Salivary lactoferrin levels, disease severity and correlates in patients with chronic periodontitis presenting to a tertiary health facility in Nigeria

Objective: This study compared the concentration of salivary lactoferrin in patients with and without chronic periodontitis and investigated correlations with clinical variables of the disease. Methods: The study included 102 participants (51 cases and 51 controls) who presented at the Periodontology Clinic of University of Benin Teaching Hospital and met the selection criteria of '4mm and above' periodontal probing depths (PPD) and positive bleeding on probing (BOP) using community periodontal index (CPI) probe. Healthy participants (controls) were patients that had PPD less than or equal to 3mm, absence of BOP and simplified oral hygiene index (OHI-S) not more than 1.2. Baseline OHI-S and CPI scores were recorded. Saliva samples were collected and analyzed using enzyme-linked immunosorbent assay. All data were analyzed with the Statistical Package for Social Sciences (SPSS) version 22.0. Results: There was a statistically significant difference between the mean (SD) lactoferrin concentration of control participants 5.27(0.59) mg/l and case participants 6.74(0.61) mg/l (p<0.001). Participants with probing pocket depths (PPD) of 6mm or more had a significantly higher mean concentration [6.85(0.06) mg/l] than that of those with PPD 4-5mm [6.71(0.67) mg/l] (p< 0.001)Lactoferrin levels were highest in participants with 'poor' oral hygiene [6.85(0.60) mg/l] and lowest in those with 'good' oral hygiene [6.65(0.83) mg/l]. Conclusion: Salivary lactoferrin levels were higher among participants with chronic periodontitis than those without chronic periodontitis and correlates positively with the main clinical characteristics of the disease

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.

Clinical Significance of Inflammatory Biomarkers in Acute Pediatric Diarrhea / 대한소아소화기영양학회지

PURPOSE: The aim of this study was to evaluate the clinical significance of inflammatory biomarkers in acute infectious diarrhea among children. METHODS: Clinical parameters including fever, bacterial and viral etiology based on stool culture and multiplex polymerase chain reaction, and nine biomarkers including C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and leukocytes in blood and calprotectin, lactoferrin, myeloperoxidase, polymorphonuclear elastase, leukocytes, and occult blood in feces were evaluated in children who were hospitalized due to acute diarrhea without underlying disease. RESULTS: A total of 62 patients were included. Among these patients, 33 had fever, 18 showed bacterial infections, and 40 patients were infected with 43 viruses. Of all the biomarkers, CRP was significantly correlated with fever (p<0.001). CRP, ESR, calprotectin, lactoferrin, myeloperoxidase, fecal leukocytes, and occult blood were significantly associated with infection with bacterial pathogens (p<0.001, p=0.04, p=0.03, p=0.003, p=0.02, p=0.03, p=0.002, respectively). The combination of CRP and fecal lactoferrin at their best cut-off values (13.7 mg/L and 22.8 µg/mL, respectively) yielded a sensitivity of 72.2%, and a specificity of 95.5% for bacterial etiology compared with their individual use. CONCLUSION: Blood CRP is a useful diagnostic marker for both fever and bacterial etiology in acute pediatric diarrhea. The combination of CRP and fecal lactoferrin yields better diagnostic capability for bacterial etiology than their use alone for acute diarrhea in children without underlying gastrointestinal disease.

Lactoferrin, chitosan and Melaleuca alternifolia-natural products that show promise in candidiasis treatment

Abstract The evolution of microorganisms resistant to many medicines has become a major challenge for the scientific community around the world. Motivated by the gravity of such a situation, the World Health Organization released a report in 2014 with the aim of providing updated information on this critical scenario. Among the most worrying microorganisms, species from the genus Candida have exhibited a high rate of resistance to antifungal drugs. Therefore, the objective of this review is to show that the use of natural products (extracts or isolated biomolecules), along with conventional antifungal therapy, can be a very promising strategy to overcome microbial multiresistance. Some promising alternatives are essential oils of Melaleuca alternifolia (mainly composed of terpinen-4-ol, a type of monoterpene), lactoferrin (a peptide isolated from milk) and chitosan (a copolymer from chitin). Such products have great potential to increase antifungal therapy efficacy, mitigate side effects and provide a wide range of action in antifungal therapy.

