Protection of red blood cell acetylcholinesterase by oral huperzine A against ex vivo soman exposure: Next generation prophylaxis and sequestering of acetylcholinesterase over butyrylcholinesterase

As part of a phase Ib clinical trial to determine the tolerability and safety of the highly specific acetylcholinesterase (AChE) inhibitor huperzine A, twelve (12) healthy elderly individuals received an escalating dose regimen of huperzine A (100, 200, 300, and 400 μg doses, twice daily for a week at each dose), with three (3) individuals as controls receiving a placebo. Using the WRAIR whole blood cholinesterase assay, red blood cell AChE and plasma butyrylcholinesterase (BChE) were measured in unprocessed whole blood samples from the volunteers following each dose, and then for up to 48 h following the final and highest (400 μg) dose to monitor the profile of inhibition and recovery of AChE. Significant inhibition of AChE was observed, ranging from 30–40% after 100 μg to >50% at 400 μg, and peaking 1.5 h after the last dose. Gradual recovery of AChE activity then occurs, but even 48 h after the last dose red blood cell AChE was about 10% below control (pre-dose) values. Huperzine A levels in plasma peaked 1.5 h after the final 400 μg dose (5.47 ± 2.15 ng/mL). Plasma BChE was unaffected by huperzine A treatment (as expected).Aliquots of huperzine A-containing (from three individuals) and placebo blood samples were exposed ex vivo to the irreversible nerve agent soman (GD) for 10 min, followed by removal of unbound huperzine and soman from the blood by passing through a small C₁₈ reverse phase spin column. Eluted blood was diluted in buffer, and aliquots taken at various time intervals for AChE and BChE activity measurement to determine the time taken to achieve full return in activity of the free enzyme (dissociation from the active site of AChE by huperzine A), and thus the proportion of AChE that can be protected from soman exposure. Huperzine A-inhibited red blood cell (RBC) AChE activity was restored almost to the level that was initially inhibited by the drug. The increased doses of huperzine A used were well tolerated by these patients and in this ex vivo study sequestered more red blood cell AChE than has been previously demonstrated for pyridostigmine bromide (PB), indicating the potential improved prophylaxis against organophosphate (OP) poisoning.     

Mechanism of red blood cell acanthocytosis and echinocytosisin vivo

Summary Patients with abetalipoproteinemia have an inborn absence of the major apoprotein of low density plasma lipoproteins, an abnormal serum and red cell lipid profile, and spiny erythrocytes, called acanthocytes. We now show that these deformed cells are reversibly converted to a normal shape, that of a biconcave disk, by incubation with 3 to 10×10^–5 m chlorpromazine. We suppose that chlorpromazine acts by expanding the cytoplasmic leaflet of the bilayer, thus promoting inward curvature. Ghosts isolated from the acanthocytes are themselves spiny but are also converted to normal, convave disks by chlorpromazine or merely by a brief incubation at 37°C in low inoic strength buffer. We attribute the latter to a redistribution of lipids between the two leaflets of the membrane bilayer. Similar observations were made with red cells and ghosts from apatient with benign echinocytosis. These observations suggest that the morphological abnormality in acanthocytes and echinocytes can be ascribed to the same mechanism as crenationin vitro; that is, a bilayer couple effect in which an excess of surface area in the outer leaflet over the inner leaflet of the membrane bilayer drives outward curvature. It is striking that cells which were chronicallyabnormal in shapein vivo contain the information to become biconcave disks immediately upon simple chemical treatmentin vitro.     

