Establishment of AIDS Animal Model with SIVmac239 Infected Chinese Rhesus Monkey

In the present research, two Chinese rhesus monkeys were inoculated intravenously with 5000 TCID50 of SIVmac239. The changes in the numbers of CD4+ T lymphocyte in peripheral blood, plasma viral loads, proviral DNA and humoral antibodies against virus were periodically monitored during 121 days. At the early stage of infection, proviral DNA had been detected in PBMCs, and infectious SIVmac239 virus had been isolated from PBMCs. At the same period, the numbers of CD4+ T lymphocytes were significantly decreased, and maintained at low level during the 121-day period of infection. Plasma viral loads reached the peak at week 2 post-inoculation and kept at a steady state subsequently. Moreover, antibodies against viral proteins were detected from plasma. All the results showed that the two Chinese rhesus monkeys had been infected with SIVmac239 successfully. This animal model can be applied for further AIDS researches.

Direct electrochemistry of glucose oxidase and biosensing for glucose based on carbon nanotubes@SnO2-Au composite

Multiwalled carbon nanotubes@SnO2-Au (MWCNTs@SnO2-Au) composite was synthesized by a chemical route. The structure and composition of the MWCNTs@SnO2-Au composite were confirmed by means of transmission electron microscopy, X-ray photoelectron and Raman spectroscopy. Due to the good electrocatalytic property of MWCNTs@SnO2-Au composite, a glucose biosensor was constructed by absorbing glucose oxidase (GOD) on the hybrid material. A direct electron transfer process is observed at the MWCNTs@SnO2-Au/GOD-modified glassy carbon electrode. The glucose biosensor has a linear range from 4.0 to 24.0 mM, which is suitable for glucose determination by real samples. It should be worthwhile noting that, from 4.0 to 12.0 mM, the cathodic peak currents of the biosensor decrease linearly with increasing the glucose concentrations in human blood. Meanwhile, the resulting biosensor can also prevent the effects of interfering species.

Determination of ranitidine in urine by capillary electrophoresis-electrochemiluminescent detection

The fast analysis of ranitidine is of clinical importance in understanding its efficiency and a patient's treatment history. In this paper, a novel determination method for ranitidine based on capillary electrophoresis-electrochemiluminescence detection is described. The conditions affecting separation and detection were investigated in detail. End-column detection of ranitidine in 5 mM Ru(bpy)(3)(2+) solution at applied voltage of 1.20 V was performed. Favorable ECL intensity with higher column efficiency was achieved by electrokinetic injection for 10 s at 10 kV. The R.S.D. values of ECL intensity and migration time were 6.38 and 1.84% for 10(-4) M and 6.01 and 0.60% for 10(-5) M, respectively. A detection limit of 7 x 10(-8) M (S/N = 3) was achieved. The proposed method was applied satisfactorily to the determination of ranitidine in urine in 6 min.

Evaluation of bioeffects of a long-term administering ChangLe on rat by proton NMR method and biochemical examination

High resolution H-1 nuclear magnetic resonance ( NMR) spectroscopy has been employed to assess long-term toxicological effects of ChangLe (a kind of rare earth complex applied in agriculture). Male Wistar rats were administrated orally with ChangLe at doses of 0, 0.1, 0.2, 2.0, 10 and 20 mg/kg body weight daily, respectively, for 6 months. Urine was collected at-day 30, 60, go and serum samples were taken after 6 months. Many low-molecular weight metabolites were identified by H-1 NMR spectra of rat urine. A decrease in citrate and an increase in ketone bodies, creatinine, DMA, DMG, TMAO, and taurine in the urine of the rats. receiving high doses were found by H-1 NMR spectra. These may mean that high-dosage of ChangLe impairs the specific region of liver and kidney, such as renal tubule and mitochondria. The decrease in citrate and the increase in succinate and alpha-ketoglutarate were attributed to a combination of the inhibition of certain citric acid enzymes, renal tubular acidosis and the abnormal fatty acid catabolism. The information of the renal capillary necrosis could be derived from the increase in DMIA, DMG and TMAO. The increase in taurine was due to hepatic mitochondria dysfunction. The conclusions were supported by the results of biochemical measure. merits and enzymatic assay.

