Plant-derived saccharides and their inhibitory potential on metastasis associated cellular processes of pancreatic ductal adenocarcinoma cells.

This study intends to investigate the inhibitory potential of different plant derived saccharides on cell migration and adhesion of pancreatic ductal adenocarcinoma (PDAC) cells to microvascular liver endothelium, particularly considering the role of transmembranous galectin-3. PDAC cell lines PancTu1 and Panc1 were characterized by considerable (transmembranous) galectin-3 (Gal3) expression. SiRNA mediated Gal3 knockdown as well as treatment with differentially processed pectins and arabinogalactan-proteins (AGPs) did not impact on cell migration of either PDAC cell line. In contrast, Gal3 knockdown reduced adhesion of PDAC cells to the liver endothelial cell line TMNK-1 being more pronounced in Panc1 cells. Similarly, plant derived substances did not impact cell adhesion of PancTu1 cells while partially hydrolyzed citrus pectin (MCP), pectinase-treated MCP (MCPPec) and partially hydrolized AGP (AGPTFA) clearly diminished adhesive properties of Panc1 cells. MCPPec or AGPTFA could not further intensify the adhesion reducing effect of galectin-3 knockdown, indicating that these plant derived polysaccharides are able to inhibit PDAC cell adhesion to liver endothelial cells in a galectin-3 dependent manner. Overall, these data suggest an inhibitory potential of plant derived processed saccharides which have undergone chemical modification in impairing PDAC cell adhesion to liver endothelium.

Chemical characterization and complement modulating activities of an arabinogalactan-protein-rich fraction from an aqueous extract of avocado leaves.

The aim of this study was to chemically characterize an arabinogalactan-protein-rich fraction (FRAGP) obtained from an aqueous extract of avocado leaves and investigate its effects on the classical pathway of the complement system. The FRAGP contained 4.6% ±â€¯1.8%, 22.5% ±â€¯4.9%, and 76.7% ±â€¯8.8% of total protein, arabinogalactan-protein, and carbohydrates, respectively. Arabinose and galactose were the main monosaccharide constituents. FT-IR and NMR data, together with linkage analyses, indicated the presence of a structure that included a (1 → 3)-linked ß-D-Galp main chain, mainly substituted at O-6 by Gal and Ara residues, which was characteristic of a type II arabinogalactan. The effect of FRAGP on the classical pathway of complement system was examined by a hemolytic fixation test and comparing with heparin, which was used as a control for inhibition. With pre-incubation, the IC50 of FRAGP was 1.90 ±â€¯1.1 µg/mL, which was similar to that of heparin (IC50 = 2.90 ±â€¯0.3 µg/mL). Without pre-incubation, the IC50 values were 18.6 ±â€¯3.7 and 8.0 ±â€¯4.1 µg/mL for FRAGP and heparin, respectively. Collectively, these results suggested that FRAGP has an inhibitory effect on the classical pathway of the complement system.

Arabinogalactan Proteins From Baobab and Acacia Seeds Influence Innate Immunity of Human Keratinocytes In Vitro.

Plant derived arabinogalactan proteins (AGP) were repeatedly confirmed as immunologically as well as dermatologically active compounds. However, little is currently known regarding their potential activity toward skin innate immunity. Here, we extracted and purified AGP from acacia (Acacia senegal) and baobab (Adansonia digitata) seeds to investigate their biological effects on the HaCaT keratinocyte cell line in an in vitro system. While AGP from both sources did not exhibit any cytotoxic effect, AGP from acacia seeds enhanced cell viability. Moreover, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that AGP extracted from both species induced a substantial overexpression of hBD-2, TLR-5, and IL1-α genes. These data suggest that plant AGP, already known to control plant defensive processes, could also modulate skin innate immune responses. J. Cell. Physiol. 232: 2558-2568, 2017. © 2016 Wiley Periodicals, Inc.

Combination therapy with lysin CF-301 and antibiotic is superior to antibiotic alone for treating methicillin-resistant Staphylococcus aureus-induced murine bacteremia.

