Objective To high express and purify toxoplasma gondii antigen GRA6 in E.coli which can be used to develop the genetic engineering diagnostic reagents.Methods The recombinant plasmid of pGEX-GRA6 was transformed to a bacterium BL21-Codon Plus(DE3)-RP and the recom- binant product was expressed under the inducement of isopropyl-beta-D-thiogalactosidase(IPTG). Cells were lysed by multiple rounds of sonication.The expression product was analyzed by using SDS- PAGE.Furthermore,it was purified by sedimentation of ammonium sulphate,desalting using Sephe- dax GS0 and affinity chromatography on glutathione-sepharose system.The immunogenicity of recom binant antigen purified was tested with Western blot.Results GRA6 was highly expressed in E.coli as fusion protein consisting of glutathione S-transferase and GRA6(GST-GRA6).The solubility anal- ysis of expression product indicated that this recombinant protein could be expressed in both superna- tant and inclusion bodies.The soluble protein of GRA6 in supernatant yield the final preparation at greater than 90% after purification.The recombinant protein purified could be recognized by human toxoplasmosis-infective sera with Western blotting analysis.Conclusions The soluble protein of GRA6 was highly expressed in E.coli and the recombinant antigen could be recognized by human tox- oplasmosis-infective serum after purification.The recombinant antigen can be used for devoloping kit to diagnose the acute and chronic infection of toxoplasma gondii.

OBJECTIVETo investigate the relationship of serum metalloproteinase with the severity of liver fibrosis and inflammation.

METHODSA total of 88 patients with HBV-related liver fibrosis and early cirrhosis were enrolled from six hospitals. Serum fibrosis markers including hyaluronic acid (HA), IV collagen (IV-C), aminoterminal propeptide of type III procollagen (PIIIP), laminin (LN), matrix metalloproteinases (MMP) 1, 2, 9 and tissue inhibitors of metalloproteinase (TIMP) 1, 2 levels were determined. Liver biopsies were assessed according to a modified Scheuer and Chevallier's scoring system.

RESULTSSerum TIMP1 (r=0.540) and MMP2 (r=0.314) were correlated positively with the degree of hepatic fibrosis, whereas serum MMP1 (r=-0.495) was correlated negatively. By receiver operating curve analysis (ROC), the sensitivity to distinguish the fibrosis stage 2 from stage 1 was 90.5% and the specificity was 52.0% if the cut-off value of MMP1 was 13.96 ng/ml, and the sensitivity was 91.6% and the specificity was 64.0% if the cut-off value of TIMP1 was 76.84 ng/ml. The sensitivity to distinguish cirrhosis (stage 4) from fibrosis (stage 3) was 70.7% and specificity was 80.9% if the cut-off value of MMP1 was 6.86 ng/ml, and the sensitivity was 60.5% and the specificity was 92.3% if the cut-off value of TIMP1 was 210.04 ng/ml.

CONCLUSIONSerum TIMP1, MMP1, MMP2 levels and TIMP1/MMP1 ratio could be used as serum fibrosis markers.

Adult ; Biomarkers ; blood ; Female ; Hepatitis B, Chronic ; blood ; complications ; Humans ; Liver Cirrhosis ; blood ; virology ; Male ; Matrix Metalloproteinase 1 ; blood ; Matrix Metalloproteinase 2 ; blood ; Middle Aged ; Tissue Inhibitor of Metalloproteinase-1 ; blood

This paper expounds how the tractor for the fracture reduction works. The clinical results show that the traction apparatus is a labour-saving and time-saving orthopedic device with simple operation and few suffering to patients.
Arm Injuries ; diagnostic imaging ; surgery ; Equipment Design ; Fracture Fixation ; instrumentation ; methods ; Fractures, Bone ; diagnostic imaging ; surgery ; Humans ; Leg Injuries ; diagnostic imaging ; surgery ; Radiography ; Traction ; instrumentation ; methods

OBJECTIVETo study the relation of the structural remodeling processes and activation of calpain I.

