Phosphatidylethanolamine is progressively exposed in RBCs during storage.

BACKGROUND: It is well established that as a blood unit ages, fewer of the unit's red blood cells (RBCs) remain in circulation post-transfusion. The mechanism for clearance is not well defined. Phosphatidylethanolamine (PE) is a phospholipid that is primarily found on the inner leaflet of healthy cells, and is an important ligand for phagocytosis of dead cells when exposed. OBJECTIVES: The objective of the present study was to measure the change in PE exposure in donor RBCs over increasing storage ages using the novel PE-specific probe, duramycin. METHODS: Five adsol (AS-1) preserved RBC units were sampled weekly for 6 weeks and were labelled with duramycin. The percentage of PE exposed on red cells in each sample was determined using flow cytometry. Surface phosphatidylserine (PS) was evaluated for comparison. RESULTS: We found that RBCs in AS-preserved donor units increasingly exposed PE, from less than 1% in freshly processed RBCs, to nearly 20% at 42 days of storage and correlated with increased relative vesiculation or microparticle concentration and release of cell-free haemoglobin. By comparison, only 5% of cells exposed PS at 42 days. CONCLUSION: We conclude that exposure of PE in the RBC outer membrane was higher than that of PS during 42 days of storage and correlated significantly with increased vesiculation and release of haemoglobin.

Maggots as a wound debridement agent for chronic venous leg ulcers under graduated compression bandages: A randomised controlled trial.

OBJECTIVES: Slough in chronic venous leg ulcers may be associated with delayed healing. The purpose of this study was to assess larval debridement in chronic venous leg ulcers and to assess subsequent effect on healing. METHODS: All patients with chronic leg ulcers presenting to the leg ulcer service were evaluated for the study. Exclusion criteria were: ankle brachial pressure indices <0.85 or >1.25, no venous reflux on duplex and <20% of ulcer surface covered with slough. Participants were randomly allocated to either 4-layer compression bandaging alone or 4-layer compression bandaging + larvae. Surface areas of ulcer and slough were assessed on day 4; 4-layer compression bandaging was then continued and ulcer size was measured every 2 weeks for up to 12 weeks. RESULTS: A total of 601 patients with chronic leg ulcers were screened between November 2008 and July 2012. Of these, 20 were randomised to 4-layer compression bandaging and 20 to 4-layer compression bandaging + larvae. Median (range) ulcer size was 10.8 (3-21.3) cm(2) and 8.1 (4.3-13.5) cm(2) in the 4-layer compression bandaging and 4-layer compression bandaging + larvae groups, respectively (Mann-Whitney U test, P = 0.184). On day 4, median reduction in slough area was 3.7 cm(2) in the 4-layer compression bandaging group (P < 0.05) and 4.2 cm(2) (P < 0.001) in the 4-layer compression bandaging + larvae group. Median percentage area reduction of slough was 50% in the 4-layer compression bandaging group and 84% in the 4-layer compression bandaging + larvae group (Mann-Whitney U test, P < 0.05). The 12-week healing rate was 73% and 68% in the 4-layer compression bandaging and 4-layer compression bandaging + larvae groups, respectively (Kaplan-Meier analysis, P = 0.664). CONCLUSIONS: Larval debridement therapy improves wound debridement in chronic venous leg ulcers treated with multilayer compression bandages. However, no subsequent improvement in ulcer healing was demonstrated.

Distinct roles for S100a8 in early embryo development and in the maternal deciduum.

S100a8 is a cytosolic protein expressed in myeloid cells where it forms a stable heterodimer with another S100 protein family member, S100a9. The S100a9(-/-) mouse is viable and phenotypically normal, whereas the S100a8(-/-) condition is embryonic lethal. We present evidence that S100a8, without S100a9, has a previously unrecognized role in embryo development between fertilization and the 8-cell stage at embryonic day (E) 2.5. S100a8 also has a second role in the maternal deciduum, where expression is associated with the vasculature from the E8.5 stage to the formation of mature placenta. Uterine natural killer cells that have a role in vascular remodelling colocalise with the S100a8 vascular expression in the metrial triangle. In inflammatory responses in peripheral tissues, S100a8 is a potent chemoattractant and also an anti-oxidant. Both roles may be important in the developing placenta. Thus we highlight two new S100a9-independent roles for S100a8 in early embryo development.

