藏医《萨滞布病隆滞布型(缺血性脑梗塞)诊断量表》条目的初步构建

目的基于藏医临床基础理论研制《藏医萨滞布病隆滞布型(缺血性脑梗塞)诊断量表》。方法检索藏医、中医及现代医学有关缺血性脑梗塞诊断的医学专著、国家及行业标准、中国知网期刊数据库1998年至今收录的相关文献,提取"萨滞布病隆滞布型(缺血性脑梗塞)"的临床诊断信息,建立该病临床表现数据库。对374份病例进行回顾性调查,应用频次分析方法对采集的临床数据进行分析;将梳理出来的症状、体征进行藏医专家咨询、赋权。结果藏医萨滞布病隆滞布型(缺血性脑梗塞)诊断常用症状、体征共79个,经专家赋权评分后,最终纳入量表条目为27个,涉及肢体表现、头面部、意识、二便、语言功能、其他、舌诊、脉诊、尿诊9个方面。结论初步构建了《藏医萨滞布病隆滞布型(缺血性脑梗塞)诊断量表》的条目。

THE INHIBITORY EFFECT OF ASTRAGALUS MEMBRANACEUS ON COXSACKIE B－3 VIRUS RNA REPLICATION

Using mice infected with coxsackie B-3 virus (CVB3) as a viral myocarditis model, we observed the inhibitory effect of Astragalus membranaceus (AM) on CVB3-RNA replication in myocardial tissue of mice by RNA-RNA in situ hybridization with negative-strand RNA Probes labelled with ￣(35)S and quantitative imaging analysis of positive signals. The mechanism of its effect on CVB3-RNA replication has been investigated by detection of beta-interferon (β-IFN) as well. Results showed that the copy numbers of CVB3-RNA as well as the histologic scores (necrosis) in myocardial tissues of infected-AM treated mice were significantly lower than those in infected and normal saline treated mice, suggesting that AM could inhibit the replication of CVB3-RVA,but its effect on CVB3-RNA replication had no correlation with induction of β-IFN.

Hand Foot and Mouth Disease Due to Enterovirus 71 in Malaysia

Hand foot and mouth disease is a febrile sickness complex characterized by cutaneous eruption (exanthem) on the palms and soles with simultaneous occurrence of muco-cutanous vesiculo-ulcerative lesions (enanthem) affecting the mouth. The illness is caused by a number of enteroviruses with coxsackievirus A16 and enterovirus 71 as the main causative agents. Human enterovirus 71 (EV71) belongs to the species Human enterovirus A under the genus Enterovirus within the family Picornaviridae. EV71 has been associated with an array of clinical diseases including hand foot and mouth disease (HFMD), aseptic meningitis, encephalitis and poliomyelitis-like acute flaccid paralysis. A large outbreak of HFMD due to highly neurovirulent EV71 emerged in Malaysia in 1997, and caused 41 deaths amongst young children. In late 2000, a recurrence of an outbreak of HFMD occurred in Malaysia with 8 fatalities in peninsular Malaysia. Outbreak of HFMD due to EV71 recurred in 2003 with an unknown number of cases and mortalities. A similar outbreak of HFMD with 2 recorded deaths in young children occurred in peninsular Malaysia in late 2005 and this was followed by a larger outbreak in Sarawak (Malaysian Borneo) with 6 reported fatalities in the early part of 2006. The current on-going outbreak of HFMD started in peninsular Malaysia in epidemiological week 12 of 2010. As with other HFMD outbreaks in Malaysia, both EV71 and CA16 were the main aetiological viruses isolated. In similarity with the HFMD outbreak in 2005, the isolation of CA16 preceded the appearance of EV71. Based on the VP1 gene nucleotide sequences, 4 sub-genogroups of EV71 (C1, C2, B3 and B4) co-circulated and caused the outbreak of hand, foot and mouth disease in peninsular Malaysia in 1997. Two sub-genogroups (C1 and B4) were noted to cause the outbreak in 2000 in both peninsular Malaysia and Sarawak. EV71 of sub-genogroup B5 with smaller contribution from sub-genogroup C1 caused the outbreak in 2003. In the 2005 outbreak, besides the EV71 strains of sub-genogroup C1, EV71 strains belonging to sub-genogroup B5 were isolated but formed a cluster which was distinct from the EV71 strains from the sub-genogroup B5 isolated in 2003. The four EV71 strains isolated from clinical specimens of patients with hand, foot and mouth disease in the Sarawak outbreak in early 2006 also belonged to sub-genogroup B5. Phylogenetic analysis of the VP1 gene suggests that the EV71 strains causing the outbreak in Sarawak could have originated from peninsular Malaysia. Epidemiological and molecular data since 1997 show the recurrence of HFMD due to EV71 in Malaysia every 2 to 4 years. In each of the past outbreaks, more than one sub-genogroup of the virus co-circulate.

