Feasibility study using a natural compound (reuterin) produced by Lactobacillus reuteri in sterilizing and crosslinking biological tissues

Bioprostheses derived from biological tissues have to be fixed and subsequently sterilized before they can be implanted in humans. Currently available crosslinking agents and sterilants used in the fixation or sterilization of biological tissues such as glutaraldehyde and formaldehyde are all highly cytotoxic, which may impair the biocompatibility of bioprostheses. Therefore, it is desirable to provide an agent suitable for use in biomedical applications that is of low cytotoxicity and may form sterile and biocompatible crosslinked products. To achieve this goal, a natural compound (reuterin), produced by Lactobacillus reuteri in the presence of glycerol, was used by our group. It is known that reuterin has antibacterial, antimycotic, and antiprotozoal activities. Additionally, as in the case with formaldehyde, reuterin may react with the free amino groups in biological tissues by using its aldehyde functional group. Therefore, it was speculated that reuterin could be used as a crosslinking agent and a sterilant for biological tissues in the same way as glutaraldehyde and formaldehyde. In the study, the production of reuterin, produced by Lactobacillus reuteri under control conditions, was reported. Preparative chromatography was used to purify reuterin. Also, the minimal inhibitory concentration and minimal bactericidal concentration of reuterin and its antimicrobial activity on a contaminated tissue were investigated. In addition, the cytotoxicity of reuterin was evaluated. Glutaraldehyde, the most commonly used sterilant in the sterilization of biological tissues, was employed as a control. Furthermore, the feasibility of using reuterin as a crosslinking agent in fixing biological tissues was studied. Fresh and the glutaraldehyde-fixed tissues were used as controls. The results obtained in the minimal inhibitory concentration and minimal bactericidal concentration studies and in the sterilization study of a contaminated tissue indicated that the antimicrobial activity of reuterin is significantly superior to its glutaraldehyde counterpart. In addition, the results obtained in the 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide assay showed that reuterin is significantly less cytotoxic than glutaraldehyde. Additionally, it was found that reuterin is an effective crosslinking agent for biological tissue fixation. The reuterin-fixed tissue had comparable free amino group content, denaturation temperature, and resistance against enzymatic degradation as the glutaraldehyde-fixed tissue. In conclusion, the results obtained in this study indicate that reuterin is an effective agent in the sterilization and fixation of biological tissues.     

A natural compound (reuterin) produced by Lactobacillus reuteri for biological-tissue fixation

The study was undertaken to examine the degree of tissue fixation by reuterin, a natural compound produced by Lactobacillus reuteri, at distinct fixation conditions (pH, temperature, and fixative concentration). Additionally, the rate of tissue fixation by reuterin was investigated using glutaraldehyde as a control. It was found by the Fourier transformed infrared spectroscopy and nuclear magnetic resonance spectroscopy that both mono- and di-aldehyde reuterin oligomers may be present in the acidic and basic aqueous reuterin solutions. Therefore, reuterin may crosslink biological tissues as glutaraldehyde (a di-aldehyde agent). The degree of tissue fixation by reuterin is significantly affected by its fixation conditions. Generally, with increasing the pH, temperature, or fixative concentration, the reduction in free-amino-group content, denaturation temperature, tensile strength, and resistance against enzymatic degradation of the reuterin-fixed tissue increased significantly. Also, the rate of tissue fixation by reuterin is significantly slower than that by glutaraldehyde. However, after fixation, it was noted that the reuterin-fixed tissue has comparable free-amino-group content, denaturation temperature, tensile strength, and resistance against enzymatic degradation as the glutaraldehyde-fixed tissue.     

