Thèses sur le sujet « Bovine lactoferrin »

Bovine lactoferrin (BLF) and bovine transferrin (BTF) are major-iron transport and regulation proteins found in bovine whey. BLF and BTF must interact with the eukaryotic cell surface to mediate their biological function of iron delivery and cellular functions of inflammatory and immunological modulation. As common components of the eukaryotic cell surface, gangliosides were used for affinity purification of BLF and BTF. Bovine gangliosides were isolated from fresh buttermilk and covalently immobilized onto controlled-pore glass beads (66 μg/g beads). After the matrix was loaded with whey protein (WPI or WPC), lactoferrin was eluted with 1 M NaCl and lll identified by N-terminal protein sequencing. Pretreated whey isolate (1 % wt/vol) showed the highest lactoferrin purity with 40% among protein sources, and whey protein isolate (10% wt/vol) showed the highest recovery with 105%. Bovine transferrin was eluted with sodium phosphate buffers at pH 7 after the immobilized matrix was loaded with a 2% (wt/vol) whey solution. The ganglioside column resulted in a 74.2% recovery of BTF from whey, and the BTF was enriched to 61% purity after Mono-Q chromatography. Bovine transferrin was identified by SDS-PAGE analysis, Western analysis, and isoelectrofocusing. In conclusion, immobilized gangliosides can be used to purify BLF and BTF from bovine whey.

A series of investigations were carried out to assessth e intestinal microbiota of rainbow trout and the potential applications of probiotics and bovine lactoferrin (M). Farm and aquarium reared rainbow trout were examined with specific emphasis on the autochthonous microbial communities. Culture-based, culture-independent and electron microscopical investigations revealed mixed, complex microbial communities in all intestinal regions. DGGE based analysis revealed unique species present either only as allochthonous populations or autochthonous populations. 16S rRNA sequence analysis allowed species level identification of a range of isolates, many of which have not been identified from the rainbow trout digestive tract previously. Two ftirther investigations were carried out to assess the potential of using commercial probiotics and bovine Lf on growth, feed utilisation, health and intestinal colonisation of rainbow trout. Standard commercial diets were supplemented with B. subtills, B. licheniformis and Enterococcus faecium either singularly or synergistically. When comparing the findings of the joint study it can be concluded that the application of probiotics with rainbow trout, and likely other finfish species, is highly complicated. Full intestinal replacement of indigenous microbiota is not likely to be a good idea when using E. faecium; the results indicate that a synergistic relationship with the indigenous microbiota is likely to be involved in providing host benefits. Bacillus probiotics only appeared to be effective at high intestinal levels indicating that a synergistic relationship with the indigenous microbiota may not be as important. T'he joint study also indicates that it is not always possible to reproduce probiotic benefits even when using the same probionts, the same fish species and similar rearing conditions. Thus, the physiological status of the fish and the indigenous microbiota are likely to play an important role in the outcome of probiotic administration. A subsequent trial was conducted to evaluate Pediococcus acidilactic! as a probiotic for rainbow trout. The experiment was conducted to supplement the diet with either vegetative cells or lyophilised powder (as commercially provided). Despite successful intestinal colonisation, irrelevant of supplementation form few significant benefits were observed. SEM of the posterior mucosa revealed a localised colonisation pattern of P. acidilactic! between the mucosal folds similar to the observed indigenous microbiota from the farmed fish. This revelation led to a further trial to investigate the nature of probiotic colonisation through the gastro-intestinal tract using electron microscopy. The study confirmed the high colonisation of P. acidilactici on the epithelium of the anterior intestine and posterior intestine. However, it was not possible to observe such colonisation with Bacillus spp. or E. faeclum; despite culture-based results to the contrary. It is likely that the true mucosal colonisation may sometimes be confused with colonisation of the mucus layer as opposed to actual attachment to the epithelium itself. Therefore, it is crucial to utilise electron microscopy in order to confirm epithelial colonisation. The nature of both the indigenous microbiota and the application of probiotics appears to be more complicated than previously thought and continued research is clearly warranted.

Methods for on-farm extraction of low-concentration (minor) proteins from raw whole bovine milk directly after milking were explored. These minor proteins have high commercial value. Lactoferrin (LF) and lactoperoxidase (LP) were used as model proteins for extraction using cation exchange chromatography. Laboratory fractionations showed that milk could be processed by conventional column chromatography without excessive column backpressures if resin with large particles sizes were used and the temperature was high enough so fat in the milk was malleable; ideally the milk should be near the secretion temperature of 37oC. Processing parameters such as equilibrium and dynamic capacities were determined for SP Sepharose ™ (GE Healthcare Technologies) and Bio Rex 70 (BioRad Laboratories) resins. SP Sepharose Big Beads (SP BB) were found to be more suitable than BR 70, for raw whole milk processing due to the larger size (200 um). Design considerations showed that column chromatography was not the most practical method for on-farm processing of fresh, raw whole milk. Trials with a single-stage stirred tank showed that SP BB resin could extract up to 65% of LF (initial LF concentration of 0.5 mg/mL) with a 10-minute adsorption time. The composite non-linear (CNL) model of Rowe et al. (1999) was used to describe LF uptake by SP BB resin in raw whole milk with initial LF concentrations of 0 to 1.0 mg/mL and resin:milk volume ratios of 0.010, 0.012, 0.017 and 0.024 over 45-minute contact times. The CNL model could be used to predict LF yields if initial feed concentration, milk and resin volumes, and contact times were known. Laboratory extractions showed that processing did not significantly affect bulk milk composition (fat, protein, lactose and total solids), indicating that the milk could be used for conventional processing after the minor proteins had been extracted. Resin cleaning and regeneration studies, using a procedure similar to that recommended by the resin supplier, showed that the Sepharose resin had not degraded and there was no significant decrease in binding capacity after 50 extraction cycles. A Protein Fractionation Robot (PFR) prototype based on a single-stage stirred tank and the operating parameters obtained from the laboratory trials was designed, assembled and coupled to an Automated Milking System (AMS) to process fresh, raw whole milk from individual cows immediately after milking. The LF and LP extracted from the milk from 16 individual cows were 19.7 - 55.2% (35.6 10.2%) and 21.2 - 99.5% (87.1 12.0%) respectively. Generally, higher extraction levels were obtained at higher resin:milk ratios. The amount of LF extracted on-farm agreed within 14.1 9.8% of those predicted by the CNL model, with predicted values generally being higher. The experimental on-farm adsorption values were calculated using data of LF recovered after elution, so differences between actual and predicted values may be due to losses during post-adsorption processing. Economic feasibility studies, based on experimental data from the PFR and realistic wholesale prices for LF and LP ($400 and $150/kg respectively) showed that PFR-based processing is economically viable if the farmer is paid for the LF and LP produced as well as the bulk milk. This system would have a payback period of approximately five years and an internal rate of return of 14.5%. Further case studies determined the sensitivity of the economics to various operating parameters and value/cost assumptions, including producing recombinant human protein from transgenic bovine milk. These studies showed that the higher the value of the processed raw milk, the higher the absorptive capacity of the resin, and the higher the value of the extracted protein, the more favourable the economics. In the extreme case of producing a very high value therapeutic protein (e.g. $20 000), the payback period could be as low as 0.3 years, with an internal rate of return of 818%.

