Effect of in vitro non-enzymatic glycosylation of human skin collagen on susceptibility to collagenase digestion

The effect of glycosylation on susceptibility of skin collagen to collagenase digestion was studied in a skin sample obtained at autopsy from the interscapular region of a 24 year old white male who had died of an acute illness and who had no history of diabetes. Homogeneous suspensions of insoluble collagen were prepared, and were incubated in 50 mmol l-1 dextrose at pH 7.35 and 37 degrees C for 7 days. Non-enzymatic glycosylation measured by the weak acid hydrolysis/thiobarbituric acid method increased from 13.1 +/- 1.0 (n = 5) to 45.2 +/- 5.5 (n = 8) nmol fructose per 10 mg collagen (P less than 0.001). Digestion of collagen using clostridial collagenase was monitored by measuring (a) hydroxyproline content and (b) absorption at 206 nm of the supernatant after centrifugation to remove substrate. The rate of digestion was similar in glycosylated and control collagen. We conclude that the ketoamine link formed in non-enzymatic glycosylation does not increase the resistance of collagen to enzymatic digestion. The possibility remains that subsequent rearrangement of this link could be important in this respect.

In vitro study of the effect of wound dressings on planktonic and biofilms from diabetic foot isolates

The management of wound bioburden has previously been evaluated using various antimicrobial wound dressings on bacterial pathogens isolated from various wounds. In this present study, the antimicrobial effect of silver-impregnated dressings (Acticoat and Silvercel) and honey-impregnated dressing (Medihoney™ Apinate) on both planktonic bacteria and quasi-biofilms by Staphylococcus aureus and Proteus mirabilis were assessed using a 6-well plate and standard agar technique. In the 6-well plate assay, a bacterial suspension of 108 colony forming unit (CFU)/mL was inoculated on each dressing in excess Luria-Bertani broth and incubated at 35 – 37°C for 30 and 60 minutes and 24 hours. After each incubation time, bacteria were recovered in sodium thioglycolate solution (STS) and the CFU/mL determined on LB agar. Dressings were cut into circular shapes (2cm diameter and placed on Mueller Hinton agar plates pre-inoculated with bacterial suspensions to determine their zones of inhibition (ZOI) after 24 hours incubation. None of the dressings was effective to significantly inhibit bacterial growth or biofilm formation at all the times tested. Acticoat and Medihoney™ Apinate produced ZOIs between 1.5 – 15 mm against both Staphylococcus aureus and Proteus mirabilis. It is possible that, dressings augmented with antibiotics can significantly reduce quasi-biofilms on standard agar.

In vitro effect of deoxynivalenol (DON) mycotoxin on porcine reproductive and respiratory syndrome virus replication.

Deoxynivalenol (DON) is a mycotoxin produced by Fusarium spp. Among monogastric farm animals, swine are the most susceptible to DON as it markedly reduces feed intake and decreases weight gain. DON has also been shown to increase susceptibility to viral infections; therefore the objective of this study was to investigate in vitro impact of DON on porcine reproductive and respiratory syndrome virus (PRRSV). Permissive cells were infected or not with PRRSV and were treated with increasing concentrations of DON. Cell survival and mortality were evaluated by determining the number of viable cells with a tetrazolium compound and by measuring lactate dehydrogenase (LDH) release, respectively. Virus titration and antiviral cytokines mRNA expression were evaluated by quantitative PCR. DON significantly affected the survival of noninfected cells in a dose dependent manner. However, DON concentrations between 140 and 280 significantly increased the survival of cells infected with PRRSV. These concentrations significantly decreased PRRSV replication by inducing a pro-inflammatory cytokines environment and an early activation of apoptosis, which in turn seem to interrupt viral replication. For the first time, this study showed that DON had significant effects on the survival of PRRSV infected cells and on virus replication, in a dose dependent manner.

Effect of deoxynivalenol (DON) mycotoxin on in vivo and in vitro porcine circovirus type 2 infections.