Development and properties of hypoallergenic infant formula

Milk proteins are composed of casein, further classified into αS1-casein, αS2-casein, β-casein, and κ-casein, and whey protein, which is separated into α-lacatalbumin, β-lactoglobulin, serum albumin, and some minor proteins, such as lactoferrin and immunoglobulin. To reduce the allergenicity of protein, heat treatment and enzymatic protein hydrolysis by endopeptidase are necessarily required. Additionally, membrane technology should be applied to produce a protein hydrolyzate, which has consistent molecular weight of peptide and low in free amino acid without allergenic peptide or protein. Extensive casein hydrolyzate and whey protein hydrolyzate are used for protein source of mainly extensively hydrolyzed protein formula (eHF) intended for the treatment of cow's milk allergy. Also, partially hydrolyzed formula (pHF) is developed, which is using a single protein source e.g., whey protein hydrolyzate. The allergenicity of infant formula can be determined according to molecular weight profile and antigenicity reduction compared to intact protein. More than 90% peptides are present in eHF have a molecular weight of 10,000 Da. Generally, antigenicity reduction in eHF and pHF is 10-6 and 10-3, respectively. Even if protein hydrolyzate is manufactured under strict quality control, there is still a risk of cross contamination of allergenic milk components through environmental conditions and the shared manufacturing process. Thus, quality assessment of protein hydrolyzate formula must be performed routinely.

Fecal lactoferrin as a new marker of disease activity in inflammatory bowel diseases

Background: Inflammatory bowel diseases [IBD] including ulcerative colitis [UC] and Crohn's disease [CD] are organic inflammatory diseases, caused by chronic mucosal inflammation of the gasrtointetinal tract. As the presenting manifestations of IBD and other diseases are similar, obtaining a clinical diagnosis can be difficult, and further invasive diagnostic procedures may be required in order to obtain a confirmed diagnosis. The aim of this study is to evaluate the diagnostic utility of measuring fecal concentrations of lactoferrin as a simple and noninvasive indicator of disease activity in patients IBD and to be correlated with endoscopic findings and disease activity index and acute inflammatory response including leucocytic count, high sensitive CRP, ESR

Methods: This study was carried on 40 patients with IBD; 24 patients with active IBD [16 UC patients and 8 CD patients] and 16 patients with inactive IBD [10 UC patients and 6 CD patients] versus 40 healthy controls. All patients underwent blood and stool sampling as well as an interview to assess the disease severity utilizing UC activity measured by the Truelove and Witts Severity Index and Crohn's Disease Activity Index. Measurement of FLA levels at different stages of inflammatory bowel disease activity to detect its role in assessment of disease severity

Results: This study showed that FLA levels were highest in patients with IBD in comparison with healthy group. FLA levels also correlated significantly with disease severity in patients with IBD where higher levels of FLA were found in patients with severe UC or Crohn's disease. At cutoff value 9.68 ug/ml FLA showed 100% sensitivity and specificity in identification of patients with IBD from healthy subjects

Conclusions: FLA is a sensitive and specific biochemical marker of inflammation for use in the diagnosis of suspected IBD cases, and its level correlates well with both clinical disease activity indices

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

BLG gene knockout and hLF gene knock-in at BLG locus in goat by TALENs / 生物工程学报

To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.

unique medicinal properties of camel products: a review of the scientific evidence

Camel milk and urine have been used as medicines in certain parts of Asia and Africa since ancient times, but only recently have scientists shown interest in exploring the claimed therapeutic benefits of camel products. Significant evidence, drawn from laboratory and limited clinical studies, has shown that camel milk on its own and occasionally mixed with camel urine is effective in the management of diverse clinical conditions such as diabetes mellitus, cancer, food allergy, autism, viral hepatitis and a host of other viral, bacterial and parasitic infections. In addition, a number of potential benefits of camel milk and urine on the cardiovascular system, particularly their antiplatelet and fibrinolytic actions, have been demonstrated. The current review presents a concise summary of the scientific evidence to support these therapeutic actions

