In vitro evaluation of a novel bioreactor based on an integral oxygenator and a spirally wound nonwoven polyester matrix for hepatocyte culture as small aggregates

BACKGROUND/AIMS: The development of custom-made bioreactors for use as a bioartificial liver (BAL) is considered to be one of the last challenges on the road to successful temporary extracorporeal liver support therapy. We devised a novel bioreactor (patent pending) which allows individual perfusion of high density cultured hepatocytes with low diffusional gradients, thereby more closely resembling the conditions in the intact liver lobuli. METHODS: The bioreactor consists of a spirally wound nonwoven polyester matrix, i.e. a sheet-shaped, three-dimensional framework for hepatocyte immobilization and aggregation, and of integrated hydrophobic hollow-fiber membranes for decentralized oxygen supply and CO2 removal. Medium (plasma in vivo) was perfused through the extrafiber space and therefore in direct hepatocyte contact. Various parameters were assessed over a period of 4 days including galactose elimination, urea synthesis, lidocaine elimination, lactate/pyruvate ratios, amino acid metabolism, pH, the last day being reserved exclusively for determination of protein secretion. RESULTS: Microscopic examination of the hepatocytes revealed cytoarchitectural characteristics as found in vivo. The biochemical performance of the bioreactor remained stable over the investigated period. The urea synthesizing capacity of hepatocytes in the bioreactor was twice that of hepatocytes in monolayer cultures. Flow sensitive magnetic resonance imaging (MRI) revealed that the bioreactor construction ensured medium flow through all parts of the device irrespective of its size. CONCLUSIONS: The novel bioreactor showed encouraging efficiency. The device is easy to manufacture with scale-up to the liver mass required for possible short-term support of patients in hepatic failure.     

Elicitor actions of N-acetylchitooligosaccharides and laminarioligosaccharides for chitinase and L-phenylalanine ammonia-lyase induction in rice suspension culture

BACKGROUND: A significant soft-tissue defect involving the helix of the external ear may present a difficult challenge to repair. OBJECTIVE: We describe our experience with the staged retroauricular to auricular pedicle flap for repair of soft-tissue defects with exposed cartilage of the helix of the ear. METHODS: The staged pedicle flap was used to repair 26 helical ear defects after excision of basal cell carcinoma (n = 16), squamous cell carcinoma (n = 3), and melanoma (n = 7). RESULTS: Defect size ranged from 1 x 2 cm to 4 x 6 cm (average 2.4 x 3.3 cm). Defects involved the superior helix in 12 patients, mid helix in 11, and inferior helix in three. No cases of infection or flap necrosis occurred. CONCLUSION: The staged retroauricular to auricular pedicle flap consistently provides a good to excellent functional and cosmetic outcome when performed on properly selected helical ear defects.     

Co-administration of a water-soluble polymer increases the usefulness of cyclodextrins in solid oral dosage forms

PURPOSE: The aim of this study was to investigate the effect of cyclodextrins (beta-CD, HP-beta-CD and (SBE)7m-beta-CD), and co-administration of a water-soluble polymer (HPMC) and cyclodextrins, on the oral bioavailability of glibenclamide in dogs. METHODS: Effects of cyclodextrins on the aqueous solubility of glibenclamide, with and without hydroxypropylmethylcellulose (HPMC), were determined by a phase-solubility method. Solid inclusion complexes were prepared by freeze-drying. Glibenclamide was administered orally and intravenously to beagle dogs. RESULTS: Aqueous solubility of glibenclamide increased as a function of cyclodextrin concentration, showing an AL-type diagram for beta-CD and an Ap-type diagrams for both of the beta-CD derivatives studied. HPMC enhanced the solubilising effect of cyclodextrins, but did not affect the type of phase-solubility diagram. Orally administered glibenclamide and its physical mixture with HP-beta-CD showed poor absolute bioavailability, while orally administered glibenclamide/cyclodextrin-complexes significantly enhanced the absolute bioavailability of glibenclamide. Orally administered glibenclamide/beta-CD/HPMC and glibenclamide/(SBE)7m-beta-CD/HPMC complexes showed similar absolute bioavailability compared to formulations not containing HPMC, even though 80% (in the case of (SBE)7m-beta-CD) or 40% (in the case of beta-CD) less cyclodextrin was used. CONCLUSIONS: The oral bioavailability of glibenclamide was significantly increased by cyclodextrin complexation. HPMC increased the solubilising effect of cyclodextrins and, therefore, the amount of cyclodextrin needed in the solid dosage form was significantly reduced by their co-administration. In conclusion, the pharmaceutical usefulness of cyclodextrins in oral administration may be substantially improved by co-administration of a water-soluble polymer.     

