Neutrophil-lymphocyte ratio and hypersensitive C-reaction protein are associated with miscarriage during the second trimester of pregnancy.

This study investigated whether biomarkers in the second trimester of pregnancy, including the white blood cell (WBC) count, neutrophil-lymphocyte ratio (NLR), hypersensitive C-reactive protein (hs-CRP) concentration, and procalcitonin (PCT) concentration, were associated with miscarriage during the second trimester of pregnancy. Sixty-two asymptomatic patients in their second trimester of pregnancy were included in the control group (group A). Among 67 patients diagnosed with late threatened miscarriage, 46 patients with ongoing pregnancy were included in group B and 21 patients with subsequent miscarriage were included in group C. The serum of these patients was collected and the biomarkers were analyzed. A paired-samples t-test was used for the comparison between the groups before and after the miscarriage. Statistical significance was set at p<0.05. Receiver operating characteristic curve (ROC) analysis was performed to evaluate the predictive value of different biomarkers for miscarriage during the second trimester of pregnancy. WBC count, neutrophil percentage, and hs-CRP levels were significantly higher in group C than in groups A and B (p<0.05). Lymphocyte percentage and albumin levels decreased significantly from group A to group C (p<0.05). In contrast, NLR increased significantly from group A to group C (p<0.05). There was a significant decrease in the WBC count, neutrophil percentage, hemoglobin concentration, and post-miscarriage NLR among the cases with miscarriage (p<0.05). The area under the curve of WBC count, NLR, hs-CRP, and the combination of these three factors for the prediction of late miscarriage varied from 78.0% to 82.6%. The combination of these three factors had the highest specificity of 91.1%, while hs-CRP had the highest sensitivity of 88.9%. WBC count, NLR, and hs-CRP levels are strongly associated with miscarriage during the second trimester of pregnancy, indicating that they are potential predictive biomarkers.

Establishment of a Gallbladder Cancer-Specific Survival Model to Predict Prognosis in Non-metastatic Gallbladder Cancer Patients After Surgical Resection.

BACKGROUND: Gallbladder cancer is an invasive cancer with a discouraging prognosis, and early detection and active intervention are of great value. AIMS: To establish a more accurate and effective survival model to predict the prognosis of patients with non-metastatic gallbladder after surgical resection. METHODS: A retrospective analysis was conducted in non-metastatic gallbladder cancer patients who were registered in the surveillance, epidemiology and end results database from 2010 to 2014. Univariate analysis and multivariate analysis were performed for the related factors that might affect the gallbladder cancer-specific survival. A prognostic gallbladder cancer-specific survival model was established using the nomogram tool. The discrimination test was measured by the c-index, and the conformance test was performed by a calibration curve. RESULTS: In all, 1422 patients with non-metastatic gallbladder cancer were identified. The prognostic factors include age, gender, lymph node dissection, postoperative chemotherapy, tumor size, histological grading, pT stage and pN stage. The gallbladder cancer-specific survival model was established based on the prognostic factors. The model's c-index was 0.775, and the 7th AJCC staging c-index was 0.649. The calibration curves showed a good correlation between prediction and actual survival. CONCLUSIONS: This study established the gallbladder cancer-specific survival model successfully. Compared with the 7th AJCC stage, this model refined the contribution of the pT stage, pN stage and other related factors and was demonstrated to be more accurate and reliable. More importantly, this model may allow clinicians to screen patients with a poor prognosis for closer follow-up or adjuvant treatment.

Changes of cortical activation in swallowing following high frequency repetitive transcranial magnetic stimulation in older adults.