Deoxynivalenol (DON) is a mycotoxin produced by Fusarium spp and is a common contaminant of grains in North America. Among farm animals, swine are the most susceptible to DON because it markedly reduces feed intake and decreases weight gain. Porcine circovirus type 2 (PCV2) is the main causative agent of several syndromes in weaning piglets collectively known as porcine circovirus-associated disease (PCVAD). The objectives of this study were to investigate the impact of DON on PCV2 replication in NPTr permissive cell line, and to determine eventual potentiating effects of DON on PCV2 infection in pigs. Noninfected and infected cells with PCV2 were treated with increasing concentrations of DON (0, 70, 140, 280, 560, 1200 ng/mL) and cell survival and virus titer were evaluated 72 h postinfection. Thirty commercial piglets were randomly divided into 3 experimental groups of 10 animals based on DON content of served diets (0, 2.5 and 3.5 mg/kg DON). All groups were further divided into subgroups of 6 pigs and were inoculated with PCV2b virus. The remaining pigs (control) were sham-inoculated with PBS. In vitro results showed that low concentrations of DON could potentially increase PCV2 replication depending on virus genotype. In vivo results showed that even though viremia and lung viral load tend to be higher in animal ingesting DON contaminated diet at 2.5 mg/kg, DON had no significant effect on clinical manifestation of PCVAD in PCV2b infected animals. DON has neither in vitro nor in vivo clear potentiating effects in the development of porcine circovirus infection despite slight increases in viral replication.

The effect of wilting and soaking Eupatorium adenophorum on its digestibility in vitro and voluntary intake by goats

An experiment was conducted to determine what effect simple treatments might have on the voluntary intake by goats in Nepal of Eupatorium adenophorum, an invasive weed that is usually only consumed by goats to a very limited extent. Samples of E. adenophorum were collected and either untreated, soaked for 2 h or wilted for 2 h before being oven dried (60 degrees C) and ground. Soaking and wilting had little effect on the chemical composition of E. adenophorum, but did increase (P=0.036) its in vitro organic matter degradability, by approximately 8%. The short-term intake rate (STIR) of treated and untreated E. adenophorum was then estimated with eight goats. Soaking time (from 2 to 24 h) was not related to STIR (r = -0.111, P=0.198), but the time E. adenophorum was left to wilt (from 2 to 48h), was positively related to STIR (r=0.521, P<0.001), with values of STIR (g dry matter/min kg goat liveweight(0.75)) being 0.405, 0.649,1.058, S.E.M. 0.088 for E. adenophorum, that had been wilted for 0, 24 and 48 h respectively (P<0.001). Liveweight change of goats and voluntary intake of E. adenophorum by goats was then estimated with 24 goats. E. adenophorum was fed either unwilted, or wilted for 24 or 48 h. It was fed as the sole forage or as a 3:1 mixture (dry matter basis) with Ficus cunia. There was a linear (P<0.001) and quadratic (P<0.01) increase in the intake of total forage and E. adenophorum with wilting time of E. adenophorum. Offering Ficus cunia increased total forage intake, but decreased E. adenophorum intake (P<0.05). After four weeks, there was virtually no change in goat liveweight and no significant difference between treatments. The results suggest that wilting E adenophorum for 24 h could increase its intake by goats, and thereby increase its usefulness, as a potential source of forage in the dry season of Nepal, when forage scarcity is a common constraint to livestock production. (C) 2007 Elsevier B.V. All rights reserved.

Metabolism of the soyabean isoflavone glycoside genistin in vitro by human gut bacteria and the effect of prebiotics

The isoflavone genistein is found predominantly in soyabeans and is thought to possess various potent biological properties, including anticarcinogenic effects. Studies have shown that genistein is extensively degraded by the human gut microflora, presumably with a loss of its anti-carcinogenic action. The aim of the present study was to investigate the potential of a prebiotic to divert bacterial metabolism away from genistein breakdown: this may be of benefit to the host. Faecal samples were obtained from healthy volunteers and fermented in the presence of a source of soyabean isoflavones (Novasoy(TM) (10 g/l); ADM Neutraceuticals, Erith, Kent, UK). Bacterial genera of the human gut were enumerated using selective agars and genistein was quantified by HPLC. The experiment was repeated with the addition of glucose (10 g/l) or fructo-oligosaccharide (10 g/l; FOS) to the fermentation medium. The results showed most notably that counts of Bifidobacterium spp. and Lactobacillus spp. were significantly increased (P<0.05 and P<0.01 respectively) under steady-state conditions in the presence of FOS. Counts of Bacteroides spp. and Clostridium spp. were, however, both significantly reduced (P<0.05) during the fermentation. A decline in genistein concentration by about 52 and 56% over the 120h culture period was observed with the addition of glucose or FOS to the basal medium (P<0.01), compared with about 91% loss of genistein in the vessels containing Novasoy(TM) (ADM Neutraceuticals) only. Similar trends were obtained using a three-stage chemostat (gut model), in which once again the degradation of genistein was about 22% in vessel one, about 24% in vessel two and about 26% in vessel three in the presence of FOS, compared with a degradation of genistein of about 67% in vessel one, about 95% in vessel two and about 93% in vessel three in the gut model containing Novasoy(TM) (ADM Neutraceuticals) only. The present study has shown that the addition of excess substrate appeared to preserve genistein in vitro. In particular, the use of FOS not only augmented this effect, but also conferred an additional benefit in selectively increasing numbers of purportedly beneficial bacteria such as bifidobacteria and lactobacilli.