Characterization of a sustained-release delivery system for combined cytokine/peptide vaccination using a poly-N-acetyl glucosamine-based polymer matrix

Identification of tumor-associated antigens (TAAs) and their class I MHC-restricted epitopes now allows for the rational design of peptide-based cancer vaccines. A biocompatible system capable of sustained release of biologically relevant levels of cytokine and TAA peptide could provide a more effective microenvironment for antigen presentation. Our goal was to test a sustained-release cytokine/TAA peptide-based formulation using a highly purified polysaccharide [poly-N-acetyl glucosamine (p-GlcNAc)] polymer. Granulocyte-macrophage colony-stimulating factor (GM-CSF; 100 microgram) and MART-1(27-35) peptide (128 microgram in DMSO) were formulated into p-GlcNAc. Peptide release was assayed in vitro using interleukin 2 production from previously characterized MART-1(27-35)-specific Jurkat T cells (JRT22). GM-CSF release was assayed via ELISA and proliferation of M-07e (GM-CSF-dependent) cells. Local bioavailability of MART-1(27-35) peptide for uptake and presentation by antigen-presenting cells was demonstrated for up to 6 days (>0.5 microgram/ml). More than 1.0 microgram/ml GM-CSF was concomitantly released over the same period. Biocompatibility and local tissue response to p-GlcNAc releasing murine GM-CSF was determined in C57BL/6 mice via s.c. injection using murine GM-CSF (0. 2 microgram/ml) in 200 microliter of a 2.5% polymer gel. Significant lymphocytic and eosinophilic infiltration was observed 2-7 days after injection with polymer containing murine GM-CSF. The results of our studies show that this biocompatible system is capable of a sustained concomitant release of biologically active peptide and cytokine into the local microenvironment. These findings support further studies to validate a p-GlcNAc delivery system vehicle for a cytokine/TAA peptide-based cancer vaccine.     

Borrelidin is an angiogenesis inhibitor; disruption of angiogenic capillary vessels in a rat aorta matrix culture model

Borrelidin, an antibiotic from Streptomyces rochei, was found to be an angiogenesis inhibitor in a rat aorta matrix culture model which forms capillary vessels in vitro. Borrelidin strongly inhibited capillary tube formation with a 50%-inhibitory concentration value of 0.8 nM, and decreased the number of capillary tubes within 24 hours when added after maturation of tube formation. Borrelidin remarkably disrupted capillary tubes in a dose-dependent manner, by inducing apoptosis of the tube-forming cells.     

On the structural preservation of recombinant human growth hormone in a dried film of a synthetic biodegradable polymer

STUDY OBJECTIVE: We sought to determine the incidence of radiographic findings of pneumonia in highly febrile children with leukocytosis and no clinical evidence of pneumonia or other major infectious source. METHODS: We conducted a prospective cohort study at a large urban pediatric hospital. Clinical practice guidelines for the use of chest radiography in febrile children were established by the emergency medicine attending staff. All records of emergency department patients with leukocytosis (WBC count >/= 20, 000/mm3), triage temperature 39.0 degreesC or higher, age 5 years or less were reviewed daily for 12 months. Physicians completed a questionnaire to note the diagnosis, the presence of respiratory symptoms and signs, and the reason for the chest radiograph (if one was obtained). Patients were excluded for immunodeficiency, chronic lung disease, or major bacterial sources of infection other than pneumonia. Pneumonia was defined by an attending radiologist's reading of the radiograph. RESULTS: We studied 278 patients. Chest radiographs were obtained in 225 for the following reasons: 79 because of respiratory findings suggestive of pneumonia and 146 because of leukocytosis and no identifiable major source of infection. Fifty-three patients did not undergo radiography. Pneumonia was found in 32 of 79 (40%; 95% confidence interval, 20% to 52%) of those with findings suggestive of pneumonia and in 38 of 146 (26%; 95% confidence interval, 19% to 34%) of those without clinical evidence of pneumonia. If patients who did not have a radiograph are assumed to not have pneumonia, the minimum estimate of occult pneumonia was 38 of 199 patients (19%; 95% confidence interval, 14% to 25%). CONCLUSION: Empiric chest radiographs in highly febrile children with leukocytosis and no findings of pneumonia frequently reveal occult pneumonias. Chest radiography should be considered a routine diagnostic test in children with a temperature of 39 degreesC or greater and WBC count of 20,000/mm3 or greater without an alternative major source of infection.     

