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We are in the midst of a biotechnology revolution. Significant advances have been made in sensors and diagnostics based on interrogation of biomolecular arrays. The surface conjugation of nucleic acids, antibodies and proteins onto 'chip' formats has resulted in new classes of high information content devices. This compilation of articles presents the emergence of a new class of such devices based on the ability to interrogate cellular or tissue microarrays. Unlike nucleic acid or antibody-based approaches, these systems enable the interrogation of more complex biological responses, and offer the potential to gather higher information content from measuring physiologic, metabolic, or network processes and responses. This endeavor presents many technological challenges but offers the promise of collecting information more closely correlated to human response and as such represents the opportunity to fabricate new sensors and diagnostics for environmental detection and medical diagnostics.

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Electron paramagnetic resonance (EPR) spectroscopy was used to compare the rates of autoxidation at 37 degrees C of acellular and liposome-encapsulated hemoglobin (LEH) crosslinked between alpha chains with bis (3,5-dibromosalicyl) fumarate (alphaalphaHb). This method avoids the difficulties inherent in using conventional ultraviolet-visible (UV-vis) spectroscopy caused by the high turbidity of liposome suspensions. Rate constants of 0.039/h and 0.065/h were obtained for the alphaalphaHb and LEH samples, respectively. Similar oxidation measurements with alphaalphaHb using UV-vis spectroscopy gave a rate constant comparable to that obtained with EPR spectroscopy. Indirect measurement of the oxidation kinetics of LEH utilizing extraction of alphaalphaHb with chloroform from partially oxidized LEH samples was unreliable because the amount of extractable hemoglobin was inversely proportional to the degree of oxidation. EPR measurements showed a shift in the g value and substantial enhancement in the intensity of the bis-histidine low-spin B complex for the encapsulated hemoglobin, indicating a perturbation of this low-spin complex. We suggest that lipid-associated perturbations are responsible for the enhancement of the oxidation observed with the LEH samples compared to the unencapsulated material.

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PURPOSE: This study investigated the vasoconstrictive effects of both stroma-free and liposome-encapsulated cross-linked hemoglobin (Hb) on vascular plexus hemodynamics, using the choroid of the rabbit eye as a model system. METHODS: Sequential subtraction of high-speed ICG fluorescence angiogram images facilitated visualization of the time-varying patterns of blood flow distribution in the choriocapillaris during the cardiac cycle. Differences between baseline and post-hemoglobin injection blood flow distributions were analyzed. Likewise, differences in the time-varying patterns of flow distribution between the particulate and liquid phases of blood during a cardiac cycle were investigated, since this may bear on differences in vasoactivity induced by circulating stroma-free vs. encapsulated Hb. RESULTS: Cross-linked Hb induced a transient, but marked, localized reduction in choriocapillaris blood flow. This effect was significantly attenuated when liposome encapsulated cross-linked hemoglobin was administered. Plexus blood flow distribution was different for particulate and liquid ICG. CONCLUSIONS: Differences in particulate and liquid ICG flow patterns suggest that one contribution to the different plexus blood flow patterns observed in the encapsulated and free Hb experiments may be due to differences in liquid and particle-bound Hb distribution within the plexus. The observed choriocapillaris blood flow reductions may be attributable to an aggregate endothelial cell contractility induced by presence of extra-cellular Hb in the choriocapillaris plexus.

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This communication describes our work in electrical, topological, and chemical micromodification of surfaces to modulate cellular form and function. We have addressed the surface physico-chemico-mechano properties of cell culture substrates that play a role in modulating cellular behavior. Single factorial model systems have been built using techniques adapted from microlithography. The tools and techniques of microfabrication, if harnessed and used correctly, can be enabling in elucidating the underlying principles and fundamental forces driving the cell-substrate interface. Additionally, the long-term practical applications of microfabrication in medicine and biomaterial/tissue engineering lie in enabling "communication" with living cells/tissues at the cellular and subcellular levels.

