A scalable microfluidic device for the mass production of microencapsulated islets.

The objective of this research was to test the viability and function of islets microencapsulated using a scalable microfluidic device that is suitable for the mass production of encapsulated islets for transplantation. A 3-D microfluidic device consisting of eight outlets with an inner fluid inlet and an outer concentric inlet to the device has been designed and fabricated using the stereolithography rapid prototyping technique. Islets were isolated from normal Wistar-Furth rat pancreas using the procedure of collagenase digestion of pancreatic tissue. Following purification, islet suspensions in 1.5% sodium alginate were pumped into the fluid inlet of the microfluidic device, which distributed the flow equally to all the eight channels according to the design. The air plenum distributed compressed air uniformly through the eight concurrent outlets, and with one fluid pump and air source, the device produced eight microencapsulations simultaneously. After encapsulation, the islets were tested for functionality using the dynamic perifusion procedure with low- and high-glucose concentrations. The device is capable of producing eight channels of steady stream of monodisperse microencapsulations of a range of diameters depending on the design and process parameters. Using this prototype device, encapsulated islets were shown to be viable in the functional tests that we performed. Thus, the mean ± standard deviation rate of insulin secretion increased from a basal rate of 0.165 ± 0.059 ng/10 islets/min to a stimulated rate of 0.422 ± 0.095 ng/10 islets/min (P < .05, n = 3), when the glucose concentration was changed from 5.5 mmol/L to 27.5 mmol/L, and this glucose stimulation index was not different from that observed with unencapsulated islets under same conditions. In summary, the high-throughput prototype device that we have designed can produce encapsulated islets that are viable and suitable for transplantation studies.

New alginate microcapsule system for angiogenic protein delivery and immunoisolation of islets for transplantation in the rat omentum pouch.

Severe hypoxia caused by a lack of vascular supply and an inability to retrieve encapsulated islets transplanted in the peritoneal cavity for biopsy and subsequent evaluation are obstacles to clinical application of encapsulation strategies for islet transplantation. We recently proposed an omentum pouch model as an alternative site of encapsulated islet transplantation and have also described a multi-layer microcapsule system suitable for coencapsulation of islets with angiogenic protein in which the latter could be encapsulated in an external layer to induce vascularization of the encapsulated islet graft. The purpose of the present study was to determine the angiogenic efficacy of fibroblast growth factor (FGF-1) released from the external layer of the new capsule system in the omentum pouch graft. We prepared 2 groups of alginate microspheres, each measuring ∼600 µm in diameter with a semipermeable poly-L-ornithine (PLO) membrane separating 2 alginate layers. While one group of microcapsules contained no protein (control), FGF-1 (1.794 µg/100 microcapsules) was encapsulated in the external layer of the other (test) group. From each of the 2 groups, 100 microcapsules were transplanted separately in an omentum pouch created in each normal Lewis rat and were retrieved after 14 days for analysis of vessel density using the technique of serial sample sections stained for CD31 with quantitative three-dimensional imaging. We found that FGF-1 released from the external layer of the test microcapsules induced a mean ± SD vessel density (mm(2)) of 198.8 ± 59.2 compared with a density of 128.9 ± 10.9 in pouches measured in control capsule implants (P = .03; n = 5 animals/group). We concluded that the external layer of our new alginate microcapsule system is an effective drug delivery device for enhancement of graft neovascularization in a retrievable omentum pouch.

The interpretation and clinical application of the word 'parity': a survey.

The objective of this study was to evaluate the use of the term 'parity' in multiple pregnancies using a questionnaire survey. One hundred and fifty obstetricians and midwives in five maternity units in southeast Thames region of the London deanery were administered the questionnaire. One hundred and twenty-six questionnaires were returned (84% response rate). Only 21% described twin delivery as a single parous experience. The improper clinical application of the word parity was shown in both obstetricians and midwives. Standardisation of the definition to ensure accuracy and uniformity in record keeping is necessary.

Human breast milk immunology: a review.

