Analysis of the human papillomavirus in laryngeal squamous cell carcinoma patients / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyze the infection of human papillomavirus in laryngeal squamous cell carcinoma patients.</p><p><b>METHODS</b>The pathological samples of 64 clinical diagnosed laryngeal squamous cell carcinoma patients were collected. Lunimex and PCR techniques were used to detect the HPV gene infection and immunohistochemistry method was used to analyze the HPV protein expression in the samples.</p><p><b>RESULTS</b>In the 64 cases, 7 were positive for HPV infection by Luminex and PCR tests. 18 were positive for HPV16/18 E6 protein expression. The total positive rate was about 39. 1%.</p><p><b>CONCLUSION</b>The high HPV infection rate in laryngeal squamous cell carcinoma patients in the study indicated indirectly that the importance of the HPV infection in pathogenesis of the laryngeal squamous cell carcinoma.</p>

The analysis of human papillomavirus infection in lip squamous cell carcinoma patients / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyze the infection of human papillomavirus in lip squamous cell carcinoma patients.</p><p><b>METHODS</b>The pathological samples of 9 clinical diagnosed lip cancer patients were collected. Lunimex and PCR techniques were used to detect the HPV gene infection and immunohistochemistry method was used to analyze the HPV protein expression in the samples.</p><p><b>RESULTS</b>In the 9 cases, 1 was positive for HPV16 gene and 7 were positive for HPV16/18 E6 protein expression. The total positive rate was about 8/9.</p><p><b>CONCLUSION</b>The high HPV infection rate in lip cancer patients in the study indicated indirectly that the importance of the direct contact to the infection of HPV what was the basis for pathogenesis of the lip squamous cell carcinoma.</p>

Contamination of live virus during tissue homogenizing by ultrasonic processor and tissue disperser / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To quantitatively evaluate the contamination area and risk of a live pathogen during tissue homogenization by either ultrasonic processor or tissue disperser.</p><p><b>METHODS</b>A recombinant Herpes Simplex Virus (rHSV) containing GFP gene was used as the index virus, and fresh liver tissue from healthy mice was used as simulated specimen. After 10% liver homogenate was mixed with rHSV (100 TCID50/0.1 mL) in a 5 mL tube, the stability of rHSV in liver homogenate and influences of an ultrasonic processor and a tissue disperser on viral infectivity were determined by GFP expressions in cell cultures. The contaminating areas of live viruses during homogenization were evaluated by a cell culture-based sedimentary. The contamination radii were counted by measurement of the distance between the operator and the farthest GFP positive well.</p><p><b>RESULTS</b>The infectivity of rHSV in 10% liver homogenate maintained almost unchanged after it was incubated at room temperature for 30 min. Treatment with an ultrasonic processor clearly dropped down the virus infectivity, while a disperser not. Obvious spills and slashes of live viruses were observed in processes of homogenization with those two apparatuses. The contamination radii are positively related with sample volume, output energy of operator and handling time.</p><p><b>CONCLUSION</b>Homogenizing infectious samples with an ultrasonic processor and a tissue disperser at commonly used conditions caused obvious spills and splashes of live viruses, which possesses high risk to induce Laboratory acquired infections (LAIs).</p>

Establishment of hamster- and human-PRNP transgenic mice / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To create transgenic mice expressing hamster- and human-PRNP as a model for understanding the physiological function and pathology of prion protein (PrP), as well as the mechanism of cross-species transmission of transmissible spongiform encephalopathies (TSEs).</p><p><b>METHODS</b>Hamster and human-PRNP transgenic mice were established by conventional methods. The copy number of integrated PRNP in various mouse lines was mapped by real-time PCR. PRNP mRNA and protein levels were determined by semi-quantitative RT-PCR, real-time RT-PCR, and western blot analysis. Histological analyses of transgenic mice were performed by hematoxylin and eosin (H & E) staining and immunohistochemical (IHC) methods.</p><p><b>RESULTS</b>Integrated PRNP copy number in various mouse lines was 53 (Tg-haPrP1), 18 (Tg-huPrP1), 3 (Tg-huPrP2), and 16 (Tg-huPrP5), respectively. Exogenous PrPs were expressed at both the transcriptional and translational level. Histological assays did not detect any abnormalities in brain or other organs.</p><p><b>CONCLUSION</b>We have established one hamster-PRNP transgenic mouse line and three human-PRNP transgenic mouse lines. These four transgenic mouse lines provide ideal models for additional research.</p>

