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To isolate human antibodies against hepatitis E virus from phage display library by a new method of panning phage antibody library based on immobilized metal affinity chromatography (IMAC).Phage antibody library was allowed to mix with hex-His tagged expressed HEV specific antigen, NE2, in solution for adequate binding before affinity resin for hex-His was added. The non-specific phage antibodies were removed by extensive washing and the specific bound phage antibodies could then be eluted to infect TG1 or repeat the binding process for subsequent rounds of purification. The specificity of the selected human antibodies were tested by antigen competitive ELISA, human sera blocking ELISA, scFv expression, and sequence analysis.His-NE2 specific recombinant phages were successfully enriched after panning procedure. Two individual phage clones, 126 and 138, showed 50% inhibition in NE2 antigen competition ELISA and obvious blocking effect by HEV positive serum in blocking ELISA. Soluble scFv of 126, 138 bound to NE2 specifically.Two specific human phage antibodies against hepatitis E virus (HEV) from phage display library were isolated by immobilized metal affinity chromatography. The immobilized metal affinity chromatography applied to phage antibody selection was a helpful supplement to the selection in solution.

Nine ducks congenitally infected with the duck hepatitis B virus (DHBV) were treated either orally (four ducks for 10 weeks) or intraperitoneally (five ducks for 12 weeks) with the Indian traditional herbal remedy Phyllanthus amarus. Compared to placebo-treated control ducks, these treatments did not result in a reduction of circulating viral DNA in the serum or in the level of viral DNA replication in the liver. In two of the five intraperitoneal-treated ducks, a reduction in the levels of duck hepatitis B surface antigenaemia (DHBsAg) was observed. The data strongly suggest that Phyllanthus amarus has no significant inhibitory effect on DHBV DNA replication and only a minor effect on DHBsAg production.

Trichomonas vaginalis is the cause of one of the most common types of vaginitis, trichomoniasis. The incidence of trichomoniasis in developed countries has decreased substantially during the past decade, but high prevalence of this disease can still be found in rural and remote areas of Australia. Clinical manifestations of symptomatic women are generally non-specific, but include vaginal discharge, vaginitis and irritation. T. vaginalis infection has also been linked to the increased risk of human immunodeficiency virus transmission. Current diagnosis of T. vaginalis relies on the visualization of motile organisms in a wet-mount preparation. Culture is used mainly in reference laboratories. The latter two methods require viable organisms and would not be suitable for use where transportation of specimens can be delayed. Two real-time fluorescence resonance energy transfer (FRET) hybridization probe PCR assays were used in this study to test for T. vaginalis DNA, targeting the β-tubulin and 18S rRNA genes. We tested 500 randomly selected female patients, in an STD setting, for T. vaginalis DNA. The FRET PCRs targeting the β-tubulin gene and the 18S rRNA gene detected 96 % (85/89) and 100 % (89/89) , respectively, of the positive specimens (first-void urine sample or genital swabs). Wet-mount microscopy was performed on 76 of these PCR-positive specimens and showed a sensitivity of 38 % (29/76). The prevalence, by PCR, of trichomoniasis was 18 % in this study. The two real-time PCRs developed in this study, targeting different genetic regions of the organism, provide a rapid, sensitive and specific diagnosis of T. vaginalis infection.

Recombinant hepatitis C virus (HCV)-like particles (HCV-LPs) containing HCV structural proteins (core, E1, and E2) produced in insect cells resemble the putative HCV virions and are capable of inducing strong and broad humoral and cellular immune responses in mice and baboons. Here, we present evidence on the immunogenicity and induction of protective immunity by HCV-LPs in chimpanzees. Chimpanzees (two in each group), were immunized with HCV-LPs or HCV-LPs plus AS01B adjuvant. After immunizations, all animals developed an HCV-specific immune response including IFN-γ+, IL-2+, CD4+, and CD8+T cell and proliferative lymphocyte responses against core, E1, and E2. Upon challenge with an infectious HCV inoculum, one chimpanzee developed transient viremia with low HCV RNA titers (103to 104copies per ml) in the third and fourth weeks after the challenge. The three other chimpanzees became infected with higher levels of viremia (104to 105copies per ml), but their viral levels became unquantifiable (3copies per ml) 10 weeks after the challenge. After the HCV challenge, all four chimpanzees demonstrated a significant increase in peripheral and intrahepatic T cell and proliferative responses against the HCV structural proteins. These T cell responses coincided with the fall in HCV RNA levels. Four naïve chimpanzees were infected with the same HCV inoculum, and three developed persistent infection with higher viremia in the range of 105to 106copies per ml. Our study suggests that HCV-LP immunization induces HCV-specific cellular immune responses that can control HCV challenge in the chimpanzee model.