BACKGROUND: This study explored whether high-frequency repetitive transcranial magnetic stimulation (rTMS) can induce positive changes in the cortical areas of older adults who do not have functional difficulties in swallowing. METHODS: Ten healthy, right-handed, elderly volunteers were subjected to 18F-labeled fluorodeoxyglucose positron emission tomography(FDG-PET) scans when at rest, swallowing before rTMS, and swallowing after rTMS. During the swallowing study, water was infused orally via a catheter at a rate of 600 mL/h. Subjects swallowed water every 20 seconds following a light flash for 30 minutes. During rest, the light source was active, but subjects were requested not to swallow. The rTMS consisted of 5 Hz applied to a pharyngeal motor hot spot in the right hemisphere for 10 minutes every weekday for 2 weeks. The intensity of the stimulation was set at 90% of the thenar motor threshold of the same hemisphere. The differences between each patient's active image and the control images (P<.05) on a voxel-by-voxel basis were examined to find significant increases in metabolism using statistical parametric mapping software. KEY RESULTS: The cortical areas activated by swallowing before rTMS included the bilateral sensorimotor cortex (Brodmann's areas 3 and 4) and showed symmetry. The cortical areas activated by swallowing after rTMS were the same as the areas before rTMS. There was no statistical difference between the two swallowing activation areas. CONCLUSIONS AND INFERENCES: Older adults displayed the symmetry of cortical control of swallowing function. High frequency rTMS did not affect the activation in the swallowing sensorimotor cortices of elderly people.

Gene therapy using plasmid DNA-encoded anti-HER2 antibody for cancers that overexpress HER2.

Plasmid DNA-encoded antibodies, or DNA-based monoclonal antibodies (dMAbs), are delivered by intramuscular injection and in vivo electroporation (EP) and are effective in virus neutralization, although they have not been evaluated for tumor gene therapy. Here we investigated whether a dMAb was appropriate for tumor gene therapy. We constructed the expression plasmids coding for the heavy or light chain of a parental murine antibody of Herceptin with the antibody genes codon- and RNA-optimized and fused to the Kozak-IgE leader sequence in pVax1. Transfection of the plasmids into human muscle RD cells resulted in functional expression of the antibody, and this exhibited the same in vitro antiproliferative activity as Herceptin. A single intramuscular injection and in vivo EP of the plasmids (100 µg per head) resulted in high and sustained antibody expression in the sera of normal mice and in effective inhibition of tumor growth in nude mice bearing HER2-positive human breast carcinoma BT474 xenografts. The antitumor efficacy of the anti-HER2 dMAb was similar to that of four doses of intravenously injected 10 mg kg-1 Herceptin. The results demonstrate that the dMAb is effective in the treatment of HER2-positive breast cancer, suggesting that this dMAb may be applicable for tumor gene therapy.

Tetrafluoroethane (R134a) hydrate formation within variable volume reactor accompanied by evaporation and condensation.

Vast size hydrate formation reactors with fast conversion rate are required for the economic implementation of seawater desalination utilizing gas hydrate technology. The commercial target production rate is order of thousand tons of potable water per day per train. Various heat and mass transfer enhancement schemes including agitation, spraying, and bubbling have been examined to maximize the production capacities in scaled up design of hydrate formation reactors. The present experimental study focused on acquiring basic knowledge needed to design variable volume reactors to produce tetrafluoroethane hydrate slurry. Test vessel was composed of main cavity with fixed volume of 140 ml and auxiliary cavity with variable volume of 0 ∼ 64 ml. Temperatures at multiple locations within vessel and pressure were monitored while visual access was made through front window. Alternating evaporation and condensation induced by cyclic volume change provided agitation due to density differences among water and vapor, liquid and hydrate R134a as well as extended interface area, which improved hydrate formation kinetics coupled with latent heat release and absorption. Influences of coolant temperature, piston stroke/speed, and volume change period on hydrate formation kinetics were investigated. Suggestions of reactor design improvement for future experimental study are also made.

Conversion of twice-daily tacrolimus to once-daily tacrolimus formulation in stable pediatric kidney transplant recipients: pharmacokinetics and efficacy.