Interaction between various 14-3-3beta segments and PrP in vitro / 中华实验和临床病毒学杂志

<p><b>UNLABELLED</b>OBJECTIVE To study the potential interaction between PrP protein.</p><p><b>METHODS</b>The supernatant of health and scrapie-infected hamsters' brain homogenate was prepared, while various recombinant 14-3-3beta or PrP proteins were purified. The possible molecular interaction between 14-3-3beta proteins and PrP was tested by pull-down and immunoprecipitation assays.</p><p><b>RESULTS</b>Both native PrP(c) and its protease-resistant isoform (PrP(Sc)) formed complexes with 14-3-3beta. The full-length recombinant 14-3-3beta proteins interacted with PrP. The domain responsible for interacting 14-3-3beta was located at N-terminal of 14-3-3beta (residues 1 to 38).</p><p><b>CONCLUSION</b>The studies of the association of PrP with 14-3-3beta may further provide insight into a potential role of 14-3-3beta in the biological function of PrP and the pathogenesis of prion disease.</p>

Establishment of a stable PrP(Sc) panel from brain tissues of experimental hamsters with scrapie strain 263K / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To establish a stable PrP(Sc) panel from brain tissues of experimental hamsters infected with scrapie agent 263K for evaluating diagnostic techniques of human and animals' prion diseases.</p><p><b>METHODS</b>Thirty brain tissue samples from hamsters intracerebrally infected with scrapie strain 263K and another 30 samples from normal hamsters were selected to prepare 10%, 1%, and 0.5% brain homogenates, which were aliquoted into stocks. PrP(Sc) in each brain homogenate was determined by proteinase K digestions followed by Western blot assay and partially by immunohistochemistry. Stability and glycoforms of PrP(Sc) were repeatedly detected by PrP(Sc)-specific Western blots in half a year and 3 years later.</p><p><b>RESULTS</b>PrP(Sc) signals were observed in all 10% brain homogenates of infected hamsters. Twenty out of 30 stocks and 19 out of 30 stocks were PrP(Sc) positive in 1% and 0.5% brain homogenatesof infected hamsters, respectively. Twenty-seven out of 30 stocks presented three positive bands in 10% brain homogenates, whereas none of 1% and 0.5% homogenates contained 3 bands. The detection of PrP(Sc)-specific signals stored in half a year and 3 years later demonstrated that the ratio of PrP(Sc) positive samples and glycoforms was almost unchanged. All normal hamsters' brain homogenates were PrP(Sc) negative.</p><p><b>CONCLUSION</b>A PrP(Sc) panel of prion disease can be established, which displays reliably stable PrP(Sc)-specific signals and glycoforms.</p>

Studies on heredity rule of the first genealogy regarding fatal familial insomnia in Henan province / 中华流行病学杂志

Objective To investigate the epidemiological,genealogic characteristic,familial history of the families with fatal familial insomnia,its clinical and pathological features as well as the heredity rule of related genes.Methods 135 familial members of 7 eras were studied.Vein blood samples from patients as well as from some familial members were collected.PRNP gene was studied with PCR,its serial was determined and then authenticated with Nsp I.Brain tissue was obtained for neuropathological test and PrPSc test with Western blot method.Results Clinical symptoms of the 2 diagnosed cases were typical.11 familial members died of similar neural disease.32 samples of their familial members,codon at D178N of PRNP of 11 members was mutated,with mutation rate as 34.38% while D129N showed as methionine.Brain tissue of both probands denaturalized into spongiform and the nerve fiber was absent but PrPSc protein was identified.Conclusion Genealogy was described in the family with fatal familial insomnia since the patients had typical clinical symptoms and pathological characteristics.It seemed necessary to confirm cases of fatal familial insomnia and their genealogy with epidemiological data and to investigate its gene characteristics as well as with neuropathological and Western blot tests.