Laser intensity and wavelength dependence of Rose-Bengal-photosensitized inhibition of red blood cell acetylcholinesterase

The intensity and wavelength-dependence of Rose-Bengal-mediated photoinhibition of red blood cell acetylcholinesterase has been studied. Irradiation of dye-membrane suspensions with 308 nm laser excitation resulted in enzyme inhibition almost 50% greater than that obtained with 514 nm laser excitation. Sodium azide and argon purging greatly decreased the photosensitized enzyme inhibition at both wavelengths. Although Rose Bengal photosensitized enzyme inhibition more efficiently upon excitation into Sn (308 nm) than into S1 (514 nm), Stern-Volmer analysis of sodium azide quenching data gave similar quenching efficiencies at both wavelengths. Irradiation of dye-membrane suspensions with increasing intensities (Nd:YAG, 532 nm, 40 ps pulse duration) resulted in a decrease in enzyme inhibition. Saturation of the Rose Bengal fluorescence intensity and light transmission occurred with nearly the same intensity-dependence, suggesting that ground-state depletion occurs at the higher intensities. Our results demonstrate that excitation of a sensitizer into higher-lying excited singlet states can result in enhanced sensitizing efficiency. However, attempts to populate such states in Rose Bengal by sequential two-photon absorption using high intensities resulted only in ground-state depletion.     

Mechanism of red blood cell acanthocytosis and echinocytosis in vivo

Patients with abetalipoproteinemia have an inborn absence of the major apoprotein of low density plasma lipoproteins, an abnormal serum and red cell lipid profile, and spiny erythrocytes, called acanthocytes. We now show that these deformed cells are reversibly converted to a normal shape, that of a biconcave disk, by incubation with 3 to 10 X 10(-5) M chlorpromazine. We suppose that chlorpromazine acts by expanding the cytoplasmic leaflet of the bilayer, thus promoting inward curvature. Ghosts isolated from the acanthocytes are themselves spiny but are also converted to normal, concave disks by chlorpromazine or merely by a brief incubation at 37 degrees C in low ionic strength buffer. We attribute the latter to a redistribution of lipids between the two leaflets of the membrane bilayer. Similar observations were made with red cells and ghosts from a patient with benign echinocytosis. These observations suggest that the morphological abnormality in acanthocytes and echinocytes can be ascribed to the same mechanism as crenation in vitro; that is, a bilayer couple effect in which an excess of surface area in the outer leaflet over the inner leaflet of the membrane bilayer drives outward curvature. It is striking that cells which were chronically abnormal in shape in vivo contain the information to become biconcave disks immediately upon simple chemical treatment in vitro.     

Mouse acetylcholinesterase unliganded and in complex with huperzine A: A comparison of molecular dynamics simulations

A 1 ns molecular dynamics simulation of unliganded mouse acetylcholinesterase (AChE) is compared to a previous simulation of mouse AChE complexed with huperzine A (HupA). Several common features are observed. In both simulations, the active site gorge fluctuates in size during the 1 ns trajectory and is completely pinched off several times. Many of the residues in the gorge that formed hydrogen bonds with HupA in the simulation of the complex now form hydrogen bonds with other protein residues and water molecules in the gorge. The opening of a “backdoor” entrance to the active site that was found in the simulation of the complex is also observed in the unliganded simulation. Differences between the two simulations include overall lower structural rms deviations for residues in the gorge in the unliganded simulation, a smaller diameter of the gorge in the absence of HupA, and the disappearance of a side channel that was frequently present in the liganded simulation. The differences between the two simulations can be attributed, in part, to the interaction of AChE with HupA. © 1999 John Wiley & Sons, Inc. Biopoly 50: 35–43, 1999     

Homocysteine induces procoagulant activity of red blood cells via phosphatidylserine exposure and microparticles generation

Increased homocysteine (Hcy) levels in plasma correlate with the risk of thromboic events. Red blood cells (RBCs), the most abundant blood cells in circulation, also play an active role in the process of thrombus formation. However, the effect of Hcy on procoagulant activity (PCA) of RBCs is unclear. In the present study, RBCs from healthy adults were treated with Hcy (8, 20, 80, 200, 800 μmol/L) for 24 h. Phosphatidylserine (PS) exposure of RBCs and red blood cell-derived microparticles (RMPs) release were detected using Alexa Fluor 488-lactadherin. PCA was assessed by coagulation time and purified clotting complexes testes. We found that Hcy treatment dose dependently enhanced PS exposure and consequent PCA of RBCs. Hcy also elevated the formation of procoagulant RMPs, with statistical significance at 800 μmol/L of Hcy. Moreover, 128 nmol/L lactadherin inhibited about 90 % PCA of RBCs and RMPs. Our data suggest that PS exposure and RMPs shedding are key sources for Hcy-induced PCA of RBCs. Lactadherin could be used to modulate the anticoagulant and procoagulant balance in this process.     

