In Vitro Fermentation of Breast Milk Oligosaccharides by Bifidobacterium infantis and Lactobacillus gasseri

It has been proposed that human milk oligosaccharides (HMO) function as a prebiotic for bifidobacteria, yet this activity has not been adequately investigated. In this study, Bifidobacterium infantis was shown to ferment purified HMO as a sole carbon source, while another gut commensal, Lactobacillus gasseri, did not ferment HMO. Our results support the hypothesis that HMO selectively amplify bacterial populations in the infant intestine.     

Variation in Consumption of Human Milk Oligosaccharides by Infant Gut-Associated Strains of Bifidobacterium breve

Human milk contains a high concentration of complex oligosaccharides that influence the composition of the intestinal microbiota in breast-fed infants. Previous studies have indicated that select species such as Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum can utilize human milk oligosaccharides (HMO) in vitro as the sole carbon source, while the relatively few B. longum subsp. longum and Bifidobacterium breve isolates tested appear less adapted to these substrates. Considering the high frequency at which B. breve is isolated from breast-fed infant feces, we postulated that some B. breve strains can more vigorously consume HMO and thus are enriched in the breast-fed infant gastrointestinal tract. To examine this, a number of B. breve isolates from breast-fed infant feces were characterized for the presence of different glycosyl hydrolases that participate in HMO utilization, as well as by their ability to grow on HMO or specific HMO species such as lacto-N-tetraose (LNT) and fucosyllactose. All B. breve strains showed high levels of growth on LNT and lacto-N-neotetraose (LNnT), and, in general, growth on total HMO was moderate for most of the strains, with several strain differences. Growth and consumption of fucosylated HMO were strain dependent, mostly in isolates possessing a glycosyl hydrolase family 29 α-fucosidase. Glycoprofiling of the spent supernatant after HMO fermentation by select strains revealed that all B. breve strains can utilize sialylated HMO to a certain extent, especially sialyl-lacto-N-tetraose. Interestingly, this specific oligosaccharide was depleted before neutral LNT by strain SC95. In aggregate, this work indicates that the HMO consumption phenotype in B. breve is variable; however, some strains display specific adaptations to these substrates, enabling more vigorous consumption of fucosylated and sialylated HMO. These results provide a rationale for the predominance of this species in breast-fed infant feces and contribute to a more accurate picture of the ecology of the developing infant intestinal microbiota.     

Probiotic Strains Influence on Infant Microbiota in the In Vitro Colonic Fermentation Model GIS1

The main goal of our study was to evaluate the effect of the individual administration of five lyophilized lactic acid bacteria strains (Lactobacillus fermentum 428ST, Lactobacillus rhamnosus E4.2, Lactobacillus plantarum FCA3, Lactobacillus sp. 34.1, Weissella paramesenteroides FT1a) against the in vitro simulated microbiota of the human colon using the GIS1 system. The influence on the metabolic activity was also assessed by quantitative determination of proteins and polysaccharides at each segment of human colon. The obtained results indicated that the lactic acid bacteria L. rhamnosus E4.2 and W. paramesenteroides FTa1 had better efficiency in synthesising exopolysaccharides and also a better probiotic potential and therefore could be recommended for use in probiotics products or food industry.     

Ability of Human Leukocytic Pyrogen to Stimulate Brain Prostaglandin Synthesis In Vitro

Abstract: Fever is thought to be mediated by leukocytic pyrogen (LP), a polypeptide synthesized by phagocytic leukocytes and which is responsible for the upwards resetting of the hypothalamic thermostat. In an attempt to study the effects of LP directly on brain tissue, purified human LP was incubated with rabbit brain slices in vitro. Because of the well-documented role of prostaglandin (PG) synthesis in both the production of fever and antipyresis, PGE levels were measured on the supernates of brain slices incubated 30 min with LP. Levels of PGE increased 3- to 4-fold in rabbit anterior and posterior hypothalami. In addition, PGE levels were similarly increased in temporal cortex slices when exposed to LP. In another set of experiments, PGE levels increased 4- to 5-fold when brain tissue was incubated with a highly purified preparation of bacterial endotoxin (ET). The ability of ET to increase brain PGE levels was not affected by moderate heating (56°C, 30 min), whereas this temperature destroyed the PGE-inducing properties of LP. The antipyretic ibuprofen markedly reduced the amount of PGE measured in the brain slice supernates after stimulation with LP, suggesting that LP brings about synthesis of PGE and not the release of preformed PG. The results demonstrate that LP is a potent inducer of PGE synthesis in rabbit brain and that receptors for LP are not restricted to the thermoregulatory center, but rather may be distributed throughout the brain.     