Experimental infection of tree shrews(Tupaia belangeri) with Coxsackie virus A16

Coxsackie virus A16(CA16) is commonly recognized as one of the main human pathogens of hand-foot-mouth disease(HFMD). The clinical manifestations of HFMD include vesicles of hand, foot and mouth in young children and severe inflammatory CNS lesions. In this study, experimentally CA16 infected tree shrews(Tupaia belangeri) were used to investigate CA16 pathogenesis. The results showed that both the body temperature and the percentages of blood neutrophilic granulocytes / monocytes of CA16 infected tree shrews increased at 4-7 days post infection. Dynamic distributions of CA16 in different tissues and stools were found at different infection stages. Moreover, the pathological changes in CNS and other organs were also observed. These findings indicate that tree shrews can be used as a viable animal model to study CA16 infection.

Replication Kinetics of Coxsackievirus A16 in Human Rhabdomyosarcoma Cells

Coxsackievirus A16(CVA16),together with enterovirus type 71(EV71),is responsible for most cases of hand,foot and mouth disease(HFMD) worldwide.Recent findings suggest that the recombination between CVA16 and EV71,and the co-circulation of these two viruses may have contributed to the increase of HFMD cases in China over the past few years.It is therefore important to further understand the virology,epidemiology,virus-host interactions and host pathogenesis of CVA16.In this study,we describe the viral kinetics of CVA16 in human rhabdomyosarcoma(RD) cells by analyzing the cytopathic effect(CPE),viral RNA replication,viral protein expression,viral RNA package and viral particle secretion in RD cells.We show that CVA16 appears to first attach,uncoat and enter into the host cell after adsorption for 1 h.Later on,CVA16 undergoes rapid replication from 3 to 6 h at MOI 1 and until 9 h at MOI 0.1.At MOI 0.1,CVA16 initiates a secondary infection as the virions were secreted before 9 h p.i.CPE was observed after 12 h p.i.,and viral antigen was first detected at 6 h p.i.at MOI 1 and at 9 h p.i.at MOI 0.1.Thus,our study provides important information for further investigation of CVA16 in order to better understand and ultimately control infections with this virus.

Expression of Coxsackievirus and Adenovirus Receptor in Human Lung Cancer： Possible Clinical Significance

OBJECTIVE To explore the relationship between CAR and the development of human lung cancer, as well as to provide the basis for the clinical treatment of lung cancer using an adenovirus vector-based gene therapy. METHODS CAR expression was assessed immunohisto- chemically in tumoral, paraneoplastic and normal samples from 112 lung cancer patients. At the same time, the mRNA and protein expression of CAR in 32 cases were determined by RT-PCR and Western blot. The relationship between CAR expression and clinicopathologic parameters was statistically analyzed. RESULTS There was no expression of CAR in normal lung tissue but a little in paraneoplastic tissue. The positive rate was 43% in squamous cell carcinoma, and 70% in adenocarcinoma. Both were much significantly higher than that in paraneoplastic tissue. The CAR expression level in adenocarcinoma was higher than that in squamous cell cancer, mRNA expression by RT-PCR and protein expression by Western blot were consistent with immunohistochemistry results. CONCLUSION CAR is overexpressed in human lung cancer, especially in adenocarcinoma. This data offer the reliable basis for adenovirus-mediated gene therapy of lung cancer; more important, CAR may take part in the formation or development of lung cancer; this may be exploitable for the development of antibody-directed therapy in human lung cancer.

5-Fluorouracil-related enhancement of adenoviral infection is Coxsackievirus-adenovirus receptor independent and associated with morphological changes in lipid membranes

AIM： To evaluate the mechanism underlying the effects of 5-Fluorouracil （5-FU） on adenoviral infection. METHODS： Low and high Coxsackievirus-Adenovirus Receptor （CAR） expressing human colon carcinoma cell lines were treated with 5-FU and two El-deleted adenoviral constructs, one transferring GFP （Ad/CMV- GFP） the other bax （Ad/CEA-bax）. The number of infected cells were monitored by GFP expression. To evaluate the effects of 5-FU in a receptor free system, Ad/GFP were encapsulated in liposomes and treated with 5-FU. Ad/GFP release was estimated with PCR and infection of 293 cells with the supernatant. Electron microscopy of the Ad5-GFP-liposome complex was made to investigate morphological changes of the liposomes after 5-FU. RESULTS： Infection rates of all cell lines increased from 50% to 98% with emerging 5-FU concentrations. The enhanced viral uptake was independent of the CAR expression. Additionally, 5-FU treated liposomes released 2-2.5 times more adenoviruses. Furthermore, 5-FU- treated liposomes appeared irregular and porous-like. CONCLUSION： adenoviral uptake is enhanced in the presence of 5-FU irrespective of CAR and is associated with morphological changes in membranes making the combination of both a promising option in gene therapy.

