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Biomedical subjects

Ling Zhou

Publications and source records attributed to Ling Zhou.

At least 55 records · Page 3Linked to original sources
PubMed2004

[Immune response induced by HIV DNA vaccine combined with recombinant adeno-associated virus].

OBJECTIVE: HIV-1 DNA vaccine and recombinant adeno-associated virus (rAAV) expressing gagV3 gene of HIV-1 subtype B were constructed and BALB/c mice were immunized by vaccination regimen consisting of consecutive priming with DNA vaccine and boosting with rAAV vaccine; the CTL and antibody response were detected and compared with those induced by DNA vaccine or rAAV vaccine separately. METHODS: HIV-1 subtype B gagV3 gene was inserted into the polyclonal site of plasmid pCI-neo, DNA vaccine pCI-gagV3 was thereby constructed; pCI-gagV3 was transfected into p815 cells, G-418-resistant cells were obtained through screening transfected cells with G418, the expression of HIV-1 antigen in G-418-resistant cells was detected by EIA; BALB/c mice were immunized with pCI-gagV3 and the immune response was tested; BALB/c mouse immunized with pCI-gagV3 and combined with rAAV expressing the same gagV3 genes were tested for antibody level in sera by EIA method and cytotoxicity response by LDH method. RESULTS: pCI-gagV3 could express HIV-1 gene in p815 cells; pCI-gagV3 could induce HIV-1 specific humoral and cell-mediated immune response in BALB/c mice. The HIV-1 specific antibody level was 1/20; when the ratio of effector cells: target cells was 50:1, the average specific cytotoxicity was 41.7%; there was no evident increase in the antibody level induced by pCI-gagV3 combined with rAAV, but there was increase in CTL response, the average specific cytotoxicity was 61.3% when effector cells: target cells ratio was 50:1. CONCLUSION: HIV-1 specific cytotoxicity in BALB/c mice can be increased by immunization of BALB/c mice with DNA vaccine combined with rAAV vaccine.

AIDS Vaccines↗
PubMed2004

[Immune potency of recombinant adeno-associated virus combined with recombinant adenovirus vaccine containing HIV-1 gp120].

OBJECTIVE: To study the immune effect of recombinant adeno-associated virus (rAAV) combined with recombinant adenovirus (rAdV) vaccine in BALB/c mice. METHODS: The codon-modified HIV-1 gp120 gene was inserted into plasmid of adeno-associated virus and adenovirus vector separately. Then the rAAV and rAdV vaccines were constructed. BALB/c mice were immunized with rAAV and rAdV vaccines in different administration scheme. The IgG antibody was detected by ELISA and CTL response was detected by intracellular cytokine stain assay. RESULTS: Both rAAV and rAdV vaccine could express gp120 gene; the mice primed with rAAV at week 0, 2 and boosted with rAdV at week 5, 14 and 20 elicited the strongest gp120 specific CTL and IgG antibody response. CONCLUSION: The mice primed with rAAV and boosted with rAdV could elicit specific CTL response and IgG antibody.

Adenoviridae↗
PubMed2003

Essential role of matrix metalloproteinases in interleukin-1-induced myofibroblastic activation of hepatic stellate cell in collagen.

