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.

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.

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.

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.

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.

A humanized anti--4-1BB monoclonal antibody suppresses antigen-induced humoral immune response in nonhuman primates.

The interaction of 4-1BB and its ligand plays an important role in the regulation of T-cell-mediated immune responses. In this study, the authors examined the effect of a humanized anti--4-1BB monoclonal antibody (H4B4) on ovalbumin-induced immune responses in baboons. Previously, a mouse monoclonal antibody, 4B4 against the human 4-1BB molecule, was generated and characterized. Based on this antibody, a humanized version of 4B4 monoclonal antibody was constructed and the resultant antibody, H4B4, showed full recovery of the binding activity of the original antibody 4B4: a 1.5-fold increase in affinity for 4-1BB. In addition, H4B4 mediated antibody-dependent cellular cytotoxicity of activated human peripheral blood T cells and CEM cells in a dose-dependent manner. Weekly administration of H4B4 at doses of 1 or 4 mg/kg could suppress immunoglobulin G production against ovalbumin. This was not a result of the overall immune suppression, because the numbers of B and T cells and the total immunoglobulin G production were not altered during treatment with H4B4. These findings suggest that treatment with H4B4 may be a valid therapeutic approach to control unwanted immune responses in persons with autoimmune diseases.

Expression and characterization of bioactive human thrombopoietin in the milk of transgenic mice.

Human thrombopoietin (hTPO) is the primary physiological regulator of platelet production and plays a pivotal role in promoting the proliferation and maturation of megakaryocytic progenitor cells and megakaryocytes. In this study, transgenic mice were produced harboring either full-length or the erythropoietin (EPO)-like amino-terminal domain of hTPO cDNA sequences fused to the regulatory elements of the bovine beta-casein gene. The transgene RNA was expressed exclusively in the mammary glands of eight transgenic mice, and a trace amount of the transgene was also found in the lungs of one mouse. The full-length form induced efficient expression of the protein with the highest expression level of 1500 microg/ml; however, the EPO-like domain alone expressed the protein at <0.1 microg/ml. The proteins from the two recombinant cDNAs have apparent molecular weights of about 74 and 17 kDa, due to glycosylation in the case of the full-length cDNA. Cell proliferation assay in vitro indicated that both of the recombinant forms stimulated proliferation of the TPO-dependent BaF3-Mpl cells. A positive correlation appeared between the amount of TPO in the milk of lactating animals and their blood platelet levels. About a twofold increase in platelet numbers in the blood was observed after direct subcutaneous injection of the recombinant hTPO at the level of 30 microg/kg of body weight. On the basis of these results, we anticipate that the recombinant hTPO produced efficiently in milk of transgenic mice will have the same activities as the native hTPO in a few in vivo as well as in vitro biochemical aspects.

Generation and characterization of a novel single-gene-encoded single-chain immunoglobulin molecule with antigen binding activity and effector functions.

Monoclonal antibody (MAb) CC49 is a murine IgG1 that reacts with tumor-associated glycoprotein (TAG)-72, a pancarcinoma antigen. Clinical trials using radiolabeled CC49 for diagnostic imaging have demonstrated specific localization of more than 90% of carcinomas. The feasibility of adopting in vivo gene inoculation methods for antibody-based immunotherapy requires introduction and expression of two genes, encoding immunoglobulin (Ig) heavy and light chains, in a single cell to generate a functional antibody. To circumvent the problems inherent in this approach, we have constructed a single-gene encoding a single-chain immunoglobulin (SCIg) that, unlike previously developed SCIgs, contains all IgG domains. To construct the novel SCIg, the carboxyl end of the constant region of the chimeric (c) CC49 kappa chain is joined, via a 30 residue Gly-Ser linker peptide, to the amino terminus of the CC49 heavy chain. To our knowledge, neither a linker peptide this long nor a linkage between the constant light (C(L)) and variable heavy domains has been reported previously. Transfectomas developed by introducing the expression construct of the amplifiable gene in dihydrofolate reductase-deficient Chinese hamster ovary (CHO dhfr-) cells secrete a 160 kDa homodimeric molecule, SCIgcCC49. The in vitro antigen binding properties of SCIgcCC49 are comparable to those of cCC49 and SCIgcCC49deltaC(H)1, a single-chain Ig deficient in constant heavy chain-1 (C(H)1) and C(L) domains. The antibody-dependent cellular cytotoxicity (ADCC) of SCIgcCC49 and cCC49 were also comparable. This single-gene approach for generating an immunoglobulin molecule may facilitate in vivo gene inoculation as well as ex vivo transfection of patients' cultured tumor-infiltrating lymphocytes for immunotherapy protocols for a variety of diseases, including cancer.

