In vitro evaluation of cytochrome P450 and glucuronidation activities in hepatocytes isolated from liver-humanized mice.

Cryopreserved human (h-) hepatocytes are currently regarded as the best in vitro model for predicting human intrinsic clearance of xenobiotics. Although fresh h-hepatocytes have greater plating efficiency on dishes and greater metabolic activities than cryopreserved cells, performing reproducible studies using fresh hepatocytes from the same donor and having an "on demand" supply of fresh hepatocytes are not possible. In this study, cryopreserved h-hepatocytes were transplanted into albumin enhancer/promoter-driven, urokinase-type plasminogen activator, transgenic/severe combined immunodeficient (uPA/SCID) mice to produce chimeric mice, the livers of which were largely replaced with h-hepatocytes. We determined whether the chimeric mouse could serve as a novel source of fresh h-hepatocytes for in vitro studies. h-Hepatocytes were isolated from chimeric mice (chimeric hepatocytes), and cytochrome P450 (P450) activities were determined. Compared with cryopreserved cells, the P450 (1A2, 2C9, 2C19, 2D6, 2E1, 3A) activities of fresh chimeric hepatocytes were similar or greater. Moreover, ketoprofen was more actively metabolized through glucuronide conjugates by fresh chimeric hepatocytes than by cryopreserved cells. We conclude that chimeric mice may be a useful tool for supplying fresh h-hepatocytes on demand that provide high and stable phase I enzyme and glucuronidation activities.

Novel humanized anti-CD20 monoclonal antibodies with unique germline VH and VL gene recruitment and potent effector functions.

The anti-CD20 chimeric monoclonal antibody (mAb) rituximab is the most widely used therapeutic antibody for B-cell malignancies. However, approximately 50% of non-Hodgkin's lymphoma (B-NHL) patients respond to treatment with this antibody. Novel humanized antibodies target membrane CD20 with enhanced effector properties should improve treatment for a broader patient population with relapsed and refractory disease. A novel chimerized form of the murine anti-CD20 1K1791 exerts more potent antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities and induces cell death by a non-caspase dependent process. Humanized mAbs derived from 1K1791 were designed using four different humanization techniques and characterized. In contrast to rituximab or 2F2 (human anti-CD20 mAb), several of these exhibited superior ADCC, CDC, inhibition of cell growth and cell death. There was a wide range of functional differences among the humanized forms of 1K1791 despite a modest replacement of amino acid residues in the CDRs. To determine whether the superior activities exhibited by parental murine mAb 1K1791 were due to differences in VH and VL rearrangement, we analyzed its germline and compared it to other anti-CD20 mAbs. A remarkable conservation of VH and Vk (VL kappa) gene usage was observed in the murine anti-CD20 mAbs. 18/23 used the same germline gene J558.42 and 4/23 used closely related genes of the 'J558' group. Thus, 22/23 belonged to VH1 family. One exception was the mAb 1K1791, which was derived from the VH9.12 germline gene. 1K1791 was also unique in its use of a Vk19/28 family gene whereas most other mAbs (21/23) used Vk4/5 family genes. A formal relationship between the particular germline gene recruitment and antibody functionality has not been established, however, the present findings identified humanized mAbs with functional activities that were superior to rituximab and 2F2. These in vitro results support future in vivo animal testing and subsequent clinical trials.

Characterization of novel murine anti-CD20 monoclonal antibodies and their comparison to 2B8 and c2B8 (rituximab).

