Degradation of partially oxidized alginate and its potential application for tissue engineering.

Alginate has been widely used in a variety of biomedical applications including drug delivery and cell transplantation. However, alginate itself has a very slow degradation rate, and its gels degrade in an uncontrollable manner, releasing high molecular weight strands that may have difficulty being cleared from the body. We hypothesized that the periodate oxidation of alginate, which cleaves the carbon-carbon bond of the cis-diol group in the uronate residue and alters the chain conformation, would result in promoting the hydrolysis of alginate in aqueous solutions. Alginate, oxidized to a low extent (approximately 5%), degraded with a rate depending on the pH and temperature of the solution. This polymer was still capable of being ionically cross-linked with calcium ions to form gels, which degraded within 9 days in PBS solution. Finally, the use of these degradable alginate-derived hydrogels greatly improved cartilage-like tissue formation in vivo, as compared to alginate hydrogels.

Treatment of head and neck melanoma, lentigo maligna subtype: a practical surgical technique.

Melanoma with the lentigo maligna histological pattern often provides a significant and difficult challenge to the head and neck surgeon. The lentigo maligna subtype is the most common type of melanoma on the head and neck. This potentially lethal form of cancer is associated with greater nonvisual lesional extension that is often not clinically apparent. Failure to excise the entire lesion results in a higher risk of local recurrence and a poorer prognosis. The staged excision technique described herein results in histological interpretation of 100% of the peripheral margins using formalin-fixed vertical sections. Definitive local excision and soft tissue reconstruction are performed in a subsequent stage, with an assurance that 100% of the peripheral margins have been evaluated and interpreted as free of disease.

Cognitive versus behavior therapy in the group treatment of obsessive-compulsive disorder.

This study examined the effects of cognitive-behavior therapy (CBT) compared with traditional behavior therapy (exposure and response prevention [ERP]) in the group treatment of obsessive-compulsive disorder. Of the 76 participants who started treatment, 38 were wait-listed for 3 months before treatment to assess possible course effects. Both treatments were superior to the control condition in symptom reduction, with ERP being marginally more effective than CBT by end of treatment and again at 3-month follow-up. In terms of clinically significant improvement, treatment groups were equivalent on the conclusion of treatment, but 3 months later significantly more ERP participants met criteria for recovered status. Only 1 of 7 belief measures changed with treatment improvement, and the extent of this cognitive change was similar between CBT and ERP groups. Discussion includes consideration of optimal formats for the delivery of different types of treatment.

Detection of biotin in individual sea urchin oocytes using a bioluminescence binding assay.

The ability to detect biomolecules in single cells is important in order to fully understand the processes by which many biochemical events occur. To that end, we have developed a bioluminescence binding assay capable of measuring the intracellular biotin content of individual cells. The assay depends on competition between an aequorin-biotin conjugate (AEQ-biotin) and free biotin within the oocytes for binding sites on the protein avidin. The assay is performed by microinjecting each component into the oocytes and following the resulting bioluminescence within the oocyte upon triggering of aequorin. Results obtained using sea urchin oocytes show that the assay performed within the cells behaves in a manner consistent with assay theory. Using the assay, the individual biotin content of the oocytes is an average of approximately 20 amol. To our knowledge, this is the first reported multicomponent binding assay to be performed inside an intact single cell.

Studies of the interactions between melatonin and 2 Hz, 0.3 mT PEMF on the proliferation and invasion of human breast cancer cells.