Quantitative Particle Characterization by Scattered Ultrasound

The topic of this thesis is an acoustic scattering technique for detennining the compressibility and density of individual particles. The particles, which have diameters on the order of 10 µm, are modeled as fluid spheres. Ultrasonic tone bursts of 2 µsec duration and 30 MHz center frequency scatter from individual particles as they traverse the focal region of two confocally positioned transducers. One transducer acts as a receiver while the other both transmits and receives acoustic signals. The resulting scattered bursts are detected at 90° and at 180° (backscattered). Using either the long wavelength (Rayleigh) or the weak scatterer (Born) approximations, it is possible to detennine the compressibility and density of the particle provided we possess a priori knowledge of the particle size and the host properties. The detected scattered signals are digitized and stored in computer memory. With this information we can compute the mean compressibility and density averaged over a population of particles ( typically 1000 particles) or display histograms of scattered amplitude statistics. An experiment was run first run to assess the feasibility of using polystyrene polymer microspheres to calibrate the instrument. A second study was performed on the buffy coat harvested from whole human blood. Finally, chinese hamster ovary cells which were subject to hyperthermia treatment were studied in order to see if the instrument could detect heat induced membrane blebbing.

HIV-1 envelope gp41 antibodies can originate from terminal ileum B cells that share cross-reactivity with commensal bacteria.

Monoclonal antibodies derived from blood plasma cells of acute HIV-1-infected individuals are predominantly targeted to the HIV Env gp41 and cross-reactive with commensal bacteria. To understand this phenomenon, we examined anti-HIV responses in ileum B cells using recombinant antibody technology and probed their relationship to commensal bacteria. The dominant ileum B cell response was to Env gp41. Remarkably, a majority (82%) of the ileum anti-gp41 antibodies cross-reacted with commensal bacteria, and of those, 43% showed non-HIV-1 antigen polyreactivity. Pyrosequencing revealed shared HIV-1 antibody clonal lineages between ileum and blood. Mutated immunoglobulin G antibodies cross-reactive with both Env gp41 and microbiota could also be isolated from the ileum of HIV-1 uninfected individuals. Thus, the gp41 commensal bacterial antigen cross-reactive antibodies originate in the intestine, and the gp41 Env response in HIV-1 infection can be derived from a preinfection memory B cell pool triggered by commensal bacteria that cross-react with Env.

A population model of folate-mediated one-carbon metabolism.

BACKGROUND: Previous mathematical models for hepatic and tissue one-carbon metabolism have been combined and extended to include a blood plasma compartment. We use this model to study how the concentrations of metabolites that can be measured in the plasma are related to their respective intracellular concentrations. METHODS: The model consists of a set of ordinary differential equations, one for each metabolite in each compartment, and kinetic equations for metabolism and for transport between compartments. The model was validated by comparison to a variety of experimental data such as the methionine load test and variation in folate intake. We further extended this model by introducing random and systematic variation in enzyme activity. OUTCOMES AND CONCLUSIONS: A database of 10,000 virtual individuals was generated, each with a quantitatively different one-carbon metabolism. Our population has distributions of folate and homocysteine in the plasma and tissues that are similar to those found in the NHANES data. The model reproduces many other sets of clinical data. We show that tissue and plasma folate is highly correlated, but liver and plasma folate much less so. Oxidative stress increases the plasma S-adenosylmethionine/S-adenosylhomocysteine (SAM/SAH) ratio. We show that many relationships among variables are nonlinear and in many cases we provide explanations. Sampling of subpopulations produces dramatically different apparent associations among variables. The model can be used to simulate populations with polymorphisms in genes for folate metabolism and variations in dietary input.

