Effect of endotoxin on lipid order and motion in erythrocyte membranes.

Electron paramagnetic resonance employing a lipid-specific spin label has been used to investigate the molecular effects of endotoxin on the physical state of bilayer lipids in rat erythrocyte membranes. When added at a concentration as low as 40 micrograms/ml to whole blood (plasma plus leukocytes present), decreased membrane lipid motion was found in subsequently washed and spin-labeled intact erythrocytes (P < 0.02). However, if endotoxin were added to washed, plasma plus leukocyte-free intact erythrocytes, no change in the motion of the spin label was found, suggesting that plasma-soluble substances and/or leukocytes are required to produce the change in the physical state of lipids. The decreased lipid motion found in these studies is discussed with reference to the known decreased deformability of endotoxin-treated red cells and to the pathogenesis of sepsis.

Analysis of S-35 labeled WR-2721 and its metabolites in biological fluids.

Studies with WR-2721 and related compounds have been hindered by the lack of a suitable assay for the drug and its major metabolites. We have developed a chromatographic method which requires no derivatization for the separation and detection of WR-2721, the free thiol, its symmetrical disulfide and other mixed disulfides. Our procedure involves ion-pairing for separation of ionizable compounds by causing polar molecules to become more lipophilic and hence separable using reverse phase HPLC. Detection is based upon liquid scintillation counting of S-35 incorporated during the synthesis of the parent compound. This method requires no pre-column preparation of samples and, by detecting the S-35 label, eliminates the chance that a coeluting species could interfere with detection, as might occur with post-column derivatization. Chromatography was done using a 10 micron C8RP column and 35% MeOH/65% 0.0113M NaH2PO4, 0.005 M hexanesulfonate, pH 5.9, flowing at 1 ml/min. Half-minute fractions were collected into scintillation vials for counting. Retention volumes for the various compounds were: column breakthrough (3.5 ml), WR-2721 (4.5 ml), WR-1065 (9 ml), and WR-33278 (24 ml). This analytical technique employing radiotracers can be used to study radioprotective mechanisms by time dependent measurements of the tissue distribution and chemical form of labeled drug. Such chemical information can then be correlated with biological measures of radiation protection.

Effect of lipopolysaccharide, leukocytes, and monoclonal anti-lipid A antibodies on erythrocyte membrane elastance.

The micropipette aspiration technique was used to quantify membrane deformability of individual red blood cells (RBCs) before and after exposing whole blood and blood free of leukocytes to lipopolysaccharide (LPS). The ability of an anti-lipid A monoclonal antibody (E5) to inhibit the effects of LPS was also investigated. In the LPS/whole blood studies, a significant increase in elasticity modulus was observed following incubation with LPS. An increase in elasticity modulus indicates a decrease in RBC membrane deformability. The effect depended on the incubation time but was not concentration-dependent in the range studied (25, 40, or 170 micrograms/mL). When incubating blood free of leukocytes with LPS, the elasticity moduli of erythrocytes did not change. Results also showed that preincubation of the LPS with E5 prior to incubation with whole blood partially inhibited the effect of LPS, suggesting a possible mechanism of the beneficial actions of monoclonal antibodies in septic shock.

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.

The temporal pattern of reduction of mortality risk after smoking cessation.

We investigated the excess mortality risks of former smokers according to the number of years since they quit smoking in a cohort of 21,112 men and women evaluated with coronary angiography and included in the Coronary Artery Surgery Study registry. There is a prompt decline in mortality risk within the first year of quitting. Thereafter, former smokers have a sustained, modestly elevated mortality risk for at least 20 years compared with people who never smoked. The pattern is similar in men and women, in 35-54, 55-64, and greater than or equal to 65 age groups, and in subcohorts of those with coronary artery disease and those without coronary artery disease.

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.

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.

Effect of lipopolysaccharide on the physical conformation of the erythrocyte cytoskeletal proteins.

Red blood cell deformability is important for effective circulation in the capillaries. It is known that red cell deformability is significantly reduced during septic shock. Surface to volume ratio, physical effects of the cytoskeletal proteins and the fluidity of lipid bilayer are some of the important intrinsic factors that regulate this mechanical function. Alterations in the physical conformation of cytoskeletal proteins in septic conditions could significantly alter their function. In this study, erythrocytes in whole blood were treated with lipopolysaccharide, the outer covering of Gram-negative bacteria released during Gram-negative sepsis. Electron paramagnetic resonance spectroscopy in conjunction with a protein-specific maleimide nitroxide spin label covalently bound to cytoskeletal proteins was used to investigate the resulting changes occurring in the physical state of cytoskeletal proteins in isolated membranes. Treatment of red blood cells with a lipopolysaccharide concentration as low as 40 micrograms/mL of blood solution for 90 minutes showed a significant decrease in the relevant EPR parameter (p < 0.01) of the spin label bound to subsequently isolated membranes, suggestive of a decreased segmental motion of the spin label and an increase in cytoskeletal protein-protein interactions. These results suggest a marked conformational alteration in the cytoskeletal proteins induced by the lipopolysaccharide and may explain, in part, the marked reduction in red blood cell deformability during septic shock. Bacterial lipopolysaccharide does not exert most of its effects on the host directly, but rather elicits the production of host factors that leads to complex septic shock. Leukocytes, endothelial tissue and many other cells release these mediators. Leukocytes are thought to be a particularly important source of such mediators, including cytokines (tumor necrosis factor, interleukins, etc.), oxygen free radicals, proteases, and hydrolyses. In order to characterize the possible mechanism by which the lipopolysaccharide acts on the physical state of the erythrocyte cytoskeleton, erythrocytes void of leukocytes and plasma were treated with lipopolysaccharide. The relevant EPR parameter showed no significant change over the control value. These results indicate that the leukocytes and their factors are responsible for the rearrangements seen in the cytoskeletal proteins of the erythrocyte membrane.

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.

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.

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.

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.

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.

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.

Development of a computerized drug interaction database (MEDICOM) for use in a patient specific environment.

Drug Interactions are a clearly defined problem that we as health professionals must deal with on a day-by-day basis. It is by far the area of health care that demands more attention today and tomorrow than was possible in the past. The amount of reference sources and text material of drug interactions is growing at such a rate that it is almost impossible to recall essential information in a reasonable time frame. If the practitioner is to continue with an uninterrupted work flow and still maintain the best possible service for the patient, an immediate and accurate method is needed in the hands of the user ... a computerized drug interaction database. The community health care standards would be ultimately raised to a level never before attainable and efficiency would continue with full utilization of professional practice. Combine then, drug interaction data with ancillary benefits such as cost containment, third party accounting, inventory control, and a multitude of other operational functions into a computerized database and the end product results in an enhanced, controlled, professional operation.