Altered Hypercoagulability Factors in Patients with Chronic Chagas Disease: Potential Biomarkers of Therapeutic Response

Thromboembolic events were described in patients with Chagas disease without cardiomyopathy. We aim to confirm if there is a hypercoagulable state in these patients and to determine if there is an early normalization of hemostasis factors after antiparasitic treatment. Ninety-nine individuals from Chagas disease-endemic areas were classified in two groups: G1, with T.cruzi infection (n = 56); G2, healthy individuals (n = 43). Twenty-four hemostasis factors were measured at baseline. G1 patients treated with benznidazole were followed for 36 months, recording clinical parameters and performance of conventional serology, chemiluminescent enzyme-linked immunosorbent assay (trypomastigote-derived glycosylphosphatidylinositol-anchored mucins), quantitative polymerase chain reaction, and hemostasis tests every 6-month visits. Prothrombin fragment 1+2 (F1+2) and endogenous thrombin potential (ETP) were abnormally expressed in 77% and 50% of infected patients at baseline but returned to and remained at normal levels shortly after treatment in 76% and 96% of cases, respectively. Plasmin-antiplasmin complexes (PAP) were altered before treatment in 32% of G1 patients but normalized in 94% of cases several months after treatment. None of the patients with normal F1+2 values during follow-up had a positive qRT-PCR result, but 3/24 patients (13%) with normal ETP values did. In a percentage of chronic T. cruzi infected patients treated with benznidazole, altered coagulation markers returned into normal levels. F1+2, ETP and PAP could be useful markers for assessing sustained response to benznidazole.     

Altered Gene Expression Pattern in Peripheral Blood Mononuclear Cells in Patients with Acute Myocardial Infarction

Background

Despite a substantial progress in diagnosis and therapy, acute myocardial infarction (MI) is a major cause of mortality in the general population. A novel insight into the pathophysiology of myocardial infarction obtained by studying gene expression should help to discover novel biomarkers of MI and to suggest novel strategies of therapy. The aim of our study was to establish gene expression patterns in leukocytes from acute myocardial infarction patients.

Methods and Results

Twenty-eight patients with ST-segment elevation myocardial infarction (STEMI) were included. The blood was collected on the 1^st day of myocardial infarction, after 4–6 days, and after 6 months. Control group comprised 14 patients with stable coronary artery disease, without history of myocardial infarction. Gene expression analysis was performed with Affymetrix Human Gene 1.0 ST microarrays and GCS3000 TG system. Lists of genes showing altered expression levels (fold change >1.5, p<0.05) were submitted to Ingenuity Pathway Analysis. Gene lists from each group were examined for canonical pathways and molecular and cellular functions. Comparing acute phase of MI with the same patients after 6 months (stable phase) and with control group we found 24 genes with changed expression. In canonical analysis three pathways were highlighted: signaling of PPAR (peroxisome proliferator-activated receptor), IL-10 and IL-6 (interleukin 10 and 6).

Conclusions

In the acute phase of STEMI, dozens of genes from several pathways linked with lipid/glucose metabolism, platelet function and atherosclerotic plaque stability show altered expression. Up-regulation of SOCS3 and FAM20 genes in the first days of myocardial infarction is observed in the vast majority of patients.     

Altered Pattern of Spontaneous Brain Activity in the Patients with End-Stage Renal Disease: A Resting-State Functional MRI Study with Regional Homogeneity Analysis

Purpose

To investigate the pattern of spontaneous neural activity in patients with end-stage renal disease (ESRD) with and without neurocognitive dysfunction using resting-state functional magnetic resonance imaging (rs-fMRI) with a regional homogeneity (ReHo) algorithm.

Materials and Methods

rs-fMRI data were acquired in 36 ESRD patients (minimal nephro-encephalopathy [MNE], n = 19, 13 male, 37±12.07 years; non-nephro-encephalopathy [non-NE], n = 17, 11 male, 38±12.13 years) and 20 healthy controls (13 male, 7 female, 36±10.27 years). Neuropsychological (number connection test type A [NCT-A], digit symbol test [DST]) and laboratory tests were performed in all patients. The Kendall''s coefficient of concordance (KCC) was used to measure the regional homogeneity for each subject. The regional homogeneity maps were compared using ANOVA tests among MNE, non-NE, and healthy control groups and post hoc t -tests between each pair in a voxel-wise way. A multiple regression analysis was performed to evaluate the relationships between ReHo index and NCT-A, DST scores, serum creatinine and urea levels, disease and dialysis duration.