Lysins are bacteriophage-derived enzymes that degrade bacterial peptidoglycans. Lysin CF-301 is being developed to treat Staphylococcus aureus because of its potent, specific, and rapid bacteriolytic effects. It also demonstrates activity on drug-resistant strains, has a low resistance profile, eradicates biofilms, and acts synergistically with antibiotics. CF-301 was bacteriolytic against 250 S. aureus strains tested including 120 methicillin-resistant S. aureus (MRSA) isolates. In time-kill studies with 62 strains, CF-301 reduced S. aureus by 3-log10 within 30 minutes compared to 6-12 hours required by antibiotics. In bacteremia, CF-301 increased survival by reducing blood MRSA 100-fold within 1 hour. Combinations of CF-301 with vancomycin or daptomycin synergized in vitro and increased survival significantly in staphylococcal-induced bacteremia compared to treatment with antibiotics alone (P < .0001). Superiority of CF-301 combinations with antibiotics was confirmed in 26 independent bacteremia studies. Combinations including CF-301 and antibiotics represent an attractive alternative to antibiotic monotherapies currently used to treat S. aureus bacteremia.

Novel chimeric lysin with high-level antimicrobial activity against methicillin-resistant Staphylococcus aureus in vitro and in vivo.

The treatment of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) is a challenge worldwide. In our search for novel antimicrobial agents against MRSA, we constructed a chimeric lysin (named as ClyH) by fusing the catalytic domain of Ply187 (Pc) with the non-SH3b-like cell wall binding domain of phiNM3 lysin. Herein, the antimicrobial activity of ClyH against MRSA strains in vitro and in vivo was studied. Our results showed that ClyH could kill all of the tested clinical isolates of MRSA with higher efficacy than lysostaphin as well as its parental enzyme. The MICs of ClyH against clinical S. aureus strains were found to be as low as 0.05 to 1.61 mg/liter. In a mouse model, a single intraperitoneal administration of ClyH protected mice from death caused by MRSA, without obvious harmful effects. The present data suggest that ClyH has the potential to be an alternative therapeutic agent for the treatment of infections caused by MRSA.

Lysins: the arrival of pathogen-directed anti-infectives.

Lysins represent a novel class of anti-infectives derived from bacteriophage. Lysins are bacterial cell-wall hydrolytic enzymes that selectively and rapidly kill (≥3 log c.f.u. in 30 min) specific Gram-positive bacteria providing a targeted therapeutic approach with minimal impact on unrelated commensal flora. The potential for bacterial resistance to lysins is considered low due to targeting of highly conserved peptidoglycan components. Through cutting-edge genetic engineering, lysins can be assembled into large libraries of anti-infective agents tailored to any bacterium of interest including drug-resistant Gram-positive pathogens such as meticillin- and vancomycin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis and Enterococcus faecium, and penicillin-resistant Streptococcus pneumoniae. Lysins can eliminate bacteria systemically and topically from mucosal surfaces and biofilms, as evidenced by experimental models of sepsis, endocarditis, pneumonia, meningitis, and nasopharyngeal, skin and vaginal decolonization. Furthermore, lysins can act synergistically with antibiotics and, in the process, resensitize bacteria to non-susceptible antibiotics. Clinical trials are being prepared to assess the safety and pharmacokinetic properties of lysins in humans.

Antitussive and immunomodulating activities of instant coffee arabinogalactan-protein.

A low molecular mass arabinogalactan-protein (AGP) composed of galactose and arabinose with a low protein content, isolated from the instant coffee powder of Coffea arabica beans, has been tested on antitussive (in vivo) and immunomodulating (ex vivo) activities. The results of antitussive tests revealed a significant dose dependant cough-suppressive effect of coffee AGP. It was observed 30 or 60 min after AGP administration and its efficacy lasted during the entire experiment course. Immunological tests showed that AGP affected some mediators of immunocompetent cells of immune system as TNF-α, IFN-γ and IL-2 cytokines. It seems that coffee AGP is a good inductor of both pro-inflammatory cytokines TNF-α and IFN-γ, however, less potent in TNF-α induction in comparison with that of ß-D-glucan. Evident induction of TNF-α, IL-2 and IFN-γ cytokines, pro-TH1 polarization supports our conclusion about bio-immunological efficacy of AGP with an emphasis on the cellular immunity.

Arabinogalactan-proteins stimulate the organogenesis of guard cell protoplasts-derived callus in sugar beet.