In vitro study of enzymatic degradation of biological tissues fixed by glutaraldehyde or epoxy compound

The study, using bacterial collagenase, was to investigate the changes in characteristics of a collagen-rich tissue, porcine pericardium, fixed by glutaraldehyde or epoxy compound (ethylene glycol diglycidyl ether) during the course of degradation. Fresh porcine pericardium was used as a control. During degradation, the heat released by the reaction of collagenase with a test sample was monitored by a highly sensitive microcalorimeter. Also, the degree of degradation of each test sample was determined by measuring its increment in free amino group content and changes in denaturation temperature and tensile strength. Microcalorimetric analysis of collagenase degradation of fresh, epoxy-fixed, and glutaraldehyde-fixed tissues revealed that the heat released during degradation correlates well with the degree of tissue degraded. The cleaving of peptide bonds in biological tissue by collagenase degradation may increase its free amino group content and reduce its denaturation temperature and tensile strength. It was noted that the fresh tissue cannot resist bacterial collagenase degradation, while the glutaraldehyde-fixed tissue had a relatively better resistance to degradation than its epoxy-fixed counterpart.     

经EX-810处理的食管用修补材料

背景：天然生物组织来源材料能逐渐被吸收,同时能够诱导组织特异性结构重塑,是理想的食管修补材料。 目的：观察环氧类交联剂EX-810处理的猪食管组织的性能特点。 方法：用4%EX-810处理猪食管组织1~120 h,通过对交联指数的测定观察交联情况;扫描电镜观察组织交联前后的形态学变化;将新鲜的,交联3,7 d的样品进行力学性能测定,观察交联对材料力学性能的影响。 结果与结论：EX-810能有效地交联组织,并能通过去除细胞、组织中的自由氨基等抗原性物质而有效降低生物组织的抗原性;同时还能保持生物组织中胶原的结构形态不受大的破坏;力学性能较处理前也有较大改善,并且还可以改变材料的应力应变曲线形状,使其更加符合食管修补材料的力学要求。提示经EX-810处理的食管修补材料既保持了天然食管材料的结构特征,同时又具有低抗原性、易于保存及更适合的力学性能等优点。     

Stability of a biological tissue fixed with a naturally occurring crosslinking agent (genipin)

The study was undertaken to investigate the stability of a biological tissue fixed with a naturally occurring crosslinking agent (genipin) at distinct elapsed storage durations. The glutaraldehyde-fixed counterpart was used as a control. Porcine pericardia procured from a slaughterhouse were used as raw materials. After fixation, the fixed tissues were sterilized in a graded series of ethanol solutions and thoroughly rinsed in phosphate buffered saline for 1 day, and then stored in a jar containing sterilized water. The samples were taken out and tested for their stability during the durations of 1day through 6 months after storage. The stability of each study group was tested by measuring its tensile strength, free-amino-group content, and denaturation temperature. Additionally, the cytotoxicity of each test sample and its corresponding storage solution were investigated in vitro using 3T3 fibroblasts. The results were examined using a microscope and 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It was found that the stability of the genipin-fixed tissue during storage was superior to its glutaraldehyde-fixed counterpart. The differences in stability between the genipin- and glutaraldehyde-fixed tissues during storage may be caused by their differences in crosslinking structure. There was no apparent cytotoxicity for both the genipin-fixed tissue and its corresponding storage solution throughout the entire course of the study, whereas significant cytotoxicity was observed for both the glutaraldehyde-fixed tissue and its storage solution. However, the cytotoxicity of the glutaraldehyde-fixed tissue decreased with increasing elapsed storage duration, whereas that of its corresponding storage solution increased. This suggested that the toxic residues remaining in the glutaraldehyde-fixed tissue leached out slowly into its corresponding storage solution during the course of storage.     