Breast feeding has been shown to enhance the development of the immune system of the newborn as well as provide protection against enteric and respiratory infections. It has been suggested that implementation of breast feeding programs has the potential to save hundreds of thousands of lives worldwide. Human milk is a bodily fluid which, apart from being an excellent nutritional source for the growing infant, also contains a variety of immune components such as antibodies, growth factors, cytokines, antimicrobial compounds, and specific immune cells. These help to support the immature immune system of the newborn baby, and protect it against infectious risks during the postnatal period while its own immune system matures. This article reviews some of the factors in human breast milk that give it these important properties.

Effect of alginate composition and purity on alginate microspheres.

BACKGROUND: Alginate is commonly used to microencapsulate islets in experiments with islet allografts and xenografts for the treatment of Type I diabetes. The purpose of the present study is to determine the effects of alginate composition and purity on the morphology and size of microspheres. METHODS: Microcapsules produced with the impure alginate types, medium-viscosity high-guluronic acid (IMVG), low-viscosity high-G (ILVG), low-viscosity high-mannuronic acid (ILVM) and medium-viscosity high-M (IMVM) were compared with one another and others generated with a highly purified LVM (HPLVM) alginate. Droplets of 1.5% alginate from an air-syringe pump were gelled in 1.1% CaCl2 solution. While leaving the alginate pressure and needle recess constant, the air-jacket pressure was varied between 9.5-10.5 PPSI to enhance stable microcapsule generation and different batches of microbeads were made from each alginate type. RESULTS: The sizes of the high-guluronic acid alginate microbeads were consistently bigger than those of the corresponding high-mannuronic acid alginate beads at all air-jacket settings. At the optimal air-jacket pressure of 9.0 PPSI, the mean+SD diameter of the IMVG microbeads was 780 + 20 microm, while that of IMVM was 607 + 44 microm (p < 0.0001, n=30). Similarly, the mean ILVG microbead diameter was 816+28 microm compared to 656+26 microm for ILVM capsules (p<0.0001, n=30). Less polymorphism was found with the HPLVM microspheres than with the ILVM microbeads. CONCLUSION: Highly purified high-mannuronic acid alginate will provide smaller, spherical microcapsules suitable for islet cell transplantation.

Oxidative stress, micronutrients, diabetes mellitus and its complications.

Oxidative stress is defined as excessive production of reactive oxygen species (ROS) in the presence of diminished antioxidant substances. It has been shown that oxidative stress has an adverse effect on glucose metabolism. Development of the disabling chronic complications of diabetes mellitus (DM) has also been attributed to oxidative stress. The body's defence against oxidative stress is accomplished by interconnecting systems of antioxidant micronutrients (vitamins and minerals) and enzymes. While the vitamins act as donors and acceptors of ROS, minerals regulate activity of the enzymes. This review article is focused on the preventive and therapeutic roles of these antioxidant micronutrients in DM. Although there is scarcity of data from controlled studies, anecdotal reports indicate that the use of certain antioxidant vitamin and mineral supplements may be beneficial as an adjunct therapy in the management of DM and its complications. In particular, it has been reported that high doses of single micronutrient antioxidant supplements, such as vitamin E, may be beneficial to patients suffering from this disease. However, micronutrient antioxidants interact with each other in a biochemical chain of defence against free radicals, and the use of high doses of a single antioxidant poses potential risks because it could perturb the antioxidant-prooxidant balance. It has, therefore, been recommended that high doses of micronutrient antioxidant vitamins should be administered in combination rather than as single supplements. There are a growing number of preparations containing mixtures of antioxidant vitamins and/or trace elements. A particularly impressive supplement is a new formula called Akesis, which is a mixture of antioxidant vitamins and antidiabetic trace elements, adequately balanced with other vitamins and minerals that enhance metabolic processes.

Islet cell transplantation for the treatment of diabetes mellitus.