Coalition of DNA polymorphisms of ApoB and ApoAI genes is related with coronary artery disease in Kazaks

Objective To explore the relationship between polymorphisms of XbaI and MspI loci of apolipoprotein B （ApoB） gene and -75 bp,＋83 bp loci of apolipoprotein AI （ApoAI） gene and coronary heart disease （CHD） in Kazaks of Xinjiang Uyghur Autonomous Region,China.Methods These loci were analyzed by PCR-restriction fragment length polymorphism （PCR-PFLP）.Two hundred and five patients with CHD and two hundred and thirty six controls were involved.Results There were significant distinctions among low-density lipoprotein cholesterol （LDL-C）,triglyceride （TG） and the ApoAI/ApoB ratio between the two groups,but no significant distinction among the polymorphism frequencies of the four sites between the two groups.The polymorphism coalition frequency of X-/Ms＋＋/M1＋-/M2＋＋ （named Coalition 11） was significantly higher in CHD compared to the control group （14.6％ vs.7.2％,P ＜ 0.05）.The level of total cholesterol （TC） in Coalition 1 1 was significantly higher and the level of the ApoAI/ApoB ratio in Coalition 11 was significantly lower than Coalition 1～10 in CHD patients.The level of the ApoAI/ApoB ratio of Coalition 11 was significantly lower than the Coalition 1～10 in control group.The levels of ApoAI/ApoB ratio of Coalition 3 were significantly higher compared to Coalition 11 in the two groups,respectively.The level of LDL-C of Coalition 3 was significantly lower than in the Coalition 11 in control group.The level of TC of Coalition 5 was significantly higher than Coalition 3 in the CHD group.The level of the ApoAI/ApoB ratio of Coalition 5 was significantly lower than in Coalition 3 or Coalition 1～10 of the two groups,respectively.The level of LDL-C of Coalition 5 was significantly higher than in Coalition 3 in control group.The ratio of ApoAI/ApoB was negatively related to TC,LDL-C and was positively related to HDL-C,both in CHD and control groups.Conclusion Coalition 11 of the 4 loci polymorphisms of the ApoB and ApoAI genes was correlated with CHD in Kazaks,and perhaps the ratio of ApoAI/ApoB was the most diagnostic parameter related with CHD among all lipid parameters.CHD may also be associated with Coalition 5,and,perhaps,Coalition 3 may have been confirmed as a protection factor against CHD,if more samples were enrolled.

EXPRESSION OF PERFORIN AND FAS LIGAND IN INFILTRATING CELLS OF MURINE MYOCARDIUM INFECTED WITH COXSACKIEVIRUS B3

Purpose. To clarify the role of perforin-and Fas ligand (L)-mediated cytotoxicity pathogenesis of viral myocarditis. Materials and methods. Forty balb/c mice were randomly divided into experimental group (n = 20) and control group (n = 20), and inoculated intraperitoneally with coxsackievirus B3(CVB3) and Eagle’s solu- tion without CVB3, respectively. The mice were sacrificed and their hearts were removed at day 7 post-in- oculation. Expression of perform and FasL were detected with immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Results. (1)Perform-and FasL-positiye cells were demonstrated in experimental murine hearts by im- munohistochemistry, however, no cells were discovered in control murine hearts; (2) The examination of RT-PCR showed the positive ratios of perform and FasL mRNA in myocardium were significantly higher in experimental group (100% and 100 % ) than that in control group (20% and 30 %, P<0.05); (3)Positive signals of perform and FasL mRNA were found in myocardium of all the experimental mice by in situ hybridization, but nothing was detected in control group. Conclusion. Perform and FasL can be expressed in infiltrating cells in murine myocardium with acute myocarditis caused by CVB3, suggesting perform and FasL might play an important role in pathogenesis of viral myocarditis.

