotic effect of lactoferrin and GOS. After 24 hours of fermentation, all tested ingredients showed significant similarities in Enterococcus for controls except for 0.20 % rhLf + 1 % GOS, which provoked a diminution of Enterococci growth. Conclusion: despite the importance of the batch culture fermentation technique in uncovering the prebiotic activity of food ingredients, it is not useful for detecting the prebiotic nature of Lf due to its nature as a protein. Thus, Lf maybe shows its prebiotic activity on the gut microbiota through other mechanisms.

Introducción: Objetivo: la lactoferrina humana (Lf) y los oligosacáridos de leche materna presentan un amplio rango de funciones. El presente estudio se centra en el papel de la Lf y/o galactooligosácridos (GOS) en la modulación de la composición de la microbiota intestinal. Métodos: se añadió lactoferrina humana recombinante (rhLf) a fórmula infantil (0,10, 0,15, 0,20 %), sola o en combinación con GOS (1 %) en botes de fermentación colónica. A lo largo de 24 horas de fermentación, se monitorizaron ácidos grasos de cadena corta, grupos de poblaciones microbianas y pH. Resultados: se observaron pequeños cambios en valores de pH y cantidad de ácido acético durante la fermentación. El contenido de ácido propiónico aumentó ligeramente, mientras que el butírico sufrió un ligero descenso. Todos los grupos bacterianos estudiados incrementaron, excepto los Bacteroides, durante la fermentación. Lactobacillus and Bifidobacterium mostraron un incremento respecto al valor inicial, demostrando el efecto prebiótico de la lactoferrina y los GOS. A las 24 horas de fermentación, todos los ingredientes estudiados mostraron similitud al control en cuanto a Enterococcus, excepto para 0,20 % rhLf + 1 % GOS, donde disminuyó el crecimiento de los enterococos. Conclusión: a pesar de la importancia de los estudios de fermentación in vitro para descubrir potenciales ingredientes prebióticos, no fue útil en el caso de lactoferrina debido a su naturaleza proteica. Por tanto, la lactoferrina podría mostrar su actividad prebiótica en la microbiota intestinal a través de otros mecanismos.

[Variation in the level of the biocomponents immunoglobulin A and lactoferrin in human milk after Holder pasteurization]. / Variación en el aporte de los biocomponentes inmunoglobulina A y lactoferrina en la leche humana después de la pasteurización Holder.

Introduction: Background: breast milk is the ideal food for newborns and infants, but there are factors that can prevent the practice of breastfeeding. Human milk banks (BLH) are a strategy to increase breastfeeding coverage; the donated milk is subjected to Holder pasteurization to guarantee its innocuousness, undergoing large changes in temperature and a decrease in the concentrations of biocomponents such as Immunoglobulin A (IgA) and lactoferrin (LF). This article describes the results of recent studies on the impact of Holder pasteurization on IgA and LF in human milk. Material and methods: a search for research articles related to the topic of interest was carried out in various databases and in accordance with inclusion criteria that considered the type of study, date of publication and quality of the journal. Results: the impact of Holder pasteurization on IgA and LF concentrations is not clear, given that the literature reports a variety of protocols and different results; however, the percentage reductions of both biocomponents are significant and consistent in the studies reviewed, suggesting the importance of establishing a standard protocol for their quantification. Conclusions: Holder pasteurization guarantees the microbiological quality of the milk distributed in HMB, but affects the amount of beneficial biocomponents for the final recipient. Government entities that regulate HMBs should evaluate the possibility of using other techniques that reduce the impact on biocomponents while preserving the microbiological quality of the product.