Diabetes mellitus is estimated to affect at least 16 million individuals in the United States and 135 million persons worldwide. It is a significant cause of morbidity and early mortality. The related expenses are astronomical with at least 15% of healthcare expenditures in the United States being used for the treatment of diabetes and its complications, a figure that approaches US$100 billion annually. The Diabetes Control and Complications Trial (DCCT) convincingly showed that intensive glucose management delays the onset and slows the progression of diabetic complications. Numerous studies have shown that pancreas transplantation not only delays the onset and progression of diabetic complications, but in some cases reverses some of the effects of diabetes. Human islet cell transplantation provides an alternative, less invasive alternative to whole organ transplantation. Human islet allotransplantation would only exacerbate the organ shortage, as recipients usually require islets from more than one pancreas. Xenotransplantation of porcine islets is a more attractive option; however, the recipient's immune response to xenografted tissue would be a formidable obstacle. Microencapsulation of the islets is a method of immunoisolation that would prevent the need for immunosuppressive drugs and the risks associated with their long-term use and have the potential to make xenoislet transplantation a clinical reality.

Prevention of morphological changes in alginate microcapsules for islet xenotransplantation.

BACKGROUND: Alginate microcapsule swelling, which occurs as a result of increased hydrophilicity owing to the Ca++ that remains after rapid chelation of the inner alginate core, is a problem in encapsulation. We have previously shown that exchange of the residual divalent Ca++ with the monovalent Na+ through the use of 6 mmol/L Na2SO4 decreases swelling in chelated alginate-polylysine-alginate microcapsules, and this process enhances their durability. The purpose of the present study was to examine the morphology of Na2SO4-treated microcapsules in long-term incubation with the use of serum-supplemented culture medium. METHODS: Spherical beads of purified alginate (3%) that were gelled with 1.1% CaCl2 were first coated with polylysine, and then with 0.24% alginate. After rapid chelation of the inner alginate core with 55 mmol/L sodium citrate, the capsules were either incubated for 30 minutes in 6 mmol/L Na2SO4 or left untreated (control). Each group of capsules was then placed in a flask containing Ham's culture medium supplemented with 20% porcine serum and incubated at 37 degrees C. RESULTS: The diameters of Na2SO4-treated capsules only increased modestly from a mean +/- SD of 635 +/- 22.08 to 684.53 +/- 17.86 microm (P<0.0001) by day 7, with no further increases thereafter. In contrast, control capsules showed a steady increase in their mean diameters, which changed from 639.55 +/- 21.44 to 735.48 +/- 108.85 microm (P < 0.0001) by day 66. In addition, whereas treated capsules remained spherical, control capsules showed progressive polymorphism. CONCLUSION: We have developed a new method of making more durable and stable microcapsules that can be used for islet cell xenotransplantation.

A reliable method for isolation of viable porcine islet cells.

HYPOTHESIS: Mechanical injury and oxidative stress caused by reoxygenation of isolated porcine islet cells result in their unresponsiveness to glucose stimulation. DESIGN: Adult pigs (weighing 25-30 kg) were anesthetized, and following intra-arterial infusion of ice-cold University of Wisconsin solution, a complete pancreatectomy was performed. The pancreatic duct was cannulated for infusion of digestion medium containing collagenase type P, 1.5 mg/mL; deoxyribonuclease I, 10 000 U; and a water-soluble analogue of vitamin E (Trolox), 1 mmol/L. After 20-minute incubations on ice, and at 37 degrees C, the pancreas was hand shaken for 1 minute, followed by filtration and separation on an automatic cell separator (COBE 2991). Islet cells, identified by dithizone staining, were perifused at 37 degrees C. RESULTS: The mean +/- SEM yield of intact purified islet cells (50-200 microm in diameter), and mostly present in clusters, was 2398 +/- 143 cells per gram (n = 12). Glucose stimulation caused a significant increase in biphasic insulin secretion in the perifusion experiments. CONCLUSION: We have developed a simple, reproducible, and reliable procedure for isolating intact and viable porcine islet cells suitable for xenotransplantation.

Durability of sodium sulfate-treated polylysine-alginate microcapsules.