Effect of fluid dynamics and device mechanism on biofluid behaviour in microchannel systems: modelling biofluids in a microchannel biochip separator

Biofluid behaviour in microchannel systems is investigated in this paper through the modelling of a microfluidic biochip developed for the separation of blood plasma. Based on particular assumptions, the effects of some mechanical features of the microchannels on behaviour of the biofluid are explored. These include microchannel, constriction, bending channel, bifurcation as well as channel length ratio between the main and side channels. The key characteristics and effects of the microfluidic dynamics are discussed in terms of separation efficiency of the red blood cells with respect to the rest of the medium. The effects include the Fahraeus and Fahraeus-Lindqvist effects, the Zweifach-Fung bifurcation law, the cell-free layer phenomenon. The characteristics of the microfluid dynamics include the properties of the laminar flow as well as particle lateral or spinning trajectories. In this paper the fluid is modelled as a single-phase flow assuming either Newtonian or Non-Newtonian behaviours to investigate the effect of the viscosity on flow and separation efficiency. It is found that, for a flow rate controlled Newtonian flow system, viscosity and outlet pressure have little effect on velocity distribution. When the fluid is assumed to be Non-Newtonian more fluid is separated than observed in the Newtonian case, leading to reduction of the flow rate ratio between the main and side channels as well as the system pressure as a whole.

Capillary Electrophoresis of Erythrocytes.

Capillary electrophoresis (CE) of erythrocytes from different sources under various conditions is reported in this paper. It was found that erythrocyte samples from sheep, duck, and human showed characteristic and reproducible elution peaks, and that the retention times of A-, B-, AB-, and O-type erythrocytes from human blood were distinctively different; even subtle differences, among individuals with the same blood type could be detected by CE. A strictly linear correlation was obtained between the peak area and the amount of human erythrocyte over a range of 4.8×102–1.9×104 cells (r=0.999), indicating that CE could be used for rapid and accurate quantification of erythrocytes. Using this CE protocol, the decrease of the surface electrical charge of erythrocyte during storage was confirmed. Therefore, this work demonstrated that CE could be a useful alternative for characterizing and quantifying erythrocytes or other cells.

The prevalence of nonadherence in difficult asthma

Rationale: With the advent of new and expensive therapies for severe refractory asthma, targeting the appropriate patients is important. An important issue is identifying nonadherence with current therapies. The extent of nonadherence in a population with difficult asthma has not been previously reported.

Objectives: To examine the prevalence of nonadherence to corticosteroid medication in a population with difficult asthma referred to a Specialist Clinic and to examine the relationship of poor adherence to asthma outcome.

Methods: General practitioner prescription refill records for the previous 6 months for inhaled combination therapy and short-acting ß-agonists were compared with initial prescriptions and expressed as a percentage. Blood plasma prednisolone and cortisol assay levels were used to examine the utility of these measures in assessing adherence to oral prednisolone. Patient demographics, hospital admissions, lung function, oral prednisolone courses, and quality of life data were analyzed to indentify the variables associated with reduced medication adherence.

Measurements and Main Results: A total of 182 patients were assessed. Sixty-three patients (35%) filled 50% or fewer inhaled medication prescriptions; 88% admitted poor adherence with inhaled therapy after initial denial. Twenty-one percent of patients filled more than 100% of presciptions, and 45% of subjects filled between 51 and 100% of prescriptions. Twenty-three of 51 patients (45%) prescribed oral steroids were found to be nonadherent.

Conclusions: A significant proportion of patients with difficult-to-control asthma remained nonadherent to corticosteroid therapy. Objective surrogate and direct measures of adherence should be performed as part of a difficult asthma assessment and are important before prescibing expensive novel biological therapies.