Arabinogalactan proteins (AGPs) represent a class of proteoglycans implicated in the development and differentiation of cells and tissues both in planta and in vitro. Here we report that AGP-rich extracts isolated from media of embryogenic and non-embryogenic suspension cultures of sugar beet (Beta vulgaris L.) are able to enhance the organogenesis of guard protoplast-derived callus and to increase the number of shoots formed, in comparison to control cultures. Immunocytochemical detection of carbohydrate antigens in the extracts revealed the presence of epitopes that typify both AGP and pectin, the latter being frequently bound to AGPs or, in some cases, even contributing to the polysaccharide structure of proteoglycan molecules. The most abundant epitopes proved to be those recognized by the JIM13, LM2, and MAC207 antibodies, whereas some others could be found only in relatively small or trace amounts--these included epitopes recognized by JIM16, JIM5, and LM6. Surprisingly, the JIM4- and JIM8-binding epitopes that are expressed in the course of in vitro morphogenetic processes of many species could not be detected at all in sugar beet AGPs. This is the first report of the improvement of sugar beet protoplast-derived callus organogenesis by exogenous AGP-rich extracts, an achievement that will have great impact on the biotechnological applications of protoplast technology in this species.

Immunomodulatory effects of arabinogalactan-proteins from Baptisia and Echinacea.

The influences of different arabinogalactan-proteins (AGPs) on proliferation and IgM-production of mouse lymphocytes as well as nitrite- and IL6-production of mouse macrophages were investigated in vitro. AGPs have been isolated and purified from roots of Baptisia tinctoria and Echinacea pallida and suspension culture of Echinacea purpurea. Comparing the AGPs, there are differences with regard to fine structure as well as to activities. AGPs from roots of B. tinctoria and E. pallida show high activity in all test systems. AGP from cell culture of E. purpurea shows no influence on proliferation of mouse lymphocytes, only weak influence on the IgM-production of mouse lymphocytes and weak stimulation of nitrite- and IL6-production in alveolar mouse macrophage culture.

Effects of Tamm-Horsfall protein on the protection of MCDK cells from oxalate induced free radical injury.

Renal cell injury and fixed particle formation is one of the theories of urinary stone formation. The exposure of renal epithelial cells to oxalate ions and calcium oxalate monohydrate crystals can cause free radical generation and increase lipid peroxidation. Tamm-Horsfall protein (THP) has a protective effect on the production of free radicals in vitro. We aimed to show that THP (and its deglycosylated products, D-THP) could protect culture cells from free radical injury in vivo as well as the possible mechanism by which this is done. Exposure of Madin-Darby canine kidney (MDCK) cells to Ox resulted in a significant increase in the release LDH, NBT and MDA, as well as an increase in caspase 3 activity, all of which were further elevated when COM crystals were added. With the addition of THP at 500 nM, there was a significant decrease in the release of LDH and the production of MDA and NBT. A decrease in capase 3 activity was observed when 500 nM THP was added to the culture medium that reached 32.7% and 40.4% of inhibition in CaOx+THP and CaOx+COM+THP, respectively. THP decreased the adhesion of COM crystals to the MDCK cells but lost its effect when THP was deglycosylated. The results indicate that both Ox and COM crystals cause the release of LDH, MDA, NBT and increase the activity of capase 3 in MDCK cells. As a free radical scavenger, THP reduces the amount of free radicals and provides significant protection at a critical concentration of 500 nM. The deglycosylated THP decreased the effect of the protection of the MDCK cells from oxalate-induced injury and an increase of adhesion of the COM crystals to the MDCK cells. Therefore, the effects of THP on the protection of oxalate induced radical injury may be partly due to its intact glycosylation and its adhesion to the cell membrane.

Differentiation between the complement modulating effects of an arabinogalactan-protein from Echinacea purpurea and heparin.

