Improved suppression of circulating complement does not block acute vascular rejection of pig-to-rhesus monkey cardiac transplants

At present, acute vascular rejection (AVR) remains a primary obstacle inhibiting long-term graft survival in the pig-to-non-human primate transplant model. The present study was undertaken to determine whether repetitive injection of low dose Yunnan-cobra venom factor (Y-CVF), a potent complement inhibitor derived from the venom of Naja kaouthia can completely abrogate hemolytic complement activity and subsequently improve the results in a pig-to-rhesus monkey heterotopic heart transplant model. Nine adult rhesus monkeys received a heterotopic heart transplant from wild-type pigs and the recipients were allocated into two groups: group 1 (n = 4) received repetitive injection of low dose Y-CVF until the end of the study and group 2 (n = 5) did not receive Y-CVF. All recipients were treated with cyclosporine A (CsA), cyclophosphamide (CyP) and steroids. Repetitive Y-CVF treatment led to very dramatic fall in CH50 and serum C3 levels (CH50 < 3 units/C3 remained undetectable throughout the experiment) and successfully prevented hyperacute rejection (HAR), while three of five animals in group 2 underwent HAR. However, the continuous suppression of circulating complement did not prevent AVR and the grafts in group 1 survived from 8 to 13 days. Despite undetectable C3 in circulating blood, C3 deposition was present in these grafts. The venular thrombosis was the predominant histopathologic feature of AVR. We conclude that repetitive injection of low dose Y-CVF can be used to continuously suppress circulating complement in a very potent manner and successfully prevent HAR. However, this therapy did not inhibit complement deposition in the graft and failed to prevent AVR. These data suggest that using alternative pig donors [i.e. human decay accelerating factor (hDAF)-transgenic] in combination with the systemic use of complement inhibitors may be necessary to further control complement activation and improve survival in pig-to-non-human primate xenotransplant model.

TMVA, a novel GPIb-binding protein, significantly prevents platelet microthrombi formation and prolongs discordant cardiac xenograft survival

In xenotransplantation, donor endothelium is the first target of immunological attack. Activation of the endothelial cell by preformed natural antibodies leads to platelet binding via the interaction of the glycoprotein (GP) Ib and von Willebrand factor (vWF). TMVA is a novel GPIb-binding protein purified from the venom of Trimeresurus mucrosquamatus. In this study, the inhibitory effect of TMVA on platelet aggregation in rats and the effect on discordant guinea pig-to-rat cardiac xenograft survival were investigated. Three doses (8, 20 or 40 mug/kg) of TMVA were infused intravenously to 30 rats respectively. Platelet aggregation rate was assayed 0.5, 12, and 24 h after TMVA administration. Wister rats underwent guinea pig cardiac cervical heterotopic transplantation using single dosing of TMVA (20 mug/kg, i.v., 0.5 h before reperfusion). Additionally, levels of TXB2 and 6-keto-PGF(1alpha) within rejected graft tissues were determined by radioimmunoassay. Treatment with TMVA at a dose of 20 or 40 mug/kg resulted in complete inhibition of platelet aggregation 0.5 h after TMVA administration. Rats receiving guinea pig cardiac xenografts after TMVA therapy had significantly prolonged xenograft survival. Histologic and immunopathologic analysis of cardiac xenografts in TMVA treatment group showed no intragraft platelet microthrombi formation and fibrin deposition. Additionally, the ratio of 6-keto-PGF(1alpha) to TXB2 in TMVA treatment group was significantly higher than those in control group. We conclude that the use of this novel GPIb-binding protein was very effective in preventing platelet microthrombi formation and fibrin deposition in a guinea pig-to-rat model and resulted in prolongation of xenograft survival. The increased ratio of PGI(2)/TXA(2) in TMVA treatment group may protect xenografts from the endothelial cell activation and contribute to the prolongation of xenograft survival.

Real-time block flow tracking of atrial septal defect motion in 4D cardiac ultrasound

Real-time cardiac ultrasound allows monitoring the heart motion during intracardiac beating heart procedures. Our application assists atrial septal defect (ASD) closure techniques using real-time 3D ultrasound guidance. One major image processing challenge is the processing of information at high frame rate. We present an optimized block flow technique, which combines the probability-based velocity computation for an entire block with template matching. We propose adapted similarity constraints both from frame to frame, to conserve energy, and globally, to minimize errors. We show tracking results on eight in-vivo 4D datasets acquired from porcine beating-heart procedures. Computing velocity at the block level with an optimized scheme, our technique tracks ASD motion at 41 frames/s. We analyze the errors of motion estimation and retrieve the cardiac cycle in ungated images. © 2007 IEEE.

