Cellular recognition and macropinocytosis-like internalization of nanoparticles targeted to integrin α2ß1.

Targeting nanoparticles to desired intracellular compartments is a major challenge. Integrin-type adhesion receptors are connected to different endocytosis routes in a receptor-specific manner. According to our previous observations, the internalization of an α2ß1-integrin-echovirus-1 complex takes place via a macropinocytosis-like mechanism, suggesting that the receptor could be used to target nanoparticles to this specific entry route. Here, silica-based nanoparticles, carrying monoclonal antibodies against the α2ß1 integrin as address labels, were synthesized. Studies with flow cytometry, atomic force microscopy and confocal microscopy showed the particles to attach to the cell surface via the α2ß1 integrin. Furthermore, quantitative analysis of nanoparticle trafficking inside the cell performed with the BioImageXD software indicated that the particles enter cells via a macropinocytosis-like process and end up in caveolin-1 positive structures. Thus, we suggest that different integrins can guide particles to distinct endocytosis routes and, subsequently, also to specific intracellular compartments. In addition, we show that with the BioImageXD software it is possible to conduct sensitive and complex analyses of the behavior of small fluorescent particles inside cells, using basic confocal microscopy images.

Effects of polyunsaturated fatty acids in growth medium on lipid composition and on physicochemical surface properties of lactobacilli.

Most probiotic lactobacilli adhere to intestinal surfaces, a phenomenon influenced by free polyunsaturated fatty acids (PUFA). The present study investigated whether free linoleic acid, gamma-linolenic acid, arachidonic acid, alpha-linolenic acid, or docosahexaenoic acid in the growth medium alters the fatty acid composition of lactobacilli and their physical characteristics. The most abundant bacterial fatty acids identified were oleic, vaccenic, and dihydrosterculic acids. PUFA, especially conjugated linoleic acid (CLA) isomers and gamma-linolenic, eicosapentaenoic, docosahexaenoic, and alpha-linolenic acids, also were identified in lactobacilli. When lactobacilli were cultured in MRS broth supplemented with various free PUFA, the incorporation of a given PUFA into bacterial fatty acids was clearly observed. Moreover, PUFA supplementation also resulted in PUFA-dependent changes in the proportions of other fatty acids; major interconversions were seen in octadecanoic acids (18:1), their methylenated derivatives (19:cyc), and CLA. Intermittent changes in eicosapentaenoic acid proportions also were noted. These results were paralleled by minor changes in the hydrophilic or hydrophobic characteristics of lactobacilli, suggesting that PUFA interfere with microbial adhesion to intestinal surfaces through other mechanisms. In conclusion, we have demonstrated that free PUFA in the growth medium induce changes in bacterial fatty acids in relation to the regulation of the degree of fatty acid unsaturation, cyclization, and proportions of CLA and PUFA containing 20 to 22 carbons. The potential role of lactobacilli as regulators of PUFA absorption may represent another means by which probiotics could redirect the delicate balance of inflammatory mediators derived from PUFA within the inflamed intestine.

Surface binding of aflatoxin B(1) by lactic acid bacteria.

Specific lactic acid bacterial strains remove toxins from liquid media by physical binding. The stability of the aflatoxin B(1) complexes formed with 12 bacterial strains in both viable and nonviable (heat- or acid-treated) forms was assessed by repetitive aqueous extraction. By the fifth extraction, up to 71% of the total aflatoxin B(1) remained bound. Nonviable bacteria retained the highest amount of aflatoxin B(1). Lactobacillus rhamnosus strain GG (ATCC 53103) and L. rhamnosus strain LC-705 (DSM 7061) removed aflatoxin B(1) from solution most efficiently and were selected for further study. The accessibility of bound aflatoxin B(1) to an antibody in an indirect competitive inhibition enzyme-linked immunosorbent assay suggests that surface components of these bacteria are involved in binding. Further evidence is the recovery of around 90% of the bound aflatoxin from the bacteria by solvent extraction. Autoclaving and sonication did not release any detectable aflatoxin B(1). Variation in temperature (4 to 37 degrees C) and pH (2 to 10) did not have any significant effect on the amount of aflatoxin B(1) released. Binding of aflatoxin B(1) appears to be predominantly extracellular for viable and heat-treated bacteria. Acid treatment may permit intracellular binding. In all cases, binding is of a reversible nature, but the stability of the complexes formed depends on strain, treatment, and environmental conditions.

Polyunsaturated fatty acids in maternal diet, breast milk, and serum lipid fatty acids of infants in relation to atopy.