Rituximab is the first anti-cancer antibody approved by the FDA for the treatment of B-cell non-Hodgkin lymphoma (B-NHL), alone or in combination with chemotherapeutic drugs. Further, rituximab is now being examined in a variety of CD20+ neoplastic diseases as well as B-cell-induced autoimmune diseases. The clinical response to rituximab is significant, resulting not only in tumor regression but also prolongation of survival. However, a subset of patients does not initially respond to rituximab or develops resistance to its further treatment. Therefore, alternative therapies for these patients are strongly desired. Rituximab activity has been thought to be by antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity and apoptosis, and studies in model systems established the role of rituximab in cell signaling-induced perturbation of anti-apoptotic survival pathways, suggesting that the patients unresponsive to rituximab may be overcome with other CD20 antibodies with different activities. This study investigated eight novel murine antibodies directed against CD20 for their physical and biological properties in comparison with 2B8 and c2B8 (rituximab). These antibodies were derived by various antigenic and immunization procedures and selected for CD20 activity. Analysis of these antibodies revealed that they all bound to various B-cell lines and CD20-transfected CHO cells. Six of the eight antibodies shared similar variable-region amino acid sequences that were also shared by 2B8 while two monoclonal antibodies did not. Of them, 1K1791 has a distinct heavy chain and both 1K1791 and 1K1782 have distinct light chains. Not all of the antibodies inhibited cell growth and only two antibodies reacted with fixed GST-CD20 recombinant fusion protein. Noteworthy, 1K1791 was found to inhibit cell proliferation and also induced caspase-independent apoptosis in the absence of cross-linker. These findings identified new antibodies with properties and epitope specificities different from 2B8. The potential clinical application of such antibodies in the treatment of B-NHL and rituximab-resistant B-NHL is discussed.

Eight genotypes (A-H) of hepatitis B virus infecting patients from San Francisco and their demographic, clinical, and virological characteristics.

Clinical lines of evidence have been accumulated that hepatitis B virus (HBV) genotypes have characteristic geographical distributions and distinct clinical impact on liver diseases. The distribution of HBV genotypes was determined with reference to hepatitis B e antigen (HBeAg) and alanine aminotransferase (ALT) levels in 165 patients with hepatitis B in San Francisco. HBV genotypes were determined by enzyme-linked immunosorbent assay (ELISA) and the unclassified samples were sequenced within the S region for phylogenetic analysis. Genotype A occurred in 60 (36%) patients, B in 16 (10%), C in 56 (34%), D in 19 (12%), E in 1 (1%), F in 1 (1%), G in 8 (5%), and H in 4 (2%). Caucasians were infected predominantly with HBV genotype A (HBV/A) (38 of 57 [67%]), Asians with HBV/C (45 of 63 [71%]), and Hispanics with HBV/F and HBV/H (4 of 9 [44%]). Serum ALT levels were higher in the patients infected with HBV/A (P = 0.03) or HBV/G (P = 0.02) than HBV/C. HBeAg was more frequent in patients infected with HBV/G than HBV/C or HBV/D (7 of 8 [88%] vs. 25 of 56 [45%] or 6 of 19 [32%], P = 0.03 or 0.01). In conclusion, eight genotypes (A-H) were identified in San Francisco in association with various ethnicities and then influenced ALT levels as well as the prevalence of serum HBeAg. HBV genotype H might be identified by combination of preS2 serotpe bksf and HBsAg serotype adw.

Frequent coinfection with hepatitis B virus strains of distinct genotypes detected by hybridization with type-specific probes immobilized on a solid-phase support.

A genotype-specific probes assay (GSPA) was developed for distinguishing the seven genotypes (A-G) of hepatitis B virus (HBV). Nucleotide (nt) sequences corresponding to preS1 region were amplified by PCR with a primer labeled with biotin, and delivered to eight wells on which complementary sequences specific to one or other genotype had been immobilized. Thereafter, hybridization of HBV DNA sequences amplified from the test serum was detected by colorimetry. When 256 sera from HBV carriers in Bangladesh, Cameroon, Japan, South Africa, USA and Uzbekistan were subjected to GSPA, genotypes were concordant with those of ELISA with monoclonal antibodies to epitopes on preS2-region products in 242 (94.6%) of them; 8 sera (3.1%) were not genotypeable by either method. Cloning analysis confirmed the presence of two distinct HBV genotypes in the seven selected sera with coinfection. There were 7 (2.7%) sera with discordant genotyping results between GSPA and ELISA. When HBV DNA clones propagated from these sera were sequenced and analyzed phylogenetically, the genotypes determined by GSPA were verified. Coinfection with HBV strains of two distinct genotypes was identified by GSPA in 28 (10.9%) sera, while it was suggested by ELISA in only 2 (0.8%) sera. The GSPA method would be particularly useful for detecting the coinfection with distinct HBV genotypes of any clinical relevance, which seems to be more frequent than reported previously.