Introducción: Antecedentes: la leche materna es el alimento ideal para neonatos e infantes, pero existen factores que puede impedir la práctica de la lactancia materna. Los bancos de leche humana (BLH) son una estrategia para aumentar la cobertura de la lactancia; la leche donada es sometida a pasteurización Holder para garantizar su inocuidad, experimentando grandes cambios de temperatura y disminución de las concentraciones de biocomponentes como la Inmunoglobulina A (IgA) y la lactoferrina (LF). Este artículo describe los resultados de estudios recientes acerca del impacto de la pasteurización Holder sobre la IgA y la LF en la leche materna. Material y métodos: se llevó a cabo una búsqueda de artículos de investigación relacionados al tema de interés en diversas bases de datos y de conformidad con criterios de inclusión que tuvieron en cuenta el tipo de estudio, la fecha de publicación y la calidad de la revista. Resultados: no está claro el impacto de la pasteurización Holder sobre las concentraciones de IgA y LF, dado que la literatura reporta variedad de protocolos y diversos resultados; no obstante, las reducciones porcentuales de ambos biocomponentes son significativas y consistentes en los estudios revisados, sugiriendo la importancia de establecer un protocolo estándar para su cuantificación. Conclusiones: la pasteurización Holder garantiza la calidad microbiológica de la leche distribuida en los BLH pero afecta la cantidad de biocomponentes beneficiosos para el receptor final. Los entes gubernamentales que rigen los BLH deberían evaluar la posibilidad de utilizar otras técnicas que disminuyan el impacto sobre los biocomponentes y conserven la calidad microbiológica del producto.

Respuesta de las citocinas a la combinación de simbióticos y lactoferrina en recién nacidos de muy bajo peso al nacer: ensayo controlado aleatorizado / Cytokine responses to symbiotic and lactoferrin combination in very low birth weight neonates: a randomized control trial

Introducción. Los probióticos y prebióticos presentan beneficios potenciales en la inflamacióncrónica de las mucosas, incluida la prevención de la enterocolitis necrosante. No obstante, los mecanismos y resultados de estos efectos inmunomoduladores son confusos. El objetivo fue investigar la respuesta de las citocinas a Lactobacillus y Bifidobacterium asociados con fructo- y galactooligosacáridos (simbióticos) y lactoferrina en recién nacidos de muy bajo peso al nacer.Población y métodos. Se asignó aleatoriamente a lactantes con ≤32 semanas de gestación y ≤1500 g de peso para recibir simbióticos o 1 ml de agua destilada como placebo desde la primera alimentación hasta el alta. Se obtuvieron muestras de sangre los días posnatales 0 ± 2, 14 ± 2 y 28 ± 2, y se midieron interferón-γ, interleucina (IL)-5, IL-10 e IL-17A.Resultados. En el grupo del estudio (n = 25), la concentración de IL-10 disminuyó a lo largo del estudio (p = 0,011), pero no cambió en el grupo de referencia. La concentración de IL-5 se mantuvo constante los primeros 14 días y luego disminuyó significativamente (p= 0,042) en el grupo del estudio, mientras que aumentó en los primeros 14 días (p = 0,019) y luego disminuyó en 28 días (p = 0,011) en el grupo de referencia (n = 25).La concentración de otras citocinas no cambió a lo largo del estudio.Conclusión. El uso combinado de probióticos con oligosacáridos y lactoferrina estuvo asociado con una disminución en la concentración de IL-10, pero no se observó un cambio en las otras citocinas.

Introduction. Probiotics and prebiotics, which are multifunctional agents, have potential benefits in chronic mucosal inflammation, including the prevention of necrotizing enterocolitis. However, the mechanisms and the results of these immunomodulatory effects are not clear. This study aimed to investigate the cytokine response to the combination of Lactobacillus and Bifidobacterium together with fructo- and galacto-oligosaccharides (symbiotic) and lactoferrin in very low birth weight neonates.Population and Methods. Infants ≤ 32 GWs and ≤ 1,500 g were randomly assigned to receive a symbiotic combination or 1 ml distilled water as placebo starting with the first feed until discharge. Blood samples were obtained at postnatal 0 ± 2, 14 ± 2, and 28 ± 2 days, and the serum levels of interferon-γ, interleukin (IL)-5, IL-10, and IL-17A were measured.Results. In the study group (n = 25), the IL-10 levels decreased throughout the study period (p = 0.011) but did not change in the control group. The IL-5 levels remained steady in the first 14 days and decreased significantly thereafter (p = 0.042) in the study group, whereas they increased in the first 14 days (p = 0.019), and then decreased in 28 days (p = 0.011) in the control group (n = 25). The levels of the other cytokines did not change throughout the study period.Conclusion.The combined use of probiotics with oligosaccharides and lactoferrin was associated with a decrease in IL-10 levels, but no change was observed in the other cytokines.