Located within the perisinusoidal space and surrounded by extracellular matrix, hepatic stellate cells (HSC) undergo phenotypic trans-differentiation called "myofibroblastic activation" in liver fibrogenesis. This study investigated the regulation of interleukin-1 (IL-1alpha) on expression of matrix metalloproteinases (MMPs) by HSC grown in three-dimensional extracellular matrix and the role of MMPs in HSC activation. To recapitulate the in vivo "quiescent" state of HSC, the isolated rat HSC were grown in three-dimensional Matrigel or type I collagen. Stimulation with IL-1alpha caused robust induction of pro-MMP-9 (the precursor of matrix metalloproteinase-9) when HSC were cultured in these matrices. IL-1alpha induced a conversion of the pro-MMP-9 to the active form only when the cells were in type I collagen. In collagen lattices, IL-1alpha provoked activation of HSC with induction of MMP-13, MMP-3, and breakdown of the matrix. The HSC activation was completely prevented by a treatment of the cells with tissue inhibitor of metalloproteinase-1 or deprivation of MMP-9. Once fully activated, HSC failed to express MMP-9 and showed attenuated induction of MMP-13 and MMP-3. Further, we demonstrated colocalization of alpha-smooth muscle actin and MMP-9 in a subpopulation of HSC in human fibrotic liver tissues. Thus, this study provides a novel model to enlighten the role of MMPs, particularly that of MMP-9, in HSC activation regulated by a specific cytokine in liver fibrogenesis.

Animals↗
PubMed2003

Immune monitoring in whole blood using real-time PCR.

There is a need for simple and sensitive assays to assess innate and adaptive immune responses to microbial agents and vaccines. Herein, we describe a whole blood method allowing to measure the induction of cytokine synthesis at the mRNA level. The originality of this method consists in the combination of PAXgene tubes containing an mRNA stabilizer for blood collection, the MagNA Pure instrument as an automated system for mRNA extraction and RT-PCR reagent mix preparation, and the real-time PCR methodology on the Lightcycler for accurate and reproducible quantification of transcript levels. We first demonstrated that this method is adequate to measure the induction of interleukin (IL)-1beta and IL-1 receptor antagonist (IL-1 RA) mRNA upon the addition of bacterial lipopolysaccharide (LPS) to whole blood. We then showed that this approach is also suitable to detect the production of mRNA encoding T cell-derived cytokines in whole blood incubated with tetanus toxoid as a model of in vitro immune response to a recall antigen. Finally, we demonstrated that this methodology can be used successfully to assess inflammatory as well as T cell responses in vivo, as it allowed to detect the induction of IL-1beta and IL-1 RA after injection of LPS in healthy volunteers, and also the induction of IL-2 upon recall immunisation with tetanus vaccine.

Adult↗
PubMed2003

Genetic variations in the internal transcribed spacer and mitochondrial small subunit rRNA gene of Naegleria spp.

Naegleria spp. are widely distributed free-living amebas, but one species in the genus, N. fowleri, causes acute fulminant primary amebic meningoencephalitis in humans and other animals. Thus, it is important to differentiate N. fowleri from the rest in the genus of Naegleria, and to develop tools for the detection of intra-specific genetic variations. In this study, one isolate each of N. australiensis, N. gruberi, N. jadini, and N. lovaniensis and 22 isolates of N. fowleri were characterized at the internal transcribed spacers (ITS) and mitochondrial small subunit rRNA (mtSSU rRNA) gene. The mtSSU rRNA primers designed amplified DNA of all isolates, with distinct sequences obtained from all species examined. In contrast, the ITS primers only amplified DNA from N. lovaniensis and N. fowleri, with minor sequence differences between the two. Three genotypes of N. fowleri were found among the isolates analyzed in both the mtSSU rRNA gene and ITS. The extent of sequence variation was greater in the mtSSU rRNA gene, but the ITS had the advantage of length polymorphism. These data should be useful in the development of molecular tools for rapid species differentiation and genotyping of Naegleria spp.

Animals↗
PubMed2003

Cryptosporidium species and genotypes in HIV-positive patients in Lima, Peru.

Cryptosporidium parasites from a cross-sectional study conducted in two national hospitals in Lima, Peru were genetically characterized to determine the diversity of Cryptosporidium spp. in HIV-positive people. A total of 2,672 patients participated in this study and provided 13,937 specimens. Cryptosporidium oocysts were detected by microscopy in 354 (13.3%) of the patients. Analysis of 951 Cryptosporidium-positive specimens from 300 patients using a small subunit rRNA-based PCR-RFLP tool identified 6 genotypes; Cryptosporidium hominis was the species most frequently detected (67.5%), followed by C. meleagridis (12.6%) and C. parvum (11.3%). Cryptosporidium canis (4.0%), C. felis (3.3%), and Cryptosporidium pig genotype (0.5%) were also found. These findings indicate that C. hominis is the predominant species in Peruvian HIV-positive persons, and that zoonotic Cryptosporidium spp. account for about 30% of cryptosporidiosis in these patients.