In Vitro Fermentation of Oat Bran Obtained by Debranning with a Mixed Culture of Human Fecal Bacteria

The prebiotic potential of oat samples was investigated by in vitro shaker-flask anaerobic fermentations with human fecal cultures. The oat bran fraction was obtained by debranning and was compared with other carbon sources such as whole oat flour, glucose, and fructo-oligosaccharide. The oat bran fraction showed a decrease in culturable anaerobes and clostridia and an increase in bifidobacteria and lactobacilli populations. A similar pattern was observed in fructo-oligosaccharide. Butyrate production was higher in oat bran compared to glucose and similar to that in fructo-oligosaccharide. Production of propionate was higher in the two oat media than in fructo-oligosaccharide and glucose, which can be used as energy source by the liver. This study suggests that the oat bran fraction obtained by debranning is digested by the gut ecosystem and increases the population of beneficial bacteria in the indigenous gut microbiota. This medium also provides an energy source preferred by colonocytes when it is metabolized by the gut flora.     

Screening for and Identification of Starch-, Amylopectin-, and Pullulan-Degrading Activities in Bifidobacterial Strains

Forty-two bifidobacterial strains were screened for α-amylase and/or pullulanase activity by investigating their capacities to utilize starch, amylopectin, or pullulan. Of the 42 bifidobacterial strains tested, 19 were capable of degrading potato starch. Of these 19 strains, 11 were able to degrade starch and amylopectin, as well as pullulan. These 11 strains, which were shown to produce extracellular starch-degrading activities, included 5 strains of Bifidobacterium breve, 1 B. dentium strain, 1 B. infantis strain, 3 strains of B. pseudolongum, and 1 strain of B. thermophilum. Quantitative and qualitative enzyme activities were determined by measuring the concentrations of released reducing sugars and by high-performance thin-layer chromatography, respectively. These analyses confirmed both the inducible nature and the extracellular nature of the starch- and pullulan-degrading enzyme activities and showed that the five B. breve strains produced an activity that is consistent with type II pullulanase (amylopullulanase) activity, while the remaining six strains produced an activity with properties that resemble those of type III pullulan hydrolase.     

Distribution of Bifidobacterial Species in Human Intestinal Microflora Examined with 16S rRNA-Gene-Targeted Species-Specific Primers

In order to clarify the distribution of bifidobacterial species in the human intestinal tract, a 16S rRNA-gene-targeted species-specific PCR technique was developed and used with DNAs extracted from fecal samples obtained from 48 healthy adults and 27 breast-fed infants. To cover all of the bifidobacterial species that have been isolated from and identified in the human intestinal tract, species-specific primers for Bifidobacterium longum, B. infantis, B. dentium, and B. gallicum were developed and used with primers for B. adolescentis, B. angulatum, B. bifidum, B. breve, and the B. catenulatum group (B. catenulatum and B. pseudocatenulatum) that were developed in a previous study (T. Matsuki, K. Watanabe, R. Tanaka, and H. Oyaizu, FEMS Microbiol. Lett. 167:113–121, 1998). The specificity of the nine primers was confirmed by PCR, and the species-specific PCR method was found to be a useful means for identifying Bifidobacterium strains isolated from human feces. The results of an examination of bifidobacterial species distribution showed that the B. catenulatum group was the most commonly found taxon (detected in 44 of 48 samples [92%]), followed by B. longum and B. adolescentis, in the adult intestinal bifidobacterial flora and that B. breve, B. infantis, and B. longum were frequently found in the intestinal tracts of infants. The present study demonstrated that qualitative detection of the bifidobacterial species present in human feces can be accomplished rapidly and accurately.     

In Vitro Characterization of Lactic Acid Bacteria Isolated from Bovine Milk as Potential Probiotic Strains to Prevent Bovine Mastitis

Probiotics and Antimicrobial Proteins - Bovine mastitis causes economic losses on dairy farms worldwide. Lactic acid bacteria (LAB) in animal health are an alternative tool to avoid antibiotic...     

Direct Evidence for the Presence of Human Milk Oligosaccharides in the Circulation of Breastfed Infants

Background

It has been hypothesized that human milk oligosaccharides (HMOs) confer systemic health benefits to breastfed infants; however, plausible mechanisms for some effects, such as systemic immunomodulation, require HMOs to access the bloodstream of the developing infant. While small concentrations of HMOs have been detected in the urine of breastfed infants there are no published studies of these oligosaccharides accessing the plasma compartment of breastfed infants. Here we determined the relative fractions of several ingested HMOs in infant urine and plasma. Plasma from formula-fed infants was used as a control.