Detección de lactoferrina en secreciones del tracto reproductivo inferior femenino / Detection of lactoferrin in female lower reproductive tract secretions

INTRODUCCIÓN La lactoferrina (LF) está presente en el tracto reproductivo y afectaría la función y la interacción entre gametas. Su expresión sería hormono-dependiente y aumentaría en procesos inflamatorios (una de las principales causas de falla del embarazo e infertilidad). Los tratamientos reproductivos de alta complejidad aún tienen una alta tasa de fracasos, sin motivos aparentes. OBJETIVO Determinar la concentración de LF en el flujo cervical de pacientes de reproducción asistida de alta complejidad y correlacionarla con el resultado de dicho tratamiento. MÉTODOS Se obtuvieron muestras de flujo cervical de pacientes previo a la transferencia embrionaria y de ovodonantes (OV, n=7). Las pacientes se dividieron en las que lograron el embarazo (E, n=16) y las que no lo hicieron (NE, n=21). Se determinó la concentración de proteínas totales y de LF (mediante un ELISA) en el flujo. Los resultados del tratamiento reproductivo se obtuvieron de las historias clínicas. La comparación entre grupos se realizó mediante el test t de Student. RESULTADOS El número de ovocitos recuperados en las E y NE fue similar (7,40 ± 1,17 y 6,33 ± 1,23, respectivamente) pero fueron menores que en las OV (15,14 ± 1,72, p<0,001). Las proteínas totales en flujo fueron más elevadas en las E (1270 ± 286,1µg/ml) con respecto a NE (658,6 ± 117,3 µg/ml, p<0,05). La concentración de LF fue más elevada en NE que en E (1,58 ± 0,56 ng/µg prot. vs. 1,08 ± 0,32 ng/µg prot., respectivamente), pero esta diferencia no fue significativa. DISCUSIÓN El mayor nivel de proteínas en el flujo de las E con respecto a las NE podría sugerir diferencias en la capacidad de secreción del epitelio cervical. La concentración de LF en el flujo fue levemente más baja en E con respecto a NE, pero la diferencia no fue significativa. El tamaño muestral pequeño podría incidir en el resultado encontrado, por lo que se continúa recolectando nuevas muestras de flujo cervical para alcanzar un mayor número de pacientes

Lactoferrina bovina disminuye la invasión de Salmonella enterica en células HEp-2 / Bovine lactoferrin decreases the invasion of Salmonella enterica to HEp-2 cells

Objetivo: Evaluar el efecto de la lactoferrina bovina (Lfb) en el proceso de invasión de Salmonella enterica ser. Typhimurium en células HEp-2. Materiales y métodos: Se infectaron células HEp-2 con 10(6) unidades formadoras de colonia (UFC) de la bacteria en ausencia y presencia de 1 y 10 mg/mL de Lfb (saturada con hierro) durante 1,5 horas a 37°C. En este estudio evaluamos 2 tratamientos: pre-infección (las células HEp-2 se incubaron con Lf 1 hora, previo a la infección con Salmonella) y post-infección (la Lfb se adicionó 15 minutos después de la infección). La capacidad de invasión de Salmonella se determinó mediante la cuantificación de las UFC recuperadas desde el interior de las células HEp-2 (después del tratamiento con 100 μg/mL y 10 μg/mL de gentamicina y Triton X-100). Resultados: En el tratamiento pre-infección se observó una disminución de 23% en la invasión de Salmonella cuando las células HEp-2 fueron pre-incubadas con 1 mg/mL de Lfb (2,8x10(5) vs 2,1x10(5), p=0,04) y una disminución de 50% cuando fueron pre-incubadas con 10 mg/mL de Lfb (2,8x10(5) vs 1,4x10(5), p=0,04). Con el tratamiento post-infección no se observaron cambios en la capacidad de invasión de Salmonella. Conclusiones: Los resultados indican que Lfb reduce la capacidad de invadir de Salmonella enterica ser. Typhimurium a células HEp-2 en el tratamiento pre-infección