Alternations of tau protein and its phosphorylated profiles in the experimental hamsters infected by scrapie agents 263K and 139A / 病毒学报

In human prion diseases, phosphorylated-tau deposition has been described in a rare genetic form, Gerstmann-Straussler-Scheinker disease, but is not considered part of the neuropathological picture of Creutzfeldt-Jakob disease. To investigate the possible changes of tau and phosphorylated tau (Ser396/Ser404) in transmissible spongiform encephalopathies (TSEs), the expressions and transcriptions of above biological factors in the brain tissues of 263K- and 139A-infected hamsters were evaluated by Western blots and Real Time PCR, respectively, followed by quantitative analyses of immunoblot images and relative transcriptional levels compared with normal animals. The contents of total tau increased, but phosphorylated tau at Ser396 and Ser404 decreased, regardless of the types of scrapie agents and clinical incubations. Transcriptions of two tau isoforms were also markedly increased. These findings suggested that dephosphorylation of tau at Ser396/Ser404 was a illness-correlative phenomenon in TSEs. Alterations of tau and phosphorylated tau (Ser396/Ser404) were either intermediate or consequent events in TSE pathogenesis and proposed the potential linkage of these bioactive proteins with the pathogenesis of prion diseases.

Molecular interaction between PrP protein and the signal protein 14-3-3 beta / 病毒学报

The molecular interaction between PrP and 14-3-3 beta and the possible interactional domain between two proteins were studied by co-immunoprecipitation, pull down and FRET assays. The results showed that PrP protein could interact with 14-3-3 beta in vitro and in vivo. The domain which responded for the interaction was located at C-terminal of PrP (amino acid residues 106 to 126). This study of the interaction between PrP and 14-3-3 protein further provided the insight into the potential role of 14-3-3 in the biological function of PrP and the pathogenesis of prion disease.

Preparations of the specific antibodies against exon 2 and exon 3 of human tau protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare the specific antibodies against exon 2 and exon 3 of human tau protein.</p><p><b>METHODS</b>Sequences encoding exon 2 and exon 3 of human tau protein were amplified from human peripheral blood DNA and cloned into a prokaryotic expression vector pGEX-2T. Fusion proteins GST-E2 and GST-E3 were expressed and purified from E. coli system. The antisera were elicited by immunization of the purified fusion proteins to rabbits and mice. The specific antibodies were purified by Protein G/A and CNBr-activated sepharose 4B coupled with GST protein. The specificity and sensitivity of the purified antibodies were evaluated by Western blotting and ELISA.</p><p><b>RESULTS</b>Recombinant fusion proteins GST-E2 and GST-E3 were expressed and purified from E. coli, which showed Mr. 29 x 10(3). Various antisera were collected from the immunized experimental animals. Reliable immunoreactive specificity and titers of the purified antibodies against exon 2 and exon 3 of human tau protein were confirmed by Western blotting and ELISA after serial purification processes.</p><p><b>CONCLUSION</b>Four specific antibodies against exon 2 and exon 3 of human tau protein have been successfully prepared, which provides basis for analyzing the role of tau in neurodegenerative diseases.</p>

Study on the characteristics of patients with Creutzfeldt-Jakob disease under 2008 surveillance data in China / 中华流行病学杂志