Le mecanisme d'echange du fer entre le nitrilotriacetato-fer(iii) et la serumtransferrine humaine ainsi que la lactoferrine bovine est determine en presence d'hydrogenocarbonate entre ph 6,5 et 9 par les techniques et methodes de la relaxation chimique associees aux spectroscopies d'absorption et d'emission de fluorescence. La serumtransferrine humaine et la lactoferrine bovine interagissent avec l'hydrogenocarbonate pour former chacune une entite proteique capable d'extraire le fer du nitrilotriacetate. Nous montrons que la capture du fer par les deux proteines carbonatees se fait d'une maniere identique et engendre des modifications de conformation suivies par des deprotonations lentes. L'echange de fer(iii) entre le chelate et le site-c de la proteine engendre la formation d'un complexe cinetique. Cette reaction est suivie par des modifications dans les conformations des proteines accompagnees par des pertes de 4 a 5 protons, cela permet aux transferrines monoferriques soit d'atteindre son etat thermodynamique final, soit de capturer un second fer. Nous avons determine toutes les constantes cinetiques et d'equilibres impliquees dans le mecanisme de capture du fer par ces deux transferrines.

La lactoferrine est une glycoproteine secretee dans les liquides de secretion, notamment dans le lait, et liberee dans le plasma par degranulation des neutrophiles. Les principales proprietes biologiques de la lactoferrine concernent les processus inflammatoire et immunitaire. La lactoferrine est, en effet, capable d'accelerer la maturation des cellules t en augmentant l'expression de l'antigene de surface cd4. Afin d'approfondir cet effet de la lactoferrine humaine sur les cellules t, nous avons utilise la lignee humaine lymphoblastique t jurkat. Nous avons ainsi montre que la lactoferrine augmente la densite de surface du cd4 en modulant l'expression du gene de ce marqueur : la synthese des arnm ainsi que l'activite du promoteur du gene du cd4 sont stimulees par la lactoferrine. Le mecanisme d'action aboutissant a cette regulation a ete elucide en etudiant le signal de transduction induit par fixation de la lactoferrine a son recepteur. Nous avons observe que la lactoferrine stimule la phosphorylation de nombreuses proteines cytosoliques, et qu'elle active une seule isoforme de la map kinase ( mitogen-activated protein kinase ). L'utilisation des inhibiteurs genisteine et pd98059 a ensuite permis de correler ces deux evenements a l'expression du cd4
Enfin, en utilisant des cellules jurkat deficientes en proteine lck, les cellules j. Cam1. 6, nous avons demontre que la kinase p56 l c k est necessaire a la regulation du cd4 par la lactoferrine. La derniere partie de nos travaux a consiste a comparer les effets des lactoferrines d'origine humaine et bovine sur certaines cellules du systeme immunitaire. Les deux proteines presentent les memes activites sur la cytotoxicite des cellules nk ( natural killer ) envers des lignees tumorales, et sur la regulation de la densite du cd4 a la surface des cellules jurkat. Par ailleurs, nos resultats indiquent que la proteine bovine regule l'expression du cd4 en stimulant egalement l'activite de la map kinase

Notre travail a consiste en l'etude du transport intestinal de la lactoferrine bovine et des effets de l'ingestion reguliere de lactoferrine bovine sur son absorption et sur la reponse immunitaire intestinale et systemique chez la souris. La lactoferrine est une glycoproteine de la famille des transferrines, presente dans le lait et diverses secretions externes de l'organisme. Elle est dotee de proprietes bacteriostatiques, de par sa capacite a lier le fer. Elle est consideree comme un facteur regulateur de croissance pour differents types cellulaires et est capable d'agir sur differentes fonctions du systeme immunitaire peripherique telles que la maturation et la differenciation des lymphocytes, la secretion de cytokines, ou la cytotoxicite. Nous avons donc evalue le role de la lactoferrine bovine vis-a-vis des cellules immunocompetentes intestinales et peripheriques. Cette etude nous a permis de montrer que chez la souris, la lactoferrine bovine est capable de franchir la barriere intestinale et gagner la circulation sanguine. L'ingestion reguliere de la proteine par des souris reduit ce passage et induit une synthese d'anticorps anti-lactoferrine dans la muqueuse intestinale et le serum (exlusion immune). Notre etude montre en outre que l'ingestion de lactoferrine bovine pendant 4 semaines stimule la reponse immunitaire non specifique intestinale et systemique. Cet effet immunomodulateur de la lactoferrine se traduit aussi bien au niveau des plaques de peyer qu'au niveau de la rate par une elevation de la distribution des lymphocytes t cd3#+cd4#+ et par une stimulation de la reponse proliferative des cellules immunocompetentes. L'etude des profils de cytokines il-2, il-4 et il-5 revele un effet preferentiel de la lactoferrine vers une reponse humorale de type th2. En conclusion, contrairement aux proteines alimentaires classiques, la lactoferrine semble aussi dotee de proprietes immunostimulantes qui lui sont inherentes et qui favorisent la reponse immune humorale.

Made available in DSpace on 2013-08-07T19:06:29Z (GMT). No. of bitstreams: 1 000446716-Texto+Completo-0.pdf: 2485126 bytes, checksum: d0edbbefdafd57b69ebba83d7dfd395f (MD5) Previous issue date: 2013
Background : Lactoferrin (LF) is a glycoprotein present in human milk with known antimicrobial effects. In vitro, LF has shown to impair the growth of respiratory syncytial virus (RSV). We sought to assess the effect of bovine (b)LF in RSV replication and different aspects of RSV disease in an in vivo murine model. Methods : BALB/C mice were inoculated with 107 PFU RSV A2 or 10% EMEM. bLF or placebo (DPBS) were administered once or twice daily by oral gavage or intraperitoneal (IP) injection at doses ranging from 2 to 10mg/animal/day, from 48h before until day 4 post-RSV inoculation. Bronchoalveolar lavage, whole lung specimens and serum samples were harvested on day 5 post inoculation to asses RSV loads, lung inflammation and cytokine concentrations. Weight loss, airway obstruction and disease severity were assessed daily in all groups. Results : On day 5 post-inoculation RSV loads, lung inflammation and serum innate, Th1, Th2 and Th17 cytokine concentrations showed no differences between RSV infected mice treated with bLF and RSV untreated mice independent of bLF dosing and administration route (p>0. 05). In addition, all bLF groups showed similar weight loss, degree of airway obstruction, and disease severity scores on days 1 to 5 post-inoculation which was comparable to infected untreated mice (p>0. 05), but higher than uninfected controls. Conclusions : Administration of oral or IP bLF at different doses did not demonstrate antiviral activity or significant effects on disease severity in the RSV mouse model. Whether these observations could be extrapolated to infants at risk of RSV infection needs to be further explored.
Introdução : Lactoferrina (LF) é uma glicoproteína presente no leite humano que possui efeitos antimicrobianos bem estabelecidos. Estudos in vitro, demonstram que a LF é capaz de desestabilizar o crescimento do vírus sincicial respiratório (VSR). Neste estudo procuramos avaliar o efeito da LF bovina (bLF) na replicação do VSR, além de avaliar diferentes aspectos da doença causada por VSR in vivo em um modelo murino.Métodos : camundongos BALB/c foram inoculados com 107 UFP de VSR A2 ou EMEM 10%. bLF ou placebo foram administrados uma ou duas vezes ao dia por gavagem ou injeção intraperitoneal (IP) em doses entre 2 e 10 mg/animal/dia, dois dias antes a 4 dias após a inoculação com VSR. Amostras de lavado broncoalveolar, pulmões e sangue foram coletadas no dia 5 após a inoculação com o objetivo de avaliar a carga viral, inflamação pulmonar e concentração de citocinas. Peso, obstrução de via aérea e gravidade da doença foram avaliados diariamente em todos os grupos do estudo. Resultados : No dia 5 após a inoculação do vírus, a carga viral, inflamação pulmonar e concentração de citocinas relacionadas a imunidade inata, respostas Th1, Th2 e Th17 no soro não demonstraram diferença, quando comparamos os animais infectados com RSV tratados com bLF e os animais infectados com RSV tratados com placebo, independente da dose ou via de administração (p>0. 05). Além disso, a perda de peso, obstrução de via aérea e gravidade da doença foi similar entre os grupos tratados com bLF nos dias 1 a 5 após a inoculação do vírus. Estes valores são comparáveis com os valores obtidos no grupo placebo, porém significativamente aumentados quando comparados aos animais não-infectados. Conclusão : a administração de bLF por via oral ou injeção IP, em diferentes doses, não demonstrou atividade antiviral ou efeitos significativos na severidade da doença em um modelo murino de VSR. Se essas observações podem ser extrapoladas para crianças em risco de desenvolver infecções por VSR, essas questões necessitam ser melhor exploradas.