Homology modeling of the receptor binding domain of human thrombopoietin.

Platelet production in blood is regulated by a lineage specific humoral factor, thrombopoietin (TPO). The amino terminal domain of TPO (TPO-N) is responsible for the signal transduction mediated by the TPO receptor, c-mpl. From the predicted length of helices we found that TPO-N belongs to the long-chain subfamily of the four-helix bundle cytokine family. We built a three dimensional model of TPO-N by a comparative homology modeling procedure. The four helices of TPO-N with an up-up-down-down topology are stabilized by a tightly packed central hydrophobic core and the extended loop AB makes an additional hydrophobic core with helices B and D outside of the four helix bundle scaffold. An interpretation of the previous site directed mutageneses results in light of the model enabled us to identify two isolated receptor binding sites. The surface made of Lys 136, Lys 138 and Lys 140 in helix D, and Pro 42 and Glu 50 in loop AB forms the first receptor binding site, while the surface of Asp 8, Arg 10 and Lys 14 in helix A represents the second binding site for the sequential receptor oligomerization.

Pyrrolidine dithiocarbamate improves the septic shock syndromes in spontaneously hypertensive rats.

1. The aim of the present study was to evaluate the effect of pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor-kappa B (NF-kappa B), on septic shock induced by Escherichia coli lipopolysaccharide (LPS) in spontaneously hypertensive rats (SHR). 2. After injection of LPS in SHR, a marked decrease in blood pressure was observed at 3 h and vascular hyporeactivity to noradrenaline (NA) was observed after 1 h. A marked increase in plasma levels of tumour necrosis factor-alpha (TNF-alpha) and nitrite (an indicator of nitric oxide) was also observed in SHR. 3. The delayed hypotension and hyporeactivity to NA induced by LPS were significantly reserved by pretreatment of rats with PDTC (10 mg/kg). The increase in plasma levels of TNF-alpha and nitrite in LPS-treated groups was also significantly suppressed by PDTC pretreatment. In addition, the survival time of SHR treated with LPS was significantly prolonged by PDTC pretreatment. 4. The present ex vivo study demonstrates that the NA-induced contraction is attenuated and the L-arginine-induced relaxation is enhanced in aortic rings obtained from LPS-treated SHR. Both the reduction of the NA-induced contraction and the increase of L-arginine-induced relaxation were reversed by pretreatment with PDTC. However, the relaxation elicited by acetylcholine (ACh) was not affected in LPS-treated SHR when compared with sham-operated SHR. In addition, the ACh-induced relaxation in LPS-treated SHR was not affected by PDTC pretreatment. 5. In normotensive Wistar-Kyoto (WKY) rats, LPS had mild effects on blood pressure, vascular hyporeactivity and plasma levels of TNF-alpha and nitrite. At a higher dose, PDTC (10 mg/kg) also prolonged survival time and improved haemodynamics in LPS-treated WKY rats. In the ex vivo study, it was noted that the relaxation elicited by ACh was significantly (P < 0.05) attenuated in LPS-treated WKY rats. This attenuation of the ACh-induced relaxation by LPS in WKY rats was significantly reversed by pretreatment with 10 mg/kg PDTC. 6. In conclusion, PDTC prolongs survival time in rats with endotoxaemia and improves the septic shock syndromes both in vivo and ex vivo. Thus, we propose that PDTC may be of use in septic patients.