Methods

Using gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry/tandem mass spectrometry (LC/MS/MS), and high performance liquid chromatography (HPLC), we analyzed the urine and plasma from 17 healthy formula-fed infants and 16 healthy breast-fed infants (and the milk from their mothers).

Results

Multiple HMOs were detected in the urine and plasma of breastfed infants, but not in formula-fed infants. Levels of 2′-fucosyllactose (2′FL), 3FL and lacto-N-neotetraose (LNnT) in both plasma (r = 0.98, p<0.001; r = 0.75, p = 0.002; r = 0.71, p = 0.004) and urine (r = 0.81, p<0.001; r = 0.56, p = 0.026; NS) correlated significantly with concentrations in the corresponding breast milk. The relative fractions of HMOs were low, 0.1% of milk levels for plasma and 4% of milk levels for urine. Within the breastfed cohort, there were significant differences between secretor and nonsecretor groups in levels of several fucosylated HMOs.

Conclusion

At least some ingested HMOs are absorbed intact into the circulation and excreted in the urine and their concentrations in these fluids correlate with levels of the corresponding mother''s milk. While relative fractions of absorbed HMOs were low, these levels have been shown to have biological effects in vitro, and could explain some of the postulated benefits of human milk.     

Syndecan-4 Is a Major Syndecan in Primary Human Endothelial Cells In Vitro,Modulated by Inflammatory Stimuli and Involved in Wound Healing

Syndecans are important cell surface proteoglycans with many functions; yet, they have not been studied to a very large extent in primary human endothelial cells. The purpose of this study was to investigate syndecan-4 expression in cultured human umbilical vein endothelial cells (HUVECs) and assess its role in inflammatory reactions and experimental wound healing. qRT-PCR analysis revealed that syndecan-3 and syndecan-4 were highly expressed in HUVECs, whereas the expression of syndecan-1 and -2 was low. HUVECs were cultured with the inflammatory mediators lipopolysaccharide (LPS) and interleukin 1β (IL-1β). As a result, syndecan-4 expression showed a rapid and strong increase. Syndecan-1 and -2 expressions decreased, whereas syndecan-3 was unaffected. Knockdown of syndecan-4 using siRNA resulted in changes in cellular morphology and focal adhesion sites, delayed wound healing and tube formation, and increased secretion of the pro-inflammatory and angiogenic chemokine, CXCL8. These data suggest functions for syndecan-4 in inflammatory reactions, wound healing and angiogenesis in primary human endothelial cells.     

Age Distribution of Human Diploid Fibroblasts: A Stochastic Model for In Vitro Aging

Variation in the lifespan of mass cultures and clones of human diploid fibroblasts can be explained on the basis of variation in the length of the mitotic cycle. This variation is of biological significance; the intrinsic standard deviation of culture lifespan is equal to about 10% of the mean. We constructed a two-parameter stochastic model based on the following assumptions: the time between successive divisions of a given cell is of random duration; cells divide or lose the ability to divide independently of one another; the probability that a cell can undergo further division is constant up to some maximum number of divisions and zero thereafter. We determined numerically the proportion of nondividing cells and the distribution of cell generations. Samples taken by Monte Carlo means from a hypothetical in vitro population were compared with clonal survival data obtained experimentally. The fit between experimental and theoretical findings was within the range of sampling variation. If we accept our model as being applicable to human diploid cell culture, we can draw the following conclusions: the proportion of dividing cells is an inadequate index of a population's age; even in populations in which almost all cells are still capable of division, a majority of the cells have less than eight generations remaining to them. At each subcultivation the ultimate fate of a culture is determined by the disposition of a relatively small number of “young” cells.     

Screening of Probiotic Activities of Forty-Seven Strains of Lactobacillus spp. by In Vitro Techniques and Evaluation of the Colonization Ability of Five Selected Strains in Humans