Analiza el efecto de la lactoferrina en la cinética de crecimiento de Salmonella typhimurium cepa SL 1344 ΔhilA, evalúa el efecto citotóxico del tratamiento con gentamicina a células HEp-2, determina el efecto in vitro de la lactoferrina sobre la adhesión de Salmonella typhimurium SL 1344 ΔhilA y especifica el tratamiento y concentración de lactoferrina que permita mayor disminución de la adherencia e invasión a las células HEp-2.
Tesis

En esta Tesis Doctoral se ha estudiado en modelos experimentales el potencial antihipertensivo de dos tipos de péptidos bioactivos: péptidos derivados de distintas zonas de la secuencia de la lactoferrina bovina (LF), incluido su dominio antimicrobiano lactoferricina (LfcinB), y heptapéptidos obtenidos mediante diseño racional a partir de hexapéptidos parentales. Se han realizado tres tipos de ensayos: ensayos in vitro para determinar los efectos inhibitorios sobre la actividad de la enzima conversora de la angiotensina I (ECA); ensayos funcionales ex vivo, usando segmentos arteriales aislados de conejo, para analizar los efectos inhibitorios de los péptidos sobre la vasoconstricción ECA-dependiente producida por angiotensina I (Ang I); y ensayos in vivo mediante la administración de los péptidos a ratas espontáneamente hipertensas (SHR) para estudiar los efectos antihipertensivos. En algunos casos, también se han realizado ensayos in vitro para determinar el potencial efecto tóxico de los péptidos no naturales y de digestión gastrointestinal simulada para analizar la biodisponibilidad de los péptidos. Se obtuvieron péptidos derivados de LfcinB mediante elongaciones tanto del extremo C-t como del N-t del péptido LfcinB20-25 (RRWQWR). Estos péptidos mostraron diferentes potencias de inhibición de la actividad ECA in vitro, e inhibieron la vasoconstricción ECA-dependiente ex vivo. No se encontró una clara correlación entre los resultados in vitro y ex vivo. Solamente LfcinB20-25 y un fragmento derivado (WQ) producido mediante digestión gastrointestinal simulada mostraron efectos antihipertensivos in vivo. Sin embargo, el fragmento no mostró efecto inhibidor de la vasoconstricción ECA-dependiente en contraste con LfcinB20-25. Por otro lado, se preparó un hidrolizado de lactoferrina bovina con pepsina y se ultrafiltró para enriquecerlo en péptidos con peso molecular menor de 3 kDa (LFH<3kDa). Este hidrolizado mostró efectos antihipertensivos mantenidos hasta 24 h tras la administración oral. LFH<3kDa se fraccionó y se identificaron 38 péptidos de los cuales se sintetizaron los 11 péptidos más abundantes. Tres de estos péptidos (LIWKL, RPYL y LNNSRAP) mostraron diferentes grados de inhibición de la actividad ECA in vitro y efectos antihipertensivos in vivo, aunque solamente dos de ellos, LIWKL y RPYL, mostraron efectos inhibidores de la vasoconstricción ECA-dependiente ex vivo. Por último, seis heptapéptidos mostraron diferentes grados de inhibición de la actividad ECA in vitro y de la vasoconstricción ECA-dependiente, pero no de la vasoconstricción ECA-independiente producida por angiotensina II. Los heptapétidos PACEI50L (RKWHFLW) y PACEI52L (RKWLFHW), y el hexapéptido parental PACEI32L (RKWHFW), mostraron efectos antihipertensivos in vivo, sin afectar a la presión arterial en ratas normotensas. Los péptidos sintetizados con D-aminoácidos mostraron mucho menos efecto inhibidor de la actividad ECA in vitro, no tuvieron efecto ex vivo, y mostraron efectos antihipertensivos in vivo tras administración intravenosa pero no oral. La toxicidad de estos péptidos no naturales para reducir la viabilidad celular in vitro se mostró a concentraciones milimolares, mucho más altas que las concentraciones micromolares con efecto inhibidor de la actividad ECA. En conclusión, se ha demostrado el potencial antihipertensivo de un péptido derivado de la lactoferricina (LfcinB20-25), de un hidrolizado con pepsina de la lactoferrina enriquecido en péptidos de bajo peso molecular (LFH<3kDa), de péptidos contenidos en dicho hidrolizado procedentes de otras zonas de la lactoferrina diferentes de LfcinB (LIWKL, RPYL y LNNSRAP), y de hexa- y heptapéptidos obtenidos mediante diseño racional (PACEI32L, PACEI50L y PACEI52L). En la mayor parte de ellos, el efecto antihipertensivo se asocia a su efecto vasoactivo por su capacidad para inhibir la actividad ECA
Ruiz Giménez, P. (2013). Efecto antihipertensivo, mediante inhibición de la enzima conversora de angiotensina I, de péptidos derivados de lactoferrina bovina y péptidos diseñados racionalmente [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31123
TESIS