To construct a human natural phage single-chain antibody (scFv) library with diversity.V(H) and V(L) genes were amplified by RT-PCR and hemi-PCR from peripheral blood lymphocytes of healthy persons. The V genes were assembled to form scFv by overlap PCR and cloned into phagemid pCANTAB-5E, and then transformed into E. coli TG1 by electroporation to construct a human natural phage scFv library. The diversity and gene family of antibody gene were analysed by sequencing and the specific antibodies against various antigens were screened through bio-panning.A human natural phage scFv library with diversity and 2x10(8) sink size was constructed successfully. The specific human scFvs against 5 antigens were obtained by bio-panning.A human natural phage scFv library with diversity is constructed successfully and can be applied to human antibody preparation.

To explore the conditions of solid-phase screening phage antibody library and to provide the experimental basis for the design of screening project.Diverse antibodies including HEV NE2-specific and non-specific humanized phage antibodies were used to study the screening conditions, such as the binding time of phage antibodies to antigen, the concentration of coating antigen, the washing times and elution method.The best binding time of positive phage antibody to antigen was 1 min. The highest positive rate of screening was obtained under the conditions of washing for 20 to 30 times and pH value of the washing solution being 5. The concentration of the coating antigen had no obvious influence on the positive rate of screening. Higher positive rate was obtained by using 10 mg/L antigen to competitively elute for 1 h.Solid-phase screening of the phage antibody library is a very complex process, in which there are close relationship between the conditions, therefore, appropriate readjustment should be made for screening conditions according to concrete conditions.

The two structures of over 650V MR & MR SLMFFP double RESURF LDMOSs with HVI are experimentally realized using SPSM BCD process for high side gate drive IC. The experimental results, coincident with the three-dimensional simulations, show that the breakdown voltage of LDMOS will increase by reducing the width of HVI metal line. The breakdown voltages of the MR double RESURF LDMOS are 670V, 760V, 990V when the widths of HVI metal lines are 20 mum, 5 mum, 0 mum, respectively. The breakdown voltages of the MR SLMFFP double RESURF LDMOS are 700V, 920V when the widths of HVI metal lines are 15 mum, 0 mum, respectively. An experimental half bridge gate drive IC is also successfully implemented, using the MR double RESURF LDMOS with HVI. As a result, the two proposed high voltage structures can be used in level shifting and HVJT for AC260V, without thick oxide and additional conductive layer

To identify the predisposing factors of deep neck abscess and review diagnosis and treatment experience.A respective review was conducted in 50 cases who were diagnosed as having deep neck abscess in this hospital from Jan. 1997 to Dec. 2002.The causes of deep neck abscess were tooth diseases (3 cases), acute tonsillitis and laryngitis (8 cases), infection of upper respiratory tract (9 cases), foreign bodies in esophagus (14 cases), diabetes mellitus (5 cases), uncertain cause (11 cases). Among 21 cases of pus bacterial cultivation, 13 (64%) cases were positive. By different ways of drainage including neck-mediastinum incision, and use of large dosage of antibiotics, 46 six cases were cured and 4 cases died. Two died of massive hemorrhage of neck blood vessel burst, one massive hemorrhage of upper digestive tract, another infective shock.Once deep neck abscess is diagnosed, early surgical drainage, appropriate use of antibiotics and control of complications and accompanying diseases are very important to improve the cure rate.

We have previously reported the production of hepatitis C virus-like particles (HCV-LP) using a recombinant baculovirus containing the cDNA of the HCV structural proteins (core, E1, and E2). These particles resemble the putative HCV virions and are capable of inducing strong and broad humoral and cellular immune responses in mice. Here we present evidence on the immunogenicity of HCV-LP and the effects of novel adjuvant systems in a nonhuman primate model, the baboon. Three groups of four baboons were immunized with HCV-LP, HCV-LP and adjuvant AS01B (monophosphoryl lipid A and QS21), or HCV-LP and the combination of AS01B and CpG oligodeoxynucleotides 10105. After four immunizations over an 8-month period, all animals developed HCV-specific humoral and cellular immune responses including antibodies to HCV structural proteins and gamma interferon + (IFN-γ + )CD4 + and IFN-γ + CD8 + T-cell responses. The immunogenicity of HCV-LP was only marginally enhanced by the use of adjuvants. The overall HCV-specific immune responses were broad and long lasting. Our results suggest that HCV-LP is a potent immunogen to induce HCV-specific humoral and cellular immune responses in primates and may be a promising approach to develop novel preventive and therapeutic modalities.