Nonlinear analysis of cardiac rhythm fluctuations using DFA method

An approach by which the detrented fluctuation analysis (DFA) method can be used to help diagnose heart failure was demonstrated. DFA was applied to patients suffering from congestive heart failure (CHF) to check correlations between DFA indices and CHF, and determine a correlation between DFA indices and mortality, with a particular attention to the residue parameter, which is a measure of the departure of the DFA from its power law approximation. DFA parameters proved to be useful as a complement to the physiological parameters weber and FE to sort out the patients into three prognostic group.

Design and formulation of functional pluripotent stem cell-derived cardiac microtissues.

Access to robust and information-rich human cardiac tissue models would accelerate drug-based strategies for treating heart disease. Despite significant effort, the generation of high-fidelity adult-like human cardiac tissue analogs remains challenging. We used computational modeling of tissue contraction and assembly mechanics in conjunction with microfabricated constraints to guide the design of aligned and functional 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues that we term cardiac microwires (CMWs). Miniaturization of the platform circumvented the need for tissue vascularization and enabled higher-throughput image-based analysis of CMW drug responsiveness. CMW tissue properties could be tuned using electromechanical stimuli and cell composition. Specifically, controlling self-assembly of 3D tissues in aligned collagen, and pacing with point stimulation electrodes, were found to promote cardiac maturation-associated gene expression and in vivo-like electrical signal propagation. Furthermore, screening a range of hPSC-derived cardiac cell ratios identified that 75% NKX2 Homeobox 5 (NKX2-5)+ cardiomyocytes and 25% Cluster of Differentiation 90 OR (CD90)+ nonmyocytes optimized tissue remodeling dynamics and yielded enhanced structural and functional properties. Finally, we demonstrate the utility of the optimized platform in a tachycardic model of arrhythmogenesis, an aspect of cardiac electrophysiology not previously recapitulated in 3D in vitro hPSC-derived cardiac microtissue models. The design criteria identified with our CMW platform should accelerate the development of predictive in vitro assays of human heart tissue function.

The profound effects of microcystin on cardiac antioxidant enzymes, mitochondrial function and cardiac toxicity in rat

Deaths from microcystin toxication have widely been attributed to hypovolemic shock due to hepatic interstitial hemorrhage, while some recent studies suggest that cardiogenic complication is also involved. So far, information on cardiotoxic effects of MC has been rare and the underlying mechanism is still puzzling. The present study examined toxic effects of microcystins on heart muscle of rats intravenously injected with extracted MC at two doses, 0.16LD(50) (14 mu g MC-LReq kg(-1) body weight) and 1LD(50) (87 mu g MC-LReq kg(-1) body weight). In the dead rats, both TTC staining and maximum elevations of troponin I levels confirmed myocardial infarction after MC exposure, besides a serious interstitial hemorrhage in liver. In the 1LD(50) dose group, the coincident falls in heart rate and blood pressure were related to mitochondria dysfunction in heart, while increases in creatine kinase and troponin I levels indicated cardiac cell injury. The corresponding pathological alterations were mainly characterized as loss of adherence between cardiac myocytes and swollen or ruptured mitochondria at the ultrastructural level. MC administration at a dose of 1LD(50) not only enhanced activities and up-regulated mRNA transcription levels of antioxidant enzymes, but also increased GSH content. At both doses, level of lipid peroxides increased obviously, suggesting serious oxidative stress in mitochondria. Simultaneously. complex I and III were significantly inhibited, indicating blocks in electron flow along the mitochondrial respiratory chain in heart. In conclusion, the findings of this study implicate a role for MC-induced cardiotoxicity as a potential factor that should be considered when evaluating the mechanisms of death associated with microcystin intoxication in Brazil. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Identification of Phenylethanoid Glycosides in Plant Extract of Plantago asiatica L. by Liquid Chromatography-Electrospray Ionization Mass Spectrometry

The present work describes a liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method for rapid identification of phenylethanoid glycosides in plant extract from Plantago asiatica L. By using a binary mobile phase system consisting of 0.2% acetic acid and acetonitrile under gradient conditions, a good separation was achieved on a reversed-phase C-18 column. The [M-H](-) ions, the molecular weights, and the fragment ions of phenylethanoid glycosides were obtained in the negative ion mode using LC-ESI-MS. The identification of the phenylethanoid glycosides (peaks 1-3) in the extract of P. asiatica L. was based on matching their retention time, the detection of molecular ions, and the fragment ions obtained by collision-induced dissociation (CID) experiments with those of the authentic standards and data reported in the literature.