Made available in DSpace on 2015-04-14T13:33:03Z (GMT). No. of bitstreams: 1 446716.pdf: 2485126 bytes, checksum: d0edbbefdafd57b69ebba83d7dfd395f (MD5) Previous issue date: 2013-03-12
Background : Lactoferrin (LF) is a glycoprotein present in human milk with known antimicrobial effects. In vitro, LF has shown to impair the growth of respiratory syncytial virus (RSV). We sought to assess the effect of bovine (b)LF in RSV replication and different aspects of RSV disease in an in vivo murine model. Methods : BALB/C mice were inoculated with 107 PFU RSV A2 or 10% EMEM. bLF or placebo (DPBS) were administered once or twice daily by oral gavage or intraperitoneal (IP) injection at doses ranging from 2 to 10mg/animal/day, from 48h before until day 4 post-RSV inoculation. Bronchoalveolar lavage, whole lung specimens and serum samples were harvested on day 5 post inoculation to asses RSV loads, lung inflammation and cytokine concentrations. Weight loss, airway obstruction and disease severity were assessed daily in all groups. Results : On day 5 post-inoculation RSV loads, lung inflammation and serum innate, Th1, Th2 and Th17 cytokine concentrations showed no differences between RSV infected mice treated with bLF and RSV untreated mice independent of bLF dosing and administration route (p>0.05). In addition, all bLF groups showed similar weight loss, degree of airway obstruction, and disease severity scores on days 1 to 5 post-inoculation which was comparable to infected untreated mice (p>0.05), but higher than uninfected controls. Conclusions : Administration of oral or IP bLF at different doses did not demonstrate antiviral activity or significant effects on disease severity in the RSV mouse model. Whether these observations could be extrapolated to infants at risk of RSV infection needs to be further explored.
Introdu??o : Lactoferrina (LF) ? uma glicoprote?na presente no leite humano que possui efeitos antimicrobianos bem estabelecidos. Estudos in vitro, demonstram que a LF ? capaz de desestabilizar o crescimento do v?rus sincicial respirat?rio (VSR). Neste estudo procuramos avaliar o efeito da LF bovina (bLF) na replica??o do VSR, al?m de avaliar diferentes aspectos da doen?a causada por VSR in vivo em um modelo murino. M?todos : camundongos BALB/c foram inoculados com 107 UFP de VSR A2 ou EMEM 10%. bLF ou placebo foram administrados uma ou duas vezes ao dia por gavagem ou inje??o intraperitoneal (IP) em doses entre 2 e 10 mg/animal/dia, dois dias antes a 4 dias ap?s a inocula??o com VSR. Amostras de lavado broncoalveolar, pulm?es e sangue foram coletadas no dia 5 ap?s a inocula??o com o objetivo de avaliar a carga viral, inflama??o pulmonar e concentra??o de citocinas. Peso, obstru??o de via a?rea e gravidade da doen?a foram avaliados diariamente em todos os grupos do estudo. Resultados : No dia 5 ap?s a inocula??o do v?rus, a carga viral, inflama??o pulmonar e concentra??o de citocinas relacionadas a imunidade inata, respostas Th1, Th2 e Th17 no soro n?o demonstraram diferen?a, quando comparamos os animais infectados com RSV tratados com bLF e os animais infectados com RSV tratados com placebo, independente da dose ou via de administra??o (p>0.05). Al?m disso, a perda de peso, obstru??o de via a?rea e gravidade da doen?a foi similar entre os grupos tratados com bLF nos dias 1 a 5 ap?s a inocula??o do v?rus. Estes valores s?o compar?veis com os valores obtidos no grupo placebo, por?m significativamente aumentados quando comparados aos animais n?o-infectados. Conclus?o : a administra??o de bLF por via oral ou inje??o IP, em diferentes doses, n?o demonstrou atividade antiviral ou efeitos significativos na severidade da doen?a em um modelo murino de VSR. Se essas observa??es podem ser extrapoladas para crian?as em risco de desenvolver infec??es por VSR, essas quest?es necessitam ser melhor exploradas.