METHODSFifteen dogs were randomly divided into three groups. The dogs in pacing group (n=5) and inhibitor group (n=5) were subjected to 3 weeks of rapid atrial pacing at 600 beats/min, control dogs (n=5) were in sham-operated group. The dogs in inhibitor group were administered intravenous N-Acetyl-Leu-Leu-Met (ALLM), a calpain inhibitor, and in pacing group and sham-operated group were administered intravenous DMSO. The activity of calpain I was measured by hydrolyzing Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl-coumarin. The ultrastructure of atrium was examined by light and electron microscopy. TnT expression was assessed by Western blot. Echocardiography examination was performed in all the three groups.

RESULTSCalpain I activity was significantly increased in pacing group (2.3-fold, P<0.01), and decreased in inhibitor group (1.1-fold, P>0.05), compared to sham-operated group respectively. The percentages of myolysis were (76.7 +/- 5.9)% and (20.8 +/- 8.1)% in pacing group and inhibitor group respectively (P<0.01). TnT expression decreased in the rapid pacing-induced persistent atrial fibrillation, and these effects were inhibited by calpain I inhibitor ALLM. The area and volume of left atrium tended to increase after 3 weeks ALLM treatment in inhibitor group, but the change was not as prominent as in pacing group (P<0.05).

CONCLUSIONSALLM can decrease calpain I activity, and prevent canine atrial cardiomyocyte structural remodeling during atrial fibrillation. This study provided a capacity of atrial cardiomyocyte protection.

Animals ; Atrial Fibrillation ; metabolism ; physiopathology ; Atrial Function, Left ; Calpain ; antagonists & inhibitors ; metabolism ; Cardiac Pacing, Artificial ; Disease Models, Animal ; Dogs ; Heart Atria ; ultrastructure ; Myocardium ; metabolism ; Troponin T ; metabolism

BACKGROUNDAtrial fibrillation (AF) is accompanied by atrial structural remodeling. Calpain activity is induced during AF. To test a causal relationship between calpain activation and atrial structural changes, N-acetyl-Leu-Leu-Met (ALLM), a calpain inhibitor, was utilized in a canine AF model.

METHODSFifteen dogs were randomly divided into 3 groups: sham-operated group, control group and calpain inhibitor group; each with 5 dogs. Sustained AF was induced by rapid right atrium pacing at 600 beats per minute for 3 weeks. ALLM was administered at a dosage of 1.0 mg x kg(-1) x d(-1) in the calpain inhibitor group. Three weeks later, the proteolysis, protein expression of TnT and myosin, calpain I localization and expression and structural changes were examined in left atrial free walls, right atrial free walls and the interatrial septum respectively. Atrial size and contractile function were also measured by echocardiography.

RESULTSLong-term rapid atrial pacing induced marked structural changes such as enlarged atrial volume, myolysis, degradation of TnT and myosin, accumulation of glycogen and changes in mitochondrial shape and size, which were paralleled by an increase in calpain activity. The positive correlation between calpain activity and the degree of myolysis (r(s) = 0.90 961, P < 0.0001) was demonstrated. In addition to structural abnormalities, pacing-induced atrial contractile dysfunction was observed in this study. The pacing-induced atrial structural alterations and loss of contractility were partially prevented by the calpain inhibitor ALLM.

CONCLUSIONSActivation of calpain represents key features in the progression towards overt structural remodeling. Calpain inhibitor, ALLM, suppressed the increased calpain activity and reversed structural remodeling caused by sustained atrial fibrillation in the present model. Calpain inhibition may therefore provide a possibility for therapeutic intervention in AF.

Animals ; Atrial Fibrillation ; pathology ; Calpain ; antagonists & inhibitors ; Cysteine Proteinase Inhibitors ; pharmacology ; Disease Models, Animal ; Dogs ; Heart Atria ; pathology ; ultrastructure ; Myosins ; analysis ; Troponin T ; analysis

OBJECTIVESTo explore the clinical and pathological features and the pathogenesis of primary biliary cirrhosis (PBC) in Chinese Mainland.

METHODS30 PBC patients were divided into the early group (Scheuer stage I and II, 19 patients) and the late group (Scheuer stage III and IV, 11 patients). The data of clinics and serology were analyzed, and the pathological features of the liver tissues were characterized. The changes of dendritic cells (DCs) and hepatic stellate cells (HSCs) were studied by immunohistochemistry.

