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PubMed2004

Pseudomonas aeruginosa biofilms exposed to imipenem exhibit changes in global gene expression and beta-lactamase and alginate production.

The lungs of cystic fibrosis (CF) patients are commonly colonized with Pseudomonas aeruginosa biofilms. Chronic endobronchial P. aeruginosa infections are impossible to eradicate with antibiotics, but intensive suppressive antibiotic therapy is essential to maintain the lung function of CF patients. The treatment often includes beta-lactam antibiotics. How these antibiotics influence gene expression in the surviving biofilm population of P. aeruginosa is not clear. Thus, we used the microarray technology to study the effects of subinhibitory concentrations of a beta-lactam antibiotic, imipenem, on gene expression in biofilm populations. Many genes showed small but statistically significant differential expression in response to imipenem. We identified 34 genes that were induced or repressed in biofilms exposed to imipenem more than fivefold compared to the levels of induction or repression for the controls. As expected, the most strongly induced gene was ampC, which codes for chromosomal beta-lactamase. We also found that genes coding for alginate biosynthesis were induced by exposure to imipenem. Alginate production is correlated to the development of impaired lung function, and P. aeruginosa strains isolated from chronically colonized lungs of CF patients are nearly always mucoid due to the overproduction of alginate. Exposure to subinhibitory concentrations of imipenem caused structural changes in the biofilm, e.g., an increased biofilm volume. Increased levels of alginate production may be an unintended adverse consequence of imipenem treatment in CF patients.

Alginates↗
PubMed2000

Survival of Bifidobacterium longum immobilized in calcium alginate beads in simulated gastric juices and bile salt solution.

Bifidobacterium longum KCTC 3128 and HLC 3742 were independently immobilized (entrapped) in calcium alginate beads containing 2, 3, and 4% sodium alginate. When the bifidobacteria entrapped in calcium alginate beads were exposed to simulated gastric juices and a bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads affected the numbers of survivors after exposure to these solutions; however, the death rates of the viable cells were not affected. Accordingly, a mathematical model was formulated which expressed the influences of several parameters (gel concentration, bead size, and initial cell numbers) on the survival of entrapped bifidobacteria after sequential exposure to simulated gastric juices followed by a bile salt solution. The model proposed in this paper may be useful for estimating the survival of bifidobacteria in beads and establishing optimal entrapment conditions.

Alginates↗
PubMed1981

Genetic mapping of chromosomal determinants for the production of the exopolysaccharide alginate in a Pseudomonas aeruginosa cystic fibrosis isolate.

Mucoid Pseudomonas aeruginosa strain FRD, a sputum isolate from a cystic fibrosis patient, was used to develop a genetic system. The mucoid appearance is due to the biosynthesis of the exopolysaccharide alginate and is a potential virulence factor of the organism. The sex factor plasmid FP2 was used for uninterrupted genetic exchange to investigate the nature of spontaneous mutations which produce frequent alginate-negative (Alg-) derivatives. Crosses between Alg+ donors and Alg- recipients demonstrated linkage between alginate genes and chromosomal markers. Crosses between an Alg- donor and Alg- recipients produced Alg+ recombinants at frequencies that varied, depending on the recipient strains used. This indicated that more than one genetic locus was associated with spontaneous mutation leading to loss of the mucoid character. Three classes of Alg- mutants were identified. Genetic exchange experiments showed that the loci of the alginate (alg) mutations of the three mutant classes are in the same region of the chromosome. The sex factor plasmid R68.45 was used for nonpolarized chromosome transfer and demonstrated close linkage between chromosomal markers (his-1, met-1) and alg markers. This was consistent with the data obtained in FP2-mediated crosses. Thus, the evidence obtained indicated that the alg genes which undergo frequent mutation are chromosomal, that several loci are involved, and that these alg loci are apparently clustered on the chromosome.

Alginates↗
PubMed2001

Role of alginate O acetylation in resistance of mucoid Pseudomonas aeruginosa to opsonic phagocytosis.

Establishment and maintenance of chronic lung infections with mucoid Pseudomonas aeruginosa in patients with cystic fibrosis (CF) require that the bacteria avoid host defenses. Elaboration of the extracellular, O-acetylated mucoid exopolysaccharide, or alginate, is a major microbial factor in resistance to immune effectors. Here we show that O acetylation of alginate maximizes the resistance of mucoid P. aeruginosa to antibody-independent opsonic killing and is the molecular basis for the resistance of mucoid P. aeruginosa to normally nonopsonic but alginate-specific antibodies found in normal human sera and sera of infected CF patients. O acetylation of alginate appears to be critical for P. aeruginosa resistance to host immune effectors in CF patients.

