Immunomodulation of monocytes by probiotic and selected lactic Acid bacteria.

Some lactic acid bacteria (LAB), especially bacteria belonging to the genus Lactobacillus, are recognized as common inhabitants of the human gastrointestinal tract and have received considerable attention in the last decades due to their postulated health-promoting effects. LAB and probiotic bacteria can modulate the host immune response. However, much is unknown about the mediators and mechanisms responsible for their immunological effect. Here, we present a study using cytokine secretion from the monocytic cell line THP-1 and NF-κB activation in the monocytic cell line U937-3xkB-LUC to elucidate immune stimulating abilities of LAB in vitro. In this study, we investigate both commercially available and potential probiotic LAB strains, and the role of putative surface proteins of L. reuteri using mutants. L. reuteri strains induced the highest cytokine secretion and the highest NF-κB activation, whereas L. plantarum strains and L. rhamnosus GG were low inducers/activators. One of the putative L. reuteri surface proteins, Hmpref0536_10802, appeared to be of importance for the stimulation of THP-1 cells and the activation of NF-κB in U937-3xkB-LUC cells. Live and UV-inactivated preparations resulted in different responses for two of the strains investigated. Our results add to the complexity in the interaction between LAB and human cells and suggest the possible involvement of secreted pro- and anti-inflammatory mediators of LAB. It is likely that it is the sum of bacterial surface proteins and bacterial metabolites and/or secreted proteins that induce cytokine secretion in THP-1 cells and activate NF-κB in U937-3xkB-LUC cells in this study.

Role of Lactobacillus reuteri cell and mucus-binding protein A (CmbA) in adhesion to intestinal epithelial cells and mucus in vitro.

Lactobacillus reuteri, a symbiotic inhabitant of the gastrointestinal tract in humans and animals, is marketed as a probiotic. The ability to adhere to intestinal epithelial cells and mucus is an interesting property with regard to probiotic features such as colonization of the gastrointestinal tract and interaction with the host. Here, we present a study performed to elucidate the role of sortase (SrtA), four putative sortase-dependent proteins (SDPs), and one C-terminal membrane-anchored cell surface protein of Lactobacillus reuteri ATCC PTA 6475 in adhesion to Caco-2 cells and mucus in vitro. This included mutagenesis of the genes encoding these proteins and complementation of mutants. A null mutation in hmpref0536_10255 encoding srtA resulted in significantly reduced adhesion to Caco-2 cells and mucus, indicating involvement of SDPs in adhesion. Evaluation of the bacterial adhesion revealed that of the five putative surface protein mutants tested, only a null mutation in the hmpref0536_10633 gene, encoding a putative SDP with an LPxTG motif, resulted in a significant loss of adhesion to both Caco-2 cells and mucus. Complementation with the functional gene on a plasmid restored adhesion to Caco-2 cells. However, complete restoration of adhesion to mucus was not achieved. Overexpression of hmpref0536_10633 in strain ATCC PTA 6475 resulted in an increased adhesion to Caco-2 cells and mucus compared with the WT strain. We conclude from these results that, among the putative surface proteins tested, the protein encoded by hmpref0536_10633 plays a critical role in binding of Lactobacillus reuteri ATCC PTA 6475 to Caco-2 cells and mucus. Based on this, we propose that this LPxTG motif containing protein should be referred to as cell and mucus binding protein A (CmbA).

Extrusion of barley and oat improves the bioaccessibility of dietary phenolic acids in growing pigs.

To evaluate the bioaccessibility of phenolic acids in extruded and nonextruded cereal grains, an in vivo experiment was carried out using growing pigs as a model system. Four diets were prepared containing either whole grain barley (BU), dehulled oat (OU), or their respective extruded samples (BE, OE) according to the requirements for crude protein, mineral, and vitamin contents in pig diets. The total contents of free phenolic acids in the OE and BE diets were 22 and 10%, respectively, higher compared with the OU and BU diets, whereas the level of bound phenolic acids was 9% higher in OE than in OU and 11% lower in BE compared with BU. The total tract bioaccessibilities of bound phenolic acids were 29 and 14% higher for the extruded BE and OE diets, respectively, compared with the nonextruded diets. The results of this study indicate an improved bioaccessibility of phenolic acids in extruded cereal grains.

Generic tools to assess genuine carbohydrate specific effects on in vitro immune modulation exemplified by ß-glucans.