Effect of pH and dose on the growth of gut bacteria on prebiotic carbohydrates in vitro

The effect of pH and substrate dose on the fermentation profile of a number of commercial prebiotics was analysed in triplicate using stirred, pH and temperature controlled anaerobic batch culture fermentations, inoculated with a fresh faecal slurry from one of three healthy volunteers. Bacterial numbers were enumerated using fluorescence in situ hybridisation. The commercial prebiotics investigated were fructooligosaccharides (FOS), inulin, galactooligosaccharides (GOS), isomaltooligosaccharides (IMO) and lactulose. Two pH values were investigated, i.e. pH 6 and 6.8. Doses of 1% and 2% (w/v) were investigated, equivalent to approximately 4 and 8 g per day, respectively, in an adult diet. It was found that both pH and dose altered the bacterial composition. It was observed that FOS and inulin demonstrated the greatest bifidogenic effect at pH 6.8 and 1% (w/v) carbohydrate, whereas GOS, IMO and lactulose demonstrated their greatest bifidogenic effect at pH 6 and 2% (w/v) carbohydrate. From this we can conclude that various prebiotics demonstrate differing bifidogenic effects at different conditions in vitro. (C) 2003 Elsevier Science Ltd. All rights reserved.

The effect of nicotine in vitro on the integrity of tight junctions in Caco-2 cell monolayers

Ulcerative colitis is characterised by impairment of the epithelial barrier and tight junction alterations resulting in increased intestinal permeability. UC is less common in smokers with smoking reported to decrease paracellular permeability. The aim of this study was thus to determine the effect of nicotine, the major constituent in cigarettes and its metabolites on the integrity of tight junctions in Caco-2 cell monolayers. The integrity of Caco-2 tight junctions was analysed by measuring the transepithelial electrical resistance (TER) and by tracing the flux of the fluorescent marker fluorescein, after treatment with various concentrations of nicotine or nicotine metabolites over 48 h. TER was significantly higher compared to the control for all concentrations of nicotine 0.01-10 M at 48 h (p < 0.001), and for 0.01 mu M (p < 0.001) and 0.1 mu M and 10 M nicotine (p < 0.01) at 12 and 24 h. The fluorescein flux results supported those of the TER assay. TER readings for all nicotine metabolites tested were also higher at 24 and 48 h only (p <= 0.01). Western blot analysis demonstrated that nicotine up-regulated the expression of the tight junction proteins occludin and claudin-l (p < 0.01). Overall, it appears that nicotine and its metabolites, at concentrations corresponding to those reported in the blood of smokers, can significantly improve tight junction integrity, and thus, decrease epithelial gut permeability. We have shown that in vitro, nicotine appears more potent than its metabolites in decreasing epithelial gut permeability. We speculate that this enhanced gut barrier may be the result of increased expression of claudin-l and occludin proteins, which are associated with the formation of tight junctions. These findings may help explain the mechanism of action of nicotine treatment and indeed smoking in reducing epithelial gut permeability. (c) 2007 Elsevier Ltd. All rights reserved.

Effect of colonic bacterial metabolites on Caco-2 cell paracellular permeability in vitro

One common effect of tumor promoters is increased tight junction (TJ) permeability. TJs are responsible for paracellular permeability and integrity of the barrier function. Occludin is one of the main proteins responsible for TJ structure. This study tested the effects of physiological levels of phenol, ammonia, primary bile acids (cholic acid, CA, and chenodeoxycholic acid, CDCA), and secondary bile acids (lithocholic acid, LCA, and deoxycholic acid, DCA) on paracellular permeability using a Caco-2 cell model. Paracellular permeability of Caco-2 monolayers was assessed by transepithelial electrical resistance (TER) and the apical to basolateral flux of [C-14]-mannitol. Secondary, but not primary, bile acids increased permeability as reflected by significantly decreased TER and increased mannitol flux. Both phenol and ammonia also increased permeability. The primary bile acid CA significantly increased occludin expression (P < 0.05), whereas CDCA had no significant effect on occludin expression as compared to the negative control. The secondary bile acids DCA and LCA significantly increased occludin expression (P < 0.05), whereas phenol had no significant effect on the protein expression as compared to the negative control. This suggests that the increased permeability observed with LCA, DCA, phenol, and ammonia was not related to an effect on occludin expression. In conclusion, phenol, ammonia, and secondary bile acids were shown to increase paracellular permeability and reduce epithelial barrier function at doses typical of levels found in fecal samples. The results contribute to the evidence these gut microflora-generated products have tumor-promoting activity.