Objective: To assess the effect of bovine lactoferrin (bLf) on the invasion of Salmonella enterica ser. Typhimurium to HEp-2 cells. Materials and methods: HEp-2 monolayers were infected with 10(6) colony forming unit (CFU) of bacteria in the absence and presence of 1 and 10 mg/mL of bLf (iron-saturated) and incubated 1.5 hours at 37°C. Two treatments were evaluated: preinfection (HEp-2 cells were incubated with bLf one hour prior to infection with Salmonella) and post-infection (bLf was added 15 minutes after the infection). Invasiveness of Salmonella was determined throgh quantification of CFU recovered from inside the HEp-2 cells (after treatment with 100 μg/mL and 10 μg/mL of gentamicin and Triton X -100). Results: In the pre-infection treatment, we observed a decrease of 23% of Salmonella invasion when HEp-2 cells were pre incubated with 1 mg/mL of bLf (2.8x105 vs 2.1x105, p=0.04) and 50% when them were pre-incubated with 10 mg/mL of bLf (2.8x10(5) vs 1.4x10(5), p=0.04). In post-infection treatment, no changes were observed in the invasiveness of Salmonella. Conclusion: The results indicated that bLf reduces the invasiveness of Salmonella enterica ser. Typhimurium to HEp-2 cells in the pre-infection treatment

Ação antifúngica da lactoferrina frente Candida non-albicans isoladas da cavidade oral de crianças infectadas pelo HIV / Antifungal action of lactoferrin against Candida non-albicans isolated from the oral cavity of HIV-infected children

A lactoferrina é uma glicoproteína de ligação ao ferro, que está presente na saliva, no leite, e em outras secreções exócrinas. Essa proteína tem inúmeras funções biológicas, incluindo efeitos antifúngicos e antibacterianos, além de imunomoduladores. O objetivo do presente estudo foi avaliar, in vitro, pela primeira vez, a atividade antifúngica da lactoferrina contra cepas de Candida nãoalbicans isoladas da cavidade oral de crianças infectadas pelo HIV e crianças saudáveis. Além disso, mensurar a degradação da lactoferrina por essas cepas através de SDS-PAGE (análise eletroforética em gel de poliacrilamida) e determinar, in vitro, a concentração mínima inibitória de lactoferrina capaz de matar 50% das células de Candida não-albicans. Cepas de Candida spp. foram obtidas através da coleta de saliva de 70 crianças infectadas pelo HIV e 50 crianças sem evidências de imunossupressão, com idade de 3 a 13 anos (ALVES, 2014). As diferentes espécies de Candida foram identificadas através de assimilação e fermentação de açúcar (API 20C, Biomérieux, França). Para o ensaio, in vitro, de morte de celular na presença de lactoferrina, 24 isolados de Candida, entre elas parapsilosis, tropicalis, krusei, guillermondi e dubliniensis, foram selecionados. Para a realização da análise por SDS-PAGE, cepas de todas as espécies consideradas resistentes nos ensaios de morte celular, foram incluídas. O porcentual de morte celular (%) de Candida não- albicans com a adição de 100 µg/ml de lactoferrina variou de 3,1% (C. dubliniensis) a 88,1% (C. tropicalis) no grupo HIV, e de 14,1% a 30,37% no grupo não-HIV (C . parapsilosis). Não houve correlação entre a densidade celular e o percentual de morte celular. C. dubliniensis foi a espécie mais resistente a lactoferrina e o porcentual de morte celular de C. parapsilosis foi significativamente maior em comparação com C. krusei na concentração de 1X104 células/ml (p = 0,033). Todos os isolados foram capazes de degradar a lactoferrina na concentração 1x108 células/ml, especialmente C. parapsilosis, pelo grupo HIV. Além disso, a lactoferrina na concentração de 500 µg/ml foi capaz de matar mais de 50% das células apenas dos isolados de C. tropicalis e C. guilliermondii. Concluiu-se que a lactoferrina tem atividade antifúngica contra Candida não-albicans de crianças infectadas pelo HIV, mas algumas espécies apresentam alguma resistência a esta proteína. Além disso, todas as Candida spp. foram capazes de degradar a lactoferrina quando estavam em concentração de 1X108 cels/ml, e a lactoferrina na concentração de 500µl/ml foi capaz de matar mais que 50% das células de C. tropicalis e C. guillermondii