Effect of oral supplementation of Lactobacillus reuteri in reduction of intestinal absorption of aflatoxin B(1) in rats

The goals of this work were to assess the ability of Lactobacillus reuteri to bind aflatoxin B(1) in the intestinal tract and determine its effect on intestinal absorption of the toxin dispensed in either single or multiple doses in a murine model. Male Wistar rats were used, and two experiments were conducted after bacteria were implanted. Experiment one involved a single-oral dose of toxin, and the subsequent flow cytometric analysis of bacteria isolated from the small intestine and treated with specific FITC-labeled AFB(1) antibodies. The second experiment was carried out supplying the toxin in 7 oral sub-doses, and the later quantification of AFB(1)-Lys adducts in blood samples by ELISA assay. The results demonstrated that L. reuteri was able to bind AFB(1) in the intestinal tract, mostly in the duodenum. Furthermore, the AFB(1)-Lys adducts were present at significantly lower levels in those animals receiving AFB(1) plus bacteria than in those receiving only AFB(1). Our findings confirm that probiotic bacteria could act as biological barriers in normal intestinal conditions thereby reducing the bioavailability of AFB(1) ingested orally in a single or multiple doses, thus avoiding its toxic effects.     

Detection of specific t(14;18) chromosomal translocations in fixed tissues

The present study was undertaken to establish the incidence of t(14;18) (q32:q21) chromosomal translocations detectable by a polymerase chain reaction (PCR) assay on fixed lymphoma biopsies. DNA samples from 113 formalin-fixed, paraffin-embedded tissue biopsies (non-Hodgkin's lymphomas, 96 cases; Hodgkin's disease, six cases; reactive, 11 cases) were amplified by the PCR. Of the 96 non-Hodgkin's lymphoma cases, 56 had a follicular pattern and 40 had a diffuse pattern. Polymerase chain reaction-amplifiable t(14;18) chromosomal translocations were detected in 23 of 43 follicular low-grade lymphomas, one of eight follicular intermediate grade lymphomas, one of five follicular high-grade lymphomas, and one of 10 diffuse large-cell lymphomas. The remaining 30 diffuse lymphomas represented the spectrum of the Working Formulation classification. There were six biopsy specimens of Hodgkin's disease and 11 biopsy specimens of follicular hyperplasia; all were negative. The translocation was not detected in 16 biopsies (non-Hodgkin's lymphomas, seven cases; follicular hyperplasia, nine cases) from patients infected with the human immunodeficiency virus. Since this procedure uses the widely available fixed paraffin-embedded material, correlative studies between histology and genetic aberrations can be readily undertaken.     

Biocompatibility of fluorinated poly(organophosphazene)

We investigated neutrophil and platelet adhesion on a fluorinated poly(organophosphazene) in vitro. The results suggested that neutrophil and platelet adhesion on the poly(organophosphazene) only occurred on a few occasions, as observed by SEM. We demonstrated that the fluorinated poly(organophosphazene) showed excellent biocompatibility compared with the poly(organophosphazene) without the fluorinated side groups or PDMS. Additionally, we estimated the competitive plasma protein adsorption to the fluorinated poly(organophosphazene) using a gold-colloid-labeled immunoassay. Interestingly, the fluorinated poly(organophosphazene) film selectively adsorbed albumin when compared with gamma-globulin and fibrinogen, suggesting that a selective albumin adsorption on the film is responsible for the suppression of platelet adhesion.     

Biocompatibility of fluorinated poly(organophosphazene)

We investigated neutrophil and platelet adhesion on a fluorinated poly(organophosphazene) in vitro. The results suggested that neutrophil and platelet adhesion on the poly(organophosphazene) only occurred on a few occasions, as observed by SEM. We demonstrated that the fluorinated poly(organophosphazene) showed excellent biocompatibility compared with the poly(organophosphazene) without the fluorinated side groups or PDMS. Additionally, we estimated the competitive plasma protein adsorption to the fluorinated poly(organophosphazene) using a gold-colloid-labeled immunoassay. Interestingly, the fluorinated poly(organophosphazene) film selectively adsorbed albumin when compared with γ-globulin and fibrinogen, suggesting that a selective albumin adsorption on the film is responsible for the suppression of platelet adhesion.     

