Blood Plasma Pseudocholinesterase as a Potential Biomarker of Autoimmune Diseases

We studied the blood plasma butyrylcholinesterase (BChE, pseudocholinesterase) activity as a potential marker for differentiating such autoimmune pathologies as type 1 diabetes mellitus (DM1), autoimmune thyroiditis (AIT) and rheumatoid arthritis (RA) from diseases having similar symptoms but different etiology. The blood plasma pseudocholinesterase activity was analyzed for patients with different types of diabetes mellitus. We showed its fivefold increase in DM1 compared to the control group, while in the second type of diabetes, which is metabolic pathology, it decreases two times. It was demonstrated that the activity of blood plasma BChE in patients with AIT and RA also increased fivefold compared to the group of patients without autoimmune disorders. Thus, the butyrylcholinesterase activity increases in diseases of an autoimmune nature, whereas in metabolic pathology it has the tendency to decrease; it indicates the adequacy of the selected criterion for the further development of a screening test-system that would allow differentiation of autoimmune diseases from the diseases of another origin, but with similar symptoms.     

Structure‐Based Design and Synthesis of Fluorene Derivatives as Novel RORγt Inverse Agonists

A new series of fluorene derivatives was designed and synthesized as novel retinoic acid receptor‐related orphan receptor gamma t (RORγt) inverse agonists utilizing a molecular hybridization approach. The new compounds 10 – 15 were evaluated for their RORγt activity using biochemical FRET and cellular reporter gene assays. Moreover, the inhibitory activity of the fluorene derivatives 10 – 15 in mouse Th17 cell differentiation assay was assessed. The hybrid compound 15 that combines both fluorene and arylsulfone moieties displayed promising RORγt activity with IC₅₀ values of 68.6 and 99.5 nm in FRET and cellular assays, respectively. In addition, molecular modeling studies were employed to investigate potential binding mode of 15 to RORγt. These results render 15 a potential lead compound for development of therapeutics for Th17‐driven autoimmune diseases.     

Flow-cytometric analysis of chicken red blood cells.

Flow-cytometric analysis of acriflavin-Feulgen stained chicken erythrocytes shows a complex distribution of amounts of deoxyribonucleic acid fluorescence, the profile consisting of a main peak and a right hand shoulder. This bimodal distribution, an artifact characteristically seen on analysis of flattened cells using orthogonal flow systems, results from fluorescence emission in preferred directions stemming from the combined effects of refractility and orientation of the cells. The shoulder disappears on analysis of lysed erythrocyte ghosts, also on analysis of cells in a medium whose refractive index approximates that the cells. An orientation effect for matrue erythrocytes was indicated by reanalysis of fractions after sorting on the basis of high and low fluorescence or scatter signals. Both fractions gave the original range of values on reanalysis, although some changes in shape of the profile and in the peak positions for the sorted cells were seen. Sodium dodecyl sulfate treatment of stained cells "loosened" the cells' structure, yielding lowered scatter values, and fluorescence values approaching those of the shoulder. The average fluorescence emission of the erythrocytes was lower than that of reticulocytes and lymphocytes. The values of the latter correspond closely, although coincidently, to that the erythrocyte shoulder values. Dual parameter analysis of forward light scatter, and fluorescence, which was detected at 90 degrees to the laser beam, showed the low fluorescence to be accompanied by low scatter signal, and the high fluorescence among the cells with the high scatter signal. The lowered forward scatter signal is due to a wider scattering of light from cells oriented edge-on to the detector, and loss of signal beyond the acceptance angle of the detector. These results suggest that the preferred directions for fluorescence are in the plane of the cells, and the values are dependent on the cells' orientation in the stream. These interpretations were supported by the results of analysis of partially oriented cells. The approaches used and conclusions arrived at are similar to those of Gledhill et al (16), Van Dilla et al (37), in their analysis of fluorescence of flat sperm cells although the affects in the case of the erythrocytes are less extreme.     