RESULTSIn all the PBC patients, the rate of the male to the female was 1 to 5, and the average age was 40.6 years. The mean levels of TBiL, ALP and GGT in the sera were (95.9+-88.5) micromol/L, (537.2+-339.2) U/L, and (582.0+-351.2) U/L, respectively. 73.3% patients showed AMA positive, and the level of GGT was positively correlated with the AMA level according to the result of statistical analysis (r=0.778, P=0.000). The symptoms of jaundice and hepatomegaly were presented more commonly in the late group than those in the early group (chi2=5.182, P<0.05; chi2=13.659, P<0.01, respectively). The main changes of morphology of PBC located in portal tracts. The liver tissues in the early stage of PBC showed the damage of bile ducts and obvious proliferation of small bile ducts. The granulomas, the lymphoid follicles and the foamy cells were found in the liver tissues of PBC (2/19 patients, 12/19 patients, and 10/19 patients in the early stage respectively, while 0/11 patients, 4/11 patients, and 3/11 patients in the late stage respectively). There was significant difference between the early stage and the late stage in presence of the lymphoid follicles and the foamy cells (t=4.489, P<0.05; t=4.019, P<0.05, respectively). The biliary pigmentary particles were mainly accumulated in the liver cells around the portal tracts in 90.0% PBC patients, and the accumulation of copper and iron increased, compared with that in normal specimens. The DCs and HSCs located mainly in the portal tracts, especially around the damaged bile ducts.

CONCLUSIONSThere are some clinical and pathological characteristics in the patients with PBC. The level of AMA has no direct relationship with the level of transaminase or bilirubin. The proliferated bile ductules may express the antigens which maybe the target of immune attack. As an antigen-presenting cell, DCs may play an important role in the pathogenesis of PBC.

Adolescent ; Adult ; Antibodies, Antinuclear ; blood ; Antigen-Presenting Cells ; immunology ; pathology ; Dendritic Cells ; pathology ; Female ; Humans ; Liver ; pathology ; Liver Cirrhosis, Biliary ; etiology ; immunology ; pathology ; Male ; Middle Aged ; Mitochondria ; immunology

BACKGROUNDTo explore the cut-off period of subclassification and pathological features of severe hepatitis (SH).

METHODSBased on combined clinical and pathological analyses, the complete clinical and biopsy or autopsy liver tissues data from 196 cases of patients with severe hepatitis were investigated. Meanwhile, proliferative hepatocytes, cholangioepithelia and collagens were identified by a panel of monoclonal antibodies such as those against albumin, cytokeratin 18,19 and collagen I, III with immunohistochemical method.

RESULTSThe clinical and pathological analyses indicated the cut-off periods of acute, subacute and chronic SH (ASH,SSH and CSH) were (13.4+/-7.2) d, (77.4+/-69.3) d and (80.5+/-63.2) d, respectively. Among all SH cases, one case of ASH patient presented clinical manifestation and pathological changes of ASH for 21 days, however, one patient with SSH was demonstrated 12 day course by histological examination. The time of cut-off period between ASH and SSH in child cases was shorter than that in adult cases. Histologically, ASH liver tissues showed massive and/or submassive necrosis caused by one attack, with congestive sinusoid frameworks and proliferative cholangioepithelium-like hepatocytes, while SSH liver tissues presented combined fresh and old submassive or massive necrosis caused by multiple attacks, accompanied by obviously proliferative bile ducts and sinusoid framework collapse.However, the pathological changes of CSH showed ASH- or SSH-like lesions on the background of chronic liver injury.

CONCLUSIONOur data indicated that the cut-off period between ASH and SSH is in accordance with the Scheme of Viral Hepatitis Prevention and Therapy, China, published in 2000, but excluded a part of child SH cases. In our study, the authors found a few pathological features in ASH and SSH.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Collagen ; metabolism ; Female ; Hepatitis ; classification ; metabolism ; pathology ; Humans ; Keratins, Type I ; metabolism ; Liver ; pathology ; Male ; Middle Aged ; Young Adult

OBJECTIVETo investigate the clinical usefulness of noninvasive diagnostic methods in evaluating liver fibrosis in hepatitis B virus (HBV) patients.