Objective To describe the epidemiological and clinical characteristics of Creutzfeldt-Jakob disease (CJD) in China. Methods Clinical and epidemical data on patients from China CJD surveillance network was analyzed. Blood and cerebral spinal fluid (CSF) specimens from these patients were collected. Western blot assay was used to detect 14-3-3 protein in CSF, PCR and sequencing assay were used for analyzing the polymorphism of 129 amino acid and mutation of PRNP gene. Results A total number of 31 probable and 11 possible sporadic CJD patients were identified. Additionally, one patient with Gerstmann-Straussler-Scheinker syndrome (GSS) and 2 familial CJD cases were identified. No geographic- or occupational-related events were observed among these cases. The mean age of onset on the probable or possible CJD patients were 56.7 and 57.4 years old, with sex ratios of the probable CJD patients as 8:9 and the possible one as 5:6 respectively. Rapid progressive dementia was the main foremost symptom, presenting in 33.3% of the CJD patients. Probable CJD patients showed more clinical manifestations than those possible ones. Conclusion Geography distribution, occupation, ratio of gender and the mean onset age of the CJD eases in 2008 were consistent with the characteristics of the sporadic CJD.

Establishment of an assay for PrP(Sc) detection based on streptomycin precipitation / 病毒学报

To establish a new Western blotting assay for PrP(Sc) detection, we optimized the Western blotting assay with a precipitation procedure of streptomycin sulfate. After digestion with PK, 10% scrapie infected hamster brain homogenates were incubated with 60 mmol/L streptomycin and the precipitated PrP(Sc) was recovered by centrifugation. The enrichment of PrP(Sc) by streptomycin sulfate precipitation was evaluated using Western blotting assay. The results showed streptomycin could bind to PK-treated PrP(Sc), forming high molecular masses, but not influence the glycosylated patterns on SDS-PAGE. Western blot assay revealed that the detective sensitivity of the streptomycin-precipitation PrP(Sc) was remarkably improved. As a sensitive, specific, rapid and flexible protocol for PrP(Sc), the protocol in this study has the potential utility, alone or combined with other techniques, for the detection of low level PrP(Sc) in the specimens from central nerve system, or from peripheral organs or body fluids.

Establishment of PrP(Sc) conversion based on serial PMCA in vitro / 病毒学报

In order to establish an amplification system in vitro with which the PrP(Sc) is able to convert PrP(C) into proteinase K-resistant isoform infinitely and whether this system is more efficient than conventional protein misfolding cyclic amplification (PMCA), scrapie strain 263K-infected hamster's brain homogenate and homologous normal brain homogenate were prepared, respectively. A new methodology, namely serial PMCA, was utilized to reveal the continuous propagation ability of PrP(Sc). Totally 8 rounds of serial PMCA were proceeded and each round contained 48 cycles of alternative sonication and incubation. Simultaneously 144 cycles of conventional PMCA was used as a control. The results showed the PrP(Sc) from hamsters' brain tissues of scrapie agent 263K could replicate efficiently and infinitely with serial PMCA compared with finite propagation of PrP(Sc) with conventional PMCA system. The study of PrP(Sc) continuous propagation in brain homogenate with serial PMCA may further provide insight into the unsettled mechanism of prions misfolding and replication and apply to detect trace amount of PrP(Sc).

Establishment of a sandwich ELISA method for detection of vascular endothelial growth factor in serum samples of hepatocellular carcinoma patients / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To establish a sandwich ELISA method for detecting vascular endothelial growth factor (VEGF) in sera of population and the patients with hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Full length and two truncated human VEGF cDNA sequences were amplified from a commercial plasmid pBLAST49-hVEGF by PCR and inserted into the prokaryotic-expression plasmid pET-32a or pGEX-2T. Various VEGF proteins were expressed and purified from E. coli in His-Trx or GST fusion forms. The specific VEGF antibodies were elicited in experimental rabbits and mice by immunization of the full length VEGF fusion protein His-Trx-VEGF1-165. After purification of antibodies with chromatograph of Protein G, a sandwich ELISA technique was established. Serum VEGF levels were evaluated in 229 adults and 291 HCC patients.</p><p><b>RESULTS</b>SDS-PAGE displayed that the molecular weights of the expressed full length (His-Trx-VEGF1-165), N-terminal (His-Trx-VEGF1-100) and C-terminal (GST-VEGF100-165) human VEGF fusion proteins were about 38KD, 31KD, and 33KD, respectively. Western blots confirmed that the prepared antisera were able to recognize both prokaryoticly and eukaryoticly expressed recombinant VEGF proteins. Assays of serially diluted His-Trx-VEGF1-100 by the established sandwich ELISA method showed that the linear range of the standard curve was 0.625-320 ng/mL, with the squared correlation coefficient R2 = 0.991. Screening of a serum panel containing 291 serum samples of HCC patients and 229 health adults revealed that the average VEGF level in HCC patients was higher than that in healthy controls, with a statically significant difference.</p><p><b>CONCLUSION</b>The established sandwich ELISA reflects the level of serum VEGF and provide scientific basis for screening metastasis and recurrence of HCC using serum VEGF as an index.</p>