Due to the important physiological role of the complement system, complement modulation, either inhibition or stimulation, is an interesting target for drug development. Several plant polysaccharides are known to exhibit complement modulating activities. Sometimes these effects are described as complement inhibition, although the basic mechanism is a stimulation of the complement activation. This misinterpretation is due to the observed reduced haemolysis in the widely used haemolytic complement assay, which does not allow to differentiate between complement activators and inhibitors, when it is performed in the classical manner. The aim of the presented study was to demonstrate that by simple modifications of the classical procedure this assay becomes an efficient tool to distinguish between real complement inhibitors and complement activating compounds without performing expensive, molecular mechanistic investigations. As practical examples heparin with proven complement inhibiting activity and AGP, a new arabinogalacatan-protein type II isolated from pressed juice of the aerial parts of Echinacea purpurea, as a potential complement activating compound were included in the study. By means of varying the preincubation time of the test compound with complement, AGP was clearly identified as a stimulator of both the classical and alternative pathway of complement activation. These findings correspond to the results of molecular mechanistic investigations. Selective removal of the arabinose side chains of AGP resulted in considerably reduced activity. Therefore, the three-dimensional structure of the polysaccharide, i. e., a backbone branched by side chains, is supposed to be important for the interactions with the complement system. The complement activating effects of AGP may contribute to the well-established immunostimulating effects of the pressed juice from Echinacea purpurea. Abbreviations. AGP:arabinogalactan-protein AGP-hydr.:hydrolysed arabinogalactan-protein AP-CA:haemolytic complement assay for the alternative pathway CP-CA:haemolytic complement assay for the classical pathway EGTA-VB:veronal buffered saline containing EGTA and Mg 2+HPS:human pooled serum RT:room temperature LPS:lipopolysaccharide RaE:rabbit erythrocytes RT:room temperature ShE(A):(sensitised) sheep erythrocytes VB:veronal buffered saline containing Ca 2+ and Mg 2+

Urothelial cytoprotective activity of Tamm-Horsfall protein isolated from the urine of healthy subjects and patients with interstitial cystitis.

PURPOSE: Tamm-Horsfall protein (THP) is a ubiquitous urinary protein with essentially no known function. We propose that THP is a cytoprotective agent that protects the urothelium from cationic species. To test this hypothesis we isolated THP from normal and interstitial cystitis urine to see if it could protect cultured cells from damage induced by the polyamine, protamine sulfate (PS). METHODS: Tamm-Horsfall protein was extracted from the urine of interstitial cystitis (IC) patients (N=28) and normal volunteers (N=5). Urothelial target cells (T24) were radiolabeled with 51Cr and then exposed to PS (0-1.0 mg/mL) for either 1.5 or 20 h. The resulting cytotoxicity data (dose-response curves) were then compared with the data obtained when PS was preincubated with 0-0.5 mg/mL of THP (IC vs normal), the semisynthetic polysaccharide, pentosan polysulfate (Elmiron), or human serum albumin. RESULTS: Toxicity of PS was significantly reduced by incubation with THP (or Elmiron) prior to evaluation by the chromium release assay, but not reduced by incubating with another protein, albumin. Tamm-Horsfall protein from IC patients' urine was less protective than an equal quantity of THP from normal urine. CONCLUSIONS: These experiments suggest that THP has an important role in bladder mucosal defense mechanisms, protecting the bladder surface from injury. Inability of THP to prevent cytotoxic damage by urinary polyamine or other urinary toxins (cationic species) may be relevant in the etiology of interstitial cystitis, as putative urinary toxic components have been described in the urine of some patients.

The effect of some bacterial products on temperature and sleep in rat.

The lipopolysaccharides from P. aeruginosa, S. minnesota and mucopeptide from Streptococcus group A injected intravenously into rats induce a dose-dependent changes of temperature. Simultaneously, a profound disturbance of sleep occurs. The administration of salicylate, which markedly suppressed the fever does not influence the mucopeptide-caused sleep disturbance. The most prominent change in the sleep pattern is a marked decrease of the total time of paradoxical sleep. The measurement of turnover rates of 5-hydroxytryptamine (5-HT) and noradrenaline (NA) in hypothalamus and midbrain, areas involved in temperature and sleep control, after injection of streptococcal mucopeptide demonstrated a significant increase of 5-HT turnover in both areas during fever and paradoxical sleep deprivation. Small electrolytic lesions of the dorsal raphe nuclei which are the largest collection of neural cells containing 5-HT completely eliminated the pyrogenic potency of mucopeptide. The findings suggest that some bacterial products might increase the body temperature through the interference with activity of 5-HT-containing neurons of the raphe complex.