Orientadores: Valdemiro Carlos Sgarbieri, Antônio Fernando Ribeiro
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
Made available in DSpace on 2018-08-16T14:45:40Z (GMT). No. of bitstreams: 1 Bernardi_DanielaMiotto_M.pdf: 2175104 bytes, checksum: cb8cf27a5c2ba0db8cf94fbc2965beec (MD5) Previous issue date: 2010
Resumo: A fibrose cística (FC) é caracterizada por intenso processo inflamatório, doença pulmonar obstrutiva, infecção das vias aéreas e má digestão/ má absorção de nutrientes e micronutrientes, sendo a nutrição determinante no prognóstico do paciente. Objetivo: Avaliar os efeitos imunológicos e nutricionais da suplementação de um concentrado protéico do soro do leite bovino (whey protein concentrate - WPC), enriquecido com TGF-ß e lactoferrina, em pacientes pediátricos com fibrose cística, bem como realizar perfil imunológico e nutricional destes pacientes. Métodos: O ensaio clínico de intervenção nutricional foi prospectivo, randomizado, duplo cego com placebo e teve duração de 4 meses. As crianças que participaram eram atendidas no Ambulatório Pediátrico de FC, do Hospital de Clínicas da UNICAMP, na faixa etária entre 3 e 12 anos e escore de Shwachman entre moderado e bom. Quarenta e cinco crianças iniciaram a suplementação e apenas 28 finalizaram, sendo 15 no grupo WPC-TGFß e 13 no grupo caseína (placebo). Foram incluídas 17 crianças saudáveis como grupo controle para as análises bioquímicas e imunológicas. Os suplementos (placebo e teste) foram submetidos às análises centesimais e microbiológicas. A avaliação dos pacientes ao longo da suplementação foi realizada em três tempos (T0=antes da suplementação, T1=depois de 2 meses e T2= no final do quarto mês). Determinaram-se os níveis de glutationa nos eritrócitos, a produção de radicais reativos de oxigênio (ROS) e de citocinas em sobrenadante de cultura de sangue periférico (TNF-a, IFN-?, IL-8, IL-6, IL-10), concentração de TGF-ß2 no soro, imunoglobulinas séricas (IgA, IgG, IgM e IgE) e IgA na saliva, cultura do escarro, níveis de albumina e pré-albumina, avaliação antropométrica e de ingestão alimentar. Em relação à análise estatística, os dados paramétricos foram analisados com o teste-T e ANOVA para amostras pareadas e os dados não paramétricos foram testados por Mann-Whitney e Friedman. O nível de significância adotado foi de 5%. Resultados: Em relação aos sistemas antioxidante e oxidante em sangue periférico, verificou-se que não houve diferença significativa entre fibrocísticos e indivíduos saudáveis. A produção espontânea de TNF-a, IL-6, IL-10 estava aumentada na FC assim como a produção de IL-6 em resposta à PHA, a concentração de TGF-ß2, IgA e IgM no soro e a concentração sanguínea de leucócitos. Por outro lado, os indivíduos saudáveis tiveram maior produção de TNF-a em resposta ao estímulo pela BCG. Houve baixo índice de desnutrição nos participantes. A suplementação com WPCTGFß não influenciou de forma significativa na concentração de GSH, imunoglobulinas, na infecção das vias aéreas e no estado nutricional. Em relação à GSH embora a mudança não tenha sido significativa, observou-se uma tendência de aumento gradual ao longo da suplementação. A suplementação também provocou um aumento significativo na produção basal de ROS pelos granulócitos, uma redução na produção de TNF-a em sobrenadante de cultura em resposta à PHA, além de reduzir momentaneamente (T1) a produção de IL-10 na presença de PHA. Observou-se atenuação da diferença estatística existente para TGF-ß2, antes da suplementação, nos dois grupos. Houve um aumento do número de eritrócitos e de hemoglobina, provavelmente pela ação da lactoferrina. Conclusão: O presente trabalho mostrou que a criança clinicamente estável com FC consegue manter um balanço oxidante e antioxidante normal. As altas concentrações de TNF-a e de IL-6 demandaram uma maior produção de IL-10, que também pode ter sido a responsável pela resposta TH1 eficiente para BCG, expresso pela produção normal de IFN-?. A maior produção de IgA e IgM confirmam um sistema imune adaptativo normal frente ao estimulo freqüente da colonização bacteriana nesses indivíduos. Existem indícios nos resultados de que a suplementação à longo prazo em crianças ainda não colonizadas poderia ser mais eficiente, o que somente poderia ser elucidado por meio de novos estudos
Abstract: Cystic fibrosis (CF) is characterized by an intense inflammatory process, pulmonary obstruction, airway infection and gastrointestinal symptoms resulting maldigestion/ malabsorption of nutrients, and as a consequence nutrition is a determinant on the patient's prognostic. The aims were to evaluate the effect of supplementation with a WPC enriched with TGF-ß and lactoferrin, in pediatric patients with cystic fibrosis. Methods: The study was a randomized, double-blind, placebo controlled, prospective clinical trial of nutritional intervention with four months of duration. Children were recruited from the Pediatric Outpatient Unit, at UNICAMP Clinical Hospital, aged between 3 and 12 years and Shwachman score between moderate and good. The study group started with 45 children, nevertheless, it ended with 28 subjects: 15 of WPC-TGF ß group and 13 of casein group (placebo). Seventeen healthy children were also used as a control group. The supplements (placebo and test) were submitted to centesimal and microbiological analysis. The supplementation was done during four months with three assessments (T0= before starting the supplementation, T1= with two months of supplementation and T2= at the end the 4th months of supplementation) with appraisal of: erythrocyte glutathione concentration, production of reactive oxygen species (ROS) by granulocytes, cytokines (TNF-a, IFN-?, IL-8, IL-6, IL-10) concentration in supernatants from cultures under spontaneous condition, PHA and Bacillus Calmette-Guérin (BCG)-stimulated peripheral blood mononuclear cells, levels of serum TGF-ß2, immunoglobulins (IgA, IgG, IgM and IgE), albumin and prealbumin, salivary IgA, sputum culture, anthropometric measurements and food intake. For statistical analysis, the parametric data were analyzed with T-test and ANOVA and nonparametric data, with Mann-Whitney and Friedman tests. The level of significance was 5%. Results: There was no statistical difference between CF patients and healthy subjects in relation to oxidant and antioxidant system in peripheral blood. The spontaneous production of TNF-a, IL-6, and IL-10, production of IL-6 in response to PHA, TGF-ß2, IgA and IgM in serum and counting white blood cells was increased in CF patients. Furthermore, healthy individuals responded better to secretion of TNF-a in response to BCG stimulation. A low incidence of malnutrition was observed. Supplementation with WPC-TGF ß did not have an influence on concentration of glutathione, immunoglobulins, infection of airways and nutritional status. Regarding GSH although the change was not significant, there was a trend of gradual increase during the supplementation. Furthermore, increased basal stimulation of granulocytes for the production of ROS reduced the production of TNF-a in culture supernatants in response to PHA, slightly reduced the production of IL-10 (T1) in response to PHA and also attenuated the statistical difference existing for TGF-ß2, prior to supplementation, in both groups. The ability to increase the total number of erythrocytes and hemoglobin was attributed to lactoferrin present in the supplement. Conclusion: This study showed that clinically stable CF children could maintain a normal oxidant and antioxidant balance. The high concentrations of TNF-a and IL-6 require a higher production of IL-10, which may have also been responsible for the efficient TH1 response to BCG, expressed by IFN-?. The higher production of IgA and IgM confirm a normal adaptive immune system against bacterial colonization in these patients. It is suggested that supplementation for longer period of children not yet colonized lead to better results than the ours obtained, however, only further study could elucidate such an hypothesis
Mestrado
Nutrição Experimental e Aplicada à Tecnologia de Alimentos
Mestre em Alimentos e Nutrição

碩士
國立嘉義大學
生物科技研究所
95
Japanese encephalitis (JE) has been an important endemic zoonoses in Taiwan. Since beginning in 1968, a mass vaccination program, using mouse-brain-derived and formalin-inactivated JEV Nakayama as vaccine, against JE for children was implemented in Taiwan. Dramatic decrease of JE virus infection has been reported, but still approximately 20-30 cases annually. Approximately 25 percent of infectious patients and 50 percent of the survivals develop permanent neurologic and/or psychiatric sequelae. Recently, the anti-JEV drug was unavailable in clinical therapy. Therefore, it is important to develop a new anti-viral drug. Lactoferrin exists in breast milk and mucous secretions; it is a iron-binding protein for transportion in intestines. This protein is also associated with non-specific immune response and inhibits a variety of microbial infections including bacterial, fungous, and viral infection. Lactoferrin can inhibit viral cell entrance into cell by binding to the virus particle directly or to membrane-bound heparan sulfate. In previous reports of our laboratory and others, the heparan sulfate on cell surface is one of possible receptors for Japanese encephalitis virus (JEV). The goal of this study was to investigate the inhibitory effect of bovine lactoferrin against JEV infection. Our results were summarized as below: (1) bovine lactoferrin (bLF) inhibits the infectious JEV, including wild-type (CJN-2K、T1P1、CC27) and laboratory-adapted strains (CJN-L1、CJN-S1、T1P1-L4、T1P1-S1、CC27-L1、CC27-L3、CC27-S6、CC27-S8); (2) bLF inhibits JEV infection by interacting with cells, not viral particle; (3) the mechanism of bLF inhibition JEV infection was not only blocking viral attachment to cellular membrane, but also reducing viral endocytosis and penetration; (4) directly interaction of heparan sulfate (HS) with bLF inhibit JEV infection. In HS-dependent CJN-S1 strain and HE-expressed CHO-K1 cell line, the professional antiviral effect of bLF has been observed; (5) in BHK-21 cells, soluble heparin inhibits the anti-JEV infection activity of bLF; (6) in addition to HS, other unknown molecules may bind to bLF and then inhibit JEV infection, because the antiviral effect of bLF has been observed in HS-independent CJN-L1 strain and HE-deficient CHO-pgsA745 cell line In this study, we demonstrated that bLF can inhibit JEV infection, however, the protection effect is still needed to study in animal experiments. In the future, it is possible to identify the receptor(s) for JEV using bLF inhibits JEV infection by binding to other unknown molecules. Study of the receptor(s) for JEV is acquired to understand the inhibition mechanism of lactoferrin on JEV infection.