The pharmacokinetics, efficacy and safety of once-daily tacrolimus formulation (Tac-OD) were assessed in 34 stable pediatric kidney transplant recipients. Enrolled patients received their dose of twice-daily tacrolimus formulation (Tac-BID) on study Days 0 through 7. On the morning of study Day 8, the total daily doses for patients were converted to Tac-OD on a 1:1 basis and maintained on a once-daily morning dosing regimen. Tacrolimus pharmacokinetic profiles were obtained on study Days 7, 14 and 28 (after dose adjustment). Although the mean C0 concentrations (4.10 ± 1.16-3.53 ± 1.10 ng/mL, p = 0.004), and AUC0-24 (151.8 ± 41.6-129.8 ± 39.3 ng h/mL, p < 0.001) were decreased significantly after a 1:1 based conversion, there was high interindividual variability. The dose of Tac-OD was decreased in 26.5% and increased in 44.1% of patients. The resultant tacrolimus dose and pharmacokinetic profiles on study Day 28 were comparable to those on Day 7. There were no serious adverse events. In conclusion, Tac-BID can be safely converted to Tac-OD in stable pediatric kidney transplant patients with the heightened therapeutic drug monitoring. Effects of drug conversion on the cardiovascular risk factors, neurological side effects and adherence should be further evaluated.

Human antibodies targeting the C-type lectin-like domain of the tumor endothelial cell marker clec14a regulate angiogenic properties in vitro.

It has been suggested that clec14a may be involved in tumor angiogenesis. However, a molecular mechanism has not been clearly identified. In this study, we show for the first time that C-type lectin-like domain (CTLD) of clec14a may be important for regulating cell migration and filopodia formation. Using phage display technology, recombinant human antibodies specific to the CTLDs of human and mouse clec14a (clec14a-CTLD (immunoglobulin G) IgG) were selected. Functional assays using the antibodies showed that clec14a-CTLD IgGs specifically blocked endothelial cell migration and tube formation without affecting cell viability or activation. Further, clec14a-CTLD IgGs inhibited clec14a-mediated cell-cell contact by blocking interaction between CTLDs. Finally, clec14a cross-linking by the clec14a-CTLD IgGs significantly downregulated clec14a expression on the surface of endothelial cells. These results strongly suggest that the clec14a-CTLD may be a key domain in angiogenesis, and that clec14a-CTLD IgGs specifically inhibit angiogenesis by modulating CTLD-mediated cell interactions and clec14a expression on the surface of endothelial cells.

Vitamin D-binding protein interacts with Aß and suppresses Aß-mediated pathology.

The level of vitamin D-binding protein (DBP) is increased in the cerebrospinal fluid of patients with Alzheimer's disease (AD), suggesting a relationship with its pathogenesis. In this study, we investigated whether and how DBP is related to AD using several different approaches. A pull-down assay and a surface plasmon resonance binding assay indicated direct interactions between purified DBP and amyloid beta (Aß), which was confirmed in the brain of AD patients and transgenic AD model mice by immunoprecipitation assay and immunohistochemical double-staining method. Moreover, atomic force microscopic examination revealed that DBP reduced Aß aggregation in vitro. DBP also prevented Aß-mediated death in cultured mouse hippocampal HT22 cell line. Finally, DBP decreased Aß-induced synaptic loss in the hippocampus and rescued memory deficits in mice after injection of Aß into the lateral ventricle. These results provide converging evidence that DBP attenuates the harmful effects of Aß by a direct interaction, and suggest that DBP is a promising therapeutic agent for the treatment of AD.

Early neurological outcomes according to CHADS2 score in stroke patients with non-valvular atrial fibrillation.