Lactoferrin is an iron-binding glycoprotein, which is present in saliva, milk and other exocrine secretions. This protein has a number of biological functions, including antifungical, antimicrobial and immunomodulatory effects. The aim of this study was to evaluate, for the first time, the antifungal activity of lactoferrin against isolates of Candida spp. from the oral cavity of HIV-infected children and children with no clinical evidence of immunosuppression. Furthermore, measure, in vitro, the degradation of lactoferrin by Candida spp. through SDS-PAGE (electrophoretic analysis on polyacrylamide gel) and determine, in vitro, the minimum inhibitory concentration of lactoferrin able to kill 50% of cells of Candida non-albicans. Strains of Candida spp. were obtained through saliva collect of 70 HIV-infected children and 50 children without evidence of immunosuppression ageing between 3 to13 years old (ALVES, 2014). All Candida species were identified by sugar assimilation and fermentation (API 20C, Biomerieux, France). For the in vitro study, death of lactoferrin of 24 Candida isolates, including parapsilosis, tropicalis, krusei, guillermondii, and dubliniensis, were selected. To perform the analysis by SDS-PAGE, strains of all available species considered resistants, which were observed at lactoferrin death`s study, were included. The cell death percentage (%) of non-albicans Candida by addition of 100 µg of lactoferrin range from 3.1% (C. dublinienes) to 88.1% (C. tropicalis) in HIV group, and 14.1% to 30.37% in N-HIV (C. parapsilosis), but there was no correlation between cell density and death %. C. dubliniensis was the most resistant specie to lactoferrin and the cell death % of C. parapsilosis was significant higher comparing to C. krusei at 1X104 cells/ml (p=0.033). All the isolates were able to degrade lactoferrin at 108 cells/ml, mainly C. parapsilosis from HIV. Furthermore, lactoferrin at 500 µg/ml concentration was able to kill more than 50% of the cells only for C. tropicalis and C. guillermondii isolates. It was concluded that lactoferrin has antifungal activity against non-albicans Candida from HIV children, but some species present some resistance to this protein. Furthermore, all Candida spp. were able to degrade lactoferrin when was at concentration of 1x108 cells / ml, also, lactoferrin at concentration 500µl / ml was able to kill more than 50% of C. tropicalis and C. guillermondii cells.

Efecto de lactoferrina bovina en la formación de biofilms en cepas clínicas de E. coli enteroagregativa / Effect of bovine lactoferrin biofilms in the formation of clinical strains of enteroaggregative E. Coli

Objetivos. Determinar el efecto de lactoferrina bovina (bLf) en la formación de biofilms en cepas clínicas de Escherichia coli enteroagregativa (EAEC), y si este efecto es independiente del hierro. Materiales y métodos. Se utilizaron dos métodos: (a) cualitativo, mediante observación directa por microscopia óptica, y (b) cuantitativo, lecturas de los valores de absorbancia mediante lector de ELISA en presencia de bLf en concentraciones de 0,01mg/mL y 1mg/mL, con y sin hierro, y no bLf (control), en 122 cepas de EAEC para el método cuantitativo (60 cepas de niños con diarrea y 62 de niños sanos) y 31 cepas para el método cualitativo. Resultados. (a) Método cualitativo: se evaluaron 31 cepas, con y sin hierro. Sin hierro la formación de biofilms fue de 77% (24/31) en el grupo control versus 58% (14/31) con bLf de 0,01 mg/mL y 4% (1/31) con 1 mg/mL. Con hierro la formación de biofilms fue 90% (28/31) en el grupo control versus 55% (17/31) con bLf de 0,01 mg/mL y 4% (1/31) a 1 mg/mL. (b) Método cuantitativo: sin hierro la absorbancia medida a OD 560 nm del grupo control fue 0,7 ± 0,5 versus 0,4 ± 0,3 con bLf 0,01mg/mL y 0,3 ± 0,2 con bLf de 1 mg/mL (p<0,0001). Esta disminución en presencia de bLf incluso se dio con hierro. Conclusiones. La bLf tiende a disminuir la formación de biofilms, mostrando un efecto inhibitorio en las cepas clínicas de EAEC, este efecto no es hierro-dependiente...