Defective chemoattractant-induced calcium signalling in S100A9 null neutrophils.

The S100 family member S100A9 and its heterodimeric partner, S100A8, are cytosolic Ca2+ binding proteins abundantly expressed in neutrophils. To understand the role of this EF-hand-containing complex in Ca2+ signalling, neutrophils from S100A9 null mice were investigated. There was no role for the complex in buffering acute cytosolic Ca2+ elevations. However, Ca2+ responses to inflammatory agents such as chemokines MIP-2 and KC and other agonists are altered. For S100A9 null neutrophils, signalling at the level of G proteins is normal, as is release of Ca2+ from the IP(3) receptor-gated intracellular stores. However MIP-2 and FMLP signalling in S100A9 null neutrophils was less susceptible than wildtype to PLCbeta inhibition, revealing dis-regulation of the signalling pathway at this level. Downstream of PLCbeta, there was reduced intracellular Ca2+ release induced by sub-maximal levels of chemokines. Conversely the response to FMLP was uncompromised, demonstrating different regulation compared to MIP-2 stimulation. Study of the activity of PLC product DAG revealed that chemokine-induced signalling was susceptible to inhibition by elevated DAG with S100A9 null cells showing enhanced inhibition by DAG. This study defines a lesion in S100A9 null neutrophils associated with inflammatory agonist-induced IP3-mediated Ca2+ release that is manifested at the level of PLCbeta.

Ligand density modulates eosinophil signaling and migration.

Eosinophils are a major component of the inflammatory response in persistent airway inflammation in asthma. The factors that determine the retention of eosinophils in the airway remain poorly understood. Elevated levels of fibronectin have been observed in the airway of patients with asthma, and the levels correlate with eosinophil numbers. To determine if fibronectin density modulates eosinophil function, we investigated the effect of fibronectin and vascular cell adhesion molecule 1 (VCAM-1) density on eosinophil migration and signaling via the p38 and extracellular regulated kinase (ERK)-mitogen-activated protein kinase (MAPK) signaling pathways. There was a dose-dependent inhibition of eosinophil spreading and migration on increasing concentrations of fibronectin but not VCAM-1. In addition, activation of p38 MAPK was inhibited at high fibronectin but not high VCAM-1 concentrations, and ERK activity was slightly reduced at high VCAM-1 and fibronectin concentrations. Together, the results demonstrate that fibronectin but not VCAM-1 inhibits eosinophil migration and signaling.

Antioxidant mechanisms of isoflavones in lipid systems: paradoxical effects of peroxyl radical scavenging.

Oxidation of lipids has been implicated in the pathophysiology of atherosclerosis. It has been suggested that scavenging of lipid peroxyl radicals contribute to the antiatherosclerotic effects of naturally occurring compounds such as the isoflavones. This group of polyphenolics includes genistein and is present in relatively high concentrations in food products containing soy. Soy isoflavones are capable of inhibiting lipoprotein oxidation in vitro and suppressing formation of plasma lipid oxidation products in vivo. However, key aspects of the antioxidant mechanisms remain unknown. In this study the antioxidant effects of genistein and other soy isoflavones on lipid peroxidation initiated by mechanistically diverse oxidants was investigated. Although isoflavones inhibited lipid peroxidation stimulated by both metal-dependent and independent processes, the concentration required for these effects were relatively high compared to those found in vivo. Interestingly, however, isoflavones were not consumed and remained in the native state over the time during which inhibition of lipid peroxidation was observed. This was also the case under conditions where synergistic inhibition of LDL oxidation was observed with ascorbate. Furthermore, in an oxidation system driven solely by peroxyl radicals, isoflavones were found to be relatively poor peroxyl radical scavengers. Consistent with the apparent lack of reactivity with lipid-derived oxidants, isoflavones were also relatively resistant to oxidation mediated by the potent oxidant peroxynitrite. The potential antioxidant mechanisms of isoflavones are discussed in the context of possible reactivities of isoflavone-derived phenoxyl radicals.