BACKGROUND AND PURPOSE: A higher CHADS(2) score or CHA(2)DS(2)-VASc score is associated with an increased risk of ischaemic stroke in patients with non-valvular atrial fibrillation (NVAF). However, there are no data regarding early neurological outcomes after stroke according to the risk levels. METHODS: In this study, a total of 649 stroke patients with NVAF were enrolled and categorized into three groups: low-risk (CHADS(2) score of 0-1), moderate-risk (CHADS(2) score 2-3), or high-risk group (CHADS(2) score ≥4). CHA(2)DS(2)-VASc score was divided into four groups including 0-1, 2-3, 4-5, and ≥6. We investigated whether there were differences in initial stroke severity, early neurological outcome, and infarct size according to CHADS(2) score or CHA(2)DS(2)-VASc score in stroke patients with NVAF. RESULTS: The initial National Institutes of Health Stroke Scale (NIHSS) score was highest in high-risk group [9.5, interquartile range (IQR) 4-18], followed by moderate-risk (8, IQR 2-17) and low-risk group (6, IQR 2-15) (P=0.012). Likewise, initial stroke severity increased in a positive fashion with increasing the CHA(2)DS(2)-VASc score. During hospitalization, those in the high-risk group or higher CHA(2)DS(2)-VASc score had less improvement in their NIHSS score. Furthermore, early neurological deterioration (END) developed more frequently as CHADS(2) score or CHA(2)DS(2)-VASc score increased. Multivariate analysis showed being in the high-risk group was independently associated with END (OR 2.129, 95% CI 1.013-4.477). CONCLUSIONS: Our data indicate that patients with NVAF and higher CHADS(2) score or CHA(2)DS(2)-VASc score are more likely to develop severe stroke and a worse clinical course is expected in these patients after stroke presentation.

Combined surgery and postoperative radiotherapy for oropharyngeal squamous cell carcinoma in Korea: analysis of 110 cases.

The treatment of oropharyngeal squamous cell carcinoma (OSCC) remains controversial. This study reviews the authors' experience of treating OSCC, evaluates the oncologic outcome and assesses the factors affecting local/regional recurrence. A retrospective analysis of 110 consecutive OSCC patients treated primarily by surgery and/or postoperative radiotherapy was carried out. 82% of patients had advanced disease (stage III or IV). The 5-year overall survival and disease specific survival rates (DSSR) were 58% and 65%, respectively. The DSSR of the soft palate or posterior pharyngeal wall, tonsillar area, and base of tongue were 80%, 62%, and 51%, respectively (P<0.05). The 5-year DSSR according to the American Joint Committee on Cancer stages was 94% for early stage and 56% for advanced stage (P<0.05). The overall recurrence rate was 38% (42 patients). The most frequent site of recurrence was the neck (46%). Only 14% of patients with recurrences were treated successfully. Positive resection margins and the presence of pathologic lymph nodes influenced the recurrence at the primary lesion and in the neck, respectively, in a statistically significant manner. Surgery and postoperative radiotherapy provided a superior outcome in patients with advanced OSCC. A randomized study is required to assess the oncologic and functional superiority of surgery or chemoradiation.

Targeted gene therapy of LS174 T human colon carcinoma by anti-TAG-72 immunoliposomes.

Anti-tumor-associated glycoprotein (TAG)-72 PEG-immunoliposomes (PILs) were prepared by conjugation of Fab' fragments of recombinant humanized monoclonal antibody, HuCC49, to sterically stabilize unilamellar liposomes (90-110 nm in diameter) to target TAG-72-overexpressing cancer cells. The liposomes consisted of 1-palmitonyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 92 mol percent, O,O'-dymyrisyl-N-lysyl aspartate (DMKD cationic lipid), 4 mol percent, distearoyl-phosphatidyl-ethanolamine-polyethylene glycol 2000 (DSPE-PEG(2000)), 3 mol percent and DSPE-maleimide (DSPE-PEG(2000)-Mal), 1 mol percent. These anti-TAG-72 PILs were able to adhere to the surface of TAG-72-overexpressing LS174 T human colon cancer cells more effectively than conventional liposomes. Also, in vitro gene transfection of the LS174 T cells by the anti-TAG-72 PILs in the presence of a high concentration of fetal bovine serum (up to 60%) was greater than that by conventional cationic lipoplexes. Intravenously administered anti-TAG-72 PILs efficiently localized in the LS174 T tumor tissues, while the non-targeted conventional liposomes did not. Intravenous administration of the anti-TAG-72 PILs containing plasmids encoding antiangiogenic proteins, such as angiostatin K1/3, endostatin and saxatilin, significantly inhibited in vivo growth of LS174 T tumors and angiogenesis in the tumor tissues. These results demonstrated the potential of TAG-72-mediated targeting of immunoliposomes as a modality for systemic gene delivery to human colon cancer cells.