Even if carbohydrate preparations from plant/fungal sources have a high degree of purity, observed immune-stimulation may be caused by minute sample contaminations. Using the example of different ß-glucans we present a range of analytical tools crucial for validation of possible immune-stimulatory effects. Two yeast (MacroGard and Zymosan) and one cereal ß-glucan (CBG40) increased IL-8 secretion by HT-29 cells considerably. Degradation of the ß-glucan samples with ß-glucan specific enzymes did hardly influence the effect of Zymosan and CBG40 but significantly decreased the effect of MacroGard. Stimulation of IL-8 secretion by CBG40 and Zymosan was hence not due to their ß-glucan content. Instead, the effect of the CBG40 sample was due to low levels of LPS despite the inability of the known LPS inhibitor Polymyxin B to supress its stimulatory effect. We conclude that targeted enzymatic degradation of samples is a powerful validation tool to investigate carbohydrate specific immune-modulation.

Cereal ß-glucan quantification with calcofluor-application to cell culture supernatants.

The specific binding of the fluorescent dye calcofluor to cereal ß-glucan results in increased fluorescence intensity of the formed complex and is in use for the quantification of ß-glucan above a critical molecular weight (MW) by flow injection analysis. In this study, this method was applied in a fast and easy batch mode. In order to emphasize the spectral information of the emission spectra of the calcofluor/ß-glucan complexes, derivative signals were calculated. A linear relationship was found between the amplitude of the second derivative signals and the ß-glucan concentration between 0.1 and 0.4 µg/mL. The low detection limit of this new method (0.045 µg/mL) enabled its use to study the transport of cereal ß-glucans over differentiated Caco-2 cell monolayers. Additionally, the method was applied to quantify ß-glucan in arabinoxylan samples, which correlated well with data by an enzyme based method.

Antiproliferative effects of fresh and thermal processed green and red cultivars of curly kale (Brassica oleracea L. convar. acephala var. sabellica).

Brassica vegetables contain a diverse range of phytochemicals with biological properties such as antioxidant and anticancer activity. However, knowledge about how biological activities are affected by processing is lacking. A green cultivar and a red cultivar of curly kale were evaluated for water/methanol-soluble phytochemicals before and after processing involving blanching, freeze storage, and boil-in-bag heat treatment. In both kale cultivars, processing resulted in a significant decrease of total phenolics, antioxidant capacity, and content and distribution of flavonols, anthocyanins, hydroxycinnamic acids, glucosinolates, and vitamin C. Interestingly, the red curly kale cultivar had a higher capacity to withstand thermal loss of phytochemicals. The extracts of both green and red curly kale inhibited the cell proliferation of three human colon cancer cell lines (Caco-2, HT-29, and HCT 116). However, extracts from fresh plant material had a significantly stronger antiproliferative effect than extracts from processed plant material.

Improved bioavailability of dietary phenolic acids in whole grain barley and oat groat following fermentation with probiotic Lactobacillus acidophilus , Lactobacillus johnsonii , and Lactobacillus reuteri.

The aim of this study was to improve the bioavailability of the dietary phenolic acids in flours from whole grain barley and oat groat following fermentation with lactic acid bacteria (LAB) exhibiting high feruloyl esterase activity (FAE). The highest increase of free phenolic acids was observed after fermentation with three probiotic strains, Lactobacillus johnsonii LA1, Lactobacillus reuteri SD2112, and Lactobacillus acidophilus LA-5, with maximum increases from 2.55 to 69.91 µg g(-1) DM and from 4.13 to 109.42 µg g(-1) DM in whole grain barley and oat groat, respectively. Interestingly, higher amounts of bound phenolic acids were detected after both water treatment and LAB fermentation in whole grain barley, indicating higher bioaccessibility, whereas some decrease was detected in oat groat. To conclude, cereal fermentation with specific probiotic strains can lead to significant increase of free phenolic acids, thereby improving their bioavailability.

In vitro testing of commercial and potential probiotic lactic acid bacteria.

Probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host. The objective of this study was to investigate the diversity of selected commercial and potential probiotic lactic acid bacteria using common in vitro screening assays such as transit tolerance in the upper human gastrointestinal tract, adhesion capacity to human intestinal cell lines and effect on epithelial barrier function. The selected bacteria include strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus farciminis, Lactobacillus sakei, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri and Pediococcus pentosaceus. Viable counts after simulated gastric transit tolerance showed that L. reuteri strains and P. pentosaceus tolerate gastric juice well, with no reduction of viability, whereas L. pentosus, L. farciminis and L. sakei strains lost viability over 180min. All strains tested tolerate the simulated small intestinal juice well. The bacterial adhesion capacity to human intestinal cells revealed major species and strain differences. Overall, L. plantarum MF1298 and three L. reuteri strains had a significant higher adhesion capacity compared to the other strains tested. All strains, both living and UV-inactivated, had little effect on the epithelial barrier function. However, living L. reuteri strains revealed a tendency to increase the transepithelial electrical resistance (TER) from 6 to 24h. This work demonstrates the diversity of 18 potential probiotic bacteria, with major species and strain specific effects in the in vitro screening assays applied. Overall, L. reuteri strains reveal some interesting characteristics compared to the other strains investigated.

Effect of processing of black currant press-residue on polyphenol composition and cell proliferation.

The press-residue of black currants provides a good source of phenolic antioxidants. The purpose of this study was to optimize the extraction of phenolic compounds from the press-residue by use of extraction conditions compatible with food use. The effects of temperature, extraction duration, and use of ultrasound-assisted extraction on the juice yield, total phenolics (TP), and anthocyanin content of aqueous extracts were studied. Within the variables and response factors tested, the optimal conditions were a 15 min extraction at 90 °C. No significant effect from ultrasound-assisted extraction was found. The composition of anthocyanins and polyphenols was highly dependent on the extraction temperature. The percentage contribution of delphinidin- and cyanidin-3-rutinoside to TP had a negative linear correlation with temperature, while delphinidin- and cyanidin-3-glucoside had a positive linear correlation with temperature, with a maximum amount obtained at 80 °C and 55 °C, respectively. Furthermore, extracts obtained at higher temperatures showed a stronger inhibition of proliferation of Caco-2, HT-29, and HCT 116 cells than extracts obtained at lower temperatures. This may be due to the decomposition of complex polyphenols at higher temperatures, making them more accessible to the cells.

Immunomodulatory activity of dietary fiber: arabinoxylan and mixed-linked beta-glucan isolated from barley show modest activities in vitro.

High intake of dietary fiber is claimed to protect against development of colorectal cancer. Barley is a rich source of dietary fiber, and possible immunomodulatory effects of barley polysaccharides might explain a potential protective effect. Dietary fiber was isolated by extraction and enzyme treatment. A mixed-linked ß-glucan (WSM-TPX, 96.5% ß-glucan, Mw 886 kDa), an arabinoxylan (WUM-BS-LA, 96.4% arabinoxylan, Mw 156 kDa), a mixed-linked ß-glucan rich fraction containing 10% arabinoxylan (WSM-TP) and an arabinoxylan rich fraction containing 30% mixed-linked ß-glucan (WUM-BS) showed no significant effect on IL-8 secretion and proliferation of two intestinal epithelial cell lines, Caco-2 and HT-29, and had no significant effect on the NF-κB activity in the monocytic cell line U937-3κB-LUC. Further enriched arabinoxylan fractions (WUM-BS-LA) from different barley varieties (Tyra, NK96300, SB94897 and CDCGainer) were less active than the mixed-linked ß-glucan rich fractions (WSM-TP and WSM-TPX) in the complement-fixing test. The mixed-linked ß-glucan rich fraction from NK96300 and CDCGainer showed similar activities as the positive control while mixed-linked ß-glucan rich fractions from Tyra and SB94897 were less active. From these results it is concluded that the isolated high molecular weight mixed-linked ß-glucans and arabinoxylans from barley show low immunological responses in selected in vitro test systems and thus possible anti-colon cancer effects of barley dietary fiber cannot be explained by our observations.

Monitoring cellular responses upon fatty acid exposure by Fourier transform infrared spectroscopy and Raman spectroscopy.