Identification of functionally important residues of human thrombopoietin.

Thrombopoietin (TPO) is a megakaryocyte growth and differentiation factor. It consists of a characteristic two domain structure. The amino-terminal domain of TPO has a sequence homology with erythropoietin and is required for the binding and activation of its receptor c-Mpl. To determine the functionally important regions interacting with its receptor, a series of site-directed mutants of TPO were constructed based on a three-dimensional model of the amino-terminal domain. Two strategies of mutagenesis were employed: 1) nonnative N-linked glycosylation scan of 12 residues predicted to be on the surface, and 2) alanine replacement scan of mostly charged 44 amino acid residues. Each TPO mutein was transiently expressed in COS7 cells, and the specific bioactivity of the TPO protein secreted into the culture medium was measured using a recombinant BaF3 cell line expressing human c-Mpl. Four alanine substitutions at Arg10, Pro42, Glu50, and Lys138 nearly or completely abolished the activity, whereas the mutation at Arg14 slightly decreased the activity, suggesting that these residues are functionally important in interacting with its receptor. These residues mapped to helix A, loop AB, and helix D. Sequence comparison between human TPO and other mammalian TPO showed that the identified residues are completely conserved among the species. However, unlike the recent report on the mutational analysis of TPO, alanine substitutions at Lys52, Lys59, Arg136, and Arg140 did not affect the TPO activity significantly in our system. The identified receptor binding regions of TPO are analogous to those of human growth hormone and erythropoietin. Based on the similarity of these three cytokines, we propose that Lys138 of helix D and Pro42 and Glu50 of loop AB may constitute one binding region, whereas Arg10 and Lys14 of helix A may constitute the other binding region to dimerize the receptors.

Characterization of chimeric antibody producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure.

Recombinant Chinese hamster ovary (CHO) cells expressing a high-level of chimeric antibody against S surface antigen of hepatitis B virus were obtained by co-transfection of heavy and light chain cDNA expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level such as 0.02, 0.08, 0.32, 1.0, and 4.0 microM. The highest producer (HP) subclone was isolated from each MTX level and was characterized with respect to cell growth and antibody production in the corresponding level of MTX. The specific growth rate of the HP subclone was inversely proportional to the MTX level. On the other hand, its specific antibody productivity (qAb) rapidly increased with increasing MTX level up to 0.08 microM, and thereafter, it gradually increased to 20 microg/10(6) cells/day at 4 microM MTX. Southern blot analysis showed that the enhanced qAb at higher MTX level resulted from immunoglobulin (Ig) gene amplification. The stability of the HP subclones isolated at 0.02, 0.08, 0.32, and 1.0 microM MTX in regard to antibody production was investigated during long-term culture in the absence of MTX. The qAb of all subclones significantly decreased during the culture. However, the relative extent of decrease in qAb was variable among the subclones. The HP subclone isolated at 1 microM MTX was most stable and could retain 59% of the initial qAb after 80 days of cultivation. Southern blot analysis showed that this decrease in qAb of the subclones resulted mainly from the loss of Ig gene copies during long-term culture. Despite the decreased qAb, the HP subclone isolated at 1 microM MTX could maintain high volumetric antibody productivity over three months because of improved cell growth rate during long-term culture.

Generation of human Fab monoclonal antibodies against preS1 of hepatitis B virus using repertoire cloning.