Chelated hollow microcapsules are unstable under in vitro conditions because of their hygroscopic nature. Nongel inducing cations, such as Na+, stabilize the outer membrane of the alginate-polylysine-alginate microcapsules leading to more stable beads. We made different batches of empty capsules with a mean +/- SEM diameter of 607+/-11 microns, and found that within 1 week of incubating these capsules in normal saline at 37 degrees C, they increased to 718+/-10 microns (p < 0.05, n = 5). In initial experiments, we made different batches of capsules and divided them into two groups. One group was left untreated (control) whereas the other was treated with 6 mM Na2SO4 for 30 min, before incubation in saline at 37 degrees C. Control capsules increased in weight and size, before beginning to melt in less than 1 week. In contrast, treated capsules rapidly lost weight and remained intact during 1 month of follow-up. In perifusion experiments, we found no deleterious effect of sodium sulfate treatment on the function of islets enclosed in the capsules.

Total antioxidant capacity of colon in patients with chronic ulcerative colitis.

It has been proposed that oxidative stress is involved in the pathophysiology of ulcerative colitis. We have reported the depletion of the nonenzymatic antioxidant, glutathione, in colon from active and inactive ulcerative colitis. The colon contains several biochemically linked antioxidant systems. We hypothesized that diminished total antioxidant capacity in active ulcerative colitis would be associated with increased colonic lipid peroxidation. This study was designed to determine total antioxidant capacity and lipid hydroperoxide levels using colon obtained at surgery from controls (N = 16; 4 females, 12 males; mean age 70 years), and active and inactive ulcerative colitis (N = 15; 3 females, 12 males; mean age 39). Total antioxidant capacity of control colon was higher in muscularis externa compared to the mucosal-submucosal layer (P < 0.05). There were no differences in colonic total antioxidant capacity or lipid hydroperoxide levels comparing control colon to inactive and active ulcerative colitis. The results did not support depletion of tissue total antioxidant capacity by free radicals. Depletion of glutathione in ulcerative colitis may be a specific disorder rather than a secondary defect attributable to global oxidative stress. Nonspecific antioxidant supplements appear unlikely to be beneficial in the treatment of ulcerative colitis.

Storage and microencapsulation of islets for transplantation.

Microencapsulation is an effective means of immunoisolation for pancreatic islet transplants. However, the process of isolating, purifying, encapsulating, and transplanting islets in a single day is labor intensive and difficult for routine use. There is an apparent need for reliable methods of islet storage, and cryopreservation has emerged as an attractive system of islet banking. While studies have shown that cryopreserved islets are viable when tested unencapsulated after thawing, it is not clear if the combination of freezing and encapsulation would affect islet function. The purpose of the present study was to determine the in vitro function of cryopreserved islets following thawing and microencapsulation. Islets were isolated from the pancreata of Sprague-Dawley rats and cryopreserved under liquid nitrogen for either 1 week or 1 month, following an overnight culture at 37 degrees C. Upon thawing, the islets were tested either unencapsulated or after encapsulation in polylysine-alginate membrane. In all experiments islets were preperifused for 1 h at 37 degrees C with a modified Krebs-Ringer bicarbonate buffer containing 3.3 mM (60 mg/dl) glucose and maintained at pH 7.4 by continuous gassing with 95% air/5% CO2. Following basal effluent sample collection on ice, the glucose concentration was raised to 16.7 mM (300 mg/dl). It was found that, within 10 min of high glucose stimulation, an average of twofold increase in insulin secretion (p < 0.01) was obtained in islets within or without microcapsules. We conclude that islets cryopreserved for 1 month prior to thawing and microencapsulation retained functional viability as determined in in vitro experiments.

Role of protein kinase C isoenzymes in fatty acid stimulation of insulin secretion.

Although hyperlipidemia is frequently associated with hyperinsulinemia. the stimulation of insulin secretion by fatty acids in the in vitro studies has remained a matter of constant debate, partly because of the uncertainty about a clearly defined mechanism to explain such a direct effect. In this study, we used a pharmacologic approach to test the hypothesis that protein kinase C (PKC) signal-transduction pathway is involved in fatty acid-stimulated insulin secretion. Isolated rat islets were perifused with either palmitate (C(16:0)) or linoleate (C(18:2)) in the absence or presence of selective inhibitors of PKC isoenzymes. Our results suggest a role for Ca2+-independent PKC isoenzymes in the signal transduction of fatty acid-stimulated insulin secretion. The data imply that either the nonconventional and/or atypical isoforms of PKC are involved in the stimulation of insulin release induced by fatty acids.