Results

Compared with healthy controls, both MNE and non-NE patients showed decreased ReHo in the multiple areas of bilateral frontal, parietal and temporal lobes. Compared with the non-NE, MNE patients showed decreased ReHo in the right inferior parietal lobe (IPL), medial frontal cortex (MFC) and left precuneus (PCu). The NCT-A scores and serum urea levels of ESRD patients negatively correlated with ReHo values in the frontal and parietal lobes, while DST scores positively correlated with ReHo values in the bilateral PCC/precuneus, MFC and inferior parietal lobe (IPL) (all P<0.05, AlphaSim corrected). No significant correlations were found between any regional ReHo values and disease duration, dialysis duration and serum creatinine values in ESRD patients (all P>0.05, AlphaSim corrected).

Conclusion

Diffused decreased ReHo values were found in both MNE and non-NE patients. The progressively decreased ReHo in the default mode network (DMN), frontal and parietal lobes might be trait-related in MNE. The ReHo analysis may be potentially valuable for elucidating neurocognitive abnormalities of ESRD patients and detecting the development from non-NE to MNE.     

成人烟雾病合并后循环病变的相关因素分析

目的:探讨成人型烟雾病合并后循环病变发生的相关危险因素,以期指导临床诊治.方法:回顾性分析2009年3月～2010年1月于解放军307医院神经外科就诊并住院的成人烟雾病患者418例,根据是否合并后循环病变(大脑后动脉病变为主)分为合并后循环病变组130例,未合并后循环病变组为288例.分别记录患者的的性别、年龄、籍贯、临床表现类型、铃木分期、既往病史(高血压、高脂血症、高血糖)、不良生活习惯(吸烟、饮酒),是否有烟雾病家族史.对其进行组间对比及logistic回归法进行变量分析.结果:阳性家族史是成人烟雾病患者合并后循环病变的独立危险因素(OR=3.898,95％CI:1.103～13.776,P=0.035),而其余观察指标均无明显相关性(P＞0.05);相对于只存在前循环病变的患者,成人烟雾病合并后循环病变的患者较易出现卒中及头痛,脑出血相对较少;此类患者于31-40岁组出现小高峰.结论:阳性家族史是成人烟雾病患者合并后循环病变的独立危险因素,患者的临床表现较重,治疗后仍可出现反复卒中.     

An Electron Microscope Study of Smooth Muscle in Pregnant Uterus of the Rabbit

In the smooth muscle of the rabbit uterus one type of myofilament can be found; this is about 50 A thick. In the sarcoplasmic reticulum there are smooth walled vesicles and many vesicles with particles attached. It is suggested that these particles may correspond to the ribonucleoprotein-containing particles of Palade and Siekevitz. The diameter of the particles is 250 A. There are also unattached particles some of which may be glycogen. The membranes of the muscle cells and the connective tissue lying between them are described.     

Inner and Outer Portions of Colonic Circular Muscle: Ultrastructural and Immunohistochemical Changes in Rat Chronically Treated with Otilonium Bromide