Lactoferricin (Lfcin), a cationic antimicrobial peptide, was purified by peptic digestion of food grade bovine lactoferrin (LF) followed by fractionation on an industrial grade cation exchange resin with stepwise salt gradient elution. A 2-step process using competitive displacement cation exchange chromatography led to 35% recovery of peptides with masses of 3124 and 3196 Da corresponding to Lfcin. Other cationic peptides produced concurrently with Lfcin were tentatively identified. Varying iron saturation levels of LF had no effect on the production of Lfcin. Two approaches were used to understand how Lfcin acts as an antimicrobial agent. Homology Similarity Analysis (HSA) was used to evaluate the impact of peptide pattern similarities of various Lfcin derivatives on their effects as antimicrobial agents. Helical property of residues 4-9 in the Lfcin sequence was the most important in determining the antimicrobial activity of Lfcin against Escherichia coli, followed by cationic charge pattern of residues 4-9 and residues 1-3. Raman spectroscopy in the C-H stretching (2800-3000 cm⁻¹) region was used to study interactions between Lfcin and bacterial membrane models. Presence of Lfcin in 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) multilamellar liposomes restricted the lateral chain-chain interaction along the acyl chain throughout the temperature range examined, and increased the main transition temperature (Tm) from 38-40°C. Lfcin had little effect on 1,2-dipalmitoyl-sn-glycerol-3-[phosphor-rac-(1-glycerol)] (DPPG) liposomes at temperatures below the Tm. In contrast, mobility of the acyl chains in 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE) was increased in the presence of Lfcin at temperatures below 32°C. Although Lfcin had little effect on the zwitterionic unsaturated phospholipids, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolamine (POPE), Lfcin lowered the lateral chain-chain interaction along the acyl chains of negatively charged 1-palmitoyl-2-oleoyl-sn-glycerol-3-[phosphor-rac-(1-glycerol)] (POPG). Raman spectral analyses also showed that the removal of arginine promoted interactions of Lfcin derivatives with hydrophobic acyl chains and methyl ends of negatively charged DPPG liposomes. Furthermore, addition of two extra tryptophan residues in Lfcin derivatives stabilized acyl chains and methyl ends in DPPE liposomes. HSA and Raman spectroscopy are two useful tools for understanding the antimicrobial mechanisms of Lfcin and will facilitate the application and utilization of Lfcin as a naturally occurring antimicrobial agent.
Land and Food Systems, Faculty of
Graduate

碩士
國立中興大學
獸醫微生物學研究所
87
Lactoferrin is an antimicrobial iron-binding glycoprotein, and the molecular weight is about 80 kDa. It is found in most mucous secretion of mammals including milk, saliva, tears, and released from activated neutrophil in the inflammatory response. The antibacterial activity and the possible mechanism of lactoferrin was studied. The results showed that lactoferrin could effectively inhibit the growth of 5 strains of E. coli that cause severe diarrhea of piglets, and 9 strains of H. pylori that cause gastrointestine disease. The effective dose of lactoferrin for antibacterial activity was 25M. We also found that this antibacterial effect would be blocked by excess of iron. In comparison of apo-transferrin, apo-ferritin, and lactoferrin showed similar activity of inhibition of E. coli. This comes to a conclusion that the inhibition effect is caused by the competition of iron in LB medium. Furthermore, we also found that in the surrounding of outer membrane from dead bacteria, the antibacterial activity of lactoferrin was diminished. Therefore we also believed that lactoferrin may act directly on the outer membrane to inhibit the gram negative bacteria growth.

碩士
國立臺灣大學
漁業科學研究所
88
To investigate the peptide-antimicrobial activity relationship of shrimp, bovine lactoferrin (bLF), the basic amino acid-rich region of lactoferricin B, RRWQWRMKKLG (N: 11 mer), yellowfin progy (Acanthopagrus latus) recombinant growth hormone (ypGH) and carp (Cyprinus carpio) recombinant interlukin-1 (cIL-1) were selected. Tiger shrimp (Penaeus monodon) hemocytes were incubated with bLF, N: 11 mer, ypGH and cIL-1, respectively, and quantified the production of superoxide anion (O2) by nitroblue tetrazolium (NBT) staining. All of the peptides could enhance the production of O2 of shrimp hemocytes, which was the first product of phagocytosis. The antimicrobial activities of the peptides were also tested by determining the minimal inhibitory concentration (MIC) of Vibrio vulnificus, Vibrio alginolyticus and Vibrio harveyi, respectively, in the presence of the peptides. In contrast to ypGH and cIL-1, bLF and N: 11 mer were successful to inhibit the growth of the pathogen of tiger shrimp. Finally, the membrane disruptions of tiger shrimp hemocytes that caused by bLF and N: 11 mer, respectively, were determined with trypan blue staining. The survival rate of bLF- and N: 11 mer-treated hemocytes was significant differ with untreated hemocytes after 3- or 1-hour (s) incubation, respectively. These results suggest that bLF and N: 11 mer have the ability to stimulate the immune responses of shrimp hemocytes and to inhibit the growth of pathogen, but the two peptides can decrease the survival rate of shrimp hemocytes.

Background: Inflammation, if uncontrolled, can promote the formation of colon cancer. Intestinal lymphocytes (IL) are immune cells that can participate in inflammation through the generation of cytokines and by causing direct cellular injury. Apoptosis, or programmed cell death, regulates the population of lymphocytes and dysregulation of this process results in the prolonged activation of IL that occurs during inflammation. Therefore, inducing apoptosis of IL is a viable mechanism by which inflammation and possibly colon carcinogenesis could be prevented. Experimentally, inflammation may be induced in mice by: 1) the addition of the chemical irritant dextran sulfate sodium (DSS) to drinking water or 2) exposure to acute exercise (AE; intense treadmill running). Bovine lactoferrin (bLf) is a dietary whey protein with demonstrated ability to promote anti-inflammatory responses by reducing pro-inflammatory or increasing anti-inflammatory cytokine concentrations within the intestine. There are also reports that bLf can alter apoptotic proteins in the intestinal epithelium to favour cell death. Moreover, athletes have been reported to supplement their diets with whey protein; it is also known clinically that after heavy competition some athletes may experience gastrointestinal distress. The role, however, of bLf in reducing inflammation by initiation of apoptosis or alteration of cytokine levels in IL has not been examined. Objectives: The primary objective of this research was to determine whether dietary bLf affects mouse IL apoptosis and cytokine concentrations in: 1) a normal, non-inflamed state, 2) following AE challenge and 3) following DSS treatment. A second objective was to directly examine the potential protective effects of dietary bLf from inflammatory damage caused by DSS and AE challenge when administered alone or in combination with a carcinogen. Methods: A total of 252 female C57BL/6 mice were used in the experiments. Apoptotic proteins (Bcl-2, caspase 3, Bax, cytochrome c), inflammatory cytokine proteins (TNF-α and IL-10) and a transcription factor for pro-inflammatory cytokines (NFκB) were determined in isolated mouse IL by Western blot analysis. Flow cytometry was used to determine the extent of apoptosis in mouse IL subsets by measuring phosphatidylserine surface expression using Annexin V+ (ANN+) and propidium iodide staining. Tissue inflammation was determined by histology (H&E staining) on segments of mouse small and large intestine. Diets were prepared and pelletted to contain 20% total protein and contained either bLf or were the control formulation (no bLf). Mice were exposed to bLf containing diets for 4 d or 12 d prior to sacrifice. DSS was provided at 5% in the drinking water for 4 consecutive days prior to sacrifice. Animals were subjected to three repeated bouts of AE (each separated by 24 h rest) involving treadmill running and sacrificed either immediately or 24 h after the final exercise bout. In the experiment involving carcinogenesis, mice were given two subcutaneous injections of azoxymethane (AOM), followed by a two week incubation period, and subsequently exposed to bLf or the control diet. Results: Results from the first experiment determined that 2.0% bLf was effective at reducing mouse IL levels of TNF-α (p<0.05) (pro-inflammatory) and increasing the percentage of apoptotic CD4+ IL (CD4+/ANN+, p<0.05) in healthy mice. Thus, 2.0% bLf was used for the subsequent experiments. Dietary bLf administration in mice exposed to AE was associated with lower levels in mouse IL of the anti-apoptotic protein Bcl-2 (p<0.01) and of TNF-α (p<0.05) and NFκB (p<0.05), both pro-inflammatory proteins. Further, the exercise protocol resulted in oxidative stress, as measured by 8-iso prostaglandin F2α levels in plasma, but did not induce intestinal inflammation, evident by the absence of both tissue damage and infiltration of immune cells. Following DSS treatment, mice supplemented with bLf enriched diets had lower levels of both TNF-α (non-significant, 34% reduction) and NFκB (p<0.05) and increased concentrations (p<0.01) of cytochrome c, a mitochondrial protein associated with cell death. DSS exposure in mice resulted in gross morphological alterations and infiltration of immune cells in the small and large intestine; these changes in tissue histology were not affected by the addition of bLf. Mice injected with AOM and then subjected to DSS, but not AE, had increased numbers (p<0.001) of aberrant crypts, preneoplastic colonic lesions, compared to animals only receiving AOM injection. Dietary bLf did not affect any of these carcinogenic processes. Conclusions: Collectively, these results suggest that dietary bLf administration reduces pro-inflammatory cytokine levels and has limited effects on apoptosis of mouse IL. Moreover, these modifying effects of bLf did not result in mucosal protection, as evident in the inability of this protein to reduce DSS-induced tissue damage or formation of aberrant crypts. Physiological and Clinical Implications: Although the long term physiological consequences of bLf supplementation in the regulation of intestinal immune homeostasis require further study, the following clinical implications are tentatively suggested by the findings from this thesis research. First, dietary bLf supplementation does not provide direct protection of the intestine during inflammation either with or without exposure to the carcinogen (i.e., AOM); hence, bLf (at least in the dietary concentration and exposure used in these experiments) may not be useful in reducing the formation of aberrant crypts and carcinogenesis. Second, dietary bLf should not be recommended as a supplement at this time for athletes experiencing intestinal distress since it had no impact on tissue indicators of disease in a model (DSS) shown to produce extensive inflammation and tissue pathology. Nonetheless, the findings raise the possibility that bLf can modify both cytokines and apoptotic protein expression in IL and may influence some aspects of inflammatory processes in the gut.