A novel monoclonal antibody of human stem cell factor inhibits umbilical cord blood stem cell ex vivo expansion

Autologous stem cell transplantation (ASCT) is the gold standard therapy for suitable multiple myeloma (MM) patients after induction with high dose therapy. To date, the evidence of a reliable marker of prognosis in these cases remains scarce. Our aim was to evaluate appearance of unrelated atypical serum immunofixation patterns (ASIPs) as a marker of prognosis in MM patients submitted to ASCT. We retrospectively analysed data from 65 patients. Interestingly, we observed that presence of ASIPs was associated with longer progression-free survival and longer overall survival. Our results suggested that presence of ASIPs could be a novel marker of good prognosis in MM patients submitted to ASCT.     

Detection of oral squamous cell carcinoma with ex vivo fluorescence confocal microscopy: Sensitivity and specificity compared to histopathology

Real‐time microscopic imaging of freshly excised tissue enables a rapid bedside‐pathology. A possible application of interest is the detection of oral squamous cell carcinomas (OSCCs). The aim of this study was to analyze the sensitivity and specificity of ex vivo fluorescence confocal microscopy (FCM) for OSCCs and to compare confocal images visually and qualitatively with gold standard histopathology. Two hundred eighty ex vivo FCM images were prospectively collected and evaluated immediately after excision. Every confocal image was blindly assessed for the presence or absence of malignancy by two clinicians and one pathologist. The results were compared with conventional histopathology with hematoxylin and eosin staining. OSCCs were detected with a very high sensitivity of 0.991, specificity of 0.9527, positive predictive value of 0.9322 and negative predictive value of 0.9938. The results demonstrate the potential of ex vivo FCM in fresh tissue for rapid real‐time surgical pathology.     

Polyphenolics enhance red blood cell resistance to oxidative stress: in vitro and in vivo

In this study we investigated the potential antioxidant properties of blueberry polyphenolics in vitro and vivo, using red blood cell (RBC) resistance to reactive oxygen species (ROS) as the model. In vitro incubation with anthocyanins or hydroxycinnamic acids (HCA) (0.5 and 0.05 mg/ml) was found to enhance significantly RBC resistance to H2O2 (100 microM) induced ROS production. This protection was also observed in vivo following oral supplementation to rats at 100 mg/ml. However, only anthocyanins were found to afford protection at a significant level, this at 6 and 24 h post supplementation. This protection was not consistent with the measured plasma levels of anthocyanins. Indeed, plasma polyphenolic concentrations were highest after 1 h, declining considerably after 6 h and not detected after 24 h. The difference in absorption between anthocyanins and HCA is likely to have contributed to the observed difference in their abilities to afford protection to RBC. This protection represents a positive role following dietary consumption of polyphenolics from blueberries, against ROS formation within RBC in vivo.     

Effects of nitric oxide and prostacyclin on deformability and aggregability of red blood cells of rats ex vivo and in vitro.

Although many diseases of the heart and circulatory system have been linked with insufficient deformability and increased aggregability of red blood cells, there are only a few drugs which can modulate these biological functions of erythrocytes. Here, we show evidences that iloprost, stable prostacyclin analogue and SIN-1, active metabolite of molsidomine which spontaneously releases NO, may be sufficient pharmacological tools for modulating red blood cell deformability and aggregability. Deformability of red blood cells was measured by shear stress laser diffractometer (Rheodyn SSD) and expressed in percent of red blood cell deformability index (DI). MA-1 (Myrenne) erythrocyte aggregometer was used for photometric measurements of aggregability in arbitrary units (MEA) of mean extent of aggregation. Experiments were carried out on rats ex vivo and in vitro using whole rat blood or isolated erythrocytes. Ex vivo SIN-1 (infusion 2 mg/kg/min i.v.) and iloprost (bolus injection 10 microg/kg i.v.) significantly improved erythrocyte deformability and aggregability at 5-15 min after administration. L-NAME (10 mg/kg i.v.)- inhibitor of nitric oxide synthase, and aspirin (1 mg/kg i.v.) caused worsening of deformability of erythrocytes in experiments ex vivo. Studies in vitro also revealed improvement of red blood cell deformability and aggregability by SIN-1 (3 microM, 15 min incubation at 22 degrees C) or iloprost (1 microM, 15 min incubation at 22 degrees C) and this phenomenon appeared not only in whole blood but also in isolated red cells. It is concluded that NO- and prostacyclin-induced improvement of red blood cell deformability and aggregability results from direct action of these compounds on erythrocytes. NO-donors and iloprost could be useful in the treatment of disorders of blood fluidity.     