X-ray scatter data for flat-panel detector CT

In modern X-ray computed tomography (CT) a trend to increased volume coverage by using multi-row detectors is apparent. Flat-panel detector CT (FPD-CT) systems provide an even larger field of measurement which, however, results in an increased scatter fraction. We investigated the scatter intensities registered in the case of FPD-CT. A hybrid model for the simulation of scatter combining deterministic and Monte Carlo methods was used for the scatter calculations. The influence of imaging parameters on the registered scatter intensity was examined both in single projections and reconstructed images. Scatter-to-primary ratios (SPRs) are given for various values of object thickness, field size, object-to-detector distance, incident energy and projection angle. For the simulations, homogeneous water phantoms and realistic patient data sets were used to produce scatter data representative for clinical situations. The SPR increases with object size, collimation and z-extent resulting in SPR ≫ 1 and respective scatter artifacts in the reconstructed images. In contrary, the scatter intensity decreases non-linearly with the object-to-detector distance. The angular and spatial distributions of scatter form a flat function as compared to the distribution of the primary signal. Single scatter appears to determine the distribution and magnitude of the total-scatter intensity at the detector.     

Discovery and pharmacological evaluation of indole derivatives as potent and selective RORγt inverse agonist for multiple autoimmune conditions

Targeting nuclear receptor RORγ is recognized to be beneficial in multiple autoimmune disorders. We disclosed new indole analogues as potent RORγ inverse agonists. RO-2 as one of the potent and orally bioavailable compounds was evaluated in various models of autoimmune disorder. It showed potent suppression of downstream markers of RORγt activity in murine and human primary cells, ex vivo PD assay and in multiple animal models of autoimmune diseases. The results indicate the potential of these indole analogues as orally bioavailable small molecule inverse agonists of RORγt, efficacious in various Th17 driven models of autoimmune disorders.     

PGE2-induced apoptotic cell death in K562 human leukaemia cells.

Prostaglandin-E2 (PGE2) is known to trigger suicidal death of nucleated cells (apoptosis) and enucleated erythrocytes (eryptosis). In erythrocytes PGE2 induced suicidal cell death involves activation of nonselective cation channels leading to Ca2+ entry followed by cell shrinkage and triggering of Ca2+ sensitive cell membrane scrambling with phosphatidylserine (PS) exposure at the cell surface. The present study was performed to explore whether PGE2 induces apoptosis of nucleated cells similarly through cation channel activation and to possibly disclose the molecular identity of the cation channels involved. To this end, Ca2+ activity was estimated from Fluo3 fluorescence, mitochondrial potential from DePsipher fluorescence, phosphatidylserine exposure from annexin binding, caspase activation from caspAce fluorescence, cell volume from FACS forward scatter, and DNA fragmentation utilizing a photometric enzyme immunoassay. Stimulation of K562 human leukaemia cells with PGE2 (50 microM) increased cytosolic Ca2+ activity, decreased forward scatter, depolarized the mitochondrial potential, increased annexin binding, led to caspase activation and resulted in DNA fragmentation. Gene silencing of the Ca2+-permeable transient receptor potential cation channel TRPC7 significantly blunted PGE2-induced triggering of PS exposure and DNA fragmentation. In conclusion, K562 cells express Ca2+-permeable TRPC7 channels, which are activated by PGE2 and participate in the triggering of apoptosis.     

Enlargement of Lyt-2-positive T cells is associated with functional impairment and autoimmune hemolytic anemia in New Zealand Black mice

We investigated the relationship between the increased cell diameter of Lyt-2+ T cells and the development of autoimmune disease in aging NZB and NZB X NZW F1 hybrid (BW) mice. Individual animals were analyzed for Lyt-2+ T cell size (by narrow-angle forward light scatter), anti-erythrocyte autoantibodies, anemia, proteinuria, and splenomegaly. The peak light scatter of the Lyt-2+ T cells correlated with the level of anti-erythrocyte autoantibodies and severity of hemolytic anemia, but not with proteinuria or splenomegaly. The cell size of this T cell subset did not increase in old BW or in NZB mice homozygous for the xid gene (NZB.xid). The in vivo administration of bacterial lipopolysaccharide to young NZB mice did not stimulate the enlargement of Lyt-2+ T cells. Ly-2+ T cells from old NZB mice could be stimulated by concanavalin A (Con A) to express interleukin 2 (IL 2) receptors and to synthesize DNA in vitro. However, in vivo administration of Con A to old NZB mice did not induce the expression of IL 2 receptors on Lyt-2+ T cells. Further, in vivo T suppressor function was impaired in old NZB mice with enlarged Lyt-2+ T cells. Thus, the enlargement of Lyt-2+ T cells in old NZB mice appears related to impaired T cell function in vivo and is associated with the development of anti-erythrocyte autoantibodies and autoimmune hemolytic anemia.     