Interactions between the hormone melatonin at pharmacological concentrations (10(-3) M) and 2 Hz, 0.3 mT pulsed electromagnetic fields (PEMF) on the proliferation and invasion of human breast cancer cells were studied in vitro. Three types of human breast cancer cells were used in this study: MDA-MB-435, MDA-MB-231, and MCF-7. Results showed that cellular growth of MDA-MB-231 cells, which were reported to be lowly metastatic, and MCF-7 cells, which were reported to be nonmetastatic, were both significantly reduced by melatonin regardless of the presence of the field. Results also showed that MDA-MB-435 and MDA-MB-231 cells were invasive, with MDA-MB-231 cells being more invasive than the MDA-MB-435 cells for both unexposed and experimental-PEMF groups. In addition, invasion studies showed that MCF-7 cells were not invasive and that melatonin did not have any effects on the invasion of these cells, with or without the PEMF. It is also suggested that since metastasis requires growth and invasion into tissue, anti-invasion agents can be used in conjunction with melatonin to prevent formation of secondary metastases. The overall studies suggest that PEMF at 2 Hz, 0.3 mT does not influence cancer metastasis; while having clinical merit in the healing of soft tissue injury, this field has shown no influence on cancer cells as 60 Hz power line fields have.

Cell-interactive alginate hydrogels for bone tissue engineering.

There is significant interest in the development of injectable carriers for cell transplantation to engineer bony tissues. In this study, we hypothesized that adhesion ligands covalently coupled to hydrogel carriers would allow one to control pre-osteoblast cell attachment, proliferation, and differentiation. Modification of alginate with an RGD-containing peptide promoted osteoblast adhesion and spreading, whereas minimal cell adhesion was observed on unmodified hydrogels. Raising the adhesion ligand density increased osteoblast proliferation, and a minimum ligand density (1.5-15 femtomoles/cm2) was needed to elicit this effect. MC3T3-E1 cells demonstrated increased osteoblast differentiation with the peptide-modified hydrogels, as confirmed by the up-regulation of bone-specific differentiation markers. Further, transplantation of primary rat calvarial osteoblasts revealed statistically significant increases of in vivo bone formation at 16 and 24 weeks with G4RGDY-modified alginate compared with unmodified alginate. These findings demonstrate that biomaterials may be designed to control bone development from transplanted cells.

Role of metastatic potential in the adhesion of human breast cancer cells to endothelial monolayers.

To gain further insight into the process of metastasis, adhesion to endothelial monolayers was compared for a nonmetastatic and a highly metastatic human breast cancer cell line. The parallel plate flow chamber was employed to quantify adhesion using an attachment assay. This assay was carried out at several physiological shear stresses both with and without endothelial TNF-alpha stimulation. At a venular shear stress of 1 dyne cm-2, the nonmetastatic cell line was more adhesive to stimulated endothelial monolayers, while no differences could be noted for resting monolayers. At a lower shear stress of 0.25 dynes cm-2, the highly metastatic cell line was more adhesive to stimulated endothelial monolayers, while the nonmetastatic cell line was more adhesive to resting monolayers. Thus, metastatic potential correlated with attachment only at low shear stresses and following endothelial stimulation. These results emphasize the importance of studying cancer cell adhesion under multiple physiological flow conditions. Furthermore, these results indicate that adhesion of these two cell lines may be controlled by two different mechanisms. Antibody blocking experiments of adhesion molecules on the endothelial cells confirmed that adhesion of the nonmetastatic cell line was mediated by E-selectin expressed on the endothelial cells and adhesion of the highly metastatic cell line was mediated by both E-selectin and VCAM-1 expressed on the endothelial cells.

Development of binding assays in microfabricated picoliter vials: an assay for biotin.

A homogeneous binding assay for the detection of biotin in picoliter vials was developed using the photoprotein aequorin as the label. The binding assay was based on the competition of free biotin with biotinylated aequorin (AEQ-biotin) for avidin. A sequential protocol was used, and modification of the assay to reduce the number of steps was examined. Results showed that detection limits on the order of 10(-14) mol of biotin were possible. Reducing the number of steps provided similar detection limits but only if the amount of avidin used was decreased. These binding assays based on picoliter volumes have potential applications in a variety of fields, including microanalysis and single-cell analysis, where the amount of sample is limited. In addition, these assays are suitable for the high-throughput screening of biopharmaceuticals.

Controlled growth factor release from synthetic extracellular matrices.