Expression and Significance of Coxsackie and Adenovirus Receptor in Renal-Cell Carcinoma

OBJECTIVE To investigate the expression of Coxsackie and Adenovirus receptor （CAR） in renal-cell carcinoma and the relationship of the CAR to the biological behavior of the carcinomas. METHODS The immunohistochemical SP method was used to detect the expression of Coxsackie and Adenovirus receptor in 48 cases of renal- cell carcinoma and in 12 cases of normal renal tissue 2 cm away from the tumor tissue. RESULTS The positive rates of CAR were 100% in 12 cases of para-tumor normal renal tissue and 35.4% in 48 cases of renal-cell carcinoma respectively. The difference of CAR expression between them was significant （P〈0.05）. The grades of the tumor were as follows： 22 in Grade Ⅰ, 17 in Grade Ⅱ and 9 in Grade Ⅲ with the CAR positive rate being 54.5%, 23.5% and 11.1%, respectively. There was a negative correlation between CAR expression and tumor grading （P〈0.05）. In addition, the number of the cases in stages I to IV were 19, 13, 11 and 5 respectively, with the respective positive rates being 57.9%, 30.8%, 18.2% and 0.0%, i.e. there also was a negative relationship between CAR expression and the stage （P〈0.05）. CONCLUSION CAR expression is down-regulated in renal-cell carcinoma compared with normal tissue. The level of CAR may be a sensitive predictor of differentiation, invasion and metastasis. Loss of CAR expression correlates with the invasive phenotype in our analysis of renal-cell carcinoma.

EFFECT OF VERAPAMIL ONCa^（2＋） INFLUX ANDCVB3－RNA REPLICATION IN CULTURED NEONATALRAT HEART CELLS INFECTED WITH CVB3

The effect of verapamil on Ca2+ influx across the myocardial plasma membrane and coxsackie virus B3 ( CVB3)-RNA replication in cultured neonatal rat heart cells infected with CVB3 was investigated. It was found that the Ca2+ influx could be inhibited significantly (P<O. 01) by verapamil (1 μmol/L) after infection of heart cells for 48h. However, when the cultured heart cells infected with CVB3 and treated with verapamil (Iμmol/L and 10 nmo/L) at the same time for 48h, the amounts of CVB3-RNA in myocytes were significantly higher than that in infected control group (P<O. 05). These phenomena suggest that the increase of Ca2+ influx of cultured heart cells infected with CVB3 could be inhibited by some calcium antagonists, e. g. verapamil at the early stage. On the other hand, verapamil might accelerate viral replication in myocardium. Thus, although verapamil could be beneficial for decreasing the secondary Ca2+ damages and improve the myocardial electric activity, it isn’t a sensible choice for therapy in early stage of virus infection with cardiac symptoms.

EFFECT OF COXSACKIEVIRUS B3 ON ION CHANNEL CURRENTS IN RAT VENTRICULAR MYOCYTES

To investigate the effects of coxsackievirus B 3(CVB 3) on ion channel currents in rat ventricular myocytes. Methods.Rat hearts were isolated with collagenase to acquire single ventricular myocytes, L type voltage dependent calcium channel(VDCC)current (I Ca ),Na + current (I Na ), outward potassium current (I out ), inwardly rectifying potassium current(I KI ) were recorded using whole cell patch clamp techniques. ResultsCVB 3 infection increased I Ca and I out , while decreased I KI ; but it had no obvious effect on I Na . Conclusion.The effects of CVB 3 on I Ca 、 I out 、 I KI may be one of the mechanisms of myocytes damage and the occurrence of abnormal electroactivities induced by CVB 3 infection.

Epstein-Barr virus-associated gastric carcinoma in Kazakhstan

AIM: To investigate the incidence of Epstein-Barr virus-associated gastric cancer (EBV-GC) in Kazakhstan and to compare it with that in Russia, Western and Asian countries in order to evaluate the significance of epidemiopathologic and ethnic factors. METHODS: In situ hybridization (ISH) of EBV-encoded small RNA-1 (EBER-1) was used to identify the presence of EBER-1 signal in 139 formalin-fixed and paraffin-embedded GC tissues from Kazakhstan. RESULTS: EBER-1 expression was observed in the nuclei of 10% of the cases of GC (14/139), but not in the surrounding normal mucosa. The incidence of the diffuse type of EBV-GC was significantly higher in Kazakhstan (14%, 13/91) than that of the intestinal type (2%, 1/48). Furthermore, the incidence was significantly higher in males (14%, 12/89) than in females (3.7%, 2/53) from all countries. The overall incidence of EBV-GC increased from 6.7% in Asian countries to 8.7% in Russia, 10.1% in Kazakhstan and 16% in Western countries. CONCLUSION: Geographical differences in the incidence of EBV-GC may reflect the epidemiologic factors and/or dietary habits independent of histological type and sex.

