Pasi Kankaanpää

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 intestines 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.

Surface Binding of Aflatoxin B1 by Lactic Acid Bacteria

ABSTRACT Specific lactic acid bacterial strains remove toxins from liquid media by physical binding. The stability of the aflatoxin B1 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 B1 remained bound. Nonviable bacteria retained the highest amount of aflatoxin B1.Lactobacillus rhamnosus strain GG (ATCC 53103) and L. rhamnosus strain LC-705 (DSM 7061) removed aflatoxin B1 from solution most efficiently and were selected for further study. The accessibility of bound aflatoxin B1 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 B1. Variation in temperature (4 to 37°C) and pH (2 to 10) did not have any significant effect on the amount of aflatoxin B1 released. Binding of aflatoxin B1 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.

Ability of Lactobacillus and Propionibacterium Strains to Remove Aflatoxin B1 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.

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.

Internalization of novel non-viral vector TAT-streptavidin into human cells

BackgroundThe cell-penetrating peptide derived from the Human immunodeficiency virus-1 transactivator protein Tat possesses the capacity to promote the effective uptake of various cargo molecules across the plasma membrane in vitro and in vivo. The objective of this study was to characterize the uptake and delivery mechanisms of a novel streptavidin fusion construct, TAT47–57-streptavidin (TAT-SA, 60 kD). SA represents a potentially useful TAT-fusion partner due to its ability to perform as a versatile intracellular delivery vector for a wide array of biotinylated molecules or cargoes.ResultsBy confocal and immunoelectron microscopy the majority of internalized TAT-SA was shown to accumulate in perinuclear vesicles in both cancer and non-cancer cell lines. The uptake studies in living cells with various fluorescent endocytic markers and inhibiting agents suggested that TAT-SA is internalized into cells efficiently, using both clathrin-mediated endocytosis and lipid-raft-mediated macropinocytosis. When endosomal release of TAT-SA was enhanced through the incorporation of a biotinylated, pH-responsive polymer poly(propylacrylic acid) (PPAA), nuclear localization of TAT-SA and TAT-SA bound to biotin was markedly improved. Additionally, no significant cytotoxicity was detected in the TAT-SA constructs.ConclusionThis study demonstrates that TAT-SA-PPAA is a potential non-viral vector to be utilized in protein therapeutics to deliver biotinylated molecules both into cytoplasm and nucleus of human cells.

BioImageXD: an open, general-purpose and high-throughput image-processing platform

BioImageXD puts open-source computer science tools for three-dimensional visualization and analysis into the hands of all researchers, through a user-friendly graphical interface tuned to the needs of biologists. BioImageXD has no restrictive licenses or undisclosed algorithms and enables publication of precise, reproducible and modifiable workflows. It allows simple construction of processing pipelines and should enable biologists to perform challenging analyses of complex processes. We demonstrate its performance in a study of integrin clustering in response to selected inhibitors.

Effects of Polyunsaturated Fatty Acids in Growth Medium on Lipid Composition and on Physicochemical Surface Properties of Lactobacilli

ABSTRACT Most probiotic lactobacilli adhere to intestinal surfaces, a phenomenon influenced by free polyunsaturated fatty acids (PUFA). The present study investigated whether free linoleic acid, γ-linolenic acid, arachidonic acid, α-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 γ-linolenic, eicosapentaenoic, docosahexaenoic, and α-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.

A Raft-derived, Pak1-regulated Entry Participates in α2β1 Integrin-dependent Sorting to Caveosomes

We have previously shown that a human picornavirus echovirus 1 (EV1) is transported to caveosomes during 2 h together with its receptor alpha2beta1 integrin. Here, we show that the majority of early uptake does not occur through caveolae. alpha2beta1 integrin, clustered by antibodies or by EV1 binding, is initially internalized from lipid rafts into tubulovesicular structures. These vesicles accumulate fluid-phase markers but do not initially colocalize with caveolin-1 or internalized simian virus 40 (SV40). Furthermore, the internalized endosomes do not contain glycosylphosphatidylinositol (GPI)-anchored proteins or flotillin 1, suggesting that clustered alpha2beta1 integrin does not enter the GPI-anchored protein enriched endosomal compartment or flotillin pathways, respectively. Endosomes mature further into larger multivesicular bodies between 15 min to 2 h and concomitantly recruit caveolin-1 or SV40 inside. Cell entry is regulated by p21-activated kinase (Pak)1, Rac1, phosphatidylinositol 3-kinase, phospholipase C, and actin but not by dynamin 2 in SAOS-alpha2beta1 cells. An amiloride analog, 5-(N-ethyl-N-isopropanyl) amiloride, blocks infection, causes integrin accumulation in early tubulovesicular structures, and prevents their structural maturation into multivesicular structures. Our results together suggest that alpha2beta1 integrin clustering defines its own entry pathway that is Pak1 dependent but clathrin and caveolin independent and that is able to sort cargo to caveosomes.

Influence of probiotic supplemented infant formula on composition of plasma lipids in atopic infants

Probiotic therapy is a new, successful approach to alleviating allergic symptoms. In this study, our aim was to investigate whether the positive results obtained with probiotic therapy would be associated with the differential absorption and utilization of dietary PUFA. 15 infants referred to a pediatric clinic on the basis of atopic eczema were weaned to Bifidobacterium Bb-12 or Lactobacillus GG supplemented infant formula, or to the same formula without probiotics (randomized, placebo-controlled, double blind study design). In plasma neutral lipids, alpha-linolenic acid (18:3 n-3) proportions were reduced by the probiotic supplementation. In phospholipids, Lactobacillus GG supplemented formula did not influence alpha-linolenic acid proportions, while Bifidobacterium Bb-12 supplemented formula increased the proportion of alpha-linolenic acid; from 0.13 +/- 0.03 to 0.24 +/- 0.03 (mean +/- SEM) (P = 0.002). These results show that some physiological effects of probiotics may be associated with physiological interactions between probiotics and dietary PUFA.

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.