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A major obstacle in the development of red cell substitutes has been overcoming their short circulation persistence. In this study, distearoyl phosphoethanolamine polyethylene glycol 5000 (PEG-PE) (10 mol%) was added to the formulation of liposome-encapsulated hemoglobin (LEH) to decrease reticuloendothelial system uptake and prolong LEH circulation persistence. PEG-LEH was radiolabeled with technetium-99m, infused into rabbits (25% of blood pool at 1 ml/min) (n = 5), and monitored by scintigraphic imaging at various times out to 48 h. At 48 h, animals were sacrificed, and tissue samples were collected for counting in a scintillation well counter. Tissue distribution data at 48 h revealed that 51.3 +/- 3.4% of the technetium-99m-PEG-LEH remained in circulation, a greater than 3-fold increase in the circulation half-life compared with circulation half-lives previously reported for non-PEG-containing LEH formulations. The liver had the greatest accumulation at 48 h (12.7 +/- 0.7%), followed by bone marrow (6.2 +/- 0.1%), whereas the spleen had only 1.4 +/- 0.2%. The addition of PEG-PE to the LEH formulation greatly prolongs the circulation persistence of LEH and represents a significant step in the development of red cell substitutes with prolonged oxygen delivery.

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The objective of this study was to explore the potential use of self-assembled monolayers (SAMs) of alkylamine and arylalkyamine as well-defined, homogeneous, tailored in vitro model surfaces for exploring the effect of hydrodynamic flow on morphology and strength of adhesion of human umbilical vein endothelial cells. The cell surface area, shape, f-actin distribution, and adhesion strength of human umbilical vein endothelial cells cultured on self-assembled monolayers of organosilanes were found to be dependent on the chemical composition of the organosilane film and the magnitude of wall shear stress. The direct effects of the differences in chemistry between the two silanes, in modulating cellular response, are probably only secondary to the modulation of cellular functions mediated by differential protein adsorption and conformation on the two silanes. For short seeding times (30 min), prior to application of flow, both substrate chemistry and shear stress modulated the cellular morphology and cytoskeletal organization. For longer seeding times (24 h), prior to application of flow, the chemistry of the underlying surface was the dominant variable in modulating cellular morphology, while the hydrodynamic shear stress modulated the cytoskeleton organization. Cells on N-(2-aminoethyl)-3-aminopropyl trimethoxysilane (EDA) were pleomorphic, while cells on ((((aminoethyl)amino)methyl)phenylethyl)trimethoxysilane (PEDA) expressed a rounded morphology. Application of an incrementally loaded flow regime (0.07-1.25 ml/s) resulted in a time- and shear stress-dependent (10-180 dyn/cm2) detachment of cells, with the cells on EDA depicting higher resistance to wall shear stress. Cellular morphology correlated with the strength of adhesion; cells with rounded morphology on a hydrophobic silane, PEDA, were less tightly bound to the silane, while spread cells on a hydrophilic silane, EDA, were more tightly bound. The higher surface free energy of EDA is speculated to influence the increased cell spreading and strength of adhesion observed in these studies. The presence of the phenyl group in PEDA reduces the surface free energy and may account for the reduced spreading and lower strength of adhesion. The use of well-defined systems, such as monolayer organosilanes, with tunable surface physicochemical properties may serve to deconstruct the complex interaction of cells with extracellular matrix components: surface charge, surface hydrophobicity, and other short- and long-range forces can be individually controlled and correlated with cellular functions. The organosilane monolayers could serve as the building blocks for sequential addition of proteins or cell adhesive/cell repulsive cues to stepwise engineering and construction of more complex systems resembling ECM matrices.

