Discovery of Lactoferrin as A Stimulant for hADSC-Derived EV Secretion and Proof of Enhancement of Resulting EVs through Skin Model

Extracellular vesicles (EVs) are secreted from hADSCs in low concentrations, which makes it difficult to utilize them for the development of therapeutic products. To overcome the problem associated with low concentration, we proposed human lactoferrin (hLF) as a stimulant for the secretion of hADSC-derived EVs. hLF has been reported to upregulate intracellular Ca2+, which is known to be capable of increasing EV secretion. We cultured hADSCs in hLF-supplemented media and analyzed the changes in intracellular Ca2+ concentration. The characteristics of hADSC-derived EVs secreted by hLF stimulation were analyzed through their number, membrane protein markers, and the presence of hLFs to EVs. The function of hADSC-derived EVs was investigated through their effects on dermal fibroblasts. We found that hLF helped hADSCs effectively uptake Ca2+, resulting in an increase of EVs secretion by more than a factor of 4. The resulting EVs had enhanced proliferation and collagen synthesis effect on dermal fibroblasts when compared to the same number of hADSC-derived EVs secreted without hLF stimulation. The enhanced secretion of hADSC-derived EVs increased collagen synthesis through enhanced epidermal penetration, which resulted from increased EV numbers. In summary, we propose hLF to be a useful stimulant in increasing the secretion rate of hADSC-derived EVs.

New immunoassay systems based on recombinant human lactoferrin

In this work, soluble and solid phase immunoreagents, including recombinant human lactoferrin (rhLF), a complex of rhLF with europium ions, rabbit antiserum to rhLF, anti-rhLF immunoglobulin purified by antigen-affinity chromatography and the conjugates of this immunoglobulin with an Eu3+ chelate or horseradish peroxidase have been obtained by a combination of biochemical and synthetic methods using rhLF as an initial compound. Biospecific interactions of the reagents in four immunochemical systems were assessed by measuring the enzyme activity or time-resolved fluorescence. The study resulted in the development of fast and precise immunoassays for biologically active rhLF in transgenic goat milk and in protein fractions obtained in the course of pure rhLF manufacture, as well as in pharmaceutical preparations and food additives.

ISOLATION AND PURIFICATION OF PROTEIN THAT IS IMMUNOLOGICALLY SIMILAR TO HUMAN LACTOFERRIN

The study aimed at isolating a substance that is immunologically similar to human lactoferrin, hereinafter — microbial-derived lactoferrin (MdLF) isolated from K.pneumoniae liquid culture. Protein extraction started with isolation of ballast proteins in 2M ammonium sulphate (p.a.). Ion chromatography with cation-exchange agents was the basic method used for isolating MLF. Proteins from the isolated protein fractions were identified with AGID method. The isolated MLF is glucoprotein with the molecular mass of about 84,000, the agarose diffusion coefficient of 3.1 × 10–7 cm2 sec–1 and the relative electrophoretic mobility of 0.42. MLF is characterized by low hydrophobicity and elutes from phenylSepharose with 0.4 ammonium sulphate. Its isoelectric point equals to 9.2.

Human lactoferrin enhances the expression of transcription factor c-Fos in neuronal cultures under stimulated conditions

Целью настоящей работы стало исследование влияния лактоферрина (Лф) человека на экспрессию транскрипционного фактора c-Fos в первичных нейрональных культурах после физиологической стимуляции, определение клеточной локализации Лф человека и возможной колокализации экзогенного белка с индуцированной экспрессией c-Fos. Методы. Первичные диссоциированные клеточные культуры получали из гиппокампа головного мозга новорожденных мышей (Р0-Р1) линии С57Вl/6. Индукцию экспрессии белка c-Fos в клетках осуществляли путем трехкратного добавления 50 мМ KСl в культуральную среду на 8-й день культивирования in vitro. Анализ содержания c-Fos проводили иммунофлюоресцентным методом через 2 часа после стимуляции. Результаты. Лф детектировался как в цитоплазме, так и в ядрах отдельных клеток культуры после стимуляции KСl. В ядрах некоторых клеток была выявлена колокализация включения Лф и экспрессии c-Fos. Было обнаружено, что предварительное введение Лф в культуральную среду увеличивало количество клеток, экспрессирующих c-Fos после добавления 50 мМ KСl. The aims of this research were 1) to study the effect of human lactoferrin (Lf) on the expression of the c-Fos transcription factor in primary neuronal cultures after physiological stimulation; 2) to determine the cellular localization of human Lf and possible colocalization of an exogenous protein with induced c-Fos expression. Methods. Primary dissociated cell cultures were obtained from the hippocampus of newborn C57Bl/6 mice (P0-P1). The expression of c-Fos was induced by addition of 50 mM KCl to the culture medium at 8 day in vitro. c-Fos content was analyzed by immunofluorescence 2 hrs after stimulation. Results. Lf was detected in cytoplasm and in nuclei after stimulation KCl. Lf inclusion and c-Fos expression were colocalized in the nuclei of some cells. Thus, results showed that pretreatment with Lf led to increase in the number of cells expressing c-Fos after exposure to 50 mM KCl.

Influence of the season and lactation on the milk composition of goats-producers of biosimilar human lactoferrin

The seasonal variability of the physicochemical parameters of milk of both ordinary goats and goats producers of a biosimilar human lactoferrin has been established. The average values of the mass fraction of fat, protein, lactose, nonfat milk solids, density index in the second and third quarters were lower than in the first and fourth quarters. At the same time, the difference between the spring-summer and autumn-winter periods in the mass fraction of fat was 25%. In the mass fraction of protein, lactose, nonfat milk solids, density it was 5–7%. Comparative analysis of milk from goats-producers of a biosimilar human lactoferrin of various lactations revealed a number of changes in the physicochemical composition of raw materials in comparison with non-transgenic animals: an increase in the mass fraction of protein by 4–6% (P˂0.05), of lactose by 2–6 (P˂0.05), nonfat milk solids by 1–6, density by 2–5% (P˂0.05), freezing temperature by 5 and a decrease in the mass fraction of fat by 5–8% (P˂0, 05). At the same time, the indicators of active and titratable acidity of milk were similar in all groups of animals. At the same time, the concentration of human lactoferrin in the milk of producers of the second and third lactation had almost identical seasonal changes in the increase and decrease in the synthesis of lactoferrin in the mammary gland during the year: in the first quarter – 2.88 and 2.97 g/l, in the second – 4.76 and 4.63, in the third – 7.44 and 7.55 and in the fourth – 7.97 and 6.72 with an average annual value of 5.84 and 5.72 g/l, respectively.