The probiotic potential of 47 selected strains of Lactobacillus spp. was investigated. The strains were examined for resistance to pH 2.5 and 0.3% oxgall, adhesion to Caco-2 cells, and antimicrobial activities against enteric pathogenic bacteria in model systems. From the results obtained in vitro, five strains, Lactobacillus rhamnosus 19070-2, L. reuteri DSM 12246, L. rhamnosus LGG, L. delbrueckii subsp. lactis CHCC 2329, and L. casei subsp. alactus CHCC 3137, were selected for in vivo studies. The daily consumption by 12 healthy volunteers of two doses of 10¹⁰ freeze-dried bacteria of the selected strains for 18 days was followed by a washout period of 17 days. Fecal samples were taken at days 0 and 18 and during the washout period at days 5 and 11. Lactobacillus isolates were initially identified by API 50CHL and internal transcribed spacer PCR, and their identities were confirmed by restriction enzyme analysis in combination with pulsed-field gel electrophoresis. Among the tested strains, L. rhamnosus 19070-2, L. reuteri DSM 12246, and L. rhamnosus LGG were identified most frequently in fecal samples; they were found in 10, 8, and 7 of the 12 samples tested during the intervention period, respectively, whereas reisolations were less frequent in the washout period. The bacteria were reisolated in concentrations from 10⁵ to 10⁸ cells/g of feces. Survival and reisolation of the bacteria in vivo appeared to be linked to pH tolerance, adhesion, and antimicrobial properties in vitro.     

人关节软骨细胞的体外分离、培养与鉴定

目的:研究人关节软骨细胞的体外分离、培养及鉴定方法,观察各代人关节软骨细胞的形态学特性。方法:取人创伤性截肢的无菌膝关节软骨,采用两步酶消化法分离培养人关节软骨细胞,并进行传代培养。通过倒置相差显微镜下观察细胞形态,绘制生长曲线,甲苯胺蓝染色及Ⅱ型胶原免疫组织化学染色对细胞进行鉴定。结果:两步酶消化法消化出的软骨细胞呈圆形,培养2-3天,细胞贴壁、变形,呈三角形或多角形,2周左右细胞融合成层,传代5次后出现去分化。软骨细胞增殖和生长缓慢。形态学、免疫组织化学染色显示细胞培养5代以内可以保持表型的稳定。结论:本研究采用胰蛋白酶及Ⅱ型胶原酶联合消化法获得大量高纯度、高活性的人关节软骨细胞。5代以内细胞生长良好,生物学特性明显,适合于实验研究,5代以后出现去分化现象。     

In Vitro Activity of Actinospectacin in Human Whole Blood

A method for testing antibacterial substances in whole blood is described. The test agent for the method was actinospectacin which reportedly has good in vivo activity, approximately in the range with chloramphenicol, but relatively poor in vitro activity in the common media. In human whole blood, however, the in vitro activity compares favorably with chloramphenicol thus indicating that whole blood may predict in vivo activity better than the usual bacteriological media.     

医学院校生物技术专业发酵工程实验体系的构建

为建设有特色的生物技术专业和培养创新复合型人才,本研究结合医学院校特点,充分利用"医"和"药"的资源,合理安排实验项目,构建了科学的发酵工程实验教学体系。结果表明该实验教学体系教学效果良好。学生的操作能力显著提高。为强化发酵工程教学,探索产学研联合培养模式将成必然。     

Human T Lymphocyte Isolation,Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration ¹. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility ². Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes ³. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 μm/min. T lymphocyte migration can be inhibited by integrin blockade ¹ or by inhibitors of the cellular actomyosin machinery that regulates cell migration ².Download video file.^{(98M, mp4)}     

In Vitro Comparison of the Effects of Probiotic,Commensal and Pathogenic Strains on Macrophage Polarization

Macrophages are important with respect to both innate and adaptive immune responses and are known to differentiate into pro-inflammatory M1- or anti-inflammatory M2-phenotypes following activation. In order to study how different bacteria affect macrophage polarization, we exposed murine RAW 264.7 macrophages to sixteen different strains representing probiotic strains, pathogens, commensals and strains of food origin. Increased inducible nitric oxide synthase (iNOS) or arginase-1 gene expression indicates M1 or M2 polarization, respectively, and was quantified by qRT-PCR. Strains of Escherichia and Salmonella elevated iNOS expression more so than strains of Enterococcus, Lactobacillus and Lactococcus, indicating that Gram-negative strains are more potent M1 inducers. However, strain-specific responses were observed. For instance, Escherichia coli Nissle 1917 was a poor inducer of iNOS gene expression compared to the other E. coli strains, while Enterococcus faecalis Symbioflor-1 was more potent in this respect compared to all the eleven Gram-positive strains tested. Macrophage polarization was further characterized by quantifying secreted pro- and anti-inflammatory cytokines. Exposure to the pathogen E. coli 042 produced a cytokine profile indicating M1 differentiation, which is in accordance with the PCR data. However, exposure to most strains resulted in either high or low secretion levels of all cytokines tested, rather than a clear M1 or M2 profile. In general, the Gram-negative strains induced high levels of cytokine secretion compared to the Gram-positive strains. Interestingly, strains of human origin had a higher impact on macrophages compared to strains of food origin.