Reaction of tetrahydrobiopterin with superoxide: EPR-kinetic analysis and characterization of the pteridine radical.

It has been shown that BH(4) ameliorates endothelial dysfunction associated with conditions such as hypertension, cigarette smoking, and diabetes. This effect has been proposed to be due to a superoxide scavenging activity of BH(4). To examine this possibility we determined the rate constant for the reaction between BH(4) and superoxide using electron paramagnetic resonance (EPR) spin trapping competition experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). We calculated a rate constant for the reaction between BH(4) and superoxide of 3.9 +/- 0.2 x 10(5) M(-1)s(-1) at pH 7.4 and room temperature. This result suggests that superoxide scavenging by BH(4) is not a major reaction in vivo. HPLC product analysis showed that 7,8-BH(2) and pterin are the stable products generated from the reaction. The formation of BH(4) cation radical (BH(4)(*+)) was demonstrated by direct EPR only under acidic conditions. Isotopic substitution experiments demonstrated that the BH(4)(*+) is mainly delocalized on the pyrazine ring of BH(4). In parallel experiments, we investigated the effect of ascorbate on 7,8-BH(2) reduction and eNOS activity. We demonstrated that ascorbate does not reduce 7,8-BH(2) to BH(4), nor does it stimulate nitric oxide release from eNOS incubated with 7,8-BH(2). In conclusion, it is likely that BH(4)-dependent inhibition of superoxide formation from eNOS is the mechanism that better explains the antioxidant effects of BH(4) in the vasculature.

A small-molecule antagonist of LFA-1 blocks a conformational change important for LFA-1 function.

Lymphocyte function-associated antigen (LFA)-1/intercellular adhesion molecule (ICAM)-1 interactions mediate several important steps in the evolution of an immune response. LFA-1 is normally expressed in a quiescent state on the surface of leukocytes and interacts weakly with its ligands ICAM-1, -2, and -3. LFA-1 activity may be regulated by receptor clustering and by increasing the affinity of LFA-1 for its ligands. Affinity modulation of LFA-1 has been shown to occur via a conformational change in the LFA-1 heterodimer that can be detected by using monoclonal antibody 24 (mAb24). We have recently described a small-molecule antagonist of LFA-1, BIRT 377, that demonstrates selective in vitro and in vivo inhibition of LFA-1/ICAM-1-mediated binding events. We now demonstrate that BIRT 377 blocks the induction of the mAb24 reporter epitope on LFA-1 on the surface of SKW-3 cells treated with various agonists known to induce high-affinity LFA-1. These data imply that BIRT 377 exerts its inhibitory effects by preventing up-regulation of LFA-1 to its high-affinity conformation.

The use of lymphocyte function-associated antigen (LFA)-1-deficient mice to determine the role of LFA-1, Mac-1, and alpha4 integrin in the inflammatory response of neutrophils.

After injury or infection, neutrophils rapidly migrate from the circulation into tissues by means of an orderly progression of adhesion receptor engagements. Neutrophils have been previously considered to use selectins exclusively to roll on vessels before an adhesion step mediated by the beta2 integrins, lymphocyte function-associated antigen (LFA)-1, and Mac-1. Here we use LFA-1(-/-) mice, function blocking monoclonal antibodies, and intravital microscopy to investigate the roles of LFA-1, Mac-1, and alpha4 integrins in neutrophil recruitment in vivo. For the first time, we show that LFA-1 makes a contribution to neutrophil rolling by stabilizing the transient attachment or tethering phase of rolling. In contrast, Mac-1 does not appear to be important for either rolling or firm adhesion, but instead contributes to emigration from the vessel. Blocking Mac-1 in the presence of LFA-1 significantly reduces emigration, suggesting cooperation between these two integrins. Low levels of alpha4beta1 integrin can be detected on neutrophils from LFA-1(+/+) and (-/-) mice. These cells make use of alpha4beta1 during the rolling phase, particularly in the absence of LFA-1. Thus LFA-1 and alpha4beta1, together with the selectins, are involved in the rolling phase of neutrophil recruitment, and, in turn, affect the later stages of the transmigration event.