Lactobacillus reuteri promotes Helicobacter hepaticus-associated typhlocolitis in gnotobiotic B6.129P2-IL-10(tm1Cgn) (IL-10(-/-) ) mice

To model inflammatory bowel disease, we assessed infection with Helicobacter hepaticus 3B1 (ATCC 51449) and a potential probiotic Lactobacillus reuteri (ATCC PTA-6475) in gnotobiotic B6.129P2-IL-10(tm1Cgn) (IL-10(-/-) ) mice. No typhlocolitis developed in germ-free controls (n=21) or in L. reuteri (n=8) or H. hepaticus (n=18) mono-associated mice for 20 weeks post-infection. As positive controls, three specific pathogen-free IL-10(-/-) mice dosed with H. hepaticus developed severe typhlocolitis within 11 weeks. Because L. reuteri PTA-6475 has anti-inflammatory properties in vitro, it was unexpected to observe significant typhlocolitis (P<0·0001) in mice that had been infected with L. reuteri followed in 1 week by H. hepaticus (n=16). The H. hepaticus colonization was not affected through 20 weeks post-infection but L. reuteri colonization was lower in co-infected compared with L. reuteri mono-associated mice at 8-11 weeks post-infection (P<0·05). Typhlocolitis was associated with an increased T helper type 1 serum IgG2c response to H. hepaticus in co-infected mice compared with H. hepaticus mono-associated mice (P<0·005) and similarly, mRNA expression in caecal-colonic tissue was elevated at least twofold for chemokine ligands and pro-inflammatory interleukin-1α (IL-1α), IL-1β, IL-12 receptor, tumour necrosis factor-α and inducible nitric oxide synthase. Anti-inflammatory transforming growth factor-β, lactotransferrin, peptidoglycan recognition proteins, Toll-like receptors 4, 6, 8 and particularly 9 gene expression, were also elevated only in co-infected mice (P<0·05). These data support that the development of typhlocolitis in H. hepaticus-infected IL-10(-/-) mice required co-colonization with other microbiota and in this study, required only L. reuteri. Although the effects other microbiota may have on H. hepaticus virulence properties remain speculative, further investigations using this gnotobiotic model are now possible.     

Social stress-enhanced severity of Citrobacter rodentium-induced colitis is CCL2-dependent and attenuated by probiotic Lactobacillus reuteri

Psychological stressors are known to affect colonic diseases but the mechanisms by which this occurs, and whether probiotics can prevent stressor effects, are not understood. Because inflammatory monocytes that traffic into the colon can exacerbate colitis, we tested whether CCL2, a chemokine involved in monocyte recruitment, was necessary for stressor-induced exacerbation of infectious colitis. Mice were exposed to a social disruption stressor that entails repeated social defeat. During stressor exposure, mice were orally challenged with Citrobacter rodentium to induce a colonic inflammatory response. Exposure to the stressor during challenge resulted in significantly higher colonic pathogen levels, translocation to the spleen, increases in colonic macrophages, and increases in inflammatory cytokines and chemokines. The stressor-enhanced severity of C. rodentium-induced colitis was not evident in CCL2^−/− mice, indicating the effects of the stressor are CCL2-dependent. In addition, we tested whether probiotic intervention could attenuate stressor-enhanced infectious colitis by reducing monocyte/macrophage accumulation. Treating mice with probiotic Lactobacillus reuteri reduced CCL2 mRNA levels in the colon and attenuated stressor-enhanced infectious colitis. These data demonstrate that probiotic L. reuteri can prevent the exacerbating effects of stressor exposure on pathogen-induced colitis, and suggest that one mechanism by which this occurs is through downregulation of the chemokine CCL2.     

Improved detection of lymphoid cell surface antigens in tissues fixed in periodate-lysine-paraformaldehyde (PLP)