Validation of precision-cut liver slices in dynamic organ culture as an in vitro model for studying CYP1A1 and CYP1A2 induction

The utilization of precision-cut liver slices in dynamic organ culture as an in vitro model was validated by comparing the induction of the biomarker responses following in vitro (rat liver slice) and in vivo exposure of rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The biomarker responses investigated were cytochrome P450s 1A1 and 1A2 (CYP1A1 and CYP1A2) mRNA, protein, and activities. Precision-cut rat liver slices were incubated in dynamic organ culture for 24 hr with medium containing 0.001-10 nM TCDD or medium without TCDD (control). The resultant mean TCDD concentration in the slices ranged from 19 to 80,925 ppt (wet wt), respectively. A concentration-dependent induction of CYP1A1 mRNA, protein, and activities and a more modest induction of CYP1A2 mRNA was observed in liver slices at all medium concentrations of TCDD. The O-demethylation of 7-methoxyresorufin, a marker for CYP1A2 activity, was induced at TCDD medium levels of 0.01 nM and greater, whereas a detectable increase in CYP1A2 protein occurred only at the higher concentrations. Comparable liver concentrations of TCDD (8-64,698 ppt wet wt) were achieved at 24 hr following a single in vivo exposure of rats to TCDD at doses ranging from 0.002 to 5 microg/kg po. Concentration-effect and dose-response relationships for induction of CYP1A1 and CYP1A2 were similar following in vitro and in vivo exposure to TCDD, although the magnitude of induction was greater for in vivo exposure. The data support the use of liver slices in dynamic organ culture for assessing the relative in vivo potency of a compound to induce CYP1A1 and CYP1A2. Human tissue can also be readily utilized in this in vitro model to predict the biological and toxicological effects of a given in vivo exposure to TCDD.     

Significance of circulating hepatocyte growth factor level as a prognostic indicator in primary breast cancer

OBJECTIVE: The purpose of the study was to develop quantitative and qualitative criteria for diagnosing fatty liver on contrast-enhanced helical CT. SUBJECTS AND METHODS: Differential liver-spleen attenuation was evaluated between 80 and 120 sec after injection in 76 patients who underwent contrast-enhanced helical CT. Unenhanced CT images had earlier established fatty liver when the liver minus spleen attenuation difference was less than or equal to -10 H (n = 18). Four observers who had not seen the unenhanced images used contrast-enhanced CT images to assess the presence of fatty liver on a five-point Likert scale, the presence of geographic areas spared from fatty infiltration, and the relative liver-spleen attenuation. The diagnostic accuracies of various imaging criteria were compared using McNemar's chi-square test (for sensitivity and specificity) and analysis of receiver operating characteristic curves. RESULTS: Sensitivity, specificity, and receiver operating characteristic curve areas for observers' qualitative judgments were 54%, 95%, and .91, respectively; for quantitative differential liver-spleen attenuation (80-100 sec; -20.5 H discriminatory value), the values were 86%, 87%, and .94, respectively; and for quantitative differential liver-spleen attenuation (101-120 sec; -18.5 H discriminatory value), the values were 93%, 93%, and .98, respectively. Differential liver-spleen attenuation was time-dependent; overlap was noted between healthy subjects and patients with fatty liver. Qualitatively, geographic sparing was highly specific (94%) for fatty liver, whereas liver attenuation greater than or equal to spleen attenuation excluded fatty liver in all but one case. CONCLUSION: Although quantitative and qualitative criteria for diagnosing fatty liver on helical CT can be determined, they are protocol-specific. Limited unenhanced hepatic CT remains the optimal technique for detection of fatty infiltration of the liver.     

Preoperative assessment and prediction of postoperative results in an artificial arterial network using computer simulation

A computer model has been developed that can be used to describe the human arterial system mathematically. It simulates the complex relationship of morphology and hydraulics in the vessel network. After entering patient data into a standard vessel model, the mean flow velocity, the flow direction, and the blood pressure at each specified point of the flow network can be calculated. The vessel picture can be altered and modified with the help of a graphic editor. Localized or diffuse stenoses, bypasses with simple or multiple anastomoses, end-to-end anastomoses, end-to-side anastomoses, etc., can be studied in terms of the hydraulic effects on the local situation or on the entire vessel system. Experimental results of ultrasonic mean flow data in vessel systems of leg and cerebral arteries of patients are compared with calculated values. The predicted and measured flow velocities show a mean difference of about 10% indicating that such a computer model may be successfully used in the optimal planning of bypass operations.     

Matrix strengthening mechanisms of an iron fiber-copper matrix composite as a function of fiber size and spacing

Copper matrix-iron fiber composites of fiber diameters from 10 to 5 × 10⁻³ mils and volume fractions from 0.03 to 0.97 were fabricated in order to study the dependence of mechanical properties on these variables. Composite elastic moduli agreed well with the predictions of the rule of mixtures. However, matrix and composite yielding and plastic flow were quite dependent on fiber diameter and spacing, exhibiting positive deviations from the simple rule of mixtures by factors of more than five in some cases. Yielding behavior may be explained by a combination of dislocation extrusion and pileup models for low volume fractions of fiber. Triaxiality generated by the difference in Poisson coefficients of the phases inhibits matrix yielding in higher volume fraction composites, allowing matrix flow only when the fibers also yield. Formerly with the Department of Metallurgy and Materials Science, M.I.T., Cambridge, Mass. Formerly with the Department of Metallurgy and Materials Science, M.I.T.     

cDNA cloning of FRIL, a lectin from Dolichos lablab, that preserves hematopoietic progenitors in suspension culture

Ex vivo culture of hematopoietic stem cells is limited by the inability of cytokines to maintain primitive cells without inducing proliferation, differentiation, and subsequent loss of repopulating capacity. We identified recently in extracts of kidney bean and hyacinth bean a mannose-binding lectin, called FRIL, and provide here evidence that this protein appears to satisfy properties of a stem cell preservation factor. FRIL was first identified based on its ability to stimulate NIH 3T3 cells transfected with Flt3, a tyrosine kinase receptor central to regulation of stem cells. Molecular characterization from polypeptide sequencing and identification of the cDNA of hyacinth bean FRIL shows 78% amino acid identity with a mannose-binding lectin of hyacinth beans. Treatment of primitive hematopoietic progenitors in suspension culture with purified hyacinth FRIL alone is able to preserve cells for 1 month without medium changes. In vitro progenitor assays for human hematopoietic cells cultured 3 weeks in FRIL displayed small blast-like colonies that were capable of serial replating and persisted even in the presence of cytokines known to induce differentiation. These results suggest that FRIL is capable of preserving primitive progenitors in suspension culture for prolonged periods. FRIL's clinical utility involving procedures for stem cell transplantation, tumor cell purging before autologous transplantation, and ex vivo cultures used for expansion and stem cell gene therapy currently are being explored.     

Efficacy of a bovine Staphylococcus aureus vaccine using interleukin-2 as an adjuvant

The purpose of this study was to examine the efficacy of a vaccination protocol using recombinant bovine interleukin-2 (rBoIL-2) as an adjuvant with a Staphylococcus aureus vaccine. Holstein dairy cows were immunized with a S. aureus vaccine in conjunction with either saline solution (n = 3), Freund's incomplete adjuvant (FIA; n = 3) or rBoIL-2 (n = 3). Whey and serum were analysed for antibody titer to specific S. aureus antigens. Isolated blood mononuclear cells (BMC) were examined for their ability to proliferate and to produce interleukin-2 (IL-2) and interferon (IFN) after either mitogenic or antigenic stimulation in vitro. Efficacy of the vaccination protocols was assessed by challenging experimental animals intramammarily with 100 colony forming units of S. aureus. Regardless of treatment, all cows exhibited similar serum antibody titers to S. aureus pseudocapsule. Cows treated with saline exhibited a significant increase in serum alpha-toxin antibody titer when compared to levels observed in FIA and rBoIL-2-treated cows. However, cows receiving rBoIL-2 treatment exhibited significantly higher lacteal pseudocapsule antibody titer compared to the other adjuvant groups. Administration of rBoIL-2 did not enhance BMC proliferative responses to the mitogens concanavalin A (ConA), phytohemagglutinin (PHA), pokeweed mitogen (PWM) or interleukin-2 (IL-2) when compared to FIA or saline treated cows. Although cows receiving rBoIL-2 treatment exhibited enhanced cytokine production upon antigenic stimulation, efficacy of the vaccination protocol was inferior compared to the protection offered by saline treatment.     

Metabolic evaluation of cultured porcine hepatocyte monolayers in human plasma from hepatic failure patients--basic study on development of a bioreactor in hybrid artificial liver

In order to develop a functional bioreactor for hybrid artificial liver, function of cultured porcine hepatocyte monolayers in human plasma from hepatic failure patients (group III, n = 5) was investigated. Culture media, Leibovitz L-15 medium (group I, n = 7) and normal human plasma (group II, n = 3), were used as controls. Morphologically, no degeneration of porcine hepatocytes in hepatic failure plasma was observed for 5 days in culture. Levels of ureogenesis showed no significant difference against the controls at day 1, 2, 5 in culture, but the level at day 3 was significantly higher than that of group II. Levels of gluconeogenesis showed a close tendency as those of ureogenesis, but the level at day 3 was significantly lower than that of group I. Levels of intracellular DNA contents, showing between 1.85 +/- 0.39 and 1.35 +/- 0.05 microgram/cm2, were compatible with those of controls during first three days in culture, but level of group III at day 5 was significantly higher than that of group I. After incubation of porcine hepatocyte in hepatic failure plasma, the amount of valine, leucine, isoleucine, glutamine, arginine, and citrulline was significantly decreased. Elevated phenylalanine and tyrosine were also decreased, but Fischer's ratio, the ratio of branched chain amino acid against aromatic amino acids, was not significantly increased. Data obtained by this investigation showed that cultured porcine hepatocytes held proper hepatic function in the hepatic failure plasma. It is concluded that culture porcine hepatocyte monolayers were a promising candidate for a bioreactor of a hybrid artificial liver.     

The influence of fluoride exposure on dentin mineralization using an in vitro organ culture model

This study aimed to characterize fluoride-induced alterations in dentin mineralization within a dentin-pulp organ culture system. Tooth sections derived from male Wistar rat incisors were cultured in Trowel-type culture for 14 days, in the presence of 0 mM, 1 mM, 3 mM and 6 mM sodium fluoride. Tooth sections were processed and analyzed for uptake of fluoride, its subsequent effect on dentin mineralization by tetracycline hydrochloride incorporation and mineral composition, expressed as calcium/phosphorous (Ca/P) ratios. Tetracycline hydrochloride incorporation was demonstrated to decrease with increased fluoride exposure, accompanied by significant increases in both Ca/P ratios and fluoride incorporation. These findings provide further evidence that the established alterations in dentin formation during fluorosis are a consequence of disruption to the mineralization process, and provide a model system with which to investigate further the potential role the extracellular matrix plays in inducing the apparent changes in mineral composition.     

Evaluation of a rapid technique for antibiotic sensitivity testing using an MS-2 automate and blood culture medium

The authors propose a rapid method for studying antibiotic sensitivity of bacteria isolated from blood cultures. An MS-2 automate is used directly with bacteria collected after centrifugation. With the exception of the sensitivity of Gram-positive cocci to the combination of sulfamethoxazole-trimethoprime and to Penicillin G and of Gram-negative bacillus to beta lactamines, the results were comparable to those obtained after isolation of bacteria on gel medium. Various causes for error are discussed.