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Infusion of liposome-encapsulated hemoglobin (LEH) induces a transient thrombocytopenia in rats (Rabinovici R, Rudolph AS, Ligler FS, Smith EF, III, Feuerstein G [1992] Biological responses to exchange transfusion with liposome-encapsulated hemoglobin. Circ Shock 37:124). A specific mechanism such as a localization of platelets during this transient LEH-induced thrombocytopenia has not been reported previously in the literature. In this study, platelets were isolated and labeled with indium-111 (111In), then retransfused into the same animal. Fifteen minutes after the 111In platelets were retransfused and allowed to equilibrate with the blood pool, a 10% top load volume of either LEH (1.8 mL, 262 mg phospholipid/kg body weight, 92 mg hemoglobin (Hb)/kg body weight), liposome vehicle (LV) (1.8 mL, 262 mg phospholipid/kg body weight), or free bovine Hb (1.8 mL, 92 mg Hb/kg body weight) (n=6 per group) was infused. Serial blood samples were drawn to determine platelet counts by radioactivity and light scattering methods. LV and Hb demonstrated no significant changes from baseline levels in circulating platelet levels, whereas LEH caused a transient 50% decrease in 111In platelet activity 2-5 minutes postinfusion, which returned to baseline levels by 15 minutes. Platelet counts based on traditional light scattering methods were not significantly different among the three treatment groups over this same time course. Localization of these 111In platelets was monitored with a gamma scintigraphic camera. After infusion of LEH, 111In platelets rapidly moved out of the circulation and sequestered in the lungs and liver with subsequent return to the circulation by 15 minutes. In contrast, no significant changes in 111In platelet activity were noted in the lungs and liver after infusion of LV and Hb. 111In platelet activity in the spleen nearly doubled 30 minutes after Hb infusion and was significantly different than spleen 111In platelet activity in the LEH and LV groups. In vitro platelet aggregation studies were also performed to determine the direct effect of LEH, LV, and Hb on platelet aggregation. The rate of thrombin-induced aggregation did not differ among control samples and samples containing LEH, LV, or Hb; none of these agents induced platelet aggregation. We conclude that LEH-induced thrombocytopenia is associated with a transient (within minutes) sequestration of platelets in the lungs and liver, with subsequent release to the circulation within 15 minutes. Tetrameric bovine Hb is associated with increased platelet accumulation in the spleen. Light scattering methods for measuring platelet levels in blood samples containing LEH are unreliable because of particulate interference.

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Intravenous administration of liposome-encapsulated hemoglobin (LEH) in rats led to an early (within 15 min) decline of hemolytic complement (C) activity in the plasma along with a significant, parallel rise in thromboxane B2 (TXB2) levels. The TXB2 response was inhibited by co-administration of soluble C receptor type 1 (sCR1) with LEH, as well as by C depletion with cobra venom factor. These observations provide evidence for a causal relationship between LEH-induced C activation and TXB2 release, and suggest that sCR1 could be useful in attenuating the acute respiratory, hematological and hemodynamic side effects of LEH described earlier in the rat.

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Free hemoglobin (Hb) induces a potent vasoconstrictor response that may limit its therapeutic application as a red blood cell replacement. We have investigated whether encapsulation of stroma-free Hb (SFHb) or cross-linked Hb (alpha alpha-Hb) in liposomes modulates Hb vasoactivity in isolated blood vessels. Relaxation of rabbit thoracic vessels was measured before and after exposure to acellular SFHb, alpha alpha-Hb, and liposome-encapsulated SFHb or alpha alpha-Hb. SFHb and alpha alpha-Hb caused significant inhibition of carbachol-induced relaxation at 0.5 mg/dl, whereas encapsulation inhibited vessel relaxation at 30- to 60-fold higher Hb concentrations. The contractile response of rabbit ear arterial segments to electrical stimulation in the presence of acellular alpha alpha-Hb resulted in a 150% increase (EC150) in contractile amplitude at 0.23 mg/dl, whereas the EC150 for encapsulated alpha alpha-Hb was 13.7 mg/dl. Mechanistic studies of the vasoconstrictor activity of Hb demonstrated that acellular alpha alpha-Hb had no effect on norepinephrine release in the rabbit ear artery. In addition, neither acellular nor encapsulated alpha alpha-Hb preparations inhibited endothelial nitric oxide (NO) synthase activity isolated from bovine pulmonary artery. However, inhibition of vessel relaxation by acellular or encapsulated alpha alpha-Hb was reversed by the NO donor S-nitrosylpenacillamine, implicating Hb-NO binding as a possible mechanism for the vasoconstrictor response. In vitro stopped-flow kinetic studies of Hb-NO binding showed similar rates of reaction for conversion of oxyhemoglobin to methemoglobin (metHb; < 2 ms), followed by rapid conversion of metHb to NO-Hb (300 ms) for both acellular and encapsulated alpha alpha-Hb, demonstrating that liposome encapsulation does not retard NO-Hb binding. The attenuated vasoactivity of encapsulated Hb may, therefore, result from the limited access of encapsulated Hb to NO imposed by the physical size of the liposome and reduced penetration of Hb across the vascular endothelium.