Polymeric matrices can be used to grow new tissues and organs, and the delivery of growth factors from these matrices is one method to regenerate tissues. A problem with engineering tissues that exist in a mechanically dynamic environment, such as bone, muscle and blood vessels, is that most drug delivery systems have been designed to operate under static conditions. We thought that polymeric matrices, which release growth factors in response to mechanical signals, might provide a new approach to guide tissue formation in mechanically stressed environments. Critical design features for this type of system include the ability to undergo repeated deformation, and a reversible binding of the protein growth factors to polymeric matrices to allow for responses to repeated stimuli. Here we report a model delivery system that can respond to mechanical signalling and upregulate the release of a growth factor to promote blood vessel formation. This approach may find a number of applications, including regeneration and engineering of new tissues and more general drug-delivery applications.

Controlled layer-by-layer immobilization of horseradish peroxidase.

Horseradish peroxidase (HRP) was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester (BcapNHS) in a controlled manner to obtain biotinylated horseradish peroxidase (Bcap-HRP) with two biotin moieties per enzyme molecule. Avidin-mediated immobilization of HRP was achieved by first coupling avidin on carboxy-derivatized polystyrene beads using a carbodiimide, followed by the attachment of the disubstituted biotinylated horseradish peroxidase from one of the two biotin moieties through the avidin-biotin interaction (controlled immobilization). Another layer of avidin can be attached to the second biotin on Bcap-HRP, which can serve as a protein linker with additional Bcap-HRP, leading to a layer-by-layer protein assembly of the enzyme. Horseradish peroxidase was also immobilized directly on carboxy-derivatized polystyrene beads by carbodiimide chemistry (conventional method). The reaction kinetics of the native horseradish peroxidase, immobilized horseradish peroxidase (conventional method), controlled immobilized biotinylated horseradish peroxidase on avidin-coated beads, and biotinylated horseradish peroxidase crosslinked to avidin-coated polystyrene beads were all compared. It was observed that in solution the biotinylated horseradish peroxidase retained 81% of the unconjugated enzyme's activity. Also, in solution, horseradish peroxidase and Bcap-HRP were inhibited by high concentrations of the substrate hydrogen peroxide. The controlled immobilized horseradish peroxidase could tolerate much higher concentrations of hydrogen peroxide and, thus, it demonstrates reduced substrate inhibition. Because of this, the activity of controlled immobilized horseradish peroxidase was higher than the activity of Bcap-HRP in solution. It is shown that a layer-by-layer assembly of the immobilized enzyme yields HRP of higher activity per unit surface area of the immobilization support compared to conventionally immobilized enzyme.

Adhesion of nonmetastatic and highly metastatic breast cancer cells to endothelial cells exposed to shear stress.

The mechanical stimulus of shear stress has to date been neglected when studying the adhesion of cancer cells to the endothelium. Confluent monolayers of endothelial cells were subjected to either 4 or 15 hours of arterial shear stress. Adhesion of nonmetastatic (MCF-7) and highly metastatic (MDA-MB-435) human breast cancer cells was then quantified using a detachment assay carried out inside the parallel plate flow chamber. Four hours of shear stress exposure had no effect on adhesion. However, 15 hours of shear stress exposure led to marked changes in the ability of the endothelial monolayer to bind human breast cancer cells. An increase in adhesive strength was observed for nonmetastatic MCF-7 cells, while a decrease in adhesive strength was observed for highly metastatic MDA-MB-435 cells. Hence, endothelial shear stress stimulation does influence the adhesion of cancer cells to the endothelium and can have different effects on the adhesion of cancer cells with different metastatic potentials. Furthermore, adhesion of nonmetastatic and highly metastatic human breast cancer cells may be controlled by two different endothelial cell adhesion molecules that are differentially regulated by shear stress. Immunohistochemistry confirmed that shear stress did in fact differentially regulate endothelial cell adhesion molecule expression.

Comparative evaluation of culture and PCR for the detection and determination of persistence of bacterial strains and DNAs in the Chinchilla laniger model of otitis media.