TMPRSS4 promotes invasion, migration and metastasis of human tumor cells by facilitating an epithelial-mesenchymal transition.

TMPRSS4 is a novel type II transmembrane serine protease found at the cell surface that is highly expressed in pancreatic, colon and gastric cancer tissues. However, the biological functions of TMPRSS4 in cancer are unknown. Here we show, using reverse transcription-PCR, that TMPRSS4 is highly elevated in lung cancer tissues compared with normal tissues and is also broadly expressed in a variety of human cancer cell lines. Knockdown of TMPRSS4 by small interfering RNA treatment in lung and colon cancer cell lines was associated with reduction of cell invasion and cell-matrix adhesion as well as modulation of cell proliferation. Conversely, the invasiveness, motility and adhesiveness of SW480 colon carcinoma cells were significantly enhanced by TMPRSS4 overexpression. Furthermore, overexpression of TMPRSS4 induced loss of E-cadherin-mediated cell-cell adhesion, concomitant with the induction of SIP1/ZEB2, an E-cadherin transcriptional repressor, and led to epithelial-mesenchymal transition events, including morphological changes, actin reorganization and upregulation of mesenchymal markers. TMPRSS4-overexpressing cells also displayed markedly increased metastasis to the liver in nude mice upon intrasplenic injection. Taken together, these studies suggest that TMPRSS4 controls the invasive and metastatic potential of human cancer cells by facilitating an epithelial-mesenchymal transition; TMPRSS4 may be a potential therapeutic target for cancer treatment.

Expression of the hepatitis B surface S and preS2 antigens in tubers of Solanum tuberosum.

In an attempt to develop an edible vaccine, we transformed a recombinant hepatitis B virus (HBV) gene encoding the middle protein of HBV that contains the surface S and preS2 antigen into potato by Agrobacterium-mediated transformation. The HBV gene was under control of either the CaMV 35S promoter, the double 35S promoter with the AlMV 5' non-translated leader sequence, or the tuber-specific patatin promoter. HBV mRNA levels were higher with the 35S promoter than with the double 35S and patatin promoters; however, the levels of the S and preS2 antigen in the transformed tubers were higher with the patatin promoter than with the CaMV 35S and double promoters. The levels of preS2 antigen produced are the highest reported to date. Transgenic potato tubers were fed to mice, and the mice showed an immune response against the HBV S antigen.

Supplemention with tetrahydrobiopterin suppresses the development of hypertension in spontaneously hypertensive rats.

It has been suggested that tetrahydrobiopterin (H(4)B), a cofactor of NO synthase, can reverse endothelial dysfunction caused by cardiovascular diseases, including atherosclerosis, coronary artery disease, and hypertension. Moreover, an impairment of H(4)B biosynthesis in spontaneously hypertensive rats (SHR) was observed. Thus, we hypothesized that the defect of the H(4)B synthesis system may play an important role in the development of hypertension in SHR. In the present study H(4)B (10 mg/kg per day IP) was used to treat SHR and Wistar-Kyoto rats (WKY) from the age of 5 through 16 weeks. Results demonstrated that chronic treatment with H(4)B significantly improved the impaired vascular responses to acetylcholine and suppressed the development of hypertension in SHR but did not affect WKY. The increase of inducible NO synthase expression, nitrotyrosine immunostaining, NO production, and superoxide anion formation in adult SHR were also significantly suppressed by chronic treatment with H(4)B. In contrast, H(4)B had no effect on WKY. In conclusion, this study demonstrated that H(4)B significantly attenuated the development of hypertension in SHR. The antihypertensive effect of H(4)B might be mediated through its direct antioxidant activity and/or decreasing oxygen free radical production from NO synthase, thereby reducing inducible NO synthase expression and peroxynitrite formation. Thus, the present study proposed that supplementation with H(4)B might be beneficial in preventing pathological conditions such as essential hypertension.