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A method for determining oxygen-carrying capacity of blood substitutes has been developed using the short-lived cyclotron-produced positron-emitting isotope 15O. This method measures the oxygen-carrying capacity of the blood substitutes in vivo in the presence of red blood cells and allows determination of changes in the oxygen-carrying capacity over time after exchange transfusion. This method is applied to the blood substitutes of liposome-encapsulated hemoglobin (LEH) and cell-free hemoglobin (Hb). We have used 15O (half-life of 2 min) to quantitate the lung uptake and tissue delivery of [15O2]LEH. Lung uptake studies were performed in intubated, catheterized rats after a 40% exchange transfusion of bovine LEH (LEBH; 0.68 g Hb/kg body wt), human hemolysate LEH (LEHH; 1.0 g Hb/kg body wt), or free bovine hemoglobin (SFHS; 0.56 g Hb/kg body wt). A bolus inhalation of 15O2 (3-5 mCi) was given at 15 min, 3 h, and 24 h post-transfusion. Arterial blood samples were collected, spun, and separated into LEH, red blood cell, and plasma fractions. 15O activity and hemoglobin content were determined for each fraction. Oxygen-carrying capacity was calculated as a percentage of the original red blood cell fraction removed. For LEBH, the carrying capacity was 15% at 15 min, 13% at 3 h, and 1% at 24 h. For LEHH, the carrying capacity was 30% at 15 min, 26% at 3 h, and 19% at 24 h. The marked decrease in carrying capacity at 24 h for LEBH compared with LEHH was attributable to the increased formation of methemoglobin in the circulating LEBH rather than increased removal from circulation, because total hemoglobin concentrations measured for both LEH samples decreased at a similar rate during the 24 h. The presence of methemoglobin reductase and other naturally occurring antioxidants in the LEHH may be responsible for maintaining the higher levels of oxyhemoglobin. Oxygen-carrying capacity for SFHS also decreased over time but at a much sharper rate compared with both LEH formulations. The carrying capacity for SFHS of 8% measured at 15 min decreased to 0.3% at 3 h and undetectable levels at 24 h. This sharper decrease in carrying capacity for SFHS is attributable to the rapid removal of the hemoglobin from circulation.