Acetylation↗
PubMed1988

Cloning of genes from mucoid Pseudomonas aeruginosa which control spontaneous conversion to the alginate production phenotype.

Strains of Pseudomonas aeruginosa causing chronic pulmonary infections in patients with cystic fibrosis are known to convert to a mucoid form in vivo characterized by the production of the exopolysaccharide alginate. The alginate production trait is not stable, and mucoid strains frequently convert back to the nonmucoid form in vitro. The DNA involved in these spontaneous alginate conversions, referred to as algS, was shown here to map near hisI and pru markers on the chromosome of strain FRD, an isolate from a cystic fibrosis patient. Although cloning algS+ by trans-complementation was not possible, a clone (pJF5) was isolated that caused algS mutants to convert to the Alg+ phenotype at detectable frequencies (approximately 0.1%) in vitro. Gene replacement with transposon-marked pJF5 followed by mapping studies showed that pJF5 contained DNA transducibly close to algS in the chromosome. Another clone was identified called pJF15 which did contain algS+ from mucoid P. aeruginosa. The plasmid-borne algS+ locus could not complement spontaneous algS mutations in trans, but its cis-acting activity was readily observed after gene replacement with the algS mutant chromosome by using an adjacent transposon as the selectable marker. pJF15 also contained a trans-active gene called algT+ in addition to the cis-active gene algS+. The algT gene was localized on pJF15 by using deletion mapping and transposon mutagenesis. By using gene replacement, algT::Tn501 mutants of P. aeruginosa were constructed which were shown to be complemented in trans by pJF15. Both algS and algT were located on a DNA fragment approximately 3 kilobases in size. The algS gene may be a genetic switch which regulates the process of alginate conversion.

Alginates↗
PubMed1999

Regulation of alginate biosynthesis in Pseudomonas syringae pv. syringae.

Both Pseudomonas aeruginosa and the phytopathogen P. syringae produce the exopolysaccharide alginate. However, the environmental signals that trigger alginate gene expression in P. syringae are different from those in P. aeruginosa with copper being a major signal in P. syringae. In P. aeruginosa, the alternate sigma factor encoded by algT (sigma22) and the response regulator AlgR1 are required for transcription of algD, a gene which encodes a key enzyme in the alginate biosynthetic pathway. In the present study, we cloned and characterized the gene encoding AlgR1 from P. syringae. The deduced amino acid sequence of AlgR1 from P. syringae showed 86% identity to its P. aeruginosa counterpart. Sequence analysis of the region flanking algR1 in P. syringae revealed the presence of argH, algZ, and hemC in an arrangement virtually identical to that reported in P. aeruginosa. An algR1 mutant, P. syringae FF5.32, was defective in alginate production but could be complemented when algR1 was expressed in trans. The algD promoter region in P. syringae (PsalgD) was also characterized and shown to diverge significantly from the algD promoter in P. aeruginosa. Unlike P. aeruginosa, algR1 was not required for the transcription of algD in P. syringae, and PsalgD lacked the consensus sequence recognized by AlgR1. However, both the algD and algR1 upstream regions in P. syringae contained the consensus sequence recognized by sigma22, suggesting that algT is required for transcription of both genes.

Alginates↗
PubMed2000

Role of Azotobacter vinelandii mucA and mucC gene products in alginate production.

Azotobacter vinelandii produces the exopolysaccharide alginate, which is essential for its differentiation to desiccation-resistant cysts. In different bacterial species, the alternative sigma factor sigma(E) regulates the expression of functions related to the extracytoplasmic compartments. In A. vinelandii and Pseudomonas aeruginosa, the sigma(E) factor (AlgU) is essential for alginate production. In both bacteria, the activity of this sigma factor is regulated by the product of the mucA, mucB, mucC, and mucD genes. In this work, we studied the transcriptional regulation of the A. vinelandii algU-mucABCD gene cluster, as well as the role of the mucA and mucC gene products in alginate production. Our results show the existence of AlgU autoregulation and show that both MucA and MucC play a negative role in alginate production.

Alginates↗
PubMed2000

The GacS sensor kinase regulates alginate and poly-beta-hydroxybutyrate production in Azotobacter vinelandii.