We investigated the applicability of FTIR-spectroscopy as a high throughput screening method for detection of biochemical changes in intact liver cells in bulk upon fatty acid exposure. HepG2 cells adapted to serum free (HepG2-SF) growth were exposed to four different fatty acids, three octadecenoic acids, differing in cis/trans-configuration or double bond position (oleic acid, elaidic acid and vaccenic acid) as well as palmitic acid in three days. High throughput FTIR spectroscopic measurements on dried films of intact cells showed spectra with high signal-to-noise ratio and great reproducibility. When applying principal component analysis (PCA) a clear discrimination between fatty acid exposures was observed. Higher levels of triacylglycerides were accumulated in cells exposed to elaidic acid than when exposed to the other fatty acids; the least accumulation appeared to be in cells exposed to palmitic acid. An increased absorption at ~966 cm(-1) corresponding to trans-double bond was detected upon elaidic acid exposure but not upon vaccenic acid exposure. Instead, upon vaccenic acid exposure two new absorption bands were observed at 981 and 946 cm(-1) due to the presence of double bond conjugation. Raman spectroscopy on single cells, with and without treatment by vaccenic acid, confirmed the presence of conjugation. By fatty acid composition analysis, the conjugation was further specified to be conjugated linoleic acid (CLA) isomers. Thus, instead of being preserved as a monounsaturated fatty acid, vaccenic acid was converted into CLA in HepG2 cells. The results demonstrate the applicability of high-throughput FTIR spectroscopy as an explorative method in in vitro systems from which biologically relevant hypotheses can be generated and further investigated.

Activation and inhibition of nuclear factor kappa B activity by cereal extracts: role of dietary phenolic acids.

The transcription factor nuclear factor kappa B (NF-kappaB) plays a critical role in stress, immune, and inflammatory responses, and the modulation of its activity can be a potentially effective preventive strategy for controlling certain diseases. Cereal grains contain phenolic compounds in concentrations comparable to those in fruits and vegetables, well-known for their beneficial effect on human health. In this study we aimed to examine the effect of different phenolic extracts from barley, oat, wheat, and buckwheat on the modulation of basal and lipopolysaccharide (LPS)-induced NF-kappaB activity and elucidate the role of phenolic acids in this modulation. Three extracts were prepared: extracts of free phenolic compounds (M1), extracts of free phenolic acids (M2), and extracts of bound phenolic acids (HY). Generally, extracts M2 showed the highest effect on modulation of NF-kappaB activity with strong inhibition of LPS-induced NF-kappaB activity at all concentrations and of the basal NF-kappaB activity at concentrations equal to or lower than 3 mg/mL. Most of extracts M1 and HY slightly increased both the basal and the LPS-induced NF-kappaB activation. However, at the highest concentrations (3 or 15 mg/mL) extracts HY inhibited LPS-induced NF-kappaB activation. Similar experiments with standard solutions of phenolic acids indicated their ability to modulate the NF-kappaB activity.

Glycosphingolipid requirements for endosome-to-Golgi transport of Shiga toxin.

Shiga toxin binds to globotriaosylceramide (Gb3) receptors on the target cell surface. To enter the cytosol, Shiga toxin is dependent on endocytic uptake, retrograde transport to the Golgi apparatus and further to the endoplasmic reticulum before translocation of the enzymatically active moiety to the cytosol. Here, we have investigated the importance of newly synthesized glycosphingolipids for the uptake and intracellular transport of Shiga toxin in HEp-2 cells. Inhibition of glycosphingolipid synthesis by treatment with either PDMP or Fumonisin B(1) for 24-48 h strongly reduced the transport of Gb3-bound Shiga toxin from endosomes to the Golgi apparatus. This was associated with a change in localization of sorting nexins 1 and 2, and accompanied by a protection against the toxin. In contrast, there was no effect on transport or toxicity of the plant toxin ricin. High-resolution mass spectrometry revealed a 2-fold reduction in Gb3 at conditions giving a 10-fold inhibition of Shiga toxin transport to the Golgi. Furthermore, mass spectrometry showed that the treatment with PDMP (DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) and Fumonisin B(1) among other changes of the lipidome, affected the relative content of the different glycosphingolipid species. The largest depletion was observed for the hexosylceramide species with the N-amidated fatty acid 16:0, whereas hexosylceramide species with 24:1 were less affected. Quantitative lipid profiling with mass spectrometry demonstrated that PDMP did not influence the content of sphingomyelins, phospholipids and plasmalogens. In contrast, Fumonisin B(1) affected the amount and composition of sphingomyelin and glycolipids and altered the profiles of phospholipids and plasmalogens.

Protein kinase Cdelta is activated by Shiga toxin and regulates its transport.