AIDS-Related Opportunistic Infections↗
PubMed2003

PCR-mediated recombination between Cryptosporidium spp. of lizards and snakes.

The presence or absence of genetic recombination has often been used as one of the criteria for Cryptosporidium species designation and population structure delineation. During a recent study of cryptosporidiosis in reptiles that were housed in the same room, 4 lizards were found to have concurrent infections of C. serpentis (a gastric parasite) and C. saurophilum (an intestinal parasite), and 6 snakes were concurrently infected with C. serpentis, C. saurophilum and a new Cryptosporidium as indicated by PCR-RFLP analysis of the SSU rRNA gene. DNA sequence analysis of cloned PCR products confirmed the diagnosis of mixed infections. Surprisingly, it appeared that 11 of the 22 clones (8 and 14 clones from a lizard and a snake, respectively) had chimeric sequences of two Cryptosporidium spp. BootScan analysis indicated the existence of recombinants among the cloned sequences and detection of the informative sites confirmed the BootScan results. Because the probability for genetic recombination between gastric and intestinal parasites is small, these hybrid sequences were likely results of PCR artifacts due to the presence of multiple templates. This was confirmed by PCR-sequencing analysis of single-copy templates using diluted DNA samples. Direct sequencing of 69 PCR products from 100- to 1,000-fold diluted DNAs from the same snake and lizard produced only sequences of C. serpentis, C. saurophilum and the unnamed Cryptosporidium sp. Thus, care should be taken to eliminate PCR artifacts when determining the presence of genetic recombination or interpreting results of population genetic studies.

Animals↗
PubMed2003

Identification of novel Cryptosporidium genotypes from the Czech Republic.

Isolates of Cryptosporidium from the Czech Republic were characterized from a variety of different hosts using sequence and phylogenetic analysis of the 18S ribosomal DNA and the heat-shock (HSP-70) gene. Analysis expanded the host range of accepted species and identified several novel genotypes, including horse, Eurasian woodcock, rabbit, and cervid genotypes.

Adolescent↗
PubMed2003

Molecular surveillance of Cryptosporidium spp. in raw wastewater in Milwaukee: implications for understanding outbreak occurrence and transmission dynamics.

Six Cryptosporidium spp. were found in 50 of 179 Milwaukee wastewater samples collected weekly over a year. Of the eight subtypes of Cryptosporidium hominis and Cryptosporidium parvum present, allele Ib was found in 14 of 16 samples, and its sequence was identical to that of the subtype in human samples from the 1993 Milwaukee outbreak of cryptosporidiosis.

Animals↗
PubMed2003

Cryptosporidium muris, a rodent pathogen, recovered from a human in Perú.

Cryptosporidium muris, predominantly a rodent species of Cryptosporidium, is not normally considered a human pathogen. Recently, isolated human infections have been reported from Indonesia, Thailand, France, and Kenya. We report the first case of C. muris in a human in the Western Hemisphere. This species may be an emerging zoonotic pathogen capable of infecting humans.

Adult↗
PubMed2003

[Gene optimization is necessary to express HPV type 6 L1 protein in the methylotrophic yeast Pichia pastoris].