Objectives. To determine the effect of bovine lactoferrin (BLF) in the formation of biofilms in clinical enteroaggregative Escherichia coli (EAEC) strains and whether this effect is independent of iron. Materials and methods. Two methods were used: (a) qualitative, by direct observation of optical microscopy, and (b) quantitative readings of the absorbance values using ELISA reader in the presence of bLf in concentrations of 0.01 mg mL and 1 mg/mL, with and without iron and no bLf (control). Analysis occurred in 122 strains of EAEC (60 strains from children with diarrhea and 62 healthy children) previously collected in a previous study of passive surveillance of diarrhea in the Southern Cone Lima. 31 strains of the same method were used for the qualitative study. Results. (A) Qualitative method: 31 strains were evaluated with and without iron. Without iron biofilm formation was 77% (24/31) in the control group versus 58% (14/31) with bLf of 0.01 mg/mL and 4% (1/31) with 1 mg/ml. Iron biofilm formation was 90% (28/31) in the control group versus 55% (17/31) with bLf of 0.01 mg/mL and 4% (1/31) with 1 mg/mL. (B) Quantitative method: without iron absorbance measured at OD 560 nm of the control group was 0.7 ± 0.5 versus 0.4 ± 0.3 with bLf 0.01 mg/mL and 0.3 ± 0.2 with bLf of 1 mg/mL (p<0.0001). This decrease in the presence of bLf included iron. Conclusions. bLf tends to decrease the formation of biofilms, showing an inhibitory effect in clinical isolates of EAEC; this effect is not iron-dependent...

Efeitos do aditivo do leite materno com ferro sobre as propriedades bacteriostáticas do leite materno / Effects of human milk fortifier with iron on the bacteriostatic properties of breast milk

OBJETIVO: Comparar o crescimento bacteriano em colostro puro e colostro com aditivo do leite materno contendo ferro. MÉTODOS: Foram comparadas 78 amostras de colostro puro ou colostro com adição de aditivo do leite materno contendo ferro para avaliar o crescimento de Escherichia coli, Staphylococcus aureus e Pseudomonas aeruginosa. Para a análise qualitativa, discos de papel-filtro foram imersos em amostras de cada grupo e incubados por 48 horas com 10¹ Unidades Formadoras de Colônias/mL de cada cepa. Para a avaliação quantitativa, 1 mL de cada cepa contendo 10(7) Unidades Formadoras de Colônias/mL foi homogeneizado com 1 mL, tanto de colostro puro quanto de colostro com aditivo do leite materno, espalhado em placa de Petri e incubado a 37ºC. O número de Unidades Formadoras de Colônias foi contado 24 horas depois. RESULTADOS: A análise qualitativa não mostrou nenhuma diferença no crescimento bacteriano. Na avaliação quantitativa, o crescimento de Escherichia coli (EC) no grupo C foi de 29,4±9,7 x 10(6) CFU/mL, enquanto no grupo FM85 foi de 31,2±10,8 x 10(6) CFU/mL. A diferença entre o crescimento médio foi de 1,9±4,9 x 10(6) CFU/mL (p = 0,001). Não houve diferenças no crescimento de Staphylococcus aureus e Pseudomonas aeruginosa. CONCLUSÃO: A adição de ferro a essa concentração reduz a ação bacteriostática do leite materno contra Escherichia coli.