Lactoferrin Combined with Retinoic Acid Stimulates B1 Cells to Express IgA Isotype and Gut-homing Molecules

It is well established that TGF-beta1 and retinoic acid (RA) cause IgA isotype switching in mice. We recently found that lactoferrin (LF) also has an activity of IgA isotype switching in spleen B cells. The present study explored the effect of LF on the Ig production by mouse peritoneal B cells. LF, like TGF-beta1, substantially increased IgA production in peritoneal B1 cells but little in peritoneal B2 cells. In contrast, LF increased IgG2b production in peritoneal B2 cells much more strongly than in peritoneal B1 cells. LF in combination with RA further enhanced the IgA production and, interestingly, this enhancement was restricted to IgA isotype and B1 cells. Similarly, the combination of the two molecules also led to expression of gut homing molecules alpha4beta7 and CCR9 on peritoneal B1 cells, but not on peritoneal B2 cells. Thus, these results indicate that LF and RA can contribute to gut IgA response through stimulating IgA isotype switching and expression of gut-homing molecules in peritoneal B1 cells.

Clinical Significance of Fecal Lactoferrin and Multiplex Polymerase Chain Reaction in Patients with Acute Diarrhea

BACKGROUND/AIMS: The diagnostic yield of fecal leukocyte and stool cultures is unsatisfactory in patients with acute diarrhea. This study was performed to evaluate the clinical significance of the fecal lactoferrin test and fecal multiplex polymerase chain reaction (PCR) in patients with acute diarrhea. METHODS: Clinical parameters and laboratory findings, including fecal leukocytes, fecal lactoferrin, stool cultures and stool multiplex PCR for bacteria and viruses, were evaluated prospectively for patients who were hospitalized due to acute diarrhea. RESULTS: A total of 54 patients were included (male, 23; median age, 42.5 years). Fecal leukocytes and fecal lactoferrin were positive in 33 (61.1%) and 14 (25.4%) patients, respectively. Among the 31 patients who were available for fecal pathogen evaluation, fecal multiplex PCR detected bacterial pathogens in 21 patients, whereas conventional stool cultures were positive in only one patient (67.7% vs 3.2%, p=0.000). Positive fecal lactoferrin was associated with presence of moderate to severe dehydration and detection of bacterial pathogens by multiplex PCR (21.4% vs 2.5%, p=0.049; 100% vs 56.5%, p=0.032, respectively). CONCLUSIONS: Fecal lactoferrin is a useful marker for more severe dehydration and bacterial etiology in patients with acute diarrhea. Fecal multiplex PCR can detect more causative organisms than conventional stool cultures in patients with acute diarrhea.

In vitro targeting effect of lactoferrin modified PEGylated liposomes for hepatoma cells / 药学学报

A lactoferrin-containing PEGylated liposome system (Lf-PLS) was developed and tested in vitro as a hepatoma-targeting drug delivery system. PEGylated liposomes (PLS) were successfully prepared using the thin film hydration method with peglipid post insertion. Lf was covalently conjugated onto the carboxyl terminal of DSPE-PEG2000-COOH on liposomes. Coumarin-6 was used to trace Lf-PLS with fluorescence. The cellular uptake of this system was carried out in asialoglycoprotein receptor (ASGPR) positive HepG2 cells via confocal microscopy and flow cytometry. The Lf-PLS liposome was observed as spherical or oval vesicles with the particle size around 130 nm, zeta potential about -30 mV and encapsulation efficiency more than 80%. The confocal microscopy images and flow cytometry data demonstrated that Lf-PLS resulted in significantly higher cell association by ASGPR positive HepG2 cells compared to PLS. The association between Lf-PLS and cells were dependent on the concentration, time and temperature, which was inhibited by pre-incubation with excessive free Lf. The results suggest that Lf-PLS has a good targeting effect on HepG2 cells in vitro. The targeting mechanism may be related to the specific binding of Lf and ASGPR on HepG2 cells, which guides Lf-PLS to the cell surface to induce an active endocytosis process. All these results demonstrated that Lf-PLS might be a potential drug delivery system in targeting hepatocellular carcinoma, which deserves more research on its targeting ability, antitumor efficiency, and metabolism in vivo for treatment of hepatomacellular carcinoma.