OBJECTIVE: Human papillomavirus 6 (HPV 6) causes genital warts, a common sexually transmitted disease. L1-capsids protein is a highly promising vaccine candidate that has entered phase II clinical trial. But the existing methodologies for producing L1-capsids in insect cells is expensive for use in developing countries. As methylotrophic yeast,the Pichia pastoris expression system offers economy,and high expression levels. Over-expression of HPV6-L1 protein in P. pastoris was the purpose of this study. METHODS: The whole L1 gene with preferred codons for P. pastoris was rebuilt and A-T rich regions were abolished, Cloning into pPIC3.5K,electroporation of KM71, in vivo screen of multiple inserts by G418 resistance, PCR analysis of pichia integrants, BMGY/BMMY are used for induction and expression of L1 proteins. RESULTS: Three clones were found to produce L1 protein which can be identified with Western blot. Compared with L1 protein from E.coli, pichia-produced L1 has some glycosylation. Reacting strongly with MabH6B10.5 in indirect immunofluorescence assay indicated that L1 protein expressed in pichia cell holds its native conformational epitopes which is important for vaccine use. A total 125 microg pure L1 protein could be obtained from 1L cultures through ion-exchange and Ni-NTA chromatography. CONCLUSION: HPV type 6 L1 protein expressed in Pichia pastoris will facilitate the HPV vaccine development and structure-function study.

Capsid Proteins↗
PubMed2003

[Studies on genetic model in familial type 2 diabetes mellitus].

This study is to explore the genetic model of type 2 diabetes mellitus (type 2 DM) among the hereditary family. One hundred and thirty-six pedigrees of familial type 2 DM were studied. The heritability of type 2 DM was estimated according to Falconer's method and the multi-factorial inheritance analyzed according to Penrose's method. Complex segregation analysis was performed using S.A.G.E-REGD. The heritability of familial type 2 DM was 94 07%-/+5.84%. Dominant major gene might influence the genesis of type 2 DM. Analysis of multi-factorial inheritance indicated that there be two genetic patterns respectively in male and female populations. By complex segregation analysis,environment,non-transmitted and co-dominant inheritance were rejected. Autosomal dominant (AD) inheritance and autosomal recessive (AR) inheritance was accepted but AR inheritance was the best pattern. This study suggested that type 2 DM had significant heritability and genetic heterogeneity,which appeared to be a disease of multi-factorial inheritance generally and autosomal dominant (AD) inheritance in part of pedigrees.

English Abstract↗
PubMed2002

Host adaptation and host-parasite co-evolution in Cryptosporidium: implications for taxonomy and public health.

To assess the genetic diversity and evolution of Cryptosporidium parasites, the partial ssrRNA, actin, and 70kDa heat shock protein (HSP70) genes of 15 new Cryptosporidium parasites were sequenced. Sequence data were analysed together with those previously obtained from other Cryptosporidium parasites (10 Cryptosporidium spp. and eight Cryptosporidium genotypes). Results of this multi-locus genetic characterisation indicate that host adaptation is a general phenomenon in the genus Cryptosporidium, because specific genotypes were usually associated with specific groups of animals. On the other hand, host-parasite co-evolution is also common in Cryptosporidium, as closely related hosts usually had related Cryptosporidium parasites. Results of phylogenetic analyses suggest that the Cryptosporidium parvum bovine genotype and Cryptosporidium meleagridis were originally parasites of rodents and mammals, respectively, but have subsequently expanded their host ranges to include humans. Understanding the evolution of Cryptosporidium species is important not only for clarification of the taxonomy of the parasites but also for assessment of the public health significance of Cryptosporidium parasites from animals.

Actins↗
PubMed2002

Temporal expression of three mouse lens fiber cell membrane protein genes during early development.