自身免疫调节因子(AIRE)的研究进展

自身免疫调节因子(autoimmune regulator,AIRE)是一种具有转录活化潜能的DNA结合蛋白。由于AIRE基因的突变可导致自身免疫病APECED(autoimmune polyendocrinopathy—candidiasis-ectodermal dystrophy,APECED),又称自身免疫性多腺体综合征I(autoimmune polyglandular syndrome typeI,APSI)。因此,这一基因在正常生理状态下很可能对维持自身免疫耐受、控制自身免疫起着重要作用。对自身免疫耐受产生机制的揭示将为自身免疫病、超敏反应、移植排斥及肿瘤的治疗提供新的策略。本文对AIRE的基因鉴定、分子结构和生化功能、亚细胞定位、组织分布及其在自身耐受产生中的作用作一综述性介绍。     

Implementation and application of a Monte Carlo model for an in vivo micro computed tomography system

Micro computed tomography (µCT) scanners are used to create high-resolution images and to quantify properties of the scanned objects. While modern µCT scanners benefit from the cone beam geometry, they are compromised by scatter radiation. This work aims to develop a Monte Carlo (MC) model of a µCT scanner in order to characterize the scatter radiation in the detector plane.The EGS++ framework with the MC code EGSnrc was used to simulate the particle transport through the main components of the XtremeCT (SCANCO Medical AG, Switzerland). The developed MC model was based on specific information of the manufacturer and was validated against measurements. The primary and the scatter radiation were analyzed and by implementing a dedicated tracing method, the scatter radiation was subdivided into different scatter components.The comparisons of measured and simulated transmission values for different absorber and filter combinations result in a mean difference of 0.2% ± 1.4%, with a maximal local difference of 3.4%. The reconstructed image of the phantom based on measurements agrees well with the image reconstructed using the MC model. The local contribution of scattered radiation is up to 10% of the total radiation in the detector plane and most of the scattered particles result from interactions in the scanned object. The MC simulations show that scatter radiation contains information about the structure of the object.In conclusion, a MC model for a µCT scanner was successfully validated and applied to analyze the characteristics of the scatter radiation for a µCT scanner.     

Coprecipitation of nonoxynol-9 with polyvinylpyrrolidone to decrease vaginal irritation potential while maintaining spermicidal potency

The aim of this study was to test the hypothesis that polyvinylpyrrolidone (PVP) would increase the critical micelle concentration (CMC) of nonoxynol-9 (N-9), providing a reduction in its irritation potential, while maintaining essential spermicidal activity. Solid coprecipitates of N-9 with PVP were manufactured with the use of a modified lyophilization process. The irritation potential of N-9 was estimated by an in vitro assay, monitoring the extent of hemolysis of red blood cells. CMCs of N-9 were measured in the presence of various concentrations of PVP. A modified Sander-Cramer assay was implemented to measure the spermicidal activity of N-9 and the N-9/PVP coprecipitates. With the use of the lyophilization process and more suitable solvents, solid coprecipitates of N-9/PVP were manufactured with no residual organic solvents. The irritation potential of N-9 was reduced when in the presence of PVP-50% hemolysis values increased from 0.054mM to more than 0.2mM. N-9 CMC values increased in the presence of PVP from 0.085mM (0% PVP) to 0.110mM (3.5% PVP) and 0.166mM (10% PVP). However, spermicidal activities ranged from 0.213mM to 0.238mM, N-9 remaining steady regardless of the amount of PVP. By use of N-9/PVP coprecipitates, the self-association properties and irritation potentials of N-9 were altered. This result suggests a process to produce a spermicidal product that reduces the detrimental implications to the vaginal epithelium while maintaining the essential spermicidal activity.     

Discovery of a novel series of hDHODH inhibitors with anti-pulmonary fibrotic activities

Human dihydroorotate dehydrogenase (hDHODH) is a flavin-dependent enzyme essential to pyrimidine de novo biosynthesis, which serves as an attractive therapeutic target for the treatment of autoimmune disorders. A novel series of hDHODH inhibitors was developed based on a lead which was obtained by a medicinal chemistry exploration. Most compounds showed moderate to significant potency against hDHODH, compounds 5d, 5e, and 6a effectively inhibited the activities of hDHODH with IC₅₀ values from 0.9 to 2.8 μM. Further studies showed that compound 5e also effectively suppressed proliferation of the activated PBMCs (IC₅₀ = 20.35 μM). Surprisingly, compound 5e also showed anti-pulmonary fibrotic activity similar to that of pirfenidone in vitro assay. Therefore, compound 5e might have potential to be developed as a novel hDHODH inhibitors for autoimmune diseases therapy.     