METHODS102 patients with chronic hepatitis B (CHB) were enrolled from Beijing Friendship Hospital Affiliated to Capital University of Medical Sciences. Noninvasive diagnostic methods including ultrasonography, CT, serum markers of liver function and fibrosis, and HBV DNA were performed and compared with histological fibrotic changes in order to establish a noninvasive method for detecting the degree of liver fibrosis.

RESULTSThe total score of liver surface, edge, parenchyma echogenicity, intrahepatic vessels, and the size of spleen had a coefficient of 0.822 with fibrotic stage. By receiver operating curve (ROC) analysis, the sensitivity to distinguish cirrhosis from CHB was 86.1% and the specificity was 95.5% if the total ultrasonic score was more than 10. The CT imaging diagnosed liver cirrhosis with a specificity of 100% and a sensitivity of 48.5%. The change of CT values in cirrhotic patients was lower than that in controls and no cirrhotic patients (F=5.805, P<0.01), when the voltage was increased from 100 KV to 140 KV. Except normal controls and S1 group, S2 and S3 group, the level of HA and collagen IV between the other groups were statistically different. The cut-off value of HA to diagnose cirrhosis was 108 (microg/L) with a sensitivity of 72.2% and a specificity of 80.3%. The cut-off value of collagen IV to diagnose cirrhosis was 188 (microg/L) with a sensitivity of 72.2% and a specificity of 78.8%. When ultrasonography was combined with serum markers, the sensitivity was 72.2% and the specificity was 80.3%.

CONCLUSIONBoth ultrasonography and serum markers are useful to diagnose cirrhosis. The combination of the two examinations is more valuable than any one alone. The characteristic CT imaging has high specificity but low sensitivity in diagnosing early cirrhosis. HA and collagen IV are correlated more closely with the stage of fibrosis, and can reflect the severity of fibrosis.

Adolescent ; Adult ; Aged ; Biomarkers ; blood ; Collagen Type IV ; blood ; Female ; Hepatitis B, Chronic ; diagnostic imaging ; pathology ; Humans ; Hyaluronic Acid ; blood ; Liver Cirrhosis ; diagnostic imaging ; pathology ; Male ; Middle Aged ; Sensitivity and Specificity ; Ultrasonography

BACKGROUNDWe hypothesize that increased atrial oxidative stress and inflammation may play an important role in atrial nerve sprouting and heterogeneous sympathetic hyperinnervation during atrial fibrillation (AF). To test the hypothesis, we examined whether the antioxidant and anti-inflammatory treatment with probucol attenuates atrial autonomic remodeling in a canine model of AF produced by prolonged rapid right atrial pacing.

METHODSTwenty-one dogs were divided into a sham-operated group, a control group and a probucol group. Dogs in the control group and probucol group underwent right atrial pacing at 400 beats per minute for 6 weeks, and those in the probucol group received probucol 1 week before rapid atrial pacing until pacing stopped. After 6-week rapid atrial pacing, general properties including left atrial structure and function, atrial hemodynamics and the inducibility and duration of AF were measured in all the groups. Atrial oxidative stress markers and serum C-reactive protein (CRP) concentration were estimated. The degree of nerve sprouting and sympathetic innervation at the right atrial anterior wall (RAAW) and the left atrial anterior wall (LAAW) were quantified by immunohistochemistry, atrial norepinephrine contents were also detected. Atrial beta-nerve growth factor (beta-NGF) mRNA and protein expression at the RAAW and LAAW were assessed by real-time quantitative RT-PCR and Western blotting respectively.

RESULTSAtrial tachypacing induced significant nerve sprouting and heterogeneous sympathetic hyperinnervation, and the magnitude of nerve sprouting and hyperinnervation was higher in the RAAW than in the LAAW. Atrial beta-NGF mRNA and protein levels were significantly increased at the RAAW and LAAW, and the upregulation of beta-NGF expression was greater at the RAAW than at the LAAW in the control group. The beta-NGF protein level was positively correlated with the density of sympathetic nerves in all groups. Probucol decreased the increase of CRP concentration and attenuated atrial oxidative stress caused by atrial tachypacing. In addition, probucol could effectively inhibit atrial beta-NGF upregulation, significantly attenuate atrial nerve sprouting and heterogeneous sympathetic hyperinnervation, and dramatically reduce the inducibility and duration of AF.

CONCLUSIONSThe atrial over-expression of beta-NGF possibly caused by increased oxidative stress and inflammation may be the main mechanism underlying atrial autonomic remodeling during AF. Probucol attenuates atrial autonomic remodeling possibly by its antioxidant and anti-inflammatory actions.

Animals ; Antioxidants ; therapeutic use ; Atrial Fibrillation ; drug therapy ; Blotting, Western ; C-Reactive Protein ; metabolism ; Cardiac Pacing, Artificial ; adverse effects ; Disease Models, Animal ; Dogs ; Electrocardiography ; Female ; Heart Atria ; Immunohistochemistry ; Male ; Nerve Growth Factor ; genetics ; metabolism ; Norepinephrine ; metabolism ; Probucol ; therapeutic use ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUNDTo explore the pathological features and pathogenesis of severe acute respiratory syndrome (SARS) to provide evidence for the clinical treatment and prevention of SARS.

METHODSPathological features of 2 cases of full autopsy and 4 cases of needle biopsy tissue samples from the patients who died from SARS were studied by light and electron microscopy. The distribution and quantity of lymphocyte subpopulations in the lungs and immune organs from SARS patients were analyzed by immunohistochemistry. The location and semi-quantitative analysis of SARS coronavirus in the tissue specimens were studied by electron microscopy, in situ hybridization and immunohistochemistry.

RESULTSIn total of 6 cases, diffuse alveolar damage and alveolar cell proliferation were common. The major pathological changes of 2 autopsy cases of SARS in lung tissues were acute pulmonary interstitial and alveolar exudative inflammation, and 2 autopsy and one biopsy lung tissues showed alveolar hyaline membrane formation. Terminal bronchiolar and alveolar desquamation of lung tissues in one autopsy and 2 biopsy cases were noted. Among 6 cases, 2 biopsy cases presented early pulmonary fibrosis and alveolar organization. Meanwhile, the immune organs, including lymph nodes and spleens from 2 autopsy cases of SARS whose disease courses were less than 12 days showed extensive hemorrhagic necrosis, reactive macrophage/histocyte proliferation, with relative depression of mononuclear and granulocytic clones in the bone marrows. However, spleen and bone marrow biopsy tissue samples from 4 dead SARS cases whose clinical course lasted from 21 to 40 days presented repairing changes. SARS coronaviruses were mainly identified in type I and II alveolar epithelia, macrophages, and endothelia; meanwhile, some renal tubular epithelial cells, cardiomyocytes, mucosal and crypt epithelial cells of gastrointestinal tracts, parenchymal cells in adrenal glands, lymphocytes, testicular epithelial cells and Leydig's cells were also detected by electron microscopy combined with in situ hybridization. The semi-quantitative analysis of lymphocyte subpopulations revealed that the proportion of CD8+ T lymphocytes were about 80% of the total infiltrative inflammatory cells in the pulmonary interstitium, with a few CD4+ lymphocytes CD3+, CD4+, CD8+ or CD20+ lymphocyte subpopulations were obviously decreased and there was imbalance in number and proportion, while CD57+, CD68+, S-100+ and HLA-DR+ cells were relatively increased in lymph nodes and spleens.

CONCLUSIONSHistologically, the pulmonary changes could be divided into acute inflammatory exudative, terminal bronchiolar and alveolar desquamative and proliferative repair stages or types during the pathological process of SARS. SARS coronavirus was found in multi-target cells in vivo, which means that SARS coronavirus might cause multi-organ damages which were predominant in lungs. There were varying degrees of decrease and imbalance in number and proportion of lymphocyte subpopulations in the immune organs of the patients with SARS. However, these changes may be reversible. It was found that cellular immune responses were predominant in the lungs of SARS cases, which might play an important role in getting rid of coronaviruses in infected cells and inducing immune mediated injury.

Aged ; Female ; Humans ; Lung ; immunology ; pathology ; virology ; Lymphocyte Subsets ; immunology ; Male ; Middle Aged ; SARS Virus ; isolation & purification ; ultrastructure ; Severe Acute Respiratory Syndrome ; immunology ; pathology ; virology