Distribution of hepatitis B virus (HBV) genotypes among HBV carriers in the Cote d'Ivoire: complete genome sequence and phylogenetic relatedness of HBV genotype E.

The characteristics of hepatitis B virus (HBV) genotype E are not well known because only a few studies have been carried out by complete genome analysis. The aim of this study was to elucidate the distribution of HBV genotypes in Cote d'Ivoire, and to clarify the genotype-related characteristics of genotype E. The distribution of HBV genotypes among 48 HBV carriers in Cote d'Ivoire was determined using serological and genetic methods. The characteristics of genotype E were evaluated by complete genome sequences, and further investigations of small S gene, basic core promoter (BCP) mutation, and precore mutation were undertaken. HBV genotype distribution among the 48 carriers was 6.3% for genotype A, 6.3% for genotype D, and 87.4% for genotype E. Complete genomes of two genotype E strains were sequenced, and found to have 98.2% to 99.2% homology at the nucleotide level when compared with genotype E strains reported previously. In 24 genotype E carriers, the precore mutation was detected in 75% of the patients without HBeAg, in contrast to only 25% of the patients with HBeAg (P < 0.05). All 24 strains have T at nucleotide 1858 in the precore region. In contrast, BCP double mutation was detected in 17% of the patients with HBeAg, and 33% of the patients without HBeAg. These results indicated as the following: (1) genotypes A, D, and E of HBV exist in Cote d'Ivoire and genotype E is the most prevalent; (2) genotype E spread with low genetic diversity over the complete genome in West Africa; (3) HBV precore and/or BCP double variants were common among the patients with genotype E infections.

Clinical outcomes of hepatitis B virus (HBV) genotypes B and C in Japanese patients with chronic HBV infection.

The aims of this retrospective survey were to determine the epidemiologic distribution of hepatitis B virus (HBV) genotypes and analyze the genotype-related clinical differences among Japanese patients with chronic HBV infection. The 158 surveyed patients with chronic HBV infection lived in Fukuoka and Okinawa were serially tested for serum alanine aminotransferase (ALT) and hepatitis B e antigen (HBeAg). Follow-up was for a period of 10.8 +/- 6.4 years (mean +/- SD). The HBV genotypes were determined in sera by an enzyme-linked immunosorbent assay and detection of HBV DNA in serum was done by the transcription-mediated amplification-hybridization protection assay. Genotypes B and C were found in 58 (36.7%) and 100 (63.3%) of the patients, respectively. Genotype B was predominant in Okinawa (B = 86.9%, C = 13.1%), whereas genotype C was predominant in Fukuoka (B = 5.2%, C = 94.8%). The HBeAg positivity and ALT abnormality rates at the start of the observation period were significantly higher in patients with genotype C (66.0% and 84.0%) than in patients with genotype B (34.5% and 22.4%) (P < 0.05, respectively). The annual rate of spontaneous HBeAg disappearance in patients with genotype B was much higher than in patients with genotype C (8.38% versus 2.34%, respectively). Patients with genotype C who were continuously HBeAg negative from entry had a significantly higher ALT abnormality (58.8%) than those with genotype B (19.2%) (P < 0.05). Interestingly, patients with genotype C who became HBeAg negative after treatment with interferon had a high ALT abnormality (58.8%). All patients with an ALT abnormality were positive for HBV DNA in their serum. These findings indicate that patients with HBV genotype C have more severe liver deterioration because of the delay of HBeAg disappearance and continued HBV replication after HBeAg disappearance.

Hepatitis B virus genotypes in Uzbekistan and validity of two different systems for genotyping.

Hepatitis B virus (HBV) has been classified into seven genotypes, designated A-G. The HBV genotype has a characteristic geographical distribution. The Republic of Uzbekistan is located in the heart of Asia and has been considered to be a region with high endemicity of hepatitis viruses. However, the present distribution of hepatitis virus infection in this region is unknown. The aim of this study was to investigate the distribution of HBV genotypes and to elucidate the validity of two genotyping systems in Uzbekistan. Fifty-four patients with hepