Probing the role of microenvironment for microencapsulated Sacchromyces cerevisiae under osmotic stress

Cell encapsulation opens a new avenue to the oral delivery of genetically engineered microorganism for therapeutic purpose. Osmotic stress is one of the universal chemical stress factors in the application of microencapsulation technology. In order to understand the effect and mechanism of the encapsulated microenvironment on protecting cells from hyper-osmotic stress, yeast cells of Saccharomyces cerevisiae Y800 were encapsulated in calcium alginate micro-gel beads (MB), alginate-chitosan-alginate (ACA) solid core microcapsules (SCM), and ACA liquid core microcapsules (LCM), respectively. The stress-induced intracellular components and enzyme activity including trehalose, glycerol and super oxide dismutase (SOD) were measured. Free cell culture was used as control. The survival of encapsulated cells and the cells released from MB, SCM and LCM after osmotic shock induced by NaCl solution (1, 2 and 3M) was evaluated. An analysis method was established to probe the effect of encapsulated microenvironment on the cell tolerance to osmotic stress. The results showed that LCM gave rise to the highest level of intracellular trehalose and glycerol, and SOD activity, as well as the highest survival rate of encapsulated cells or cells released from microcapsule. It was demonstrated that LCM was able to induce the highest stress response and stress tolerance of cells, which was adapted during culture, while SCM failed. The theoretical analysis revealed that it was the liquid alginate matrix in microcapsule that played a central role in domesticating the cells to adapt to hyper-osmotic stress. This finding provides a very useful guideline to cell encapsulation.     

Encapsulation of parathyroid cells in hollow fibers: a preliminary report

The purpose of experiments was to evaluate the survival and functioning of human parathyroid cells after encapsulation in hollow fibers (HFs). The polypropylene HFs K600(PP Accurel (Akzo-Nobel, Germany) of inner diameter 0.6 mm, wall thickness 0.2 mm, original or surface modified were used for encapsulation. Production of parathormone (PTH) by encapsulated cells was measured in vitro. HF were filled with parathyroid cell suspension and tightly closed. Encapsulated cells were cultured for 9 or 33 days in RPMI 1640 containing 10% FCS or in Chang's medium. The level of PTH, produced by encapsulated cells was evaluated in the culture medium with radioimmunoassay test (RIA). The assays were performed every 2-4 days. The result of PTH assay was similar in both types of tested media as well as with unmodified and modified HFs, being 2-4 pg/ml of culture medium per 10(3) encapsulated cells. In conclusion, encapsulation in original or modified HFs ensures diffusion of nutrients from culture medium to encapsulated cells and allows for functioning of cells for at least 33 days in vitro.     

Calcium-dependent regulation of NF-(kappa)B activation in cystic fibrosis airway epithelial cells

Dysregulation of nuclear factor kappa B (NF-(kappa)B) and increased Ca(2+) signals have been reported in airway epithelial cells of patients with cystic fibrosis (CF). The hypothesis that Ca(2+) signaling may regulate NF-(kappa)B activation was tested in a CF bronchial epithelial cell line (IB3-1, CFTR genotype DeltaF508/W1282X) and compared to the CFTR-corrected epithelial cell line S9 using fluorescence microscopy to visualized in situ NF-(kappa)B activation at the single cell level. Upon stimulation with IL-1beta,we observed a slow but prolonged [Ca(2+)](i) increase (up to 10 min) in IB3-1 cells compared to S9 cells. The IL-1beta-induced [Ca(2+)](i) response was accompanied by an activation of NF-(kappa)B in IB3-1 but not in S9 cells. Pretreatment of IB3-1 cells with the ER Ca(2+) pump inhibitor thapsigargin inhibited the IL-1beta-induced [Ca(2+)](i) response. Treatment with either the calcium chelator BAPTA or an inhibitor of I(kappa)Balpha phosphorylation (digitoxin) led to a drastic [Ca(2+)](i) decrease accompanied by an inhibition of NF-(kappa)B activation of IL-1beta-stimulated IB3-1 cells in comparison to untreated cells. In IB3-1 cells cultured at low temperature (26 degrees C) for 16 h, the IL-1beta-induced [Ca(2+)](i) response was inhibited and no significant NF-(kappa)B activation was observed. To our knowledge, this is the first report of visualization of the Ca(2+)-mediated activation of NF-(kappa)B in individual living airway epithelial cells. Our results support the concept that [Ca(2+)](i) is a key regulator of NF-(kappa)B activation in CF airway epithelial cells.     

An improved method of microencapsulation and its evaluation to protect Lactobacillus spp. in simulated gastric conditions

An improved method of microencapsulation was developed to increase the efficacy of capsules in protecting the encapsulated bacteria under simulated gastric conditions. Lactobacillus acidophilus CSCC 2400 was encapsulated in calcium alginate and tested for its survival in simulated gastric conditions. The effects of different capsule sizes (200, 450, 1000 microm), different sodium alginate concentrations (0.75%, 1%, 1.5%, 1.8% and 2% w/v) and different concentrations of calcium chloride (0.1, 0.2, 1.0 M) on the viability of encapsulated bacteria were investigated. The viability of the cells in the microcapsules increased with an increase in alginate capsule size and gel concentration. There was no significant difference (p>0.05) in the viability of encapsulated cells when the concentration of calcium chloride was increased. Increase in cell load during encapsulation increased the number of bacterial survivors at the end of 3-h incubation in simulated gastric conditions. Hardening the capsule in calcium chloride solution for a longer time (8 h) had no impact on increasing the viability of encapsulated bacteria in a simulated gastric environment. The release of encapsulated cells at different phosphate buffer concentrations was also studied. When encapsulated L. acidophilus CSCC 2400 and L. acidophilus CSCC 2409 were subjected to low pH (pH 2) and high bile concentration (1.0% bile) under optimal encapsulation conditions (1.8% (w/v) alginate, 10(9) CFU/ml, 30 min hardening in 0.1 M CaCl(2) and capsule size 450 microm), there was a significant increase (p<0.05) in viable cell counts, compared to the free cells under similar conditions. Thus the encapsulation method described in this study may be effectively used to protect the lactobacillus from adverse gastric conditions.     

Improved activity of streptozotocin-selected insulinoma cells following microencapsulation and transplantation into diabetic mice

We have recently shown that repeated streptozotocin (STZ) treatment induces the selection of insulinoma cells (RINmS) with both improved resistance to diabetogenic toxins and functional activity, compared to parental RINm cells. The aim of the present study was to estimate the potential of RINmS cells to maintain their engineered characteristics during in vivo hyperglycemic conditions. It was found that microencapsulation and transplantation into diabetic mice preserved a three-fold higher level of insulin content in selected RINmS cells when compared to the parental ones. Retrieval of transplanted encapsulated cells from the peritoneal cavity of diabetic mice had a significantly higher insulin content and a more intense insulin response to secretogogues in selected RINmS cells when compared to retrieved RINm cells. In conclusion, our results show that RINmS cells do not lose their improved functional characteristics after encapsulation and transplantation into diabetic mice.     

cDNA cloning and mapping of a novel islet-brain/JNK-interacting protein

IB1/JIP-1 is a scaffold protein that regulates the c-Jun NH(2)-terminal kinase (JNK) signaling pathway, which is activated by environmental stresses and/or by treatment with proinflammatory cytokines including IL-1beta and TNF-alpha. The JNKs play an essential role in many biological processes, including the maturation and differentiation of immune cells and the apoptosis of cell targets of the immune system. IB1 is expressed predominantly in brain and pancreatic beta-cells where it protects cells from proapoptotic programs. Recently, a mutation in the amino-terminus of IB1 was associated with diabetes. A novel isoform, IB2, was cloned and characterized. Overall, both IB1 and IB2 proteins share a very similar organization, with a JNK-binding domain, a Src homology 3 domain, a phosphotyrosine-interacting domain, and polyacidic and polyproline stretches located at similar positions. The IB2 gene (HGMW-approved symbol MAPK8IP2) maps to human chromosome 22q13 and contains 10 coding exons. Northern and RT-PCR analyses indicate that IB2 is expressed in brain and in pancreatic cells, including insulin-secreting cells. IB2 interacts with both JNK and the JNK-kinase MKK7. In addition, ectopic expression of the JNK-binding domain of IB2 decreases IL-1beta-induced pancreatic beta-cell death. These data establish IB2 as a novel scaffold protein that regulates the JNK signaling pathway in brain and pancreatic beta-cells and indicate that IB2 represents a novel candidate gene for diabetes.     

Cationic phospholiposomes: efficient delivery vehicles of anticancer derivatives of ATP to multiple myeloma cells

Analogs of adenosine triphosphate (ATP) with substitutions at the 8-position have been shown to be cytotoxic to multiple myeloma, one of the most prevalent and serious blood cancers. However, these drugs do not readily cross biological membranes and are very sensitive to phosphatases present in body fluids. To circumvent these disadvantages, 8-substituted ATPs were encapsulated into cationic phospholiposomes generated from cationic phosphatidylcholines (EDOPC; 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, and EDPPC, the corresponding dipalmitoyl homolog), compounds with low toxicity that readily form liposomes. Vortexing was an efficient encapsulation procedure, more so than freeze-thawing. At the lipid:drug ratio of 5:1 (mol/mol), 20% of 8-Br-ATP was encapsulated within EDOPC liposomes. Efficient encapsulation and retention of 8-NH?-ATP required the inclusion of cholesterol. Liposomes of EDOPC:cholesterol (55:45 mole/mole), at a lipid:drug mole ratio of 10:1, captured ~40% of the drug presented. Cytotoxicity assays of this formulation on multiple myeloma cells in culture showed encapsulated drug to be up to 10-fold more effective than free drug, depending upon dose. Intracellular distribution studies (based on fluorescent derivatives of lipids and of ATP) revealed that both liposomes and drug were taken up by multiple myeloma cells, and that uptake of a fluorescent ATP derivative was significantly greater when encapsulated than when free. Liposomes prepared from EDPPC, having a higher phase-transition temperature than EDOPC, captured 8-NH?-ATP satisfactorily and released it more slowly than the unsaturated formulations, but were also less cytotoxic. The superior encapsulation efficiencies of the positively charged liposomes can be understood in terms of the electrostatic double layer due to a very high positive charge density on their inner surface. Electrostatic augmentation of encapsulation for small vesicles can be dramatic, easily exceeding an order of magnitude.     

Cationic phospholiposomes: efficient delivery vehicles of anticancer derivatives of ATP to multiple myeloma cells

Analogs of adenosine triphosphate (ATP) with substitutions at the 8-position have been shown to be cytotoxic to multiple myeloma, one of the most prevalent and serious blood cancers. However, these drugs do not readily cross biological membranes and are very sensitive to phosphatases present in body fluids. To circumvent these disadvantages, 8-substituted ATPs were encapsulated into cationic phospholiposomes generated from cationic phosphatidylcholines (EDOPC; 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, and EDPPC, the corresponding dipalmitoyl homolog), compounds with low toxicity that readily form liposomes. Vortexing was an efficient encapsulation procedure, more so than freeze-thawing. At the lipid:drug ratio of 5:1 (mol/mol), 20% of 8-Br-ATP was encapsulated within EDOPC liposomes. Efficient encapsulation and retention of 8-NH₂-ATP required the inclusion of cholesterol. Liposomes of EDOPC:cholesterol (55:45 mole/mole), at a lipid:drug mole ratio of 10:1, captured ~40% of the drug presented. Cytotoxicity assays of this formulation on multiple myeloma cells in culture showed encapsulated drug to be up to 10-fold more effective than free drug, depending upon dose. Intracellular distribution studies (based on fluorescent derivatives of lipids and of ATP) revealed that both liposomes and drug were taken up by multiple myeloma cells, and that uptake of a fluorescent ATP derivative was significantly greater when encapsulated than when free. Liposomes prepared from EDPPC, having a higher phase-transition temperature than EDOPC, captured 8-NH₂-ATP satisfactorily and released it more slowly than the unsaturated formulations, but were also less cytotoxic. The superior encapsulation efficiencies of the positively charged liposomes can be understood in terms of the electrostatic double layer due to a very high positive charge density on their inner surface. Electrostatic augmentation of encapsulation for small vesicles can be dramatic, easily exceeding an order of magnitude.     

Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference

Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation.Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or after malignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper we demonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression in airway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targeting human IL-8 in cultured airway epithelial cells (IB3-1, Cftr; C38, Cftr-corrected) stimulated with TNF-α, IL-1β or heat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reduced by shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-κB, or on the protein levels of IκB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for the treatment of inflammatory diseases.     

γ-Glutamyltransferase catabolism of S-nitrosoglutathione modulates IL-8 expression in cystic fibrosis bronchial epithelial cells

S-nitrosoglutathione (GSNO) is an endogenous nitrosothiol involved in several pathophysiological processes. A role for GSNO has been envisaged in the expression of inflammatory cytokines such as IL-8; however, conflicting results have been reported. γ-Glutamyltransferase (GGT) enzyme activity can hydrolyze the γ-glutamyl bond present in the GSNO molecule thus greatly accelerating the release of bioactive nitric oxide. Expression of GGT is induced by oxidative stress, and activated neutrophils contribute to GGT increase in cystic fibrosis (CF) lung exudates by releasing GGT-containing microvesicles. This study was aimed at evaluating the effect of GSNO catabolism mediated by GGT on production of IL-8 in CF transmembrane regulation protein-mutated IB3-1 bronchial cells. The rapid, GGT-catalyzed catabolism of GSNO caused a decrease in both basal and lipopolysaccharide-stimulated IL-8 production in IB3-1 cells, by modulating both NF-κB and ERK1/2 pathways, along with a decrease in cell proliferation. In contrast, a slow decomposition of GSNO produced a significant increase in both cell proliferation and expression of IL-8, the latter possibly through p38-mediated stabilization of IL-8 mRNA. Our data suggest that the differential GSNO catabolism mediated by GGT enzyme activity can downregulate the production of IL-8 in CF cells. Hence, the role of GGT activity should be considered when evaluating GSNO for both in vitro and in vivo studies, the more so in the case of GSNO-based therapies for cystic fibrosis.     

Cell viability of microencapsulated Bifidobacterium animalis subsp. lactis under freeze-drying, storage and gastrointestinal tract simulation conditions

The purpose of this study was to improve the survival of Bifidobacterium animalis subsp. lactis 10140 during freeze-drying process by microencapsulation, using a special pediatric prebiotics mixture (galactooligosaccharides and fructooligosaccharides). Probiotic microorganisms were encapsulated with a coat combination of prebiotics–calcium-alginate prior to freeze-drying. Both encapsulated and free cells were then freeze-dried in their optimized combinations of skim milk and prebiotics. Response surface methodology (RSM) was used to produce a coating combination as well as drying medium with the highest cell viability during freeze-drying. The optimum encapsulation composition was found to be 2.1 % Na-alginate, 2.9 % prebiotic, and 21.7 % glycerol. Maximum survival predicted by the model was 81.2 %. No significant (p?>?0.05) difference between the predicted and experimental values verified the adequacy of final reduced models. The protection ability of encapsulation was then examined over 120 days of storage at 4 and 25 °C and exposure to a sequential model of infantile GIT conditions including both gastric conditions (pH 3.0 and 4.0, 90 min, 37 °C) and intestinal conditions (pH 7.5, 5 h, 37 °C). Significantly improved cell viability showed that microencapsulation of B. lactis 10140 with the prebiotics was successful in producing a stable symbiotic powdery nutraceutical.     

Increased NaCl-induced interleukin-8 production by human bronchial epithelial cells is enhanced by the DeltaF508/W1282X mutation of the cystic fibrosis transmembrane conductance regulator gene

A satisfactory model describing the airway surface fluid (ASF) in the airways of persons with cystic fibrosis (CF) remains to be established due to theoretical challenges to both the "Hydration Hypothesis" and the "Salt Hypothesis." Irrespective of these models, inhaled hypertonic saline is often used to facilitate clearance of inspissated secretions. Hypertonicity induces interleukin-8 (IL-8) expression, a potent chemokine for neutrophils. The objectives of this study were: (i) to determine the relative contribution of three potential cis-regulatory elements in the regulation of NaCl-induced IL-8 production in BEAS-2B human bronchial epithelial cells, (ii) to compare NaCl-induced IL-8 expression in IB3-1 bronchial epithelial cells, which have the DeltaF508/W1282X mutation of the CF transmembrane conductance regulator (CFTR) gene, with that in C38 cells, which are IB3-1 cells stably transfected with a truncated but functional CFTR gene, and (iii) to compare equal osmolar concentrations of NaCl and D-sorbitol in the induction of IL-8 in all three cell types. In human bronchial epithelial cells, binding sites for NFkappaB, AP-1, and NF-IL6 in the 5'-flanking region of the IL-8 promoter are necessary for optimal NaCl induction of IL-8. Human bronchial epithelial cells with the DeltaF508/W1282X CFTR mutation produce an exaggerated amount of basal and NaCl-induced IL-8.     

Liposome-mediated delivery of pteridine antifolates to cells in vitro: potency of methotrexate, and its alpha and gamma substituents

We have examined the growth-inhibitory potency of several pteridines encapsulated in negatively charged liposomes, including methotrexate, methotrexate-gamma-methylamide, methotrexate-gamma-dimethylamide, methotrexate-alpha-aspartate, and a lipophilic methotrexate-phosphatidylethanolamine conjugate. The potency of encapsulated methotrexate is greater than the potency of the free drug for CV1-P cells, but not for other cell lines. The potency of methotrexate-gamma-methylamide and methotrexate-gamma-dimethylamide is only minimally improved by encapsulation. The potency of methotrexate-alpha-aspartate is increased by encapsulation. In addition, the lipophilic methotrexate derivative has demonstrable potency when incorporated in liposomes. We have also examined the potency of several pteridines under conditions where the cells are exposed to the drug for periods shorter than the entire growth assay. Reduction of the exposure time decreases the potency of both encapsulated and free drugs. However, the difference in potency between the encapsulated and free drug is increased, because the potency of the encapsulated drug is affected less. Consequently, encapsulated methotrexate-gamma-aspartate is 300-fold more potent than free drug, if CV1-P cells are exposed to drug for 4 h. Moreover, encapsulated methotrexate is more potent than free methotrexate for growth inhibition of L929 fibroblasts, if the term of exposure is less than 8 h. Potency is least affected by reduction of exposure length for the lipophilic methotrexate derivative.     