Analyses of the expressions of GFAP in the brain tissues of hamsters infected with various amounts of scrapie strain 263K at terminal stage / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate whether gliosis in the brain tissues of the hamsters infected with various amounts of scrapie strain 263K is correlated with the inoculation doses or the incubation times.</p><p><b>METHODS</b>The total values of glial fibrillary acidic protein (GFAP) in brains were evaluated by Western Blots and the GFAP-stained cells were detected by immunohistochemistry (IHC). The characteristics of GFAP distributions among various groups were defined by quantitive and statistic analyses.</p><p><b>RESULTS</b>Compared with the brain tissues of normal hamsters, remarkably higher total GFAP levels and more GFAP-stained cells were observed in the brain tissues of infected ones, howbeit, no significant difference was addressed among the infected groups.</p><p><b>CONCLUSION</b>Inoculations of various amounts of scrapie strain 263K into experimental hamsters intracerebrally induced the similar patterns of gliosis in the brains at the clinically terminal stage, regardless of infectious doses and incubation times.</p>

Establishment of a prion disease PrP(Sc) panel from the brain tissues of experimental hamsters infected with scrapie agent 263K / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a prion disease PrP(Sc) panel from the brain tissues of experimental hamsters and to address the stability of the panel conserved under the specific condition, for evaluating the diagnostic techniques of human and animal's prion diseases.</p><p><b>METHODS</b>30 brain tissues of hamsters infected with scrapie strain 263K intracerebrally and 30 ones of normal hamsters were enrolled in this panel. Each brain sample was prepared to 10%, 1% and 0.5% homogenates and aliquoted into stocks. The presences of PrP(Sc) in each brain sample were evaluated with PrP-specific Western Blots and partially with immunohistochemistry, and the stability of PrP(Sc) signals in each sample were repeatedly assessed half a year later and 3 years later.</p><p><b>RESULTS</b>PrP(Sc) signals were detected in all stocks of 10% brain homogenates from infected hamsters, 26 out of 30 stocks of 1% homogenates and 19 out of 30 stocks of 0.5% homogenates. The assessments of PrP(Sc) signals in all samples half-year and three years later demonstrated almost unchanged. All homogenates of brain tissues of normal hamsters were PrP(Sc) negative.</p><p><b>CONCLUSION</b>A prion disease PrP(Sc) panel of the brain tissues, which includes 90 PrP(Sc) positive stocks and PrP(Sc) negative ones, was successfully established, with a reliable stability of PrP(Sc) signals.</p>