Expression of coxsackie and adenovirus receptor in small cell lung cancer

Objective: We explored the expression of coxsackie and adenovirus receptor (CAR) in small cell lung cancer (SCLC) tissue. Methods: CAR expression in 31 SCLC was assessed in formaldehyde-fixed, paraffin-embedded tissue according to the EnVision immunohistochemistry procedure, while 3 samples of surgical specimens of non-malignant lung disease were taken as the negative control. Results: We observed that the expression of CAR was detectable positive in all the 31 cases from the small cell lung cancer tissue, in contrasting that non-malignant lung tissue control. Conclusion: The high expression of CAR appeared in SCLC tissue indicates that it play an important role in of adenovirus vector-based gene therapy in SCLC.

PROTECTIVE ROLE OF SOPHOCARPINE IN COXSACKIEVIRUS B3 INFECTION IN CULTURED RAT CARDIOMYOCYTES

Objective To observe the anti-CVB3 （ Coxsackievirus B3 ） effect of sophocarpine （SC） extracted from Sophora flavescens, a traditional Chinese herb in vitro. Methods Cardiomyocytes from the neonatal rat were cultured to establish the viral myocarditis model The cells were divided into four groups： infected group （ infected by CVB3 ） , SC treated group （ added SC 100 μg/mL after viral infection ）, SC control group （ added SC 100 μg/mL only）, and normal control group. The cytopathic effect （CPE） and the beating frequency of the myocardial cells were observed and the LDH levels in the supernatant were measured at day 2,3, and 5. The cultured myocytes were added different concentrations of SC （ 12. 5 -400 μg/mL ） after infection with CVB3, the CPE was observed and the concentrations of LDH were measured and compared at day 2, 3, and 5. Results In the SC treated group （ 100 μg/mL ） , the cytopathic effect was lighter and the LDH level was lower than the infected group. SC in a concentration of 12. 5 - 300 μg/mL could relieve the CPE and lower the LDH level, while in a higher concentration （400 μ/m ） , it exacerbated the CPE caused by the virus, and the LDH levels were higher than the infected cells. Conclusion SC in certain concentration could protect the cultured rat cardiomyocytes from CVB3 infection.

ROLE OF COXSACKIEVIRUS AND ADENOVIRUS RECEPTOR (CAR)IN CARDIOTOXICITY INFECTED BY COXSACKIEVIRUS B3

Objective To explore the role of coxsackievirus and adenovirus receptor（CAR） in cardiotoxicity infected by coxsackieviras B3. Methods A toxic cellular model was established in vitro by adding myocarditic coxsackievirus B3 （CVB3m） into the culture of neonatal mouse cardiomyocytes. 48 h later, the cardiomyocytes were divided into control, CVB3m, and CAR antibody ＋ CVB3m groups. CVB3m-mediated myocytopathic effect of above three groups was observed after further culturing for 48h. At the same time, the cardiomyocytes＇ viability of above three groups was assessed by MTT assay. Results The degree of cytopathic effect（CPE） of CAR antibody ＋ CVB3m group was significantly lower than CVB3m group （ P 〈 0. 01 ） and there was a significant increase in cell viability in CAR antibody ＋ CVB3m group compared with CVB3m group（ P 〈 0. 01 ）. No significant difference was found between CAR antibody ＋ CVB3m group and control group. Conclusion CAR antibody possesses a protective effect on CVB3m infected cardiomyoctyes, which indicates that CAR may play an important role in mediating cardiotoxicity infected by CVB3m.

Characterization of Pigeon Paramyxoviruses (Newcastle disease virus) Isolated in Kazakhstan in 2005

Isolates of Newcastle disease virus (NDV) from deceased wild and domestic pigeons in Kazakhstan were obtained from the Almaty region during 2005 and were genotypically analyzed by using reverse transcription polymerase chain reaction (RT-PCR) with primers specific to the viral fusion (F) protein gene. Part of the amplified F protein DNA product (nucleotide sequence 47-422) and the deduced amino acid sequences were compared phylogenetically with those from strains previously reported in other geographic regions. Phylogenetic analysis indicated that the Kazakhstanian pigeon paramyxovirus type 1 (PPMV-1) isolates belong to genotype VI or 4bii. To our knowledge, this is the first reported VI isolates that possess the sequences of 112 GKRQKR116* F117 within the F0 protein. The information is fundamental to improving the efficiency of control strategies and vaccine development for NDV.