Tese de doutoramento em Engenharia Biomédica
Lactoferrin (LF) is an iron-binding protein predominantly found in mammalian secretions. This protein and its variants have been proposed for cancer therapy for many years owing to their tumor-targeting properties. Previous studies showed that LF and its derived peptides inhibit the proliferation of cancer cells. However, the detailed mechanisms by which LF exerts its effect are still fairly unknown. Moreover, there are few reports concerning LF effect on breast cancer cells, which is one of the most common malignant tumors in the World. In this sense, the present thesis aimed to investigate the cytotoxicity of bovine lactoferrin (bLF) and its variants against several breast cancer cells, namely T-47D, MDA-MB-231, Hs578T and MCF-7 cell lines. The results showed that bLF at concentrations of 1.875 μM, 3.75 μM, 7.5 μM, 15 μM and 30 μM could efficiently inhibit the growth of cancer cells but showed a very low effect on normal breast cells (MCF-10-2A). Moreover, its variants (apo-bLF, holo-bLF and LfcinB17-41) were also able to inhibit cancer cells’ growth, except for LfcinB26-36. Additionally, bLF, apo-bLF and holo-bLF did not promote the proliferation of breast cancer cells at low concentrations (0.25 μM, 0.5 μM and 1 μM) as reported for other cancer cell lines. Simultaneously, the degradation assay excluded the possibility that bLF anticancer effects could be due to its degraded peptides under cell culture conditions. On the other hand, it was found that most of the bLF was blocked outside the cells, despite that a few amount was able to be internalized to the cytoplasm. Its peptide LfcinB17-41 also succeeded in penetrating the cell membrane but could not enter the nucleus. Subsequently, we found that the inhibitory effects of bLF on the breast cancer cells resulted from the cell cycle arrest without effects in cell death by apoptosis. Depending on the cell lines, this prevention of cell cycle progression induced by bLF occurred at different phases. Nevertheless, the MAPK/ERK and PI3K/AKT signaling pathways were not implicated in the cell cycle arrest observed. bLF anticancer effect was associated, however, with an increase of AMPKα phosphorylation and a decrease in the levels of mTOR and its phosphorylation. To our knowledge this is the first time this pathway has been implicated in the mechanisms underlying bLF cytotoxicity against cancer. These findings suggest that bLF could be a new mTOR-targeting drug in cancer therapy. However, it is important to notice that no apoptotic cells could be found in bLF-treated cancer cells. The use of higher bLF concentrations (12.5 μM, 50 μM, 125 μM and 175 μM) was expected to exhibit different effects on the breast cancer cells as compared with the low concentrations range. In fact, in the high range of concentrations bLF selectively induced cell death by apoptosis in MCF-7 cells. The mechanisms of bLF-induced apoptosis included the intrinsic pathway since it was observed the mitochondrial membrane depolarization and a decrease in Bcl-2 levels. In addition, bLF also induced significantly the cell cycle arrest of these cells at the G1 phase, while the same concentration of another protein source (bovine serum albumin - BSA) did not affected significantly the cells. This suggests that bLF cytotoxicity is not due to the addition of great amounts of exogenous proteins in the cell microenvironment. The western bolt analysis confirmed that bLF blocked the cell cycle progression by adjusting cell cycle related regulators, such as CDC25c. Additionally, bLF showed a clear inhibitory effect on the MCF-7 cells ability to form colonies, which is one of the favorite features of anticancer drugs for preventing metastasis. We also found that the promoting effect on the migration of MCF-7 cells may be due to the fact that bLF changes cell microenvironment positively for cell migration similarly to BSA. The results gathered in this thesis demonstrated the potential of bLF as an anticancer agent and provided some new insights on its mechanisms of action. However, further work is still required before bLF can be considered for clinical applications. Being a food-derived protein, bLF is commonly consumed in the daily life, as well as in supplements for health care. Nevertheless, the relation between its consumption and cancer prevention remains to be elucidated.
A Lactoferrina (LF) é uma proteína com alta afinidade para ligação ao ferro, predominantemente encontrada nas secreções dos mamíferos. Esta proteína e as suas variantes têm vindo a ser propostas como agentes interessantes para a terapia do cancro. Estudos anteriores mostraram que a LF e os seus péptidos inibem a proliferação de células cancerígenas. No entanto, os mecanismos detalhados pelos quais a LF exerce o seu efeito são pouco conhecidos. Além disso, há poucos estudos sobre os efeitos da LF em células de cancro da mama, que constitui um dos tumores malignos mais comuns a nível mundial. Neste sentido, na presente tese pretendeu-se estudar a citotoxicidade da lactoferrina de origem bovina (bLF) e das suas variantes contra várias linhas celulares de cancro da mama, nomeadamente T-47D, MDA-MB-231, Hs578T e MCF-7. Os resultados mostraram que a bLF em concentrações de 1,875 μM , 3,75 μM, 7,5 μM, 15 μM e 30 μM inibe eficientemente o crescimento de células cancerígenas, mas apresenta um efeito muito pouco pronunciado nas células normais da mama (MCF-10-2A). Além disso, as suas variantes (apo-bLF, holo-bLF e LfcinB17-41) também foram capazes de inibir o crescimento das células cancerígenas, exceto o péptido LfcinB26-36. Adicionalmente, a bLF, apo-bLG e holo-bLF não promoveram a proliferação das células cancerígenas a baixas concentrações (0,25 μM , 0,5 μM e 1 μM) tal como foi relatado para outras linhas celulares. Adicionalmente, excluiu-se a possibilidade de que os efeitos anti-cancerígenos da bLF possam ser devidos aos seus péptidos resultantes da degradação da proteína sob as condições de cultura das células. Por outro lado, verificou-se que a maior parte da bLF é bloqueada no exterior das células, apesar de uma pequena quantidade internalizar a célula para o espaço citoplasmático. O péptido LfcinB17-41 também conseguiu penetrar a membrana celular mas não o núcleo. Subsequentemente, verificou-se que os efeitos inibidores da bLF sobre as células cancerígenas da mama resultaram da paragem do ciclo celular, sem se ter observado um efeito na morte celular por apoptose. Dependendo das linhas celulares, esta paragem do ciclo celular induzida pela bLF ocorreu em diferentes fases. No entanto, não foi possível associar as vias de sinalização MAPK/ERK e PI3K/AKT ao efeito observado no ciclo celular. Por outro lado, o efeito anti-cancerígeno da bLF foi associado a um aumento da fosforilação da AMPKα e a uma diminuição dos níveis de mTOR e da sua fosforilação. Esta é a primeira vez que esta via foi associada aos mecanismos subjacentes à citotoxicidade da bLF contra células de cancro. Estes resultados sugerem que a bLF poderá ser uma nova droga, cujo alvo é a proteína mTOR, a explorar na terapia do cancro. No entanto, é importante notar que não se observaram células apoptóticas em nenhuma das linhas celulares tratadas com bLF. Aquando da utilização de concentrações de bLF mais elevadas (12,5 μM, 50 μM, 125 μM e 175 μM) esperava-se que as mesmas exibissem diferentes efeitos sobre as células cancerígenas comparativamente com a gama de baixas concentrações. Na verdade, para concentrações de bLF elevadas observou-se uma indução selectiva de morte celular por apoptose nas células MCF-7. Os mecanismos de apoptose induzida pela bLF incluiram a via intrínseca no sentido em que se detectou a despolarização da membrana mitocondrial e a diminuição dos níveis de Bcl-2. Adicionalmente, a bLF também induziu significativamente a paragem do ciclo celular destas células na fase G1, enquanto que uma concentração similar de outra proteína (albumina do soro bovino - BSA) não afectou significativamente as células. Isto sugere que a citotoxicidade da bLF não é devida ao facto de se adicionarem grandes quantidades de proteína exógena ao microambiente celular. Pela técnica de Western blot confirmou-se que a bLF bloqueia a progressão do ciclo celular, tal como mostrou a diminuição dos níveis da proteína CDC25c. Por outro lado, a bLF mostrou um efeito inibidor evidente sobre a células MCF-7 no que se refere à sua capacidade para formar colónias, o que constitui uma das características desejadas em fármacos anti-cancerígenos para prevenir as metástases. O efeito promotor da bLF sobre a migração de células MCF-7 pode ser devido ao facto desta proteína modificar o microambiente celular de uma forma positiva para a migração celular, tal como se observou com a utilização de BSA. Os resultados obtidos nesta tese demonstraram o potencial da bLF como agente anti-cancerígeno e permitiram esclarecer possíveis mecanismos envolvidos na sua atividade. Todavia, é ainda necessário conduzir mais trabalho de investigação antes que bLF possa ser considerada para aplicações clínicas. Sendo uma proteína derivada de alimentos, a bLF é vulgarmente consumida na alimentação humana, bem como em suplementos para a saúde. No entanto, a relação entre o seu consumo e a prevenção do cancro continua por elucidar.