Shape and shift changes related to the function of leukocyte integrins LFA-1 and Mac-1.

Integrin activity on leukocytes is controlled tightly, ensuring that ligand binding occurs only when leukocytes are in contact with their targets. For an integrinlike LFA-1, this ligand-binding activity comes about as a result of increased integrin clustering. Affinity regulation of integrins also plays a role, but the conformational changes giving rise to increased affinity appear to be secondary to clustering. Conformationally altered LFA-1 can be created artificially by deletion of the I domain, which is the key domain involved in ligand binding for many but not all integrins. Although I domain-deleted LFA-1 (DeltaI-LFA-1) cannot bind ligand, it is able to signal constitutively into the cell. One measure of this signaling activity is the ability of DeltaI-LFA-1 to activate beta1 integrins on the same T lymphocyte. Leukocytes use LFA-1 to migrate across the endothelium. Active beta1 integrins may be required subsequently to bind the matrix proteins encountered by leukocytes as they continue their voyage into the tissue interior.

Nitration and oxidation of a hydrophobic tyrosine probe by peroxynitrite in membranes: comparison with nitration and oxidation of tyrosine by peroxynitrite in aqueous solution.

It has been reported that peroxynitrite will initiate both oxidation and nitration of tyrosine, forming dityrosine and nitrotyrosine, respectively. We compared peroxynitrite-dependent oxidation and nitration of a hydrophobic tyrosine analogue in membranes and tyrosine in aqueous solution. Reactions were carried out in the presence of either bolus addition or slow infusion of peroxynitrite, and also using the simultaneous generation of superoxide and nitric oxide. Results indicate that the level of nitration of the hydrophobic tyrosyl probe located in a lipid bilayer was significantly greater than its level of oxidation to the corresponding dimer. During slow infusion of peroxynitrite, the level of nitration of the membrane-incorporated tyrosyl probe was greater than that of tyrosine in aqueous solution. Evidence for hydroxyl radical formation from decomposition of peroxynitrite in a dimethylformamide/water mixture was obtained by electron spin resonance spin trapping. Mechanisms for nitration of the tyrosyl probe in the membrane are discussed. We conclude that nitration but not oxidation of a tyrosyl probe by peroxynitrite is a predominant reaction in the membrane. Thus, the local environment of target tyrosine residues is an important factor governing its propensity to undergo nitration in the presence of peroxynitrite. This work provides a new perspective on selective nitration of membrane-incorporated tyrosine analogues.

Metabolism of S-nitrosoglutathione by endothelial cells.

S-nitrosoglutathione (GSNO) is an inhibitor of platelet aggregation and has also been shown to protect the ischemic heart from reperfusion-mediated injury. Although GSNO is often used in cell culture as a source of nitric oxide, the mechanisms of GSNO metabolism are not well established. We show here that GSNO decomposition by bovine aortic endothelial cells has an absolute dependence on the presence of cystine in the cell culture medium. In addition, GSNO decay is inhibited by diethyl maleate, an intracellular glutathione scavenger, but not by buthionine sulfoximine, a glutathione synthesis inhibitor. This indicates that thiols in general, rather than specifically glutathione, are the major factors that influence GSNO decay. Only 40% of the nitroso group of GSNO could be recovered as nitrite/nitrate, suggesting that the primary route of GSNO decay is reductive and that nitric oxide is only a minor product of GSNO decay. We conclude that the intracellular thiol pool causes the reduction of extracellular disulfides to thiols, which then directly reduce GSNO.