This study was designed to determine the persistence of culturable bacteria versus DNA in the presence of a middle ear effusion in a chinchilla model of otitis media. Cohorts of animals were either infected with an ampicillin-resistant Haemophilus influenzae strain or injected with a tripartite inoculum consisting of freeze-thawed Streptococcus pneumoniae; pasteurized Moraxella catarrhalis; and DNA from H influenzae. The H influenzae-infected animals displayed culture positivity and polymerase chain reaction positivity through day 35. In the chinchillas infected with the low-copy number inocula of S pneumoniae, DNA was not detectable after day 1 from the co-inoculated pasteurized M catarrhalis bacteria or the purified H influenzae DNA; however, amplifiable DNA from the live low-copy number bacteria persisted through day 21 even though they were not culture-positive past day 3. These results demonstrate that DNA, and DNA from intact but nonviable bacteria, does not persist in an amplifiable form for more than a day in the presence of an effusion; however, live bacteria, while not culturable, persist in a viable state for weeks.

Detecting biomolecules in picoliter vials using aequorin bioluminescence.

The quantitative determination of proteins in picoliter-volume vials is described. The assay is based on the bioluminescence of the photoprotein aequorin along with photon-counting detection. Using this approach, avidin can be detected at femtomole levels by taking advantage of its inhibitory effect on the bioluminescence signal generated by biotinylated recombinant aequorin. The picoliter vials were fabricated on glass substrates using a laser ablation technique. Parameters that affect the reproducibility of the assay such as the fabrication and calibration of the pipets, the fabrication of the vials, and the composition of the assay solutions were studied.

Cognitive restructuring in the treatment of social phobia. Efficacy and mode of action.

Cognitive restructuring (CR) is commonly used to treat social phobia, although its contribution to treatment efficacy has not been established. CR requires the person to think about and discuss feared social events with his or her therapist and thus entails some degree of exposure to social stimuli. CR also is thought to enhance the efficacy of therapeutic exposure exercises (EXP). Four predictions were tested based on this model: Relative to a control intervention matched for the exposure inherent in CR, CR is more effective in (1) reducing social phobia, (2) reducing negative social cognitions, (3) increasing positive cognitions, and (4) enhancing the effects of subsequent EXP. People with generalized social phobia (N = 60) were randomly assigned to CR followed by EXP or to a control intervention followed by EXP. Support was found for predictions 1 to 3, but not 4.

Calibration of micropipets using the bioluminescent protein aequorin.

A new method for calibrating micropipets and determining accurate injection volumes using a pressure-based injector has been developed. This method employs the bioluminescent protein aequorin and can be used to determine injection volumes as small as 3 pL. The calibration plots are linear over at least 3 orders of magnitude. In contrast to conventional micropipet calibration methods that employ fluorescent molecules, the present method produces small background signals.

Determination of the extent of protein biotinylation by fluorescence binding assay.

A method was developed to determine the total amount of biotin present in biotinylated protein conjugates. Conjugates of bovine serum albumin, alkaline phosphatase, and horseradish peroxidase were used in this case study. The extent of biotinylation was determined by complete acid hydrolysis or by enzymatic digestion using proteinase K to release biotin from the biotinylated proteins, followed by sensitive detection of biotin using a coupled HPLC-binding assay system. This detection system is based on the enhancement of the fluorescence of streptavidin-FITC by biotin. The extent of biotinylation determined by this method was compared with the values obtained by a conventional colorimetric method that is based on the displacement of the dye 4-hydroxyazobenzene-2-carboxylic acid (HABA) from the binding sites of avidin. It was found that, because the described method determines the amount of liberated biotin after hydrolysis, it does not suffer from steric hindrance problems associated with the ability of biotin on a protein surface to displace HABA from avidin. Therefore, this method can provide a more accurate determination of the extent of biotinylation. It was also determined that the acid hydrolysis of the biotinylated protein was more effective in releasing the conjugated biotin compared to enzymatic digestion by proteinase K.