Research progress of lactoferrin as drug carriers / 药学学报

Lactoferrin (Lf) is one of the food protein belonged to the innate immune system. Apart from its main biological function of binding and transport of iron ions, lactoferrin also has many other functions and properties such as antibacterial, antiviral, antiparasitic, catalytic, anti-cancer, anti-allergic and radioprotecting. Lf is usually used as additives of food and cosmetics. The research of lactoferrin has been increasingly reported, and the application of lactoferrin as a drug carrier has drawn extensive attention over the recent year. In this paper, researches of lactoferrin as drug carriers are classified and summarized in brain targeting, liver tumor targeting, lung tumor targeting and oral delivery systems according to their different characteristics.

Effects of decitabine against acute T lymphoblastic leukemia cell line Molt4 / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effects and possible mechanisms of decitabine on Molt4 in vitro.</p><p><b>METHODS</b>Effects of decitabine on cells proliferation were detected by using CCK-8, the apoptosis by Annexin V-FITC, cell cycles by propidium iodide-FACS. Discrepancy genes were screened by RNA-seq technique. The CpG methylation of lactoferrin (LTF) gene in Molt4 cells were identified by Bisulfite sequencing PCR (BSP). The expression of LTF mRNA in Molt4 by RT-PCR and LTF protein expression were analyzed by Western blot.</p><p><b>RESULTS</b>Decitabine effectively inhibited proliferation and induced apoptosis for Molt4 cells by an time- and dose-dependent manners. Cell cycles were arrested at the G₀/G₁ phase. The promoter methylation degree of LTF gene in Molt4 cells was 72.3% before decitabine treatment and decreased to 45.0% after treatment with 0.50 μmol/L decitabine for 72 h. After the reduction of methylation, expression of its mRNA and protein increased, meanwhile caspase 3 and caspase 9 protein expression levels increased.</p><p><b>CONCLUSION</b>The demethylating drug decitabine can induce apoptosis, detain cell cycle at phase G₀/G₁, inhibit proliferation and up-regulate LTF gene expression in Molt4 cells. LTF may become a new target for acute T lymphoblastic leukemia.</p>

The effect of two artificial salivas on the adhesion of Candida albicans to heat-polymerized acrylic resin

PURPOSE: Xerostomia can diminish the quality of life, leads to changes in normal chemical composition of saliva and oral microbiata, and increases the risk for opportunistic infections, such as Candida albicans. Various artificial salivas have been considered for patients with xerostomia. However, the knowledge on the antifungal and antiadhesive activity of artificial saliva substitutes is limited. The aim of the present study was to evaluate influence of two artificial salivas on the adhesion of Candida albicans to the polymethylmethacrylate disc specimens. MATERIALS AND METHODS: Two commercial artificial salivas (Saliva Orthana and Biotene Oral Balance Gel) were selected. 45 polymethylmethacrylate disc specimens were prepared and randomly allocated into 3 groups; Saliva Orthana, Biotene-Oral Balance gel and distilled water. Specimens were stored in the artificial saliva or in the sterile distilled water for 60 minutes at 37degrees C. Then they were exposed to yeast suspensions including Candida albicans. Yeast cells were counted using x40 magnification under a light microscope and data were analysed. RESULTS: Analysis of data indicated statistically significant difference in adhesion of Candida albicans among all experimental groups (P=.000). Findings indicated that Saliva Orthana had higher adhesion scores than the Biotene Oral Balance gel and distilled water (P<.05). CONCLUSION: In comparison of Saliva Orthana, the use of Biotene Oral Balance Gel including lysozyme, lactoferrin and peroxidase may be an appropriate treatment method to prevent of adhesion of Candida albicans and related infections in patients with xerostomia.

Differential toxicological endpoints of di(2-ethylhexyl) phthalate (DEHP) exposure in MCF-7 and MDA-MB-231 cell lines: Possible estrogen receptor α (ERα) independent modulations.