C19H17CdN3O5, monoclinic, C2/c (no. 15), a = 27.369(3) Å, b = 9.126(1) Å, c = 15.326(2) Å, β = 104.285(1)°, V = 3709.6 Å3, Z = 8, Rgt(F) = 0.0288, wRref(F2) = 0.0626, T = 296 K

Frequency spectrum analysis is introduced to eddy current nondestructive testing. From the equivalent circuit of the eddy current testing system, the eddy current testing system is derived to be a second-order system and the characteristic of the output response signal for the square wave of the eddy current testing system is given. Then the frequency spectrum of the eddy current testing system output signal is analyzed. Finally, experimental results are given to show that the frequency analysis method for testing multiparameters is feasible and practical in use.

We have previously described the generation of hepatitis C virus–like particles (HCV-LPs) in insect cells and shown that immunization with HCV-LPs elicited both humoral and cellular immune responses in mice. To further characterize the HCV-LPs as a vaccine candidate, we evaluated the effects of adjuvant AS01B (monophosphoryl lipid A [MPL] and QS21), CpG 10105, and the combination of the 2 adjuvants on the immunogenicity of HCV-LPs in AAD mice (transgenic for HLA-A2.1). All HCV-LP–immunized mice (with or without adjuvant) developed high titers of anti-HCV E1/E2 antibodies after 4 injections intramuscularly. However, antibody titers in mice immunized with HCV-LP plus AS01B, plus CpG 10105, or plus the combination of AS01B and CpG 10105 were 4, 3, and 10 times higher, respectively, than that of HCV-LP alone. Isotype analysis of the induced anti-envelope antibodies showed that HCV-LP alone induced a predominant immunoglobulin (Ig) G1 response. In contrast, when the 2 adjuvants AS01B and CpG 10105 were combined, the response became predominantly IgG2a whereas HCV-LP plus AS01B or CpG 10105 gave a mixed IgG1 and IgG2a response, indicating that AS01B and CpG 10105 promote a more T-helper type 1 (Th1) response and that combining the 2 adjuvants results in an additive or synergistic interaction. These observations were further confirmed by the results of CD4+ enzyme-linked immunospot assay for interferon (IFN)-γ and interleukin (IL)-4 and intracellular cytokine staining of IFN-γ producing CD8+ cells. In conclusion, HCV-LP is a promising vaccine candidate against HCV infection and the adjuvants used are potent immune enhancers for this approach. (HEPATOLOGY 2003;37:52-59.)

Epidermal growth factor (EGF) plays an important role in the regulation of gastrointestinal tissue growth and development, and it can stimulate epithelial proliferation, cell differentiation and growth. It has been established that the EGF can promote gastric cytoprotection and ulcer healing. But the potential ability of EGF to regulate the gastric cancer growth is unknown. This study is to investigate the influence of EGF on human gastric cancer cell and the implanted tumor growth of nude mice.The cell growth rates of human gastric adenocarcinoma cell lines MKN-28, MKN-45, SGC-7901 and normal human gastric epithelial cells 3T3 were assessed when incubated with recombinant human EGF (rhEGF, 0.05, 0.1, 0.5, 1.0, 10, 50, 100 mg.L(-1)) using MTT method. The cells of MKN-28, MKN-45, SGC-7901 (gastric cancer tissue 1.5mm(3)) were implanted in the BALB/cA nude mice for 10 days. The EGF was given intraperitoneally (15, 30, 60 microg.kg(-1)) for 3 weeks. The body weights of the tumor-bearing animals and their tumor mass were measured afterwards to assess the mitogenic effect of rhEGF in the nude mice.Within the concentration range of 0.05-100mg.L(-1), rhEGF could increase the cell growth of normal 3T3 cells (cell growth rate 100% vs 102.8%, P0.05), but partially restrain the gastric cancer cell growth. The latter effect was related to cell differentiation. In 15-60 microg/kg rhEGF groups, the mean implanted tumor mass of MKN-28 cell were 1.75 g, 1.91 g, 2.08 g/NS group 1.97 g (P0.05), the mean tumor mass of SGC-7901 cell were 1.53 g, 1.07 g, 1.20 g/NS group 1.07 g (P0.05), and for MKN-45 cell, the tumor mass were respectively 1.92 g, 1.29 g, 1.77 /NS group 1.82 g (P0.05). So rhEGF had no obvious effect on implanted MKN-28, SGC-7901 and MKN-45 tumor growth.EGF has no stimulating effect on the human gastric cancer cell growth neither in vitro nor in vivo.