Azotobacter vinelandii produces two polymers: the extracellular polysaccharide alginate and the intracellular polyester poly-beta-hydroxybutyrate (PHB). A cosmid clone (pSMU588) from an A. vinelandii gene library diminished alginate production by A. vinelandii mucoid strain ATCC 9046. The nucleotide sequence and predicted amino acid sequence of the locus responsible for the mucoidy suppression revealed 65% identity to Pseudomonas GacS, a transmembrane sensor kinase of the two-component regulators, whose cognate response regulator, GacA, is a global activator regulating several products and virulence factors. Plasmid pMC15, harboring gacS, and a strain carrying a gacS nonpolar mutation were constructed. Either pMC15 or the gacS mutation significantly reduced alginate production and transcription of algD, the gene coding for the key enzyme GDP-mannose dehydrogenase of the alginate biosynthetic pathway. We found that the gacS mutation also reduced PHB accumulation and impaired encystment. Taken together, these data indicate that in A. vinelandii the gacSA global system regulates polymer synthesis.

Alginates↗
PubMed1989

Comparative evaluation of mitogenicity and basement-membrane-degrading activity of Pseudomonas aeruginosa slime glycolipoprotein and alginate.

Alginate from a heavily mucoid Pseudomonas aeruginosa strain and slime glycolipoprotein obtained from the revertant nonmucoid variant of the mucoid strain were tested for mitogenic activity on human peripheral lymphocytes and for degradation of 3H-labeled basement membranes of the anterior lens capsule of bovine eyes. Slime glycolipoprotein exerted mitogenic activity in concentrations from 50 to 200 micrograms/ml, whereas alginate was not mitogenic as shown by [3H]thymidine uptake. Alginate did not show any basement membrane degradation, whereas slime glycolipoprotein exhibited basement-membrane-degrading activity from 35 to 450 micrograms/ml in a dose-related manner. This activity was inhibited by metal chelators but not thiol protease inhibitors. The results suggest that alginate lacks the mitogenic and biodegrading activities of slime glycolipoprotein; these activities nevertheless need further investigation.

Alginates↗
PubMed2000

ALGINATE LYASE: review of major sources and enzyme characteristics, structure-function analysis, biological roles, and applications.

Alginate lyases, characterized as either mannuronate (EC 4.2.2.3) or guluronate lyases (EC 4.2.2.11), catalyze the degradation of alginate, a complex copolymer of alpha-L-guluronate and its C5 epimer beta-D-mannuronate. Lyases have been isolated from a wide range of organisms, including algae, marine invertebrates, and marine and terrestrial microorganisms. This review catalogs the major characteristics of these lyases, the methods for analyzing these enzymes, as well as their biological roles. Analysis of primary sequence data identifies some markedly conserved motifs that should help elucidate functional domains. Information about the three-dimensional structure of a mannuronate lyase from Sphingomonas sp., combined with various mutagenesis studies, has identified residues that are important for catalytic activity in several lyases. Characterization of alginate lyases will enhance and expand the use of these enzymes to engineer novel alginate polymers for applications in various industrial, agricultural, and medical fields. In this review, we explore both past and present applications of this important enzyme and discuss its future prospects.

Alginates↗
PubMed2001

In vitro effects of transcatheter injection on structure, cell viability, and cell metabolism in fibroblast-impregnated alginate microspheres.

PURPOSE: To determine if microsphere-encapsulated cell preparations can be delivered through a microcatheter without compromising microsphere structure, cell viability, or metabolism. MATERIALS AND METHODS: Fibroblast-impregnated microspheres were fabricated by using 1.0% alginate and rabbit synovial fibroblasts. Fibroblast-impregnated alginate microspheres injected through microcatheters were analyzed in parallel with identical noninjected microspheres. The effects of transcatheter injection on structure and cell viability (percentage of viable cells per microsphere) were correlated with microsphere size. Structural effects were analyzed by using light microscopy, and 7-day percentage (ratio of live cells to dead cells) cell viability was assessed with confocal microscopy and fluorescent staining. In a second series of experiments, the metabolism of small microspheres was studied during a course of 7 days by using a spectrophotometric bioanalyzer. RESULTS: Transcatheter injection caused fracturing and/or fragmentation of large (800-1,000 microm) and medium (500-750 microm) microspheres, while small (250-400 microm) microspheres were structurally unaffected by transcatheter injection. Fracturing and fragmentation were associated with cell release from the alginate matrix. Although transcatheter injection reduced cell viability by 17%-23% in all size categories, it did not cause a detectable alteration in the rate of glucose metabolism. CONCLUSION: Transcatheter injection was physiologically well tolerated by fibroblasts encapsulated in alginate microspheres; however, when microsphere diameter exceeded the catheter diameter, fracturing and fragmentation of microspheres compromised the sequestration function of the microsphere vector.