Protein kinase C (PKC) isozymes regulate different vesicular trafficking steps in the recycling or degradative pathways. However, a possible role of these kinases in the retrograde pathway from endosomes to the Golgi complex has previously not been investigated. We report here the involvement of a specific PKC isozyme, PKCdelta, in the intracellular transport of the glycolipid-binding Shiga toxin (Stx), which utilizes the retrograde pathway to intoxicate cells. Upon binding to cells, Stx was shown to specifically activate PKCdelta and not PKCalpha. The involvement of PKCdelta and PKCalpha in the retrograde transport of Stx was then monitored biochemically and by immunofluorescence after inhibition or depletion of the isozymes. PKCdelta, but not PKCalpha, was shown to selectively regulate the endosome-to-Golgi transport of StxB. Upon inhibition or knockdown of PKCdelta, StxB molecules colocalized less with giantin and more with EEA1, indicating that the molecules were accumulated in endosomes, unable to reach the Golgi complex. The inhibition of Golgi transport of Stx was reflected by a strong reduction in the toxic effect, demonstrating that transport of Stx to the cytosol is dependent on PKCdelta activity. These results are in agreement with our previous data, which show that Stx is able to stimulate its own transport.

Annexin A2 recognises a specific region in the 3'-UTR of its cognate messenger RNA.

Annexin A2 is a multifunctional Ca(2+)- and lipid-binding protein. We previously showed that a distinct pool of cellular Annexin A2 associates with mRNP complexes or polysomes associated with the cytoskeleton. Here we report in vitro and in vivo experiments showing that Annexin A2 present in this subset of mRNP complexes interacts with its cognate mRNA and c-myc mRNA, but not with beta(2)-microglobulin mRNA translated on membrane-bound polysomes. The protein recognises sequence elements within the untranslated regions, but not within the coding region, of its cognate mRNA. Alignment of the Annexin A2-binding 3'-untranslated regions of annexin A2 mRNA from several species reveals a five nucleotide consensus sequence 5'-AA(C/G)(A/U)G. The Annexin A2-interacting region of the 3'-untranslated region can be mapped to a sequence of about 100 nucleotides containing two repeats of the consensus sequence. The binding elements appear to involve both single and double stranded regions, indicating that a specific higher order mRNA structure is required for binding to Annexin A2. We suggest that this type of interaction is representative for a group of mRNAs translated on cytoskeleton-bound polysomes.

Depletion of sphingolipids facilitates endosome to Golgi transport of ricin.

It has been previously demonstrated that depletion of cholesterol inhibits endosome to Golgi transport. Whether this inhibition is due to disruption of sphingolipid- and cholesterol-containing lipid rafts that are selected for Golgi transport or whether there is a physical requirement of cholesterol for either membrane deformations, facilitating formation of transport vesicles, or for recruitment of cytosolic constituents is not obvious. To investigate this in more detail, we have studied endosome to Golgi transport of ricin in sphingolipid-deficient cells using either a mutant cell line that does not express serine palmitoyltransferase, the first enzyme in sphingolipid biosynthesis, or a specific inhibitor, myriocin, of the same enzyme. Depletion of sphingolipids gave an increased sensitivity to ricin, and this increased sensitivity was inhibited by addition of sphingolipids. Importantly, endosome to Golgi transport of ricin, measured as sulfation of a modified ricin molecule, was increased in sphingolipid-deficient cells. No effect was seen on other pathways taken by ricin. Interestingly, cholesterol depletion inhibited endosome to Golgi transport even in cells with reduced levels of sphingolipids, suggesting that cholesterol as such is required for formation of transport vesicles. Our results indicate that the presence of sphingolipids actually limits and may function to control endosome to Golgi transport of ricin.

Golgi vesiculation induced by cholesterol occurs by a dynamin- and cPLA2-dependent mechanism.

It was recently demonstrated that an increase in the cellular cholesterol level leads to vesiculation of the Golgi apparatus. This vesiculation affects the entire Golgi apparatus and is a reversible process. We have now started to elucidate the mechanism behind this cholesterol-induced vesiculation of the Golgi apparatus. Transient transfection of cells with dominant negative mutant constructs of dynamin 1 and 2 inhibited the vesiculation; expression of dynK44A in HeLa cells stably transfected with this construct had the same effect. However, the vesiculation seems to be independent of clathrin, as cholesterol-induced vesiculation still occurred following knock down of clathrin heavy chain in HeLa cells using RNA interference as well as in BHK cells where expression of antisense to clathrin heavy chain had been induced. Importantly, the cPLA2 inhibitor MAFP and the chelator BAPTA-AM that binds cytosolic Ca2+ inhibited the cholesterol-induced vesiculation, suggesting involvement of a cPLA2 that requires cytosolic Ca2+ for translocation to membranes. Furthermore, in response to an increased cellular cholesterol level, an EGFP-cPLA2 fusion protein translocated to the Golgi apparatus. Thus, our results demonstrate that the cholesterol-induced vesiculation of the Golgi apparatus is mediated by a cPLA2- and dynamin-dependent mechanism.