In this paper, a novel substrate termination technology (STT) for lateral double-diffused MOSFET (LDMOS) based on curved junction extension is proposed and experimentally demonstrated. A low-doped P-substrate, which is inserted in the curved region of the LDMOS, adjusts the high-doped abrupt P-body/N-drift junction with small curvature radius to low-doped P-sub/N-drift junction with large curvature radius, thus reducing peak electric field and avoiding premature avalanche breakdown at the curved abrupt P-body/N-drift junction. The proposed STT has been applied to a super junction (SJ) LDMOS. The experimental results show that the SJ LDMOS with the proposed STT exhibits off-state breakdown voltage (BV) of 800 V, which is the highest BV for the reported SJ LDMOS by far.

The genome of an Australian strain of duck hepatitis B virus (AusDHBV) was cloned from a pool of congenitally DHBV-infected-duck serum, fully sequenced and found by phylogenetic analyses to belong to the ‘Chinese’ DHBV branch of the avian hepadnaviruses. Sequencing of the Pre-S/S gene of four additional AusDHBV clones demonstrated that the original clone (pBL4.8) was representative of the virus present in the pool, and a head-to-tail dimer of the clone was infectious when inoculated into newly hatched ducks. When the published sequences of 20 avian hepadnaviruses were compared, substitutions or deletions in the polymerase (POL) gene were most frequent in the 500 nt segment encoding the ‘spacer’ domain that overlaps with the Pre-S domain of the Pre-S/S gene in a different reading frame. In contrast, substitutions and deletions were rare within the adjacent segment that encodes the reverse transcriptase domain of the POL protein and the S domain of the envelope protein, presumably because they are more often deleterious.

The efficacy of DNA vaccines encoding the duck hepatitis B virus (DHBV) pre-S/S and S proteins were tested in Pekin ducks. Plasmid pcDNA I/Amp DNA containing the DHBV pre-S/S or S genes was injected intramuscularly three times, at 3-week intervals. All pre-S/S and S-vaccinated ducks developed total anti-DHBs and specific anti-S antibodies with similar titers reaching 1/10,000 to 1/50,000 and 1/2,500 to 1/4,000, respectively, after the third vaccination. However, following virus challenge, significant differences in the rate of virus removal from the bloodstream and the presence of virus replication in the liver were found between the groups. In three of four S-vaccinated ducks, 90% of the inoculum was removed between

A quantitative assay of hepatitis B virus (HBV) binding to hepatocyte plasma membranes was adapted to show that leucocyte plasma membranes bind serum-derived HBV saturably, and that this binding is inhibited using synthetic peptides representative of the large envelope protein of HBV. Using a panel of ligand-blocking monoclonal antibodies (mAb) to opsonin receptors, it was shown that the three classes of Fc gamma R and CR3 are not major receptors for HBV on leucocytes or hepatocytes. It was also shown that HBV does not utilize the receptor for IgA, Fc alpha R, for attachment to leucocytes, despite reported sequence homology between the large envelope protein of HBV and the Fc portion of human IgA. Evidence is presented that the receptor for HBV on leucocytes may differ from the hepatocyte receptor(s), based on synthetic peptide inhibition assays of HBV binding. Furthermore, it was observed that glycosaminoglycans influence the HBV-liver and leucocyte interactions, providing evidence that HBV attachment may be a multi-stage process.

This paper discusses the breakdown mechanism and proposes a new simulation and test method of breakdown voltage (BV) for an ultra-high-voltage (UHV) high-side thin layer silicon-on-insulator (SOI) p-channel lateral double-diffused metal-oxide semiconductor (LDMOS). Compared with the conventional simulation method, the new one is more accordant with the actual conditions of a device that can be used in the high voltage circuit. The BV of the SOI p-channel LDMOS can be properly represented and the effect of reduced bulk field can be revealed by employing the new simulation method. Simulation results show that the off-state (on-state) BV of the SOI p-channel LDMOS can reach 741 (620) V in the 3-μm-thick buried oxide layer, 50-μm-length drift region, and at −400 V back-gate voltage, enabling the device to be used in a 400 V UHV integrated circuit.

A novel high concentration linear variable doping interface thin silicon layer (TSL) silicon-on-insulator (SOI) super junction (SJ) LDMOS is proposed. The design of the linear variable doping can deplete the high drift concentration. The proposed structure uses a TSL to achieve charge balance and eliminate substrate-assisted depletion effect. The dielectric electric field (EI) and the breakdown voltage (BV) of the TSL SOI SJ are 530 V/μm and 552 V with 30 µm length drift region and 1 µm-thick dielectric layer, respectively, and the specific on-resistance (Ron, sp) is 0.03403 Ω·cm2 and FOM (FOM=BV2/Ron,sp) is 8.95 MW/cm2, when gate voltage is 5 V.