Alginates↗
PubMed1993

Effect of Pseudomonas aeruginosa alginate on Escherichia coli- and Staphylococcus aureus-induced inflammatory mediator release from human cells.

We analyzed the role of soluble purified Pseudomonas aeruginosa alginate on Escherichia coli K-12 (pANN5211) as well as on Staphylococcus aureus 121c-induced inflammatory mediator release from human platelets, granulocytes, and basophils. In the presence of alginate (1-4 mg/ml), the mucoid exopolysaccharide of P. aeruginosa, the bacteria-induced inflammatory mediator release was modulated as follows: (1) the E. coli- as well as S. aureus-induced chemiluminescence response from human neutrophils increased 2- to 3-fold and 5- to 6-fold, respectively; (2) the E. coli-induced leukotriene B4 formation from human neutrophils was enhanced (from 29.17 +/- 1.8 up to 36.9 +/- 4 ng/10(7) cells) as was also observed for the E. coli- and S. aureus-induced histamine release (3- to 4-fold) and the 12-hydroxyeicosatetraenoic acid generation from human platelets (2-fold), and (3) prolonged duration of the E. coli-induced increase in CD11b expression was observed. Alginate by itself was inactive. Our results provide evidence that alginate interacts with hemolysin-producing E. coli and S. aureus bacteria and thus leads to a modulation of the cellular response pattern, which leads to the local destruction of the lung in cystic fibrosis.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed2005

Quantitative chemical mapping of sodium acrylate- and N-vinylpyrrolidone-enhanced alginate microcapsules.

Alginate microcapsules enclosing recombinant cells secreting therapeutic products have been used successfully to treat several murine models of human diseases. The mechanical and chemical properties of these alginate capsules can be improved by the addition and in situ photo-polymerization of sodium acrylate and N-vinylpyrrolidone in the alginate capsule. The purpose of this modification was to form additional covalent cross-links. In this work we have used scanning transmission X-ray microscopy (STXM) to probe the nature and location of the chemical modifications in the modified capsules by comparison with unmodified capsules. Analysis of X-ray image sequences and selected area spectra has been used to map the calcium gradient in capsules, to identify the presence of polyacrylate throughout the capsules and the localization of poly-N-vinylpyrrolidone in the outer regions of the alginate capsules. The differences in the spatial distributions of these species have led to better understanding of the chemical modifications that provide a mechanically more stable capsule structure.

Acrylates↗
PubMed2005

Alginate beads as immobilization matrix for hepatocytes perfused in a bioreactor: a physico-chemical characterization.

Because of their peculiar physico-chemical properties, alginate beads have often been proposed as an alternative cell immobilization matrix for many biotechnological applications. For entrapped hepatocytes perfused in a bioreactor, alginate beads have been demonstrated to promote viability and three-dimensional cell organization. In order to optimise the hepatocyte cell culture, we investigated the relationship between alginate beads properties, at high and low content of guluronic acid (G), and the relative cell viability and reorganization when perfused in a bioreactor. The primary structure of alginates did not apparently influence the hepatocytes culture in 8 h of perfusion in a bioreactor. However, our results confirm a preference for beads with a high content of G due to their superior mechanical resistance.

Alginates↗
PubMed1993

In vitro activation of human macrophages by alginate-polylysine microcapsules.

Microencapsulated islets of Langerhans have been proposed as a bioartificial pancreas. However, foreign body reaction with fibrosis has been observed around implanted microcapsules. Since macrophages are present in this reaction and interleukin-1 (IL-1), a cytokine released by activated macrophages, may induce fibrosis, we tested the capacity of alginate-polylysine microcapsules to activate macrophages. Human monocytes were isolated from whole blood of healthy donors by a Ficoll density gradient and adherence to a plastic support. Monocytes were cultured for 24 h with: (1) alginate-polylysine microcapsules; (2) lipopolysaccharide (LPS) (positive control group); and (3) alone (negative control group). Monocyte activation was evaluated by measuring the secretion of IL-1 beta and the production of intracellular IL-1 alpha and IL-1 beta. Macrophages characterization was performed by immunocytological subtyping. IL-1 beta release and intracellular IL-1 beta and IL-1 alpha production were significantly higher when macrophages were cultured with alginate-polylysine microcapsules than when macrophages were cultured alone. In conclusion, macrophages are activated in vitro by alginate-polylysine microcapsules. This effect may be involved in the fibrosis observed in vivo around implanted microcapsules. In addition, interleukin-1, released during macrophage activation, may cross the microcapsule membrane and impair islet function.