Rat colonic circular muscle, main target of otilonium bromide (OB) spasmolytic activity, is subdivided in an inner and outer portion. Since the inner one is particularly rich in organelles involved in calcium availability (caveolae, smooth endoplasmic reticulum, mitochondria), the expression of specific markers (Caveolin-1, eNOS, calreticulin, calsequestrin) in comparison with the outer portion was investigated. The possible changes of these organelles and related markers, and of muscarinic receptors (Mr2) were then studied after OB chronic exposition. Rats were treated with 2–20 mg/kg/OB for 10 or 30 days. Proximal colon was processed by electron microscopy, immunohistochemistry, and western blot. In colon strips the stimulated contractility response to muscarinic agonist was investigated. The inner portion showed a higher expression of Caveolin-1 and Mr2, but not of eNOS, calreticulin and calsequestrin, compared to the outer portion. Chronic OB treatment caused similar ultrastructural and immunohistochemical changes in both portions. Organelles and some related markers were increased at 10 days; Mr2 expression and muscle contractility induced by methacholine was increased at 30 days. The present findings: 1) provide new information on the immunohistochemical properties of the inner portion of the circular layer that are in favour of a role it might play in colonic motility distinct from that of the outer portion; 2) demonstrate that chronically administered OB interferes with cell structures and molecules responsible for calcium handling and storage, and modifies cholinergic transmission. In conclusion, chronic OB administration in the colonic circular muscle layer directly interacts with the organelles and molecules calcium-related and with the Mr2.     

Pattern of Skeletal Muscle Differentiation in Fish: Molecular Biological Approaches

The initial stages of myogenesis going in myoblasts include the stages of induction, determination, and differentiation. The induction and determination of cells in the myotomes are controlled by morphogenetic signals from neighboring tissues of the notochord and neural tube manifested as expression of genes of Shh and Wnt families, respectively. In fish (at the example of danio), this signal is passed to somite cells neighboring the notochord; later the cells migrate to the embryo surface and differentiate into slow muscle fibers. Synthesis of the main contractile proteins, primarily the components of myosin molecule—heavy chain (MHC) and individual isoforms of light chains (MLC1, MLC2, and MLC3)—are encoded by different genes during different ontogenetic stages. The peptide maps obtained after -chymotrypsin digestion of MHCs from larvae, fast and slow skeletal muscle of loach are different, which points to differences in their primary structure. In addition, considerable differences were revealed in the structure of MLC isoforms at different ontogenetic stages. The definitive fast muscle contained three light chain types, MLC1, MLC2, and MLC3; slow muscle, MLC1 and MLC3; while the larval muscle fibers included a specific larval MLCL in addition to MLC3.     

Identification of a Novel Marker for Primordial Smooth Muscle and Its Differential Expression Pattern in Contractile vs Noncontractile Cells

The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375–392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, M_r = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, M_r = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle α-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle α-actinin. Our results suggest that the 1E12 antigen is a member of the α-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.     

细胞因子对血管平滑肌细胞MMP-2基因表达的诱导及其作用机制研究

为了探讨血管平滑肌细胞 ( VSMC)基质金属蛋白酶 - 2 ( MMP- 2 )基因的表达调控机制 ,利用Northern印迹杂交和 MMP- 2活性酶图分析检查 b FGF、TNF- α和 IL- 1 β对 VSMC MMP- 2基因表达的影响 ,应用电泳迁移率改变实验 ( EMSA)和 CAT分析对其作用机制进行研究 .结果证实 ,3种细胞因子均能显著诱导 MMP- 2基因表达 ,其作用强度依次为 b FGF>TNF-α>IL - 1β.将 MMP-2基因 5′侧翼 - 61 9～ 1 9bp调控序列克隆进携带报告基因的重组质粒 p SV0 - CAT后 ,经转染VSMC及 CAT分析显示 ,在上述 3种细胞因子的作用下 ,该调控序列可激活 cat基因表达 ,三者促进 cat表达的活性与其诱导 VSMC表达 MMP- 2的结果相一致 ;EMSA结果显示 ,被 b FGF和TNF- α刺激的 VSMC中产生与该基因调控区序列特异结合的转录调控因子 .提示细胞因子除可激活 VSMC细胞周期调节基因表达外 ,还可通过诱导 MMP- 2表达而发挥其对细胞外基质代谢的调节作用及参与 VSMC迁移的启动过程 ;细胞因子对 VSMC MMP- 2基因表达的诱导作用是通过促进转录调控因子的合成或活化而实现的 .     

Immunoglobulin Deposition in Liver of Patients with Active Chronic Hepatitis and Antibody against Smooth Muscle

Liver biopsies from 33 patients with miscellaneous liver diseases were studied by direct immunofluorescence. Three patients with active chronic hepatitis possessed a distinctive pattern of staining with anti-IgG conjugate. They were relatively young women and all three possessed the anti-smooth-muscle antibody. In addition, all three were untreated at the time of study. It is suggested that direct immunofluorescence on liver biopsies may help in the investigation of liver disease and that humoral immunity may participate in the pathogenesis of active chronic hepatitis.     

An Immunohistochemical Study of Leu 7 and PCNA Expression in Thyroid Neoplasms

Monoclonal Antibody (MoAb) HNK, or anti-leu-7, is reactive with several neuroendocrine and nonneuroendocrine tumors. The aim of this study is to examine anti-leu-7 reactivity in thyroid neoplasms and its relationship to cellular proliferation as determined by anti-PCNA reactivity. The expression of anti-leu-7 in 56 thyroid neoplasms (24 papillary carcinomas, 14 follicular carcinomas, two medullary carcinomas and 16 follicular adenomas) was examined immunohistochemically. Papillary and follicular thyroid carcinomas reacted with anti-leu-7 in a membranous and cytoplasmic pattern in 88% and 93% of cases, respectively. The adjacent benign tissues were nonreactive. Only eight cases diagnosed as follicular adenomas were reactive with anti-leu-7. Furthermore, the mean proliferative index (PI), as measured by the percentage of nuclei immunoreactive with anti-PCNA, was greater than 30% in all thyroid neoplasms reactive with anti-leu-7. The PI was 58% for papillary carcinomas and 68% and 48% for follicular carcinomas, and follicular adenomas, respectively. Lesions originally classified as follicular adenomas that were nonreactive with anti-leu-7 had a PI of 24% and were reclassified as hyperplastic nodules. These data suggest that anti-leu-7 may be useful for characterizing thyroid neoplasia.     

An Immunohistochemical Study of Leu 7 and PCNA Expression in Thyroid Neoplasms

Monoclonal Antibody (MoAb) HNK, or anti-leu-7, is reactive with several neuroendocrine and nonneuroendocrine tumors. The aim of this study is to examine anti-leu-7 reactivity in thyroid neoplasms and its relationship to cellular proliferation as determined by anti-PCNA reactivity. The expression of anti-leu-7 in 56 thyroid neoplasms (24 papillary carcinomas, 14 follicular carcinomas, two medullary carcinomas and 16 follicular adenomas) was examined immunohistochemically. Papillary and follicular thyroid carcinomas reacted with anti-leu-7 in a membranous and cytoplasmic pattern in 88% and 93% of cases, respectively. The adjacent benign tissues were nonreactive. Only eight cases diagnosed as follicular adenomas were reactive with anti-leu-7. Furthermore, the mean proliferative index (PI), as measured by the percentage of nuclei immunoreactive with anti-PCNA, was greater than 30% in all thyroid neoplasms reactive with anti-leu-7. The PI was 58% for papillary carcinomas and 68% and 48% for follicular carcinomas, and follicular adenomas, respectively. Lesions originally classified as follicular adenomas that were nonreactive with anti-leu-7 had a PI of 24% and were reclassified as hyperplastic nodules. These data suggest that anti-leu-7 may be useful for characterizing thyroid neoplasia.     

Expression of Ser729 Phosphorylated PKC Epsilon in Experimental Crescentic Glomerulonephritis: An Immunohistochemical Study

PKCε, a DAG-dependent, Ca²⁺- independent kinase attenuates extent of fibrosis following tissue injury, suppresses apoptosis and promotes cell quiescence. In crescentic glomerulonephritis (CGN), glomerular epithelial cells (GEC) contribute to fibro-cellular crescent formation while they also transdifferentiate to a mesenchymal phenotype. The aim of this study was to assess PKCε expression in CGN. Using an antibody against PKC-ε phosphorylated at Ser⁷²⁹, we assessed its localization in rat model of immune-mediated rapidly progressive CGN. In glomeruli of control animals, pPKCε was undetectable. In animals with CGN, pPKCε was expressed exclusively in glomerular epithelial cells (GEC) and in GEC comprising fibrocellular crescents that had acquired a myofibroblasttype phenotype. In non-immune GEC injury induced by puromycin aminonucleoside and resulting in proteinuria of similar magnitude as in CGN, pPKCε expression was absent. There was constitutive pPKCε expression in distal convoluted tubules, collecting ducts and thick segments of Henley’s loops in both control and experimental animals. We propose that pPKC_ε expression occurring in GEC and in fibrocellular crescentic lesions in CGN may facilitate PKCε dependent pathologic processes.     

不同时相血管平滑肌细胞受促分裂原刺激后基因表达谱及迁移活性的比较

探讨处于细胞周期不同时相的血管平滑肌细胞 (vascular smooth muscle cell,VSMC)受促分裂原刺激后基因表达谱及迁移行为的变化 .采用细胞平面及跨膜迁移试验 ,观察处于静止期和增殖状态的 VSMC在相同因素作用下迁移活性的变化 ;应用 Northern印迹杂交和体外转录分析 ,检测 VSMC迁移及增殖相关基因表达谱 .结果显示 ,处于静止期的 VSMC,在具有促分裂作用的细胞因子 b FGF、IL- β和 TNF- α刺激下 ,所迁移的距离分别是对照组的 5.8± 1 .53、4.68± 0 .89和 4.1 5± 1 .0 4倍 ,在相同条件下 ,处于增殖状态的细胞其迁移距离仅为静止期细胞的 1 /4;b FGF可诱导VSMC降解胶原成分进行跨膜迁移 ;杂交及体外转录分析证实 ,静止期的 VSMC被 b FGF诱导后 ,迁移相关基因基质金属蛋白酶 - 2 (matrix metalloproteinase 2 ,MMP- 2 )和组织基质金属蛋白酶 - 2抑制剂 (tissue inhibitor of metalloproteinase 2 ,TIMP- 2 )及增殖相关基因骨桥蛋白 (osteopontin,OPN)和 c- jun同时被活化 ;相反 ,处于增殖时相的细胞在该因子作用下 ,仅有 OPN和 c- jun基因进行转录 .提示 ,处于细胞周期不同时相的 VSMC受促分裂原刺激后迁移活性和迁移及增殖相关基因表达谱明显不同 ,在相同因素作用下 ,静止期细胞的迁移相关基因表达水     

Increased Expression of Colonic Mucosal Melatonin in Patients with Irritable Bowel Syndrome Correlated with Gut Dysbiosis

Dysregulation of the gut microbiota/gut hormone axis contributes to the pathogenesis of irritable bowel syndrome (IBS). Melatonin plays a beneficial role in gut motility and immunity. However, altered expression of local mucosal melatonin in IBS and its relationship with the gut microbiota remain unclear. Therefore, we aimed to detect the colonic melatonin levels and microbiota profiles in patients with diarrhea-predominant IBS (IBS-D) and explore their relationship in germ-free (GF) rats and BON-1 cells. Thirty-two IBS-D patients and twenty-eight healthy controls (HCs) were recruited. Fecal specimens from IBS-D patients and HCs were separately transplanted into GF rats by gavage. The levels of colon mucosal melatonin were assessed by immunohistochemical methods, and fecal microbiota communities were analyzed using 16S rDNA sequencing. The effect of butyrate on melatonin synthesis in BON-1 cells was evaluated by ELISA. Melatonin levels were significantly increased and negatively correlated with visceral hypersensitivity in IBS-D patients. GF rats inoculated with fecal microbiota from IBS-D patients had high colonic melatonin levels. Butyrate-producing Clostridium cluster XIVa species, such as Roseburia species and Lachnospira species, were positively related to colonic mucosal melatonin expression. Butyrate significantly increased melatonin secretion in BON-1 cells. Increased melatonin expression may be an adaptive protective mechanism in the development of IBS-D. Moreover, some Clostridium cluster XIVa species could increase melatonin expression via butyrate production. Modulation of the gut hormone/gut microbiota axis offers a promising target of interest for IBS in the future.     

Precipitating Factors of Acute Illness in Patients with Chronic Disease

The precipitating causes of acute illness in patients with chronic organic disease can often be prevented, reversed or controlled. The various kinds of precipitating factors are described, and their anticipation and the prompt institution of remedial measures are emphasized.