Induction of enlarged intestinal lymphoid aggregates during acute glutathione depletion in a murine model.

Glutathione is a nonenzymatic antioxidant synthesized by most animal cells and is depleted in inflammatory bowel disease. The effects of glutathione depletion on intestinal histology and inhibitory neurochemicals was examined in a mouse model. Glutathione depletion in A/J mice involved inhibition of gamma-glutamylcysteine synthetase using L-buthionine-(S,R)-sulfoximine (BSO) for 10 days. Ileum and colon were obtained from saline-control mice, BSO-treated mice, and BSO-treated mice receiving ascorbate or glutathione monoethylester. Glutathione, lipid peroxides, and nicotineamide adenine dinucleotide phosphate diaphorase activity were measured by colorimetric assays. Vasoactive intestinal peptide was measured by radioimmunoassay. Glutathione depletion induced enlargement of mucosal-submucosal lymphoid aggregates without germinal centers in ileum and colon. These aggregates were prevented by supplementation with glutathione monoethylester but not ascorbate. Tissue levels of inhibitory neurochemicals were unchanged. Depletion of glutathione appears to induce enlarged lymphoid aggregates by recruitment of lymphocytes from the peripheral circulation. A component of the inflammation that develops in inflammatory bowel disease could be related to depletion of tissue levels of glutathione.

Depletion of total antioxidant capacity in type 2 diabetes.

The purpose of the study was to examine the relationship between antioxidant depletion, glycemic control, and development of chronic complications in a controlled population of type 2 diabetic patients. Fifty age-matched type 2 diabetic patients receiving sulfonylureas but not insulin treatment were screened and assigned to two groups based on the presence or absence of proteinuria. A third group of normal subjects without diabetes were also enrolled in the study. All subjects in the three groups were Egyptians who were matched for body weight, and the two diabetic groups were also age-matched. Plasma glucose and fructosamine levels were higher in the two groups of diabetic patients versus the control group, but lipid peroxide levels were higher only in the patients with proteinuria. Compared with the control group, the total antioxidant capacity was depleted in the two diabetic groups, but the depletion was more severe in patients with proteinuria. Thus, the mean Trolox equivalent antioxidant capacity (TEAC) of the control group was 2.7+/-0.45, versus 1.7+/-0.5 (P < .001) in the patients without proteinuria. Furthermore, the TEAC measured in patients with proteinuria, who also had more diabetic complications, was lower (1.4+/-0.5, P < .001) than the TEAC in patients without urinary protein. In conclusion, a depletion of the total antioxidant capacity is associated with a higher incidence of diabetic complications.

An In Vitro Cell Based Model for Assessing the Potential of Allergens to Release Mediators Through the Cross-linking of IgE.

The aim of this study was to examine the capacity of an in vitro model to test the potential of an allergen to cause cross-linking of IgE bound to the surface of mast cells. The model involved the passive sensitization of murine mast cells, with zearalenone IgE and subsequent exposure to anti IgE, zearalenone, zearalenone-BSA (with up to nine bound zearalenone molecules) or peanut lectin. The extent of cross-linking was determined by measuring the release of IgE mediators TNFalpha and histamine. Release of TNFalpha from IgE sensitized cells increased following exposure to zearalenone-BSA, but not following exposure to zearalenone alone or to peanut lectin. Histamine release could not be quantified against background. The results suggested that the model could be used to test allergenic potential through the availability of epitopes to bind and cross-link IgE on the surface of mast cells. As IgE is species specific, the model was adapted for use with a human cell system employing mast cells in lung fragments. TNFalpha release was measured, and the system was calibrated with the inhalant allergen from Timothy Grass and Timothy Grass specific IgE.