Molecular analysis of endothelial progenitor cell (EPC) subtypes reveals two distinct cell populations with different identities

BACKGROUND: The term endothelial progenitor cells (EPCs) is currently used to refer to cell populations which are quite dissimilar in terms of biological properties. This study provides a detailed molecular fingerprint for two EPC subtypes: early EPCs (eEPCs) and outgrowth endothelial cells (OECs). METHODS: Human blood-derived eEPCs and OECs were characterised by using genome-wide transcriptional profiling, 2D protein electrophoresis, and electron microscopy. Comparative analysis at the transcript and protein level included monocytes and mature endothelial cells as reference cell types. RESULTS: Our data show that eEPCs and OECs have strikingly different gene expression signatures. Many highly expressed transcripts in eEPCs are haematopoietic specific (RUNX1, WAS, LYN) with links to immunity and inflammation (TLRs, CD14, HLAs), whereas many transcripts involved in vascular development and angiogenesis-related signalling pathways (Tie2, eNOS, Ephrins) are highly expressed in OECs. Comparative analysis with monocytes and mature endothelial cells clusters eEPCs with monocytes, while OECs segment with endothelial cells. Similarly, proteomic analysis revealed that 90% of spots identified by 2-D gel analysis are common between OECs and endothelial cells while eEPCs share 77% with monocytes. In line with the expression pattern of caveolins and cadherins identified by microarray analysis, ultrastructural evaluation highlighted the presence of caveolae and adherens junctions only in OECs. CONCLUSIONS: This study provides evidence that eEPCs are haematopoietic cells with a molecular phenotype linked to monocytes; whereas OECs exhibit commitment to the endothelial lineage. These findings indicate that OECs might be an attractive cell candidate for inducing therapeutic angiogenesis, while eEPC should be used with caution because of their monocytic nature.

Outgrowth Endothelial Cells: Characterization and Their Potential for Reversing Ischemic Retinopathy

Purpose. Endothelial progenitor cells (EPCs) have potential for promoting vascular repair and revascularization of ischemic retina. However, the highly heterogeneous nature of these cells causes confusion when assessing their biological functions. The purpose of this study was to provide a comprehensive comparison between the two main EPC subtypes, early EPCs (eEPCs) and outgrowth endothelial cells (OECs), and to establish the potential of OECs as a novel cell therapy for ischemic retinopathy.

Methods. Two types of human blood-derived EPCs were isolated and compared using immunophenotyping and multiple in vitro functional assays to assess interaction with retinal capillary endothelial cells and angiogenic activity. OECs were delivered intravitreally in a mouse model of ischemic retinopathy, and flat mounted retinas were examined using confocal microscopy.

Results. These data indicate that eEPCs are hematopoietic cells with minimal proliferative capacity that lack tube-forming capacity. By contrast, OECs are committed to an endothelial lineage and have significant proliferative and de novo tubulogenic potential. Furthermore, only OECs are able to closely interact with endothelial cells through adherens and tight junctions and to integrate into retinal vascular networks in vitro. The authors subsequently chose OECs to test a novel cell therapy approach for ischemic retinopathy. Using a murine model of retinal ischemia, they demonstrated that OECs directly incorporate into the resident vasculature, significantly decreasing avascular areas, concomitantly increasing normovascular areas, and preventing pathologic preretinal neovascularization.

Conclusions. As a distinct EPC population, OECs have potential as therapeutic cells to vascularize the ischemic retina.

Functional effect of the C242T polymorphism in the NAD(P)H oxidase p22phox gene on vascular superoxide production in atherosclerosis.

BACKGROUND:

Increased superoxide anion production increases oxidative stress and reduces nitric oxide bioactivity in vascular disease states. NAD(P)H oxidase is an important source of superoxide in human blood vessels, and some studies suggest a possible association between polymorphisms in the NAD(P)H oxidase CYBA gene and atherosclerosis; however, no functional data address this hypothesis. We examined the relationships between the CYBA C242T polymorphism and direct measurements of superoxide production in human blood vessels.