Wide spread use of Di-(2-ethylhexyl) phthalate (DEHP) has made it a ubiquitous contaminant in today’s environment, responsible for possible carcinogenic and endocrine disrupting effects. In the present investigation an integrative toxico-proteomic approach was made to study the estrogenic potential of DEHP. In vitro experiments carried out with DEHP (0.1-100 μM) induced proliferations (E-screen assay) in human estrogen receptors-α (ERα) positive MCF-7 and ERα negative MDA-MB-231 breast cancer cells irrespective of their ERα status. Further, DEHP suppressed tamoxifen (a potent anti-breast cancer drug) induced apoptosis in both cell types as shown by flowcytometric cell cycle analysis. Label-free quantitative proteomics analysis of the cell secretome of both the cell lines indicated a wide array of stress related, structural and receptor binding proteins that were affected due to DEHP exposure. The secretome of DEHP treated MCF-7 cells revealed the down regulation of lactotransferrin, an ERα responsive iron transport protein. The results indicated that toxicological effects of DEHP did not follow an ERα signaling pathway. However, the differential effects in MCF-7 and MDA-MB-231 cell lines indicate that ERα might have an indirect modulating effect on DEHP induced toxicity.

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...

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.

Lactoferrin Constitutively Enhances Differentiation of Osteoblastic MC3T3-E1 Cells in Vitro

During bone remodeling, there is requirement of differentiation of osteoblastic cells. Previously, we identified proteins differentially expressed in soft tissue during bone healing. Of these proteins, we focused the effect of LTF on differentiation of osteoblast. In order to analyze the osteogenic ability of LTF, we treated conditioned media collected from human LTF-stably transfected HEK293T cells into osteoblastic MC3T3-E1. The results showed that the activity and expression of alkaline phosphatase were increased in MC3T3-E1 cells treated with conditioned media containing LTF in dose- and time-dependent manner. At the same time, we observed the significant increase of the expression of osteoblastic genes, such as ALP, BSP, COL1A1, and OCN, and along with matrix mineralization genes, such as DMP1 and DMP2, in LTF conditioned media-treated groups. Moreover, the result of treating recombinant human LTF directly into osteoblastic MC3T3-E1 showed the same pattern of treating conditioned media containing LTF. Our study demonstrated that LTF constitutively enhances osteoblastic differentiation via induction of osteoblastic genes and activation of matrix mineralization in MC3T3-E1 cells.

Comparison of expression and antibacterial activities of recombinant porcine lactoferrin expressed in four Lactobacillus species / 生物工程学报

The coding sequence for the mature peptide of porcine lactoferrin (Plf) was synthesized according to the codon usage of lactobacillus, to establish optimized porcine lactoferrin Lactobacillus expression system. The gene was ligated into the Xho I/BamH I site of Lactobacillus expression vector pPG612.1 and the recombinant plasmid pPG612.1-plf was transformed individually into Lactobacillus casei ATCC393, Lactobacillus pentosus KLDS1.0413, Lactobacillus plantarum KLDS1.0344 or Lactobacillus paracasei KLDS1.0652 by electroporation. After induction with xylose, expression of the recombinant proteins was detected by Western blotting and confocal laser scanning microscopy. Secretion of recombinant Plf proteins from four recombinant species was determined quantitatively by ELISA. The antibacterial activities of recombinant proteins were measured by agar diffusion method. The result shows that Plf was correctly expressed in four species of recombinant lactobacillus, with molecular weight of about 73 kDa. The expression levels in recombinant Lactobacillus casei, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus paracasei were 9.6 μg/mL, 10.8 μg/mL, 12.5 μg/mL and 9.9 μg/mL, respectively. Antimicrobial activity experiment shows that the recombinant proteins were active against E. coli, Staphylococcus aureus, Salmonella typhimurium, Listeria, Pasteurella. The recombinant Plf expressed by recombinant Lactobacillus plantarum showed the best antibacterial activity among all recombinant lactobacillus species. These data represent a basis for the development and application of porcine lactoferrin from recombinant lactobacillus.