Analysis on Iridoid Glycosides in Crude and Processed Extracts from Cornus Officinals by Liquid Chromatography-electrospray Ionization Mass Spectrometry

The iridoid glycosides in crude and processed extracts from cornus officinals have been analyzed by high performance liquid chromatography-electrospray ionization mass spectrometry. Samples were analyzed by a reversed-phase C18 column using a binary eluent under gradient conditions. Seven iridoid glycosides could be separated and detected. The [M-H](-) ions of iridoid glycosides in the negative ion mode were observed, which reflect their molecule mass information. An in-source collision induced dissociation (in-source CID) experiment was carried out in order to identify the structures and to measure the contents of iridoid glycosides. The epimers were discovered in the experiment for the first time, namely 7 alpha-O-ethyl-morroniside and 7 beta-O-ethylmorroniside.

Structural analysis of monoterpene glycosides extracted from Paeonia lactiflora Pall. using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Paeoniflorin standard was first investigated by electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry (ESI-FTICR-MS/MS) using a sustained off-resonance irradiation (SORI) collision-induced dissociation (CID) method at high mass resolution. The experimental results demonstrated that the unambiguous elemental composition of product ions can be obtained at high mass resolution. Comparing MS/MS spectra and the experimental methods of hydrogen and deuterium exchange, the logical fragmentation pathways of paeoniflorin have been proposed. Then, the extracts of the traditional Chinese medicine Paeonia lactiflora Pall. were analyzed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). By comparison with the ESI-FTICR-MS/MS data of paeoniflorin, the isomers paeoniflorin and albiflorin in Paeonia lactiflora Pall. have been identified using HPLC/MS with CID in an ion trap and in-source CID. Furthermore, using the characteristic fragmentation pathways, the retention times (t(R)) in HPLC and MS/MS spectra, the structures of three other kinds of monoterpene glycoside compounds have been identified on-line without time-consuming isolation.

Investigation of heptakis(2,6-di-O-methyl)-beta-cyclodextrin inclusion complexes with flavonoid glycosides by electrospray ionization mass spectrometry

The non-covalent complexes between three flavonoid glycosides (quercitrin, hyperoside and rutin) and heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD) were investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). The 1:1 complexation of each flavonoid glycoside (guest) to the DM-beta-CD (host) was monitored in the negative ion mode by mixing each guest with an up to 30-fold molar excess of the host. The binding constants for all complexes were calculated by a linear equation in the order: DM-beta-CD:quercitrin > DM-beta-CD:rutin > DM-beta-CD:hyperoside. A binding model for the complexes has also been proposed based on the binding constants and tandem mass spectrometric data of these complexes.

Separation and determination of flavone and xanthone glycosides in Tibetan folk medicinal species Swertia mussotii and S-franchetiana by capillary electrophoresis

The contents of five pharmacologically active flavone and xanthone glycosides, namely, swertianolin, swertisin, isoorientin, mangiferin, and 7-O-[alpha-L-rhamnopyranosyl-(1 -> 2)-beta-D-xylopyranosyl]-1,8-dihydroxy-3-methoxyxanthone, extracted from Tibetan folk medicinal species Swertia mussotii and S. franchetiana were determined by capillary electrophoresis with diode-array detection. The separation of five components has been optimized with a capillary column with a total length of 48.5 cm and effective length of 40 cm (50 mu m i.d). The influence of the running buffer, the sodium dodecyl sulfonate (SDS) concentration, organic modifier, etc. on the resolution was evaluated. The background electrolyte contained 30 mM borate buffer, 28 mM SDS, 1.0% (v/v) acetonitrile, and was adjusted to pH 9.0 with 0.1 M NaOH. A good baseline resolution was obtained for the separation of five components within 5 min with the working voltage of 24 kV and a column temperature of 25 degrees C. The established method was rapid and reproducible for the separation and determination of five flavone and xanthone glycosides from the extracts of S. mussotii and franchetiana plant samples.

The glycosides from Lomatogonium rotatum

A new phenyl glycoside, 2-(3'-O-beta-D-glucopyranosyl) benzoyloxygentisic acid (1), along with seven known glycosides 2-8 was isolated from Tibetan herbal medicine Lomatogonium rotatum. The structures of the compounds were elucidated by spectroscopic methods including 1D and 2D NMR techniques and MS data.

Two new benzochromone glycosides from the stem of Berchemia racemosa

Two new benzochromone glycosides, rubrofusarin 6-O-alpha-L-rhamnosyl- (1 -> 6)-O-beta-D-glucopyranoside (1) and demethylflavasperone 10-O-beta-D-glucopyranoside (2), have been isolated from the stem of Berchemia racemosa Sieb. et Zucc. (Rhamnaceae). Their structures were elucidated on the basis of spectroscopic evidence.