METHODS AND RESULTS:

Vascular NAD(P)H oxidase activity was determined in human saphenous veins obtained from 110 patients with coronary artery disease and identified risk factors. Immunoblotting, reverse-transcription polymerase chain reaction, and DNA sequencing showed that p22phox protein, mRNA, and 242C/T allelic variants are expressed in human blood vessels. Vascular superoxide production, both basal and NADH-stimulated, was highly variable between patients, but the presence of the CYBA 242T allele was associated with significantly reduced vascular NAD(P)H oxidase activity, independent of other clinical risk factors for atherosclerosis.

CONCLUSIONS:

Association of the CYBA 242T allele with reduced NAD(P)H oxidase activity in human blood vessels suggests that genetic variation in NAD(P)H oxidase components may play a significant role in modulating superoxide production in human atherosclerosis.

Endothelial Progenitors as Tools to Study Vascular Disease

Endothelial progenitor cells (EPCs) have great clinical value because they can be used as diagnostic biomarkers and as a cellular therapy for promoting vascular repair of ischaemic tissues. However, EPCs also have an additional research value in vascular disease modelling to interrogate human disease mechanisms. The term EPC is used to describe a diverse variety of cells, and we have identified a specific EPC subtype called outgrowth endothelial cell (OEC) as the best candidate for vascular disease modelling because of its high-proliferative potential and unambiguous endothelial commitment. OECs are isolated from human blood and can be exposed to pathologic conditions (forward approach) or be isolated from patients (reverse approach) in order to study vascular human disease. The use of OECs for modelling vascular disease will contribute greatly to improving our understanding of endothelial pathogenesis, which will potentially lead to the discovery of novel therapeutic strategies for vascular diseases.

Structure-activity relationships of FMRFamide-related peptides contracting Schistosoma mansoni muscle

This study reports the potent myoactivity of flatworm FMRFamide-related peptides (FaRPs) on isolated muscle fibers of the human blood fluke, Schistosoma mansoni. The turbellarian peptides YIRFamide (EC50 4 eta M), GYIRFamide (EC50 1 eta M). and RYIRFamide (EC50 7 eta M), all induced muscle contraction more potently than the cestode FaRP GNFFRFamide (EC50 500 eta M). Using a series of synthetic analogs of the flatworm peptides YIRFamide, GYIRFamide and RYIRFamide, the structure-activity relationships of the muscle FaRP receptor were examined. With a few exceptions, each residue in YIRFamide is important in the maintenance of its myoactivity. Alanine scans resulted in peptides that were inactive (Ala(1), Ala(2), Ala(3) and Ala(4) YIRFamide; Ala(4) and Ala(5) RYIRFamide) or had much reduced potencies (Ala(1), Ala(2) and Ala(3) RYIRFamide). Substitution of the N-terminal (Tyr(1)) residue of YIRFamide with the non-aromatic residues Thr or Arg produced analogs with greatly reduced potency. Replacement of the N-terminal Tyr with aromatic amino acids resulted in myoactive peptides (FIRFamide, EC50 100 eta M; WIRFamide, EC50 0.5 eta M). The activity of YIRFamide analogs which possessed a Leu(2), Phe(2) or Met(2) residue (EC50's 10, 1 and 3 eta M, respectively) instead of Ile(2) was not significantly altered, whereas, YVRFamide had a greatly reduced (EC50 200 eta M) activity. Replacement of the Phe(4) with a Tyr(4) (YIRYamide) also greatly lowered potency. Truncated analogs were either inactive (FRFamide, YRFamide, HRFamide, RFamide, Famide) or had very low potency (IRFamide and MRFamide), with the exception of nLRFamide (EC50 20 eta M). YIRF free acid was inactive. In summary, these data show the general structural requirements of this schistosome muscle FaRP receptor to be similar, but not identical, to those of previously characterized molluscan FaRP receptors. (C) 1997 Elsevier Science Inc.