Granzyme B-induced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs

Granzyme B (gzmB) of cytotoxic T lymphocytes (CTL) is essential for recovery from intracellular pathogens, but the molecular basis of its action is still unresolved. Here, we analyzed gzmB-mediated death pathways under physiological conditions using ex vivo virus-immune CTLs that express perf and gzmB, but not gzmA (gzmB(+)CTL). We show that gzmB(+)CTL abrogate target cell proliferation most likely by inducing cell death, independent of caspases and mitochondrial signaling. In addition, the data reveal that gzmB(+)CTL independently induce pro-apoptotic processes either via caspase-3/-7, leading to plasma membrane perturbance and ROS production or via Bid/Bak/Bax, resulting in cytochrome c release and that both pathways elicit loss of DeltaPsi(m). Our data provide evidence for a pleiotropic pro-apoptotic function of gzmB presumably to counteract evasion strategies of pathogens and to control tumors.     

Proton HR-MAS MR spectroscopy of oral squamous cell carcinoma tissues: an ex vivo study to identify malignancy induced metabolic fingerprints

Oral squamous cell carcinoma (SCC) represents more than 90% of all head and neck cancers as reported by Hermans (Cancer Imaging, 5(Spec No A), S52–S57, 2005), which draws attention of investigative research for novel predictive metabolic biomarkers to understand the malignancy induced biochemical perturbations occurring at molecular level. In the present work, proton HR-MAS NMR spectroscopic studies have been performed on resected human oral SCC tumor tissues, its neighboring margins and bed tissues (n = 159), obtained from 36 patients (n = 27 training set; n = 9 unknown test set), for the identification of metabolic fingerprints. The proton NMR spectra were then subjected to chemometric unsupervised PCA and supervised OSC-filtered PCA and PLS-DA multivariate analysis. Application of PLS-DA on orthogonally signal corrected training data-set (n = 120 tissue specimens; 27 patients) allowed >95% correct classification of malignant tissues from benign samples with >98% specificity and sensitivity. The OSC-PLS-DA model thus constructed was used to predict the class membership of unknown tissue specimens (n = 39) obtained from nine patients. These tissue samples were correctly predicted in its respective histological categories with 97.4% diagnostic accuracy. The regression coefficients obtained from OSC-filtered PLS-DA model indicated that malignant tissues had higher levels of glutamate, choline, phosphocholine, lactate, acetate, taurine, glycine, leucine, lysine, isoleucine and alanine, and lower levels of creatine and PUFA, representing altered metabolic processes (lipidogenesis, protein synthesis, and volume regulation) during tumor progression. Thus proton HR-MAS MR spectroscopy could efficiently identify the metabolic perturbations of malignant tumor from non-malignant bed and margins tissue specimens, which may be helpful in understanding the extent of tumor penetration in neighboring tissues.     