Treatment of scrapie pathogen 263K with tetracycline partially abolishes protease-resistant activity in vitro and reduces infectivity in vivo / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To study the possible effect of tetracycline on protease-resistant activity in vitro and infectivity in vivo of a scrapie strain 263K.</p><p><b>METHODS</b>Scrapie pathogens were incubated with tetracycline at different concentrations for various periods of time and protease-resistant PrP signals were evaluated with proteinase K-treatment and Western blots. The preparations treated with tetracycline were intracerebrally inoculated into golden hamsters and typical TSE manifestations were noted. PrPSc in brain tissues of the infected animals was detected by PrP specific Western blot assays.</p><p><b>RESULTS</b>Protease-resistant PrP was significantly reduced in or removed from the preparations treated with tetracycline in a dose-dependant manner. Compared with the control group after incubated for 53.75 +/- 0.50 days, the preparations treated with 5 mmol/L and 20 mmol/L tetracycline prolonged the incubation time of 61.5 +/- 1.73 and 59.5 +/- 0.58 days (P < 0.05).</p><p><b>CONCLUSION</b>Treatment of scrapie pathogen 263K with tetracycline reduces or removes its protease-resistant activity in vitro.</p>

Establishment of a protein misfolding cyclic amplification for PrPSc / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a methodology of protein misfolding cyclic amplification (PMCA) and utilize in the detection of PrP(Sc) in brain tissues from prion diseases.</p><p><b>METHODS</b>Different amounts of Scrapie 263K agent bulk were mixed with brain homogenates of health hamsters and treated with repeated incubation/sonication for 10 to 15 cycles. The proteinase K-resistant PrP(Sc) was evaluated with Western Blot.</p><p><b>RESULTS</b>In this experimental situation, 263K agent replicated rapidly in vitro, utilizing hamsters' brains as the medium. With the established PrP(Sc)-PMCA technique, PrP(Sc) signals in the preparations containing less than 10(-5) diluted 263K bulk could be detected. Compared with conveniently used immuno-blot assay, the sensitivity of PrP(Sc)-PMCA for PrP(Sc) was 10(5) to 10(6)-fold increased. It has been also shown that homogenates of cerebellar and brain stem could be used as the medium for PrP(Sc) replication.</p><p><b>CONCLUSION</b>A rapidly replicating method for PrP(Sc), PrP(Sc)-PMCA, was successfully established, providing a new approach for early diagnosis of prion diseases and research on the biological features of prion.</p>

Study on the characteristic of Surveillance Creutzfeldt-Jakob disease patients from January to August in 2006 in China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To describe the incidence condition and characteristic of Creutzfeldt-Jakob disease (CJD) in China.</p><p><b>METHODS</b>The clinical and epidemical information of patients from China CJD surveillance network was analyzed. Blood and cerebrospinal fluid (CSF) specimens from these patients were differently collected to be used to detect the 14-3-3 protein in the CSF and to analyze the PRNP gene.</p><p><b>RESULTS</b>Ten possible and 8 probable clinically diagnosed CJD patients were found. These patients had sporadic CJD. There were no geographic clustering and occupational risk with these patients. The mean age at onset of disease was approximately 60 years. The male to female ratio was approximately 1:1; rapidly progressive dementia was the main early symptom, which was present in 44% of patients.</p><p><b>CONCLUSION</b>The geographic distribution, occupation, the ratio of male to female and the mean age of onset were consistent with the characteristics of sporadic CJD.</p>

Generation and identification of phage engineering antibodies library against hamster prion protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Generation and Identification of Phage Engineering Antibodies Library against Hamster Prion Protein.</p><p><b>METHODS</b>Fab antibodies were identified and confirmed. BALB/c mice were immuned with PrP proteins. After the third immunization, the total RNA was extracted from the mice spleens. The genes of heavy Fd Fragments and light chain of antibodies amplified by a series of specific primers of human IgG Fab fragment were cloned into phagemid vector pComb3.</p><p><b>RESULTS</b>The combinatorial Fab library were constructed successfully and the cloning efficiencies both of light chain and Fd fragments were about near 10(6). The Fab library were panned by four cycles and screened with purified haPrP23-231 antigen on microtiter plates. 12 mAbs were isolated after four cycles of panning, five of which were sequenced and resulted sequence data were analyzed by alignment with GenBank immunoglobulin genes. Two strain of new heavy and light chain genes of Fab antibodies were identified and confirmed.</p><p><b>CONCLUSION</b>The research in this article will provide foundation for study of diagnosis and therapy of prion.</p>