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Previous studies have documented that liposome-encapsulated hemoglobin (LEH) can cause a rapid and transient thrombocytopenia following intravenous injection into small animals. The present study evaluated the role of complement during the LEH-induced thrombocytopenia in rats. We have compared changes in platelet levels in the blood, platelet organ distribution, and total hemolytic complement levels following intravenous administration of LEH in control and complement-depleted rats. Changes in platelet organ distribution at various times after LEH administration were monitored by labeling autologous platelets with indium-111 (111In)-oxine and imaging the 111In-platelets with a gamma camera after reinjection. Platelet counts were determined by light-scattering methods and by following 111In radioactivity at various times after LEH administration. Platelet levels did not significantly change for the complement-depleted rats during the 60 min following an injection of LEH, whereas thrombocytopenia (40% decrease) was noted within 4 min post-LEH-injection for control rats with a gradual return to baseline circulating platelet levels within 60 min. This drop in circulating platelets was correlated with a rapid redistribution of 111In-platelets from the circulation to the lungs and liver, whereas complement-depleted rats showed no transient movement of the 111In-platelets from the circulation. Baseline complement levels of 21.6 +/- 2.2 CH50/ml for control rats and 0.2 +/- 0.1 CH50/ml for complement-depleted rats did not significantly change during the 60 min following LEH administration. This study suggests that complement must be present during LEH-induced transient thrombocytopenia, as complement-depleted rats underwent no thrombocytopenia, and that the transient LEH-induced thrombocytopenia may be associated with complement activation.

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OBJECTIVE: To investigate the effect of liposome-encapsulated hemoglobin, an experimental blood substitute, on the function of the mononuclear phagocytic system in normovolemic mic, in ex vivo murine splenocytes and in a transformed murine monocytic cell line, RAW 264.7. DESIGN: Prospective, randomized trial. SETTING: Center for Biomolecular Science and Engineering, Naval Research Laboratory, and the Thomas Jefferson University. SUBJECTS: Female Balb/c mice (n = 27). INTERVENTIONS: Mice were injected into the tail vein with liposome-encapsulated hemoglobin or liposome vehicle and were killed at varying time points for blood sampling and splenocyte isolation and culture. MEASUREMENTS AND MAIN RESULTS: Injection of liposome-encapsulated hemoglobin in mice (2.2 of lipid/kg and 0.56 g of hemoglobin/kg, n = 9) did not increase serum tumor necrosis factor (TNF)-alpha concentrations at 2, 8, 15, and 24 hrs after administration. In the ex vivo procedure, lipopolysaccharide (1 microgram/mL)-induced TNF-alpha production by splenocytes from mice injected with liposome-encapsulated hemoglobin was attenuated at 2 and 4 hrs (73%, p = .002 at 2 hrs), compared with TNF-alpha production by splenocytes from sham animals challenged with the same concentration of lipopolysaccharide. In the in vitro procedure, simultaneous exposure of liposome-encapsulated hemoglobin (0.88 to 8.8 mg/mL) and lipopolysaccharide (0.125 to 1 microgram/mL) to the murine-derived, peritoneal monocytic RAW 264.7 cell line showed significantly reduced TNF-alpha peptide, but not messenger RNA, 1 to 4 hrs after exposure as compared with cells challenged with lipopolysaccharide alone. This effect correlated with the rapid phagocytosis (1 hr to 4 hrs) of liposome-encapsulated hemoglobin by RAW 264.7 cells. Phagocytic activity in RAW 264.7 cells exposed to both liposome-encapsulated hemoglobin and lipopolysaccharide showed reduced uptake compared with uptake of liposome-encapsulated hemoglobin. The liposome-induced reduction in TNF-alpha peptide production elicited by lipopolysaccharide was countered by extending the time period to an overnight delay between liposome-encapsulated hemoglobin exposure and lipopolysaccharide challenge. Liposome-encapsulated hemoglobin incubated with lipopolysaccharide in vitro, and subsequently washed to remove free lipopolysaccharide, stimulated TNF-alpha expressed by RAW 264.7 cells. Incubation with liposome-encapsulated hemoglobin alone did not evoke TNF-alpha production in these cells. CONCLUSIONS: These data suggest that liposome-encapsulated hemoglobin modulates the response of the mononuclear phagocyte system to endotoxin, possibly through binding of lipopolysaccharide, presentation to macrophages with subsequent phagocytosis, and modulation of cytokine response by a posttranscriptional mechanism. This effect is attenuated by extending the period between exposure to liposome-encapsulated hemoglobin and endotoxin. The clinical relevance of these findings awaits further investigation.