Discrimination between red blood cell and platelet components of blood clots by MR microscopy

Magnetic resonance imaging (MRI) of pulmonary emboli obtained ex vivo, verified by immunohistochemistry, showed that platelet layers display brighter signal intensity than areas containing predominantly red blood cells (RBC) in T (1)-weighted MRI. These results were surprising since platelets do not contain paramagnetic haemoglobin that would enhance magnetic relaxation. Our assumption was that the fibrin meshwork areas with entrapped RBC retain abundant extracellular space filled with serum, whereas platelets regroup into tight aggregates lacking serum, essentially mimicking solid tissue structure, rich with cellular proteins that enhance T (1)-relaxation. Our hypothesis was examined by MRI and NMR relaxometry of in vitro RBC suspensions and sedimented platelets, as well as by MRI of model clots and pulmonary emboli obtained ex vivo. Pure sedimented platelets exhibited shorter proton spin lattice relaxation times (T (1) = 874 +/- 310 ms) than those of venous blood of a healthy male with 40% haematocrit (T (1) = 1277 +/- 66 ms). T (1)-values of RBC samples containing high haematocrit (>/=80%) resembled T (1) of platelet samples. In T (1)-weighted spin-echo MRI echo time and repetition time (TE/TR = 10/120 ms) the ratio of signal intensities between a non-retracted whole blood clot (with a haematocrit of 35%) and a pure platelet clot was 3.0, and the ratio between a retracted whole blood clot with an estimated haematocrit of about 58% and a pure platelet clot was 2.6. We conclude that T (1)-weighted MRI can discriminate between platelet layers of thrombi and RBC-rich areas of thrombi that are not compacted to a haematocrit level of >/=80%.     

Uptake of lactate by the liver: effect of red blood cell carriage

Multiple-indicator dilution experiments with labeled lactate were performed in the livers of anesthetized dogs. A mixture of (51)Cr-labeled erythrocytes, [(3)H]sucrose, and L-[1-(14)C]lactate or a mixture of (51)Cr-labeled erythrocytes, [(14)C]sucrose, and L-[2-(3)H]lactate was injected into the portal vein, and samples were obtained from the hepatic vein. Data were evaluated using a model comprising flow along sinusoids, exchange of lactate between plasma and erythrocytes and between plasma and hepatocytes, and, in the case of L-[1-(14)C]lactate, metabolism to H[(14)C]O(-)(3) within hepatocytes. The coefficient for lactate efflux from erythrocytes was 0.62 +/- 0.24 s(-1), and those for influx into and efflux from hepatocytes were 0.44 +/- 0.13 and 0.14 +/- 0.07 s(-1), respectively. The influx permeability-surface area product of the hepatocyte membrane for lactate (P(in)S, in ml x s(-1) x g(-1)) varied with total flow rate (F, in ml s(-1) x g(-1)) according to P(in)S = (3.1 +/- 0.5)F + (0.021 +/- 0.014). Lactate in plasma, erythrocytes, and hepatocytes was close to equilibrium, whereas lactate metabolism was rate limiting.     

Evaluation of copper toxicity in isolated human peripheral blood mononuclear cells and it's attenuation by zinc: ex vivo

Copper and zinc act as a cofactor of over 300 mammalian proteins. Both have same electronic configuration therefore they are antagonist at higher individual concentration. The present study was designed with the aim to investigate the mechanisms pertaining to toxic effects of copper on human peripheral blood mononuclear cells (PBMCs) and to evaluate the cytoprotective effect of zinc on copper-induced cytotoxicity. The copper uptake into PBMCs was progressively increased with increasing concentration of metal in the growth medium. However, no significant effect on copper uptake was observed in the presence of zinc. Cell proliferation rate was decreased with increasing copper concentration. Interestingly, the proliferation rate of zinc treated PBMCs remained nearly the same as that of control cells. LD₅₀ of copper (115 μM) was increased six times (710 μM) in presence of zinc for PBMCs. At higher concentrations of copper (> 100 μM) decrease level of GSH was noticed. Increased levels of metallothionein in PBMCs were observed in response to zinc. DNA fragmentation studies also showed that copper produced DNA fragmentation at LD₅₀ (115 μM). Subsequently, zinc showed protection against DNA fragmentation caused by copper. Cell structure of PBMCs at LD₅₀ (115 μM copper) showed membrane bound cystic spaces and mitochondria having disrupted cristae and few myelin figures. In presence of zinc at LD₅₀ of copper (115 μM) cells showed improvement in mitochondrial structure and membrane bound cystic spaces. Taken together, the results of our study demonstrates that zinc play an important role in prevention of copper toxicity in peripheral blood mononuclear cells.