OBJECTIVE: To compare bacterial growth in pure colostrum versus colostrum with human milk fortifier (HMF) containing iron. METHODS: The growth of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa in 78 samples of pure colostrum or colostrum with added iron-containing HMF was compared. For qualitative analysis, filter paper discs were immersed in samples from each group and incubated for 48 hours with 10¹ colony forming units (CFUs)/mL of each strain. For quantitative assessment, 1 mL of each strain containing 10(7) CFUs/mL was homogenized with 1 mL of either colostrum or colostrum with human milk fortifier, seeded into a Petri dish, and incubated at 37ºC. Twenty-four hours later, the number of CFUs was counted. RESULTS: The qualitative analysis showed no difference in bacterial growth. In the quantitative evaluation, E. coli growth in the control group was 29.4±9.7 x 10(6) CFU/ mL, while in the HMF group it was 31.2±10.8 x 10(6) CFU/mL. The difference between the average growth was 1.9±4.9 x 10(6) CFU/mL (p = 0.001). There were no differences in S. aureus and P. aeruginosa growth. CONCLUSION: Addition of iron at this concentration reduces breast milk bacteriostatic action against E. coli.

Effects of human milk fortifier with iron on the bacteriostatic properties of breast milk.

OBJECTIVE: To compare bacterial growth in pure colostrum versus colostrum with human milk fortifier (HMF) containing iron. METHODS: The growth of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa in 78 samples of pure colostrum or colostrum with added iron-containing HMF was compared. For qualitative analysis, filter paper discs were immersed in samples from each group and incubated for 48 hours with 10(1) colony forming units (CFUs)/mL of each strain. For quantitative assessment, 1 mL of each strain containing 10(7) CFUs/mL was homogenized with 1 mL of either colostrum or colostrum with human milk fortifier, seeded into a Petri dish, and incubated at 37°C. Twenty-four hours later, the number of CFUs was counted. RESULTS: The qualitative analysis showed no difference in bacterial growth. In the quantitative evaluation, E. coli growth in the control group was 29.4±9.7×10(6)CFU/mL, while in the HMF group it was 31.2±10.8×10(6)CFU/mL. The difference between the average growth was 1.9±4.9×10(6)CFU/mL (p=0.001). There were no differences in S. aureus and P. aeruginosa growth. CONCLUSION: Addition of iron at this concentration reduces breast milk bacteriostatic action against E. coli.

Efeito protetor da lactoferrina humana no trato gastrintestinal / Protective effect of human lactoferrin in the gastrointestinal tract / Efecto protector de la lactoferrina humana en el sistema gastrointestinal

OBJETIVO: Descrever os mecanismos de ação da lactoferrina humana na proteção de morbidades gastrintestinais. FONTES DE DADOS: Revisão não sistemática da literatura utilizando como estratégia de busca pesquisa bibliográfica em bases de dados, as quais incluíram SciELO, Lilacs e MedLine entre 1990 e 2011. Os descritores utilizados foram: lactoferrina, leite materno/humano, gastrintestinal e imunidade, nos idiomas português e inglês. SÍNTESE DOS DADOS: A lactoferrina é a segunda proteína predominante no leite humano, com concentrações mais elevadas no colostro (5,0 a 6,7mg/mL) em relação ao leite maduro (0,2 a 2,6mg/mL). Em contraste, o leite de vaca contém teores inferiores, 0,83mg/mL no colostro e 0,09mg/mL no leite maduro. A lactoferrina desempenha diversas funções fisiológicas na proteção do trato gastrintestinal. A atividade antimicrobiana está relacionada à capacidade de sequestrar ferro dos fluidos biológicos e/ou de desestruturar a membrana de micro-organismos. A lactoferrina possui também a capacidade de estimular a proliferação celular. A ação anti-inflamatória desempenhada pela lactoferrina está associada à capacidade de penetrar no núcleo do leucócito e bloquear a transcrição do fator nuclear Kappa B. Diante da importância da lactoferrina na prevenção de doenças infecciosas em crianças aleitadas ao peito, a indústria vem, por meio da engenharia genética, desenvolvendo tecnologias para expressar esta proteína recombinante humana em plantas e animais, na tentativa de adequar a composição das fórmulas infantis àquela do leite humano. CONCLUSÕES: A lactoferrina humana é um peptídeo com potencial para prevenir morbidades, especialmente às gastrintestinais. Evidências científicas dos efeitos protetores da lactoferrina humana fortalecem ainda mais a recomendação para prática do aleitamento materno.