Cholesterol loading induces a block in the exit of VSVG from the TGN.

Recent work from our laboratory demonstrated that increased cellular cholesterol content affects the structure of the Golgi apparatus. We have now investigated the functional consequences of the cholesterol-induced vesiculation of the Golgi apparatus and the role of actin for these changes. The results showed that cholesterol-induced vesiculation and dispersion of the Golgi apparatus is a reversible process and that reversal can be inhibited by cytochalasin D, an actin-disrupting reagent. Furthermore, electron microscopy revealed that jasplakinolide, which stabilizes actin filaments, prevented the dispersion, but not the vesiculation of the Golgi cisternae. Importantly, the different Golgi markers seemed to be separated even after vesiculation. To investigate whether transport through the different steps of the exocytic pathway was affected in cholesterol-treated cells, we visualized ER to plasma membrane transport by using ts045-VSVG-GFP. In COS-1 cells expressing ts045-VSVG-GFP increased cholesterol levels did not affect transport of VSVG into the vesiculated Golgi apparatus. However, increased levels of cholesterol resulted in retention of the nascent G protein in vesicles with the TGN-marker TGN46. Biotinylation of cell surface molecules to quantify arrival of VSVG at the plasma membrane confirmed that cholesterol treatment inhibited export of the VSVG protein. In conclusion, the data show that transport of VSVG into/through a vesiculated Golgi is feasible, but that cholesterol loading inhibits exit of VSVG from the vesicles containing TGN markers. Furthermore, the data illustrate the importance of actin filaments for Golgi structure.

Membrane ruffling and macropinocytosis in A431 cells require cholesterol.

Cholesterol is important for the formation of caveolea and deeply invaginated clathrin-coated pits. We have now investigated whether formation of macropinosomes is dependent on the presence of cholesterol in the plasma membrane. Macropinocytosis in A431 cells was induced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, a potent activator of protein kinase C (PKC). When cells were pretreated with methyl-beta-cyclodextrin to extract cholesterol, the phorbol ester was unable to induce the increased endocytosis of ricin otherwise seen, although PKC could still be activated. Electron microscopy revealed that extraction of cholesterol inhibited the formation of membrane ruffles and macropinosomes at the plasma membrane. Furthermore, cholesterol depletion inhibited the phorbol ester-induced reorganization of filamentous actin at the cell periphery, a prerequisite for the formation of membrane ruffles that close into macropinosomes. Under normal conditions the small GTPase Rac1 is activated by the phorbol ester and subsequently localized to the plasma membrane, where it induces the reorganization of actin filaments required for formation of membrane ruffles. Cholesterol depletion did not inhibit the activation of Rac1. However, confocal microscopy showed that extraction of cholesterol prevented the phorbol ester-stimulated localization of Rac1 to the plasma membrane. Thus, our results demonstrate that cholesterol is required for the membrane localization of activated Rac1, actin reorganization, membrane ruffling and macropinocytosis.

Pathways followed by ricin and Shiga toxin into cells.

The plant toxin ricin and the bacterial toxin Shiga toxin belong to a group of protein toxins that inhibit protein synthesis in cells enzymatically after entry into the cytosol. Ricin and Shiga toxin, which both have an enzymatically active moiety that inactivates ribosomes and a moiety that binds to cell surface receptors, enter the cytosol after binding to the cell surface, endocytosis by different mechanisms, and retrograde transport to the Golgi apparatus and the endoplasmic reticulum (ER). The toxins can be used to investigate the various transport steps involved, both the endocytic mechanisms as well as pathways for retrograde transport to the ER. Recent studies show that not only do several endocytic mechanisms exist in the same cell, but they are not equally sensitive to removal of cholesterol. New data have revealed that there is also more than one pathway leading from endosomes to the Golgi apparatus and retrogradely from the Golgi to the ER. Trafficking of protein toxins along these pathways will be discussed in the present article.