Inhibitory effect of endothelin-1 on the isoproterenol-induced chloride current in human cardiac myocytes.

It is still controversial whether the cAMP-activated Cl(-) current (I(Cl,cAMP)) is expressed in human cardiomyocytes. The whole-cell configuration of the voltage-clamp technique was used to examine in detail the I(Cl,cAMP) in single human atrial and ventricular myocytes. Human cardiomyocytes were enzymatically isolated from atrial or ventricular specimens obtained from open-heart surgery or cardiac transplantation, respectively. Isoproterenol (1 microM) or forskolin (10 microM) was used to activate the cAMP second-messenger system. The isoproterenol- or forskolin-induced Cl(-) current was elicited in 12 of 54 atrial myocytes but was completely absent from ventricular myocytes. The isoproterenol-induced Cl(-) current in atrial myocytes was time-independent and had a reversal potential close to zero. Endothelin-1 (30 nM) inhibited the isoproterenol-induced Cl(-) current by 75+/-6% (n=4). This inhibitory effect of endothelin-1 was attenuated by pretreating atrial myocytes with the endothelin ET(A) receptor antagonist, BQ485, but not with the ET(B) receptor antagonist, BQ-788. The results provide evidence that the I(Cl,cAMP) exists in human atria, but not ventricle, and is inhibited by endothelin-1.

Purification and structural analysis of the hepatitis B virus preS1 expressed from Escherichia coli.

The preS1 of hepatitis B virus (HBV) is located at the outermost part of the envelope protein and possesses several functionally important regions such as hepatocyte receptor-binding site and virus-neutralizing epitopes. As the first step to understand the structure-function relationship for the preS1 antigen, we have purified the preS1 and performed its structural characterization by circular dichroism (CD) spectroscopy. The preS1 was purified to near homogeneity from bacterially expressed glutathione S-transferase (GST)-preS1 fusion protein by two-step purification, affinity chromatography on glutathione-agarose column, and cation-exchange chromatography on Mono S column. The CD analysis showed that the purified preS1, which was largely unstructured in aqueous solution, acquired a significant (16%) alpha-helical structure when analyzed in 50% trifluoroethanol or 20 mM SDS. The results suggest that the preS1 assumes a mainly unstructured conformation and may form induced secondary structures upon binding to target proteins or under hydrophobic environment.

Modulation of actinorhodin biosynthesis in Streptomyces lividans by glucose repression of afsR2 gene transcription.

While the biosynthetic gene cluster encoding the pigmented antibiotic actinorhodin (ACT) is present in the two closely related bacterial species, Streptomyces lividans and Streptomyces coelicolor, it normally is expressed only in S. coelicolor-generating the deep-blue colonies responsible for the S. coelicolor name. However, multiple copies of the two regulatory genes, afsR and afsR2, activate ACT production in S. lividans, indicating that this streptomycete encodes a functional ACT biosynthetic pathway. Here we report that the occurrence of ACT biosynthesis in S. lividans is determined conditionally by the carbon source used for culture. We found that the growth of S. lividans on solid media containing glucose prevents ACT production in this species by repressing the synthesis of afsR2 mRNA; a shift to glycerol as the sole carbon source dramatically relieved this repression, leading to extensive ACT synthesis and obliterating this phenotypic distinction between S. lividans and S. coelicolor. Transcription from the afsR2 promoter during growth in glycerol was dependent on afsR gene function and was developmentally regulated, occurring specifically at the time of aerial mycelium formation and coinciding temporally with the onset of ACT production. In liquid media, where morphological differentiation does not occur, ACT production in the absence of glucose increased as S. lividans cells entered stationary phase, but unlike ACT biosynthesis on solid media, occurred by a mechanism that did not require either afsR or afsR2. Our results identify parallel medium-dependent pathways that regulate ACT biosynthesis in S. lividans and further demonstrate that the production of this antibiotic in S. lividans grown on agar can be modulated by carbon source through the regulation of afsR2 mRNA synthesis.