Stabilization of cell surface antigens and preservation of tissue morphologic characteristics are important for diagnostic immunologic studies. Current reports continue to regard unfixed frozen sections as the material of choice for immunoperoxidase studies of lymphoproliferative diseases. In this study, periodate-lysine-paraformaldehyde (PLP) is shown to be a valuable fixative for the improved detection of surface antigens in lymphoid tissue. In cases of non-Hodgkin's lymphoma and Hodgkin's disease, more frequent detection of diagnostic markers and ease of interpretation was demonstrated by use of PLP-fixed frozen tissue as compared with unfixed frozen tissue. Immunoglobulin staining was more easily interpreted in 30% of B-cell non-Hodgkin's lymphoma. In Hodgkin's disease, Ki-1 antigen, a diagnostic marker of Reed-Sternberg cells, was found in PLP-fixed tissue from two cases in which this antigen was not detected in corresponding unfixed frozen tissue. The authors have demonstrated that PLP-fixed tissue can be sent to a central reference laboratory at ambient or room temperature, avoiding the expense and inconvenience of transporting specimens on dry ice. The authors conclude that PLP fixation is the preferred method for immunopathologic study of human lymphomas.     

Characterization of an antimicrobial substance produced by Lactobacillus plantarum NTU 102

Background/PurposeLactic acid bacteria (LAB) are used in a variety of bio-industrial processes, including milk fermentation, and have been reported to have bactericidal activities. We previously isolated Lactobacillus plantarum NTU 102 from homemade Korean-style cabbage pickles. The aims of this work were to perform a screen of the antimicrobial substances produced by L. plantarum NTU 102 and to characterize it.MethodsIn this study, we investigated the bactericidal activity of this LAB strain and demonstrated that the cell-free supernatant of L. plantarum NTU 102 had antimicrobial activity.ResultsThe antibacterial activity was significantly decreased by proteolytic enzymes, including pepsin, proteinase K, and trypsin, suggesting that the antimicrobial substance had proteinaceous properties. Additionally, this activity was heat stable and not affected by alterations in the pH from 1.0 to 4.0. The antibacterial substance produced by L. plantarum NTU 102, which we named LBP102, exhibited a broad inhibitory spectrum. The active compound was identified by nuclear magnetic resonance (NMR) techniques (¹H NMR and ¹³C NMR). The IUPAC name was 2-(2-1 mino-1-hydroxyethoxy) ethyl 2-methylpropanoate. The substance showed antibacterial activity against Vibrio parahaemolyticus, and completely inhibited the growth of V. parahaemolyticus on agar plates at a concentration of 75 μg/mL.ConclusionThe proposed antimicrobial substance, LBP102 was found to be effective against V. parahaemolyticus BCRC 12864 and Cronobacter sakazakii BCRC 13988. The remarkable effects of LBP102 against this and other pathogens indicated its potential as a natural preservative/food additive.     

Lactobacillus reuteri DSM 17938 in the prevention of antibiotic-associated diarrhoea in children: a randomized clinical trial

ObjectivesTo assess the effectiveness of Lactobacillus reuteri DSM 17938 for the prevention of diarrhoea and antibiotic-associated diarrhoea (AAD) in children.MethodsHospitalized children who received antibiotics were assigned by a computer-generated list to receive L. reuteri (at 2 × 10⁸ CFU) or placebo, twice daily, for the duration of antibiotic treatment. Follow up was for 1 week after antibiotic cessation. The primary outcome measures were diarrhoea and AAD. Both were defined according to one of three definitions (i) three or more loose or watery stools per day for ≥48 h; (ii) three or more loose or watery stools per day for ≥24 h; or (iii) two or more loose or watery stools per day for ≥24 h. For AAD, it had to be diarrhoea caused by Clostridium difficile or otherwise unexplained diarrhoea.ResultsA total of 250 children were randomized and 247 were analysed (L. reuteri n = 123, placebo n = 124; median age 4 months). The occurrences of diarrhoea and AAD were similar in both groups, regardless of the definition used. Using the strictest definition (i.e. definition (i)), the occurrence of diarrhoea in the L. reuteri group was 25 (20%) compared with 16 (13%) in the placebo group (absolute risk reduction –0.07 (–0.17 to 0.02). The occurrence of AAD was 14 (11.4%) in the L. reuteri group compared with 8 (6.5%) in the placebo group (absolute risk reduction –0.05 (–0.13 to 0.02)). The groups were similar with respect to all secondary outcome measures, including adverse events.ConclusionsLactobacillus reuteri was not effective in the prevention of diarrhoea or AAD in children.     