BACKGROUND: The increased consumption of n-6 polyunsaturated fatty acids (PUFA) has been shown to coincide with the increased prevalence of atopic diseases. We aimed to investigate whether maternal diet and atopic status influence the PUFA composition of breast milk and the serum lipid fatty acids of infants. METHODS: Maternal diet was assessed by a food questionnaire. The PUFA composition of breast milk obtained at 3 months from 20 allergic and 20 healthy mothers and of their infants' (10 atopic and 10 nonatopic/group of mothers) serum lipids was analyzed. RESULTS: Although no differences in maternal PUFA intake were observed, the breast milk of allergic mothers contained less gamma-linolenic acid (18:3 n-6) than that of healthy mothers. Similarly, atopic infants had less gamma-linolenic acid in phospholipids than healthy infants, although n-6 PUFA were elevated in other serum lipid fractions in atopic infants. The serum lipid fatty acids in atopic infants did not correlate with those in maternal breast milk. CONCLUSION: Our results suggest that dietary n-6 PUFA are not as readily transferred into breast milk or incorporated into serum phospholipids, but may be utilized for other purposes, such as eicosanoid precursors, in allergic/atopic individuals. Subsequently, high dietary proportions of n-6 PUFA, or reduced proportions of regulatory PUFA, such as gamma-linolenic acid and n-3 PUFA, may be a risk factor for the development of atopic disease.

Probiotics: effects on immunity.

The gastrointestinal tract functions as a barrier against antigens from microorganisms and food. The generation of immunophysiologic regulation in the gut depends on the establishment of indigenous microflora. This has led to the introduction of novel therapeutic interventions based on the consumption of cultures of beneficial live microorganisms that act as probiotics. Among the possible mechanisms of probiotic therapy is promotion of a nonimmunologic gut defense barrier, which includes the normalization of increased intestinal permeability and altered gut microecology. Another possible mechanism of probiotic therapy is improvement of the intestine's immunologic barrier, particularly through intestinal immunoglobulin A responses and alleviation of intestinal inflammatory responses, which produce a gut-stabilizing effect. Many probiotic effects are mediated through immune regulation, particularly through balance control of proinflammatory and anti-inflammatory cytokines. These data show that probiotics can be used as innovative tools to alleviate intestinal inflammation, normalize gut mucosal dysfunction, and down-regulate hypersensitivity reactions. More recent data show that differences exist in the immunomodulatory effects of candidate probiotic bacteria. Moreover, distinct regulatory effects have been detected in healthy subjects and in patients with inflammatory diseases. These results suggest that specific immunomodulatory properties of probiotic bacteria should be characterized when developing clinical applications for extended target populations.

The influence of polyunsaturated fatty acids on probiotic growth and adhesion.

The establishment of the intestinal microflora, and probiotic bacteria, may control the inflammatory conditions in the gut. As polyunsaturated fatty acids (PUFA) possess antimicrobial activities, they may deter the action of probiotics. We assessed whether free linoleic, gamma-linolenic, arachidonic, alpha-linolenic and docosahexaenoic acids at physiological concentrations in the growth media would influence the growth and adhesion of Lactobacillus GG (probiotic), Lactobacillus casei Shirota (probiotic) and Lactobacillus bulgaricus (dairy strain). Higher concentrations of PUFA (10-40 microg PUFA ml(-1)) inhibited growth and mucus adhesion of all tested bacterial strains, whilst growth and mucus adhesion of L. casei Shirota was promoted by low concentrations of gamma-linolenic acid and arachidonic acid (at 5 microg ml(-1)), respectively. PUFA also altered bacterial adhesion sites on Caco-2 cells. Caco-2 cells grown in the presence of arachidonic acid were less adhered to by all three bacterial strains. Yet, L. casei Shirota adhered better on Caco-2 cells grown in the presence of alpha-linolenic acid. As the adhesion to mucosal surfaces is pivotal in health promoting effects by probiotics, our results indicate that the action of probiotics in the gut may be modulated by dietary PUFA.

Ability of Lactobacillus and Propionibacterium strains to remove aflatoxin B, from the chicken duodenum.

The ability of Lactobacillus rhamnosus strains GG and LC-705 to remove AFB1 from the intestinal luminal liquid medium has been tested in vivo using a chicken intestinal loop technique. In this study, the GG strain of L. rhamnosus decreased AFB1 concentration by 54% in the soluble fraction of the luminal fluid within 1 min. This strain was more efficient in binding AFB1 compared with L. rhamnosus strain LC-705 (P < 0.05) that removed 44% of AFBl under similar conditions. Accumulation of AFB1 into the intestinal tissue was also determined. There was a 74% reduction in the uptake of AFB1 by the intestinal tissue, in the presence of L. rhamnosus strain GG compared with 63% and 37% in the case of Propionibacterium freudenreichii ssp. shermanii JS and L. rhamnosus strain LC-705, respectively. The complexes formed in vitro between either L. rhamnosus strain GG or L. rhamnosus strain LC-705 and AFB1 were stable under the luminal conditions for a period of 1 h.

Binding of aflatoxin B1 alters the adhesion properties of Lactobacillus rhamnosus strain GG in a Caco-2 model.