PURPOSE: Three different lens fiber cell intrinsic membrane proteins, MIP (Major Intrinsic Protein), MP19, and connexin50 (Cx50), have separately been implicated as causative candidates for congenital cataracts. The aim of this study was to determine gene transcript expression of these three proteins during successive stages of mouse embryonic development. METHODS: Total RNA was prepared from mouse embryos taken at days 9-10 (E9-E10) of gestation, heads of day 11-13 (E11-E13) embryos, and lenses of adult mice. Coupled reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine gene transcript expression of MIP, Cx50, and MP19 during embryonic development. The products of RT-PCR were further cloned into the TOPO(TM) TA vector, and further analyzed by double strand nucleotide sequencing. RESULTS: Cx50 gene expression was observed throughout the developmental period observed (E9-E13). MIP transcripts were first observed at mouse embryonic day 11.5 (E11.5) and synthesis continued throughout the developmental period observed. The gene for MP19 (Lim2) begins to express at mouse embryo day 12 (E12) and synthesis continued throughout the developmental period observed. mRNA levels for all three proteins appear to remain steady from these early embryonic stages through adulthood. CONCLUSIONS: The identified early expression of Cx50, MIP, and Lim2 transcripts in mouse embryonic stages suggests that all three proteins play very important, probably quite different, roles in lens fiber cell differentiation. Variation in the temporal expression of these three genes during the course of development suggests a critical gene-coordinated regulation throughout lens fiber cell development. These three genes clearly play important roles in early normal lens development since it is known that mutations in the sequence of each membrane protein results in cataractogenesis.

Animals↗
PubMed2002

Analysis of spontaneous mRNA cytokine production in peripheral blood.

The quantification of the cytokine mRNAs synthesised by peripheral blood cells should make it possible to estimate a "peripheral immune statute". However, an accurate quantification can only be performed from a fresh whole blood sample in which mRNA is protected against nuclease digestion, and where gene transcription is inhibited. As discussed in this note, this has been made possible by the use of surfactant reagents such as tetradecyltrimethylammonium oxalate. We performed RT-PCR for the quantification of IL-10 and IFN-gamma mRNAs spontaneously produced in peripheral blood. The results showed pronounced higher IFN-gamma transcript levels in whole blood compared to peripheral blood mononuclear cells (PBMC) from the same individuals, while no significant difference was observed for IL-10 mRNA. The higher amounts of IFN-gamma mRNA observed in blood can be attributed at least to mRNA degradation. Using a real time PCR technique, we indeed demonstrated that blood IFN-gamma mRNA is rapidly degraded in vitro, the t 1/2 being worth approximately one hour at room temperature.

Cytokines↗
PubMed2002

DNA cleavage potency, cytotoxicity, and mechanism of action of a novel class of enediyne prodrugs.

We have discovered a novel class of (E)-3-acyloxy-4-(arylmethylidene)cyclodeca-1,5-diynes which exhibit promising enediyne-like DNA cleavage and cytotoxic activities. LC-MS analysis of the incubation mixture (pH 8.5, 37 degrees C) confirmed formation of 10-membered ring enediyne presumably via an allylic cation and suggested that the 1,4-benzenoid diradical might be one of the active species for DNA damage and cytotoxicity.

Alkynes↗
PubMed2002

Cytokine mRNA quantification by real-time PCR.

Real-time PCR represents a new methodology that accurately quantifies nucleic acids. This has been made possible by the use of fluorogenic probes, which are presented in two forms, namely hydrolysis probes (also called TaqMan probes) and hybridisation probes. We decided to apply this methodology to cytokine mRNA quantification and this led us to the development of a protocol that provides an easy way to develop and perform rapidly real-time PCR on a Lightcycler instrument. It was made possible by the use of freely available software that permits a choice of both the hydrolysis probe and the primers. We firstly demonstrated that the reproducibility of the method using hydrolysis probes compares favourably with that obtained with hybridisation probes. We then applied this technique to determine the kinetics of IL-1ra, IL-1beta, IL-5, IL-13, TNF-alpha and IFN-gamma induction upon stimulation of human peripheral blood mononuclear cells (PBMC) by phytohaemagglutinin (PHA). Finally, the method was also used successfully to demonstrate that IFN-alpha induces IL-10 mRNA accumulation in human monocytes.

Cytokines↗