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BACKGROUND: Liposome-encapsulated hemoglobin (LEH) has been developed as an emergency blood substitute, yet its effect on human complement has never been explored. Considering that complement activation is a major pathogenic factor in the respiratory distress syndrome that often develops in trauma and shock, LEH-induced complement activation may be a critical safety issue. STUDY DESIGN AND METHODS: Various LEH and corresponding empty liposomes were incubated with normal human sera, and various markers of complement activation (serum levels of C4d, Bb, SC5b-9, and CH50; C5a-induced granulocyte aggregation; membrane deposition of C3b) were measured. Incubations were also performed in the presence of (ethylene-bis[oxyethylenenitrilo]tetraacetic acid) (EGTA) and Mg++ (EGTA/Mg++) and soluble complement receptor type 1. RESULTS: LEH and liposomes activated human complement, as indicated by significant changes in one or more markers. The effect was primarily due to the presence of the phospholipid vehicle; small, unilamellar, highly homodispersed vesicles induced the greatest degree of complement activation. Complement activation was partially inhibited by EGTA/Mg++. The latter finding, together with the parallel increases in serum C4d and Bb, suggests activation of both the classical and alternative pathways. Soluble complement receptor type 1 (0.05-20 micrograms/mL) efficiently inhibited all vesicle-induced complement activation. CONCLUSION: Because of complement activation, the use of LEH for transfusion may require careful evaluation of safety. Soluble complement receptor type 1 may be useful as a prophylactic agent for complement activation-related complications of liposome infusions.

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In exploring the occurrence and mechanism of liposome-encapsulated hemoglobin (LEH)-induced complement (C) activation, we found that normal human serum contained low titers of IgG and IgM class natural antibodies with reactivity against LEH, and that the amount of vesicle-bound IgM significantly correlated with LEH-induced C consumption. IgM binding to LEH was inhibited by phosphocholine and ATP, but not by choline chloride. These data suggest that naturally occurring antibodies play a key role in LEH-induced C activation, and that a major portion of these antibodies are directed against the phosphate moiety on the phospholipid headgroups of liposome bilayers.

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The effects of encapsulating bovine hemoglobin (BHb) in the bicontinuous cubic phase formed by monooleoylglycerol and water was investigated with Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction. Cubic phase was formed in the presence of 1-10 wt% BHb. Studies using X-ray diffraction reveal that at 0.5-2.5 wt% BHb, the cubic phase structure is characterized by the double diamond lattice (Pn3m). At 2.5-5 wt% BHb, coexistence of two cubic phase structures, Pn3m and the gyroid lattice (Ia3d), was observed while at BHb, concentrations higher than 5 wt% the gyroid structure persists. FTIR shows there is an increase in intensity of the free nu C = O (1745 cm-1) and a corresponding decrease in the intensity of the hydrogen bonded nu C = O (1720 cm-1) as the BHb concentration is increased. The nu C-O-CO peak shifts from 1183 cm-1 to 1181 cm-1 as the concentration of BHb raised from 2.5 to 10 wt% indicating BHb may induce subtle changes in the interfacial region of cubic phase monoolein. The bandwidth of the nu asCH2 stretch (2926 cm-1) increased in the presence of 5 wt% BHb compared to samples with 2.5 or 10 wt% BHb. The increase in frequency of the nu sCH2 stretch (2854 cm-1) induced by increasing temperature 20 to 60 degrees C was dampened when BHb was present compared to samples heated in isotonic buffer. Analysis of the amide I band at 1650 cm-1 showed that the secondary structure of BHb is not affected by encapsulation in monoolein. In vitro release studies showed that 45% of the entrapped BHb was released after 144 h at 37 degrees C. The porous nature of bulk cubic phase was further demonstrated by diffusion of K2Fe(CN)6 and conversion of 73% of the oxyhemoglobin to methemoglobin after 1 h. These results suggest that the cubic phase may be useful for encapsulation of Hb as a red cell substitute and for the encapsulation and delivery of other bioactive agents.