Alginates↗
PubMed1998

Comparative study of the use of poly(glycolic acid), calcium alginate and pluronics in the engineering of autologous porcine cartilage.

New cartilage formation has been successfully achieved by technology referred to as tissue engineering. Polymers and hydrogels such as poly(glycolic acid), calcium alginate, and poly(ethylene) and poly(propylene) hydrogels have been used as cell carriers to regenerate cartilage in the nude mouse model. The next step toward human applications of engineered cartilage is to demonstrate their potential in immunocompetent animal models. This study compared the suitability of three polymers for generating tissue engineered elastic cartilage using autologous cells in an immuno-competent porcine animal model. Auricular cartilage was obtained from pigs. Chondrocytes were isolated onto fiber based poly(glycolic acid) (PGA) scaffolds or suspended in calcium alginate or pluronic F127 gel at constant concentrations. Chondrocyte-polymer constructs were either implanted (PGA) or injected (calcium alginate and pluronic) as autologous implants subcutaneously into the pigs from which the cells had been isolated. Specimens were harvested and analyzed grossly and historically after 6 weeks in vivo. All explants demonstrated cartilage formation to a variable degree. When using PGA or calcium alginate, the overall histological appearance of the tissue formed is that of fibrocartilage with thick bundles of collagen dispersed in the tissue. When using pluronics as scaffold, histologic features resemble those of native elastic cartilage, showing a more organized arrangement of the cells, which seems to correlate to functional properties as elastin presence in the tissue engineered cartilage. Elastic cartilage engineered in an immunocompetent animal model varies with the type of polymer used. The behavior of the cell-polymer constructs is not fully understood and outcome seems to be related to several factors, including inflammatory reaction. Further studies with similar models are needed to determine the feasibility of engineering tissue generated from different cell-polymer constructs prior to human application.

Alginates↗
PubMed2000

Cartilage generation using alginate-encapsulated autogenous chondrocytes in rabbits.

In this study, we investigated the possible use of calcium alginate as a matrix for cartilage generation with autogenous chondrocytes, and examined whether the generated cartilage could keep its original volume over time when used as an implant for filling and contour restoration in the host body. Biodegradable, biocompatible, and injectable calcium alginate impregnated with isolated autogenous chondrocytes from the auricle was injected into the gluteus muscle of 12 New Zealand White rabbits. The volume of injected calcium alginate was always 3 mL, and the density of chondrocytes was 10 x 10(6) cells per milliliter. At 4 weeks (short-term period, n = 6) and 20 weeks (long-term period, n = 6) after injection, the histologic findings and the volume of the generated cartilaginous nodules were analyzed. At the time of harvest, 10 of the 12 specimens revealed findings characteristic of natural cartilage. However, histologic examination demonstrated scanty vascular and fibrous tissue ingrowth. Many osteoid matrices, including marrow-like cells, were noted in the vicinity of the neocartilage. The approximate original volume of the injected material was maintained over 20 weeks. These results suggest that although complete cartilage replacement was not always achieved, calcium alginate-autogenous chondrocytes may represent an injectable implant that can generate new autogenous fibro-osteo-cartilaginous tissue for volume augmentation.

Alginates↗
PubMed2006

Chitosan-coated alginate membranes for cultivation of limbal epithelial cells to use in the restoration of damaged corneal surfaces.

Some chemicals or thermal burns may result in abnormal reepithelialization by conjunctival epithelial cells and it causes different types of damage on the cornea surface. When reepithelialization does not occur, chronic inflammation and neovascularization develop, often leading to stroma scarring and/or ulceration. The aim of this study is to restore the human corneal surface with autologous corneal epithelial sheets generated by serial cultivation of the limbal epithelial cells over the different compositions of composite membranes. The composite membranes were prepared by coating the alginate membrane with chitosan. In this method, alginate membrane was prepared by precipitation of the sodium alginate solution in calcium chloride solution. Alginate membranes were washed, dried and immersed into the chitosan solutions to prepare composite membranes. The composite membranes were characterized based on their morphology, hydrophilicity, swellability, and chemical structure. In the last part of the study, composite membranes were used as base matrices for limbal epithelial cell cultivation. The cell cultivation on polymeric membranes was investigated as the in vitro studies. In these studies cell attachment, spreading and growth on polymeric membranes were evaluated.

Alginates↗