Biocompatibility of poly(ether)urethane-gold nanocomposites

We have prepared the nanocomposites of a polyether-type waterborne polyurethane (PU) incorporated with different amounts (17.4-174 ppm) of gold (Au) nanoparticles ( approximately 5 nm). The nanocomposite containing a certain amount (43.5 ppm) of gold was previously demonstrated to possess the optimal thermal and mechanical properties, as well as much reduced foreign body reactions in subcutaneous rats. In this study, the surface morphology, biocompatibility, oxidative degradation, and free radical scavenging ability of the nanocomposites were characterized in vitro. The nanocomposite at 43.5 ppm of gold ("PU-Au 43.5 ppm") exhibited different surface morphology confirmed by the atomic force microscope. PU-Au 43.5 ppm also showed enhanced cellular proliferation, reduced platelet and monocyte activation, and much less bacterial adhesion, relative to PU alone or nanocomposites at the other Au contents, in general. This better biocompatibility was associated with the surface morphological change in the presence of Au. The oxidative degradation in PU-Au 43.5 ppm was also inhibited. The increased oxidative stability corresponded to the greater free radical scavenging ability of the nanocomposites.     

Effects of heparin immobilization on the surface characteristics of a biological tissue fixed with a naturally occurring crosslinking agent (genipin): an in vitro study

Heparinized biomaterials have been used to manufacture blood-contacting prostheses. The present study was intended to characterize the surface properties of a genipin-fixed biological tissue immobilized with heparin using the methods of ionic binding (the /h-i tissue) or covalent binding via multi-point attachment (the /h-m tissue) or end-point attachment (the /h-e tissue). The surface characteristics of test tissues evaluated were water contact angle, surface tension, protein adsorption, platelet adhesion, and cellular compatibility. Nonheparinized and the glutaraldehyde-fixed counterparts were used as controls. It was found that immobilization of heparin on the glutaraldehyde- and genipin-fixed tissues increased their hydrophilicity and surface tension and suppressed their mole ratio of adsorbed fibrinogen to adsorbed albumin and the amount of platelets adhered. Among the heparinized tissues, the /h-m tissue was more hydrophobic and had a higher mole ratio of adsorbed fibrinogen to adsorbed albumin and a greater amount of platelets adhered than the /h-i and /h-e tissues. In general, the surface characteristics of the /h-i tissue were comparable to the /h-e tissue. However, it is known that the ionically immobilized heparin may be displaced from the surface by an ion-exchange mechanism when exposed to blood. There were no significant differences in hydrophilicity, surface tension, the mole ratio of adsorbed fibrinogen to adsorbed albumin, and the amount of platelet adhesion between the glutaraldehyde- and genipin-fixed tissues in comparison with their respective counterparts. However, the cellular compatibility of the genipin-fixed tissues with or without heparinization was significantly superior to its glutaraldehyde-fixed counterparts.     

Inhibition of eosinophil chemotaxis by a new antiallergic compound (cetirizine)

The in vivo inhibitory effect of a new antiallergic, anti-H1 drug, cetirizine, on eosinophil attraction at skin sites challenged with various stimuli has been recently suggested. In the present work, we confirmed that this molecule, at therapeutical concentration, has a potent inhibitory action on eosinophil response to different chemoattractant mediators such as platelet-activating factor (PAF acether) and N-formyl methionyl leucyl phenyl alanyl in vitro. Another anti-H1 drug, polaramine, did not show this effect at the same concentration. These findings suggest that cetirizine in addition to its antihistaminic effect could also play a direct inhibitory effect on eosinophil recruitment. Moreover, cetirizine was not toxic for eosinophils and did not induce degranulation, as shown by the absence of peroxidase release. Comparison between cetirizine and a PAF acether antagonist (BN 52021) suggested that cetirizine did not act by a PAF receptor-blocking activity.