Inhibition of oxidized low-density lipoprotein-induced apoptosis in endothelial cells by nitric oxide. Peroxyl radical scavenging as an antiapoptotic mechanism.

Proatherogenic oxidized low-density lipoprotein (oxLDL) induces endothelial apoptosis. We investigated the anti-apoptotic effects of intracellular and extracellular nitric oxide (*NO) donors, iron chelators, cell-permeable superoxide dismutase (SOD), glutathione peroxidase mimetics, and nitrone spin traps. Peroxynitrite (ONOO-)-modified oxLDL induced endothelial apoptosis was measured by DNA fragmentation, TUNEL assay, and caspase-3 activation. Results indicated the following: (i) the lipid fraction of oxLDL was primarily responsible for endothelial apoptosis. (ii) Endothelial apoptosis was potently inhibited by *NO donors and lipophilic phenolic antioxidants. OxLDL severely depleted Bcl-2 levels in endothelial cells and *NO donors restored Bcl-2 protein in oxLDL-treated cells. (iii) The pretreatment of a lipid fraction derived from oxLDL with sodium borohydride or potassium iodide completely abrogated apoptosis in endothelial cells, suggesting that lipid hydroperoxides induce apoptosis. (iv) Metalloporphyrins dramatically inhibited oxLDL-induced apoptosis in endothelial cells. Neither S-nitrosation of caspase-3 nor induction of Hsp70 appeared to play a significant role in the antiapoptotic mechanism of *NO in oxLDL-induced endothelial apoptosis. We propose that cellular lipid peroxyl radicals or lipid hydroperoxides induce an apoptotic signaling cascade in endothelial cells exposed to oxLDL, and that *NO inhibits apoptosis by scavenging cellular lipid peroxyl radicals.

Preliminary investigation of the biomechanics of internal fixation of sagittal split osteotomies with miniplates using a newly designed in vitro testing model.

PURPOSE: This article reports a preliminary study undertaken to investigate the biomechanics of internal fixation systems for sagittal split ramus osteotomy of the mandible with advancement, using a newly designed 3-point bovine rib testing model. MATERIALS AND METHODS: After 1 of 5 different miniplates internal fixation systems was placed, a vertical load was applied just below the superior border of sagittal split osteotomy gaps in bovine ribs positioned in a custom-made cradle using a compound cantilered bar device. Load/displacement data were gathered, and the mean elastic limits of the 5 miniplate designs were compared by using a 1-way analysis of variance (ANOVA) with Tukey multiple comparisons. RESULTS: The elastic limit of the rigid fixation system was higher when using a curved plate design than with a straight plate design. CONCLUSION: The 3-point bovine rib model used in this study is an inexpensive, discriminating, and reproducible method for testing internal fixation systems on sagittal ramus osteotomies under conditions that more accurately represent the human mandible in function.

The regulation of integrin function by Ca(2+).

Integrins are metalloproteins whose receptor function is dependent on the interplay between Mg(2+) and Ca(2+). Although the specificity of the putative divalent cation binding sites has been poorly understood, some issues are becoming clearer and this review will focus on the more recent information. The MIDAS motif is a unique Mg(2+)/Mn(2+) binding site located in the integrin alpha subunit I domain. Divalent cation bound at this site has a structural role in coordinating the binding of ligand to the I domain containing integrins. The I-like domain of the integrin beta subunit also has a MIDAS-like motif but much less is known about its cation binding preferences. The N-terminal region of the integrin alpha subunit has been modelled as a beta-propeller, containing three or four 'EF hand' type divalent cation binding motifs for which the function is ill defined. It seems certain that most integrins have a high affinity Ca(2+) site which is critical for alphabeta heterodimer formation, but the location of this site is unknown. Finally intracellular Ca(2+) fluxes activate the Ca(2+) requiring enzyme, calpain, which regulates cluster formation of leucocyte integrins.