Human monoclonal antibodies (MAbs) have considerable potential in the prevention and treatment of many viral diseases. A combinatorial antibody library of heavy (Fd)- and light-chain genes derived from peripheral blood lymphocytes of a volunteer with high antibody titer to preS1 of HBV was constructed and expressed on the surface of filamentous phages. The library contained 7 x 10(9) independent clones. A phage antibody population from the third panning against preS1 was converted to one expressing soluble Fabs by removal of the g3 sequences from the pComb3 phagemid vector and subsequent transformation into E. coli TG1 cells. Screening of the library led to the identification of two clones, 3DW and 8GW, showing high reactivity toward preS1. The authenticity of the Fabs was confirmed by immunoblot analysis which yielded approximately 60 and approximately 30 kDa bands under nonreducing and reducing conditions, respectively. The soluble Fabs of 3DW and 8GW exhibited relative affinities of 6 x 10(5) and 8 x 10(6) M(-1), respectively. The sequencing results demonstrate that all Fd sequences belong to subgroup II and all light chain sequences belong to subgroup I. There are differences in CDR length and composition, especially in the FW3 and CDR3 regions of the heavy- and light-chain genes. These human Fab MAbs specific to preS1, generated from a combinatorial library, represent prototypes of passive immunotherapy candidates for viral hepatitis B.

In vitro neutralization of hepatitis B virus by monoclonal antibodies against the viral surface antigen.

In vitro HBV infection and neutralization were assayed using an anti-preS1 murine monoclonal antibody (1B3) and anti-preS2 (H69K) and anti-S (CS131A) murine-human chimeric antibodies. The 1B3 (IgG1) and H69K (IgG1) was constructed previously and the CS131A was constructed for this study by expressing stably the chimeric heavy and light chains in Chinese hamster ovary cells and purifying from the culture supernatant. Previous study showed that the H69K and CS131A recognize known virus-neutralizing epitopes, while the 1B3 does not. For the assays, adult human hepatocyte primary culture was infected with the adr or ayw subtype of HBV, and the infectivity and subsequent replication was confirmed both by measuring the kinetics of HB-sAg secretion by the infected cells and detecting the intermediate replicative form of HBV DNA in the cells. Next, the hepatocytes were infected with the adr or ayw subtype of the virus that had been preincubated with various concentrations of each of the antibodies and the neutralization of HBV was analyzed. The results showed that the anti-preS2 and anti-S chimeric antibodies exhibited neutralizing activity against both the adr and ayw subtypes of the virus, with approximately 1,000 and 2,000 times higher specific activity than polyclonal hepatitis B immune globulin, respectively, but the anti-preS1 antibody scarcely neutralized the infection. The neutralizing activities of the antibodies were consistent with their epitope specificity and antigenbinding affinity, suggesting that this neutralization assay is specific. The in vitro neutralization assay will be useful for evaluating the neutralizing activity of anti-HBV antibodies before in vivo testing in chimpanzees.

Development of an in vitro bioassay system for human thrombopoietin by constructing a recombinant murine cell line expressing human thrombopoietin receptor.

We have developed a simple and rapid in vitro bioassay system for human thrombopoietin (hTPO) by constructing a recombinant murine BaF3 cell line expressing the hTPO receptor. The cDNA encoding hTPO receptor (c-Mpl) was cloned from human erythroleukemia (HEL) cells by reverse transcription-polymerase chain reaction (RT-PCR) and linked to the human cytomegalovirus promoter in pcDNA3 to yield expression plasmid phTR. The expression plasmid was stably transfected into BaF3 cells. The resulting transformants were initially selected in RPMI medium containing G418 and murine IL-3 (MuIL-3) and subjected to positive selection in the medium containing hTPO. Finally, cell proliferation of the selected clones in response to hTPO was measured using a colorimetric MTT assay. Most transformants showed a dose-dependent proliferation in response to 0.1 to 100 ng/ml hTPO, among them a cell clone (BaF-mpl), that showed a saturation density of 1.0 x 10(6) cells/ml and a doubling time of 16 h in the log growth phase. This clone was chosen for further characterization of hTPO-dependent proliferation. The BaF-mpl cells showed specificity for TPO, and they died within 24 h in the absence of TPO, which enabled us to complete the assay within 2 days. In addition, optimal MTT assay conditions were established for MTT treatment time and the number of cells to be added in the assay.