Lactic acid bacteria have been previously reported to possess antimycotoxigenic activities both in vitro and in vivo. The objective of this study was to investigate the effect of aflatoxin B1 on adhesion capability of Lactobacillus rhamnosus strain GG using a Caco-2 adhesion model. Removal of aflatoxin B1 by L. rhamnosus strain GG reduced the adhesion capability of this strain from 30% to 5%. It is therefore concluded that aflatoxins may influence the adhesion properties of probiotics able to sequester them, and subsequently these bacteria may reduce the accumulation of aflatoxins in the intestine via increased excretion of an aflatoxin-bacteria complex.

Dietary fatty acids and allergy.

The increase in the prevalence of atopic diseases has recently been linked to altered consumption of polyunsaturated fatty acids (PUFAs). As typical Western diets contain almost 10 times more linoleic acid (18:2 omega-6) than alpha-linolenic acid (18:3 omega-3), it is the metabolism of the former that predominates. Subsequently produced arachidonic acid-derived eicosanoids alter the balance of T-helper cells type 1 and type 2 thus favouring the production of immunoglobulin (Ig)E. In atopic subjects, the impact of this excessive eicosanoid production may be further strengthened as a result of changes in cyclic nucleotide metabolism exacerbated by substrate availability. Dietary omega-3 fatty acids can have marked influence on both specific and nonspecific immune responses in modifying eicosanoid production and replacing omega-6 fatty acids in cell membranes. Therefore, it is concluded that careful manipulation of dietary PUFAs may play a key role in the successful management of inflammation associated with atopic diseases.

Physicochemical alterations enhance the ability of dairy strains of lactic acid bacteria to remove aflatoxin from contaminated media.

Lactobacillus rhamnosus GG and Lactobacillus rhamnosus LC-705, previously shown to effectively bind to aflatoxin B1, were subjected to various chemical and physical treatments to examine the effects of these treatments on the binding affinity of these strains toward aflatoxin B1. Treatment of bacterial pellets of both strains with hydrochloric acid significantly (P < 0.05) enhanced the binding ability to bind aflatoxin B1 was also observed when the bacterial pellets were subjected to heat treatment by either autoclaving or boiling at 100 degrees C in a water bath, put the impact of these two treatments was not as effective as the acid treatment. Ethanol, UV radiation, sonication, alkaline, or pH treatments either had not effect or reduced the binding ability of the bacteria.

Ability of dairy strains of lactic acid bacteria to bind a common food carcinogen, aflatoxin B1.

This study was conducted to examine the ability of selected dairy strains of lactic acid bacteria to remove aflatoxin B1 (AFB1) from liquid media. Both Lactobacillus rhamnosus strain GG (LBGG) and L. rhamnosus strain LC-705 (LC705) can significantly (P > 0.05) remove AFB1 when compared with that by other strains of either Gram-positive or Gram-negative bacteria. Removal of AFB1 by LBGG and LC705 was a rapid process with approximately 80% AFB1 removed at 0 hr. Removal of AFB1 by these two strains was both temperature and bacterial concentration dependent.

Determination of complement-mediated killing of bacteria by viability staining and bioluminescence.

Complement-mediated killing of bacteria was monitored by flow cytometric, luminometric, and conventional plate counting methods. A flow cytometric determination of bacterial viability was carried out by using dual staining with a LIVE/DEAD BacLight bacterial viability kit. In addition to the viable cell population, several other populations emerged in the fluorescence histogram, and there was a dramatic decrease in the total cell count in the light-scattering histogram in the course of the complement reaction. To permit luminometric measurements, Bacillus subtilis and Escherichia coli were made bioluminescent by expressing an insect luciferase gene. Addition of substrate after the complement reaction resulted in bioluminescence, the level of which was a measure of the viable cell population. All three methods gave essentially the same killing rate, suggesting that the bacteriolytic activity of serum complement can be measured rapidly and conveniently by using viability stains or bioluminescence. In principle, any bacterial strain can be used for viability staining and flow cytometric analysis. For the bioluminescence measurements genetically engineered bacteria are needed, but the advantage is that it is possible to screen automatically a large number of samples.

The significance of 3H-thymidine degradation in cell culture experiments with special reference to rheumatoid arthritis.

The degradation of 3H-thymidine under various cell culture conditions was analysed. It was found that a half of 3H-thymidine was degraded to 3H-thymine during 24 hours in PHA stimulation of blood lymphocytes. A control culture in which PHA was not added also caused 3H-thymidine degradation. 3H-thymidine degradation was prevented by adding 5-nitrouracil to the incubation medium at a concentration of 0.577 mg/ml. At the same time 5-NU increased 3H-thymidine incorporation into lymphocytes by 47%. 5-NU also eliminated the inhibitory effect of rheumatoid arthritis synovial tissue eluate on PHA stimulation. In addition 5-NU and nonradioactive thymine increased the 3H-thymidine labelling index of fresh rheumatoid arthritis synovial membrane biopsies, and also more of the isotope was accumulated in the individual cells of the membrane. These studies demonstrate that 3H-thymidine degradation is an important phenomenon in cell cultures and that it can be prevented effectively by using 5-nitrouracil with 3H-thymidine.