Towards Quantitative Optical Cross Sections in Entomological Laser Radar – Potential of Temporal and Spherical Parameterizations for Identifying Atmospheric Fauna

In recent years, the field of remote sensing of birds and insects in the atmosphere (the aerial fauna) has advanced considerably, and modern electro-optic methods now allow the assessment of the abundance and fluxes of pests and beneficials on a landscape scale. These techniques have the potential to significantly increase our understanding of, and ability to quantify and manage, the ecological environment. This paper presents a concept whereby laser radar observations of atmospheric fauna can be parameterized and table values for absolute cross sections can be catalogued to allow for the study of focal species such as disease vectors and pests. Wing-beat oscillations are parameterized with a discrete set of harmonics and the spherical scatter function is parameterized by a reduced set of symmetrical spherical harmonics. A first order spherical model for insect scatter is presented and supported experimentally, showing angular dependence of wing beat harmonic content. The presented method promises to give insights into the flight heading directions of species in the atmosphere and has the potential to shed light onto the km-range spread of pests and disease vectors.     

Human placenta contains an epithelial scatter protein

Scatter factor (SF) is a protein produced by cultured fibroblasts which causes epithelia to "scatter" into isolated cells. We found significant scatter activity in vivo in second trimester (but not term) human amniotic fluid and in human placenta. Placental SF was purified 500,000-fold and identified as a protein with Mr 78 kd. Factor scattered Madin-Darby canine kidney and human squamous carcinoma cells at 15 pM. Amino acid sequences from tryptic peptides did not match any known protein. Human placental fibroblasts produced high titers of scatter activity. SF may be involved in development and may enhance carcinoma invasion.     

Purified scatter factor stimulates epithelial and vascular endothelial cell migration

Fibroblasts and smooth muscle cells release a protein activity which causes epithelial sheets to "scatter" into isolated cells. Purification of scatter factor (SF) activity from ras-transformed 3T3 cells was reported recently. We purified ras-3T3 SF by a slightly different method with essentially similar findings. Purified factor showed a single band at 77 +/- 3 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Scatter activity was eluted from gel slices at this molecular size. Reduction with mercaptoethanol caused the loss of activity and the appearance of two bands (58 and 31 kDa). We report the amino acid composition of ras-3T3 SF and sequences of several tryptic peptides. These sequences were not similar to the known proteins in the Protein Database. We have shown previously that partially purified ras-3T3 scatter activity stimulates migration of epithelial and vascular endothelial cells in a new migration assay utilizing microcarrier beads. We now demonstrate that the same purified ras-3T3 protein scatters epithelial cells and stimulates epithelial and endothelial migration in microcarrier bead and Boyden chamber assays. Partially purified human smooth muscle scatter activity shares these activities, but the protein(s) responsible has not been isolated. Migration-stimulating activity was maximal at ras-3T3 protein concentrations less than 10 ng/ml (0.13 nM). ras-3T3 SF had no collagenolytic activity and did not stimulate DNA synthesis in fibroblast growth factor-responsive human melanocytes. ras-3T3 SF appears to be a new protein which regulates endothelial and epithelial mobility; and, therefore, it may be involved in vascular repair and wound healing.     

Selenium, glutathione and glutathione peroxidases in blood of patients with chronic liver diseases

Disturbances in the antioxidant system could play a role in pathogenesis of chronic liver disease. The aim of our study was to evaluate the levels/activities of antioxidants in blood of patients with chronic liver disease. We estimated selenium and glutathione concentrations and glutathione peroxidase activities in blood of 59 patients with chronic hepatitis B or C virus infection (group 1) and 64 patients with alcoholic, autoimmune or cryptogenic chronic liver disease (group 2). The results were compared with 50 healthy controls. Whole blood and plasma selenium and red cell glutathione concentrations were significantly lower in the patients compared with the controls. Red cell glutathione peroxidase activity was slightly reduced in both subgroups of group 1 and in group 2 with normal alanine aminotransferase values. Plasma glutathione peroxidase activity was slightly but significantly higher in patients with elevated aminotransferase values. The findings suggest that disturbances in antioxidant parameters in blood of patients with chronic liver disease may be the cause of the peroxidative damage of cells.     

基于近红外漫反射光谱快速测定淫羊藿蛋白质含量

采用近红外漫反射光谱技术对淫羊藿(Epimedium)的蛋白质含量进行快速且无损检测。近红外漫反射光谱经二阶导数处理、标准多元离散校正及主成分分析聚类处理后, 采用改进最小二乘法回归得到的定标模型预测效果最佳, 定标决定系数、交互验证标准差及交互验证相关系数分别为0.923、0.554和0.717。近红外光谱分析法的测定结果与用凯氏定氮法所得结果无显著差异, 两种方法测定值的相关性较高(R²=0.933 9)。重复性实验表明, 近红外光谱分析法的相对标准偏差为0.937%。该研究首次采用近红外光谱分析法测定了8种淫羊藿的蛋白质含量。该方法简便、精确, 在淫羊藿资源开发利用和药材质量控制方面具有参考意义。     

Leukocyte differential analysis in multiple laboratory species by a laser multi-angle polarized light scattering separation method.

Leukocyte differential analysis was performed in various species, particularly laboratory animals, by the laser multi-angle polarized light scattering separation method. Venous blood specimens were drawn from the following subjects: healthy adult men and women ("humans"); cynomolgus monkeys ("monkeys"); common marmosets ("marmosets"); beagle dogs ("dogs"); miniature potbelly pigs ("swine"); Japanese white rabbits ("rabbits"); Hartley guinea pigs ("guinea pigs"); and Sprague-Dawley rats ("rats"). 90 degrees/10 degrees scatter plot: Basophils and mononuclear-polymorphonuclear cells were separated in all subjects, but individual 10 degrees and 90 degrees scatter plots overlapped in dogs and guinea pigs, respectively. 90 degrees depolarized /90 degrees scatter plot: Neutrophils and eosinophils were clearly separated in human, monkey, guinea pig, swine and rat subjects. The eosinophil cluster was not clearly plotted in marmoset, dog, or rabbit. 0 degree/10 degrees scatter plot: Regarding this plot for monocytes and lymphocytes, cells were plotted in the following order in all subjects: lymphocytes < basophils < or = monocytes in the 0 degree (size) scatter; and lymphocytes [symbol: see text] monocytes < or = basophils in the 10 degrees (complexity) scatter. Compared to other species, the rat scatter showed a tendency to overlapping plots in both the 0 degree and 10 degrees scatters in the monocyte and lymphocyte clusters. In both dog and guinea pig, the monocyte and neutrophil plots overlapped in the 0 degree and 10 degrees scatters. Basobox: In the human and rabbit subjects, the basophil cluster was plotted within the established basobox, but no clear cell cluster was plotted in the other subjects. As a result of comparing the percentage values for leukocytes in various species obtained by using the CD3500 apparatus versus the corresponding values obtained manually, good correspondence was found in the monkey, and eosinophils in the marmoset were lower with CD3500 than manually. In the rabbit, the mean measured value for basophils matched in the manual and CD3500 findings. In the guinea pig, the CD3500 values were lower than the manual values for lymphocytes, but higher for monocytes and neutrophils. The above findings suggest that the laser multi-angle polarized light scattering separation method is indeed capable of analyzing leukocytes from various species based on cell size and cell complexity, i.e., the presence or absence of nuclei, granules and cell enclosures.     

Identification of novel quinazolinedione derivatives as RORγt inverse agonist

Novel small molecules were synthesized and evaluated as retinoic acid receptor-related orphan receptor-gamma t (RORγt) inverse agonists for the treatment of inflammatory and autoimmune diseases. A hit compound, 1, was discovered by high-throughput screening of our compound library. The structure–activity relationship (SAR) study of compound 1 showed that the introduction of a chlorine group at the 3-position of 4-cyanophenyl moiety increased the potency and a 3-methylpentane-1,5-diamide linker is favorable for the activity. The carbazole moiety of 1 was also optimized; a quinazolinedione derivative 18i suppressed the increase of IL-17A mRNA level in the lymph node of a rat model of experimental autoimmune encephalomyelitis (EAE) upon oral administration. These results indicate that the novel quinazolinedione derivatives have great potential as orally available small-molecule RORγt inverse agonists for the treatment of Th17-driven autoimmune diseases. A U-shaped bioactive conformation of this chemotype with RORγt protein was also observed.