Yeast cells for encapsulation of bioactive compounds in food products: A review

Nowadays bioactive compounds have gained great attention in food and drug industries owing to their health aspects as well as antimicrobial and antioxidant attributes. Nevertheless, their bioavailability, bioactivity, and stability can be affected in different conditions and during storage. In addition, some bioactive compounds have undesirable flavor that restrict their application especially at high dosage in food products. Therefore, food industry needs to find novel techniques to overcome these problems. Microencapsulation is a technique, which can fulfill the mentioned requirements. Also, there are many wall materials for use in encapsulation procedure such as proteins, carbohydrates, lipids, and various kinds of polymers. The utilization of food-grade and safe carriers have attracted great interest for encapsulation of food ingredients. Yeast cells are known as a novel carrier for microencapsulation of bioactive compounds with benefits such as controlled release, protection of core substances without a significant effect on sensory properties of food products. Saccharomyces cerevisiae was abundantly used as a suitable carrier for food ingredients. Whole cells as well as cell particles like cell wall and plasma membrane can act as a wall material in encapsulation process. Compared to other wall materials, yeast cells are biodegradable, have better protection for bioactive compounds and the process of microencapsulation by them is relatively simple. The encapsulation efficiency can be improved by applying some pretreatments of yeast cells. In this article, the potential application of yeast cells as an encapsulating material for encapsulation of bioactive compounds is reviewed.     

Insulin secretion dynamics of free and alginate-encapsulated insulinoma cells

This study investigates the effect of alginate/poly-l-lysine/alginate (APA) encapsulation on the insulin secretion dynamics exhibited by an encapsulated cell system. Experiments were performed with the aid of a home-built perfusion apparatus providing a 1 min temporal resolution. Insulin profiles were measured from: (i) murine insulinoma βTC3 cells encapsulated in calcium alginate/poly-l-lysine/alginate (APA) beads generated with high guluronic (G) or high mannuoric (M) content alginate, and (ii) murine insulinoma βTC-tet cells encapsulated in high M APA beads and propagated in the presence and absence of tetracycline. Results show that encapsulation in APA beads did not affect the insulin secretion profile shortly post-encapsulation. However, remodeling of the beads due to cell proliferation affected the insulin secretion profiles; and inhibiting remodeling by suppressing cell growth preserved the secretion profile. The implications of these findings regarding the in vivo function of encapsulated insulin secreting cells are discussed.     

Evaluation of a system producing the hemopoietic factor. WEHI-3B cell line function, when encapsulated in a polypropylene hollow fibre

The purpose of our study was evaluation of functioning of WEHI-3B (an mouse cell line producing IL-3) cells encapsulated in hollow fibers (HF). In vitro: the WEHI-3B cells were encapsulated in HF of polypropylene K600 silikonized, and cultured over two weeks. In vivo: the encapsulated WEHI-3B after weeks culture, were implanted subcutaneously into mice for 1 week. After explantation encapsulated WEHI 3-B were cultured again in culture medium for one week. The production of IL-3 by encapsulated WEHI-3B cells was assessed by evaluation of IL-3 dependent, BaF3 cells viability. The percent number of one day survival of BaF3 cells in the culture medium supplemented with 15% of encapsulated WEHI-3B in vitro or encapsulated WEHI-3B after in vivo conditioned medium was comparable with positive control. Possible replacement of recombinant cytokines with HF encapsulated cytokine-producing cells may be a chance for continous supplementation of the factors for hematopoietic stem cells differentiation.     

Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms

Methods to microencapsulate enzyme, cells, and genetically engineered cells have been described in this article. More specific examples of enzyme encapsulation include the microencapsulation of xanthine oxidase for Lesch-Nyhan disease; phenylalanine ammonia lyase for pheny, ketonuria and microencapsulation of multienzyme systems with cofactor recycling for multistep enzyme conversions. Methods for cell encapsulation include the details for encapsulating hepatocytes for liver failure and for gene therapy. This also includes the details of a novel two-step method for encapsulation of high concentrations of smaller cells. Another new approach is the detailed method of the encapsulation of genetically engineered Escherichia coli DH5 cells for lowering urea, ammonia, and other metabolites in kidney or, liver failure and other diseases.