OBJECTIVE: To describe mechanisms of action of human lactoferrin to protect gastrointestinal morbidities. DATA SOURCES: Nonsystematic literature review using the following databases: SciELO, Lilacs and Medline from 1990 to 2011. The key-words used were lactoferrin, human milk/breastfeeding, gastrointestinal, and immunity, in Portuguese and English. DATA SYNTHESIS: Lactoferrin is the second predominant protein in the human milk, with higher concentrations in the colostrum (5.0 to 6.7mg/mL) if compared to mature milk (0.2 to 2.6mg/mL.) In contrast, cow's milk has lower levels, with 0.83mg/mL in the colostrum and 0.09mg/mL in the mature milk. Lactoferrin has several physiological functions to protect the gastrointestinal tract. The antimicrobial activity is related to the ability to sequester iron from biological fluids and/or to destruct the membrane of microorganisms. Lactoferrin also has the ability to stimulate cell proliferation. The anti-inflammatory action exercised by lactoferrin is associated with its ability to penetrate the core of the leukocyte and to block the Kappa B nuclear factor transcription. Given the importance of lactoferrin to prevent infectious diseases for breastfed children, the industry is using genetic engineering techniques to develop the expression of recombinant human lactoferrin in animals and plants, attempting to adjust the composition of infant formulas to that of human milk. CONCLUSIONS: Human lactoferrin is a peptide with great potential for preventing morbidity, especially in the gastrointestinal tract. Scientific evidence of the protective effects of human lactoferrin strengthens even more the recommendation for breastfeeding.

OBJETIVO: Describir los mecanismos de acción de la lactoferrina humana en la protección de morbilidades gastrointestinales. FUENTES DE DATOS: Revisión no sistemática de la literatura utilizando como estrategia de búsqueda investigación bibliográfica en bases de datos, que incluyeron SciELO, Lilacs y MedLine entre 1990 y 2011. Los descriptores utilizados fueron: lactoferrina, leche materna/humana, gastrointestinal e inmunidad, en los idiomas portugués e inglés. SÍNTESIS DE LOS DATOS: La lactoferrina es la segunda proteína predominante en la leche humana, con concentraciones más elevadas en el calostro (5,0 a 6,7mg/mL) respecto a la leche madura (0,2 a 2,6mg/mL). En contraste, la leche de vaca contiene tenores inferiores, 0,83mg/mL en el calostro y 0,09mg/mL en la leche madura. La lactoferrina desempeña diversas funciones fisiológicas en la protección del sistema gastrointestinal. La actividad antimicrobiana está relacionada a la capacidad de secuestrar hierro de los fluidos biológicos y/o de desestructurar la membrana de microorganismos. La lactoferrina posee además la capacidad de estimular la proliferación celular. La acción antiinflamatoria desempeñada por la lactoferrina está asociada a la capacidad de penetrar en el núcleo del leucocito y bloquear la transcripción del nuclear factor Kappa B. Frente a la importancia de la lactoferrina en la prevención de enfermedades infecciosas en niños amamantados al pecho, la industria viene, por medio de ingeniería genética, desarrollando tecnologías para expresar esta proteína recombinante humana en plantas y animales en el intento de adecuar la composición de las fórmulas infantiles a aquella de la leche humana. CONCLUSIONES: La lactoferrina humana es un péptido con potencial para prevenir morbilidades, especialmente las gastrointestinales. Evidencias científicas de los efectos protectores de la lactoferrina humana fortalecen todavía más la recomendación para la práctica de la lactancia materna.

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.