Thesis (M.S.)--University of Wisconsin--Madison, 1996.
Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 56-60).

碩士
東海大學
食品科學系
95
In this study, lactoferrin hydrolysate (bLFH) was prepared by pepsin cleavage of bovine lactoferrin (bLF) at 37°C and pH 2.5, and the purposes of this study are to investigate the anti-microbial and anti-oxidative properties of bLFH and EDTA. The study was divided into three parts. The first part is to investigate the antimicrobial activities of bLF and bLFH solution. Preparing bLFH solution with different hydrolyzing time (0, 10, 20, 30, 40, 50 min and 1, 2, 3, 4, 5, 6 h) to investigate their degree of hydrolysis (DH). Results indicted that the DH of bLFH solution would be increased while hydrolyzing time increasing. After 4 h (DH was 6.88%), the DH was not changed significantly. The SDS-PAGE of bLFH showed that increased in hydrolyzing time, 2.55 kD polypeptides within bLFH solution would be increased. bLFH solution hydrolyzed for 4 hours contained more polypeptides with low molecular weight. The minimal inhibitory concentrations (MICs) of bLF and bLFH solution hydrolyzed for 4 hours toward Staphylococcus aureus ATCC 9144 were 0.4 mg bLF/mL and 0.2 mg bLFH/mL, respectively. bLF showed no antibacterial activity to Pseudomonas aeruginosa ATCC 14207, but the MIC of bLFH solution to this strain is 1.6 mg bLFH/mL. The antibacterial activity of bLFH solution over 4 h-reaction would be increased slightly but there was no significant difference with bLFH solution hydrolyzed for 4 hours. The second part is to investigate the microbial and chemical properties of non-vacuum-package ground hams added 250 ppm EDTA and/or 4 mg bLFH/g meat and all of them named EDTA treatment, bLFH treatment and bLFH+EDTA treatment were stored at 4°C for 0, 2, 4 and 6 days. Results indicted that bLFH had pro-oxidative action. And perhaps owing to EDTA the TBARS value of bLFH+EDTA treatment was the lowest. The nonheme iron of bLFH treatment was more than control, but bLFH+EDTA treatment and control were not significantly different. The nonheme iron of EDTA treatment was the least. The total plate counts, lactic acid bacterial counts and coliforms in all treatments were lower than control which indicted that adding bLFH and/or EDTA could inhibit the growth of microbe. The third part is to investigate the microbial properties of non-vacuum-package Chinese meat balls added 250 ppm EDTA and/or 4 mg bLFH/g meat and all of them named EDTA treatment, bLFH treatment and bLFH+EDTA treatment were stored at 7°C for 0, 2, 4, 6 and 8 days. The results showed that bLFH did not have any antioxidative activity. And perhaps owing to EDTA the TBARS value of bLFH+EDTA treatment was the lowest The total plate counts and the lactic acid bacterial counts in all treatments were lower than control which indicted that adding bLFH and/or EDTA into cooked meat products could also inhibit the growth of microbe.