Suppression of the development of hypertension by the inhibitor of inducible nitric oxide synthase.

Our previous study demonstrated that the aortic inducible nitric oxide synthase (iNOS) expression and the plasma nitrite level in spontaneously hypertensive rats (SHR) were greater than that in age-matched Wistar-Kyoto rats (WKY). We subsequently hypothesized that the over-expression of iNOS might play an important role in the pathogenesis of hypertension in SHR. In the present study, pyrrolidinedithiocarbamate (PDTC, 10 mg kg(-1) day(-1), p.o., antioxidant and nuclear factor-kappa B inhibitor) and aminoguanidine (15 mg kg(-1) day(-1), p.o., selective inhibitor of iNOS) was used to treat SHR and WKY from age of 5 weeks through 16 weeks. We found that PDTC and aminoguanidine significantly suppressed the development of hypertension and improved the diminished vascular responses to acetylcholine in SHR but not in WKY. Likewise, the increase of iNOS expression, nitrotyrosine immunostaining, nitric oxide production and superoxide anion formation in adult SHR were also significantly suppressed by chronic treatment with PDTC and aminoguanidine. In conclusion, this study demonstrated that both PDTC and aminoguanidine significantly attenuated the development of hypertension in SHR. The results suggest that PDTC suppresses iNOS expression due to its anti-oxidant and/or nuclear factor-kappa B inhibitory properties. However, the effect of aminoguanidine was predominantly mediated by inhibition of iNOS activity, thereby reducing peroxynitrite formation. We propose that the development of a more specific and potent inhibitor of iNOS might be beneficial in preventing pathological conditions such as the essential hypertension.

Mouse monoclonal antibodies to hepatitis B virus preS1 produced after immunization with recombinant preS1 peptide.

We have efficiently generated mouse monoclonal antibodies (MAbs), which bind specifically to amino acids 21-47 of the preS1 domain of hepatitis B virus (HBV) by immunizing mice with the preS1 peptide (amino acids, aa 1-56) conjugated to keyhole limpet hemocyanin. Hybridomas were screened by an indirect enzyme-linked immunosorbent assay (ELISA) using the purified preS1 peptide as a coated antigen. Eighteen positive hybridomas were selected and subjected to isotyping. Of these, 5 clones secreted immunoglobulin G (IgG) and 13 clones secreted IgM. Four (KR1, KR2, KR3, and KR4) of the 5 IgG MAbs bound to preS1 peptide (aa 21-47). Epitope mapping using bacterially expressed GST fusion proteins revealed that three clones (KR2, KR3, KR4) (IgG1, K) recognize aa 21-35, while KR1 (IgG2a, K) recognizes aa 35-47 of the preS1. These MAbs immunoprecipitated HBV particles, demonstrating that they bind to native HBV particles.

Fine mapping of virus-neutralizing epitopes on hepatitis B virus PreS1.

We identified the epitopes on the preS1 which induce antibodies that neutralize both ad and ay subtypes of hepatitis B virus (HBV). Previously we generated murine monoclonal antibodies KR359 and KR127 that bind specifically to the preS1 of HBV. In this study we have performed fine mappings of the epitopes of the antibodies by examining their reactivity with GST fusion proteins, which contain a series of deletion mutants of the preS1. KR359 and KR127 specifically recognize aa 19-26 and 37-45 of the preS1, respectively. The antibodies neutralized both adr and ayw subtypes of the virus in an in vitro neutralization assay using in vitro infection of adult human hepatocyte primary culture by HBV. The epitopes showed little sequence divergence and the antibodies bound to the preS1 of all the HBV subtypes and variants tested.