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Liposome-encapsulated hemoglobin (LEH) has been tested in animals as an oxygen-carrying red cell substitute and has been shown to be beneficial in the treatment of hemorrhagic shock. The effects of LEH on immune responses have not been studied thoroughly in any well-controlled model. Using a murine model, we evaluated nephrotoxicity and hepatotoxicity as well as immune function parameters following LEH administration. Following intravenous administration of LEH, 1) a serum spike of interleukin-6 (IL-6) occurred in mice at 4-8 hours, with no elevation of IL-1, tumor necrosis factor (TNF), or interferon-gamma (IFN-gamma); 2) the serum liver function enzymes SGOT (AST, aspartate aminotransferase) and SGPT (ALT, alanine aminotransferase) were elevated at 48 hours; 3) only a slight increase in serum antibody to bovine hemoglobin was observed; and 4) increased hematopoietic activity was observed in the spleen and bone marrow. The finding that only IL-6 but not the associated TNF, IL-1, or IFN-gamma is secreted in vivo following LEH administration is novel and may have significance in defining the mechanisms underlying specific adverse responses observed with LEH administration in animals.

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A new method is described for producing biomedically relevant polymers with precisely defined micron scale surface texture in the x, y, and z planes. Patterned Si templates were fabricated using photolithography to create a relief pattern in photoresist with lateral dimensions as small as 1 micron. Electroless Ni was selectively deposited in the trenches of the patterned substrate. The Ni served as a resilient mask for transferring the patterns onto the Si substrate to depths of up to 8.5 microns by anisotropic reactive ion etching with a fluorine-based plasma. The 3-dimensional (3-D) textured silicon substrates were used as robust, reusable molds for pattern transfer onto poly (dimethyl siloxane), low density poly (ethylene), poly (L-lactide), and poly (glycolide) by either casting or injection molding. The fidelity of the pattern transfer from the silicon substrates to the polymers was 90 to 95% in all three planes for all polymers for more than 60 transfers from a single wafer, as determined by scanning electron microscopy and atomic force microscopy. Further, the 3-D textured polymers were selectively modified to coat proteins either in the trenches or on the mesas by capillary modification or selective coating techniques. These selectively patterned 3-D polymer substrates may be useful for a variety of biomaterial applications.

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Physiological responses and circulation properties of liposome-encapsulated hemoglobin (LEH) labeled with technetium-99m (99mTc) were measured in rats after a 10% (170 mg/kg hemoglobin; 430 mg/kg phospholipid) or a 50% (450 mg/kg hemoglobin, 2.3 g/kg phospholipid) hypovolemic exchange transfusion (n = 5 per exchange group). Mean arterial pressure returned to baseline values (105 +/- 8 mmHg) by 90 min post-infusion for both groups. By 20 h, mean arterial pressure remained at baseline values for the 10% group, but dropped to 30 +/- 14 mmHg for the 50% group. For both groups, bradycardia was seen after the exchange period, but heart rate recovered by 30 min for the 10% group and by 90 min for the 50% group. The 99mTc-LEH remained in circulation longer for the 50% group (18.2 h half-life) than for the 10% group (2.4 h half-life). Removal of 99mTc-LEH from the bloodstream was via the liver and spleen. At 20 h, 99mTc-LEH accumulation in these organs was greater for the 10% group (liver, 36.2 +/- 1.7%; spleen, 37.5 +/- 2.5%) than for the 50% group (liver, 17.0 +/- 1.4%; spleen, 17.1 +/- 1.4%). The data show that there is less clearance of 99mTc-LEH from the bloodstream by the reticuloendothelial system after a 50% hypovolemic exchange transfusion, thus supporting the possible use of LEH as an oxygen-carrying resuscitative fluid in situations of severe blood loss.

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