The second domain of intercellular adhesion molecule-1 (ICAM-1) maintains the structural integrity of the leucocyte function-associated antigen-1 (LFA-1) ligand-binding site in the first domain.

The first domain of intercellular adhesion molecule-1 (ICAM-1) binds to the leucocyte function-associated antigen-1 (LFA-1) I domain, which contains the principal ligand-binding site of this leucocyte integrin. Whether the function of the second domain is also to directly bind LFA-1 has been unclear. Our data show that mutation in the hydrophilic EF loop of ICAM-1 domain 2 resulted in impaired binding of the isolated I domain when compared with wild-type ICAM-1. LFA-1 on T-cells also binds with reduced affinity to this ICAM-1 mutant. A hybrid construct containing the first domain of vascular cell-adhesion molecule-1 joined to domains 2-5 of ICAM-1 was unable to bind to the I domain, showing that there is no direct interaction between the second domain of ICAM-1 and the I domain. This construct was also not bound by LFA-1 expressed in T-cells. Function-blocking monoclonal antibodies that map to domain 2 of ICAM-1, implicating this domain in ligand binding, were found to act indirectly. In summary our data suggest that the second domain of ICAM-1 has a role in maintaining the structure of the LFA-1 ligand-binding site in the first domain of ICAM-1 but does not appear to have a direct role in ligand binding.

Epidemiology of maxillofacial injuries at trauma hospitals in Ontario, Canada, between 1992 and 1997.

BACKGROUND: The purpose of this study was to review the epidemiology of maxillofacial skeletal injuries in severely injured patients admitted to trauma hospitals in Ontario, Canada, with an Injury Severity Score > 12. METHODS: The Ontario Trauma Registry was accessed to examine the epidemiology of maxillofacial skeletal injuries in severely injured patients treated at 12 trauma hospitals in the province of Ontario, Canada, between 1992 and 1997. Data were collected prospectively, and a descriptive analysis was performed to determine the pattern of maxillofacial injuries, including patient age, sex distribution, etiology of injury, time of injury, and injury profile. RESULTS: There were 2,969 patients that met the inclusion criteria. The median age was 25 years, and men were injured at a 3:1 ratio over women. Most severely injured patients with maxillofacial fractures were injured as a result of motor vehicle collision (70%), with only 33% of the patients restrained with a seat-belt. The temporal distribution of injuries showed that most injuries occurred during evening hours, on weekends, and in the summer. The largest number of fractures was found in the maxilla and orbital bones. The Injury Severity Score of the patients in this study ranged from 13 to 75, with a median of 25. The injury most commonly associated with maxillofacial fractures was injury to the head and neck area. Of patients with injury to the head and neck, most had an altered level of consciousness or injuries to the skull, brain, or cranial vessels. CONCLUSION: Many severely injured patients have maxillofacial injuries. Long-term collection of epidemiologic data regarding maxillofacial fractures is important for the evaluation of existing preventative measures and useful in the development of new methods of injury prevention. Furthermore, insight into the epidemiology of facial fractures and concomitant injuries is an integral component in evaluating the quality of patient care, developing optimal treatment regimens, and making decisions regarding appropriate resource and manpower allocations.

A comparison of human S100A12 with MRP-14 (S100A9).

MRP-8 and -14 are two S100 proteins highly expressed as a complex by neutrophils, and to a lesser extent by monocytes and certain squamous epithelia. However, less is known about the close homologue S100A12. This S100 protein is expressed by neutrophils and here we show that it is also expressed by monocytes, but not lymphocytes. An absence of coimmunoprecipitation of MRP-14 and S100A12 indicates that S100A12 is not associated with the MRP proteins in vivo. When directly compared to MRP-14, S100A12 expression by squamous epithelia is more restricted. In esophagus and psoriatic skin, S100A12 is differentially regulated, like MRP-14, but the expression pattern of the two S100 proteins is quite different.