Empirical analysis of factors in depressive cognition: the cognitive triad inventory.

The purpose of this study was to examine the factor analytic evidence for the three components of Beck's cognitive triad (view of self, world, and future). Two hundred sixty college undergraduates participated in the study. Factor analytic results indicated that the three scales of the CTI generate five factors, which represent positively phrased "future" items, negatively phrased "future" items, positively phrased "world" items, negatively phrased "world" items, and positively phrased "self" items. The negatively phrased "self" items were distributed among the negatively phrased "world" and negatively phrased "future" factors. These findings partially support the utility of categorizing depressogenic cognitions into three constructs (self, world, and future) and also the notion that different cognitive operations or response styles may be elicited from positive vs. negative items.

Research methods for investigating causal relations between SLE disease variables and psychiatric symptomatology.

People with systemic lupus erythematosus (SLE) frequently experience psychiatric problems. Some researchers and clinicians presume that these psychiatric problems are a direct manifestation of the disease, while others suggest that psychosocial and environmental factors have greater etiological significance. The majority of the studies addressing these issues in patients with SLE are too methodologically limited to make confident conclusions regarding etiology. More methodologically sound research in this area is needed. This article describes some of the limitations in past research in the areas of sampling, measurement, research design, data analyses and data presentation. Suggestions for improved methodology in future research are offered.

Preincubation of endotoxin with monoclonal anti-lipid A (E5), but not in vivo treatment, inhibits circulatory dysfunction.

Monoclonal antibodies (mAb) directed against the toxic lipid A portion of lipopolysaccharide (LPS) have been shown to bind lipid A in vitro, but clinical trials of such mAbs have yielded mixed results. In 53 rats instrumented for macrocirculatory and cremaster muscle microcirculatory measurements, we examined whether E5, a murine-derived anti-lipid A mAb, could inhibit LPS-induced circulatory dysfunction when incubated with LPS in vitro or given separately in vivo prior to LPS administration. Compared with Control rats (Group I), rats infused with 10 mg/kg Escherichia coli LPS (Group II) displayed marked decreases in arterial pressure and cardiac output and marked decreases in erythrocyte velocity in second, third, and fourth order skeletal muscle arterioles. Infusion of 2 mg/kg E5 90 min prior to LPS infusion (Group III) did not improve cardiovascular performance. In contrast, incubation of LPS with either 2 mg/kg (Group IV) or 10 mg/kg (Group V) E5 prior to infusion significantly attenuated LPS-induced changes in both macrocirculatory and microcirculatory function. Further investigation of the disparity between the in vitro and in vivo neutralizing capacity of anti-lipid A mAbs may aid interpretation of the variable clinical results achieved with these preparations.

Combination therapy with zidovudine prevents selection of human immunodeficiency virus type 1 variants expressing high-level resistance to L-697,661, a nonnucleoside reverse transcriptase inhibitor.

L-697,661 is a human immunodeficiency virus type 1 (HIV-1)-specific nonnucleoside reverse transcriptase (RT) inhibitor. Its tolerability and activity in combination with zidovudine were evaluated in a 48-week double-blind study. One hundred nineteen zidovudine-naive HIV-1-infected patients with CD4 cell counts of 200-500/mm3 received either combination therapy, L-697,661 alone, or zidovudine alone. Activity was assessed by CD4 cell count changes. Selection for L-697,661-resistant virus was monitored by susceptibility testing of RT expressed by circulating viral RNA. Therapy was generally well tolerated. All groups receiving zidovudine exhibited transient increases in CD4 cell counts, while the L-697,661 monotherapy group showed a significant decline and yielded RT > 100-fold resistant to L-697,661 and associated with substitutions at RT residue 181. The RT from patients receiving combination therapy was maximally 15-fold less susceptible to L-697,661. Hence, cotreatment with zidovudine prevents selection of HIV-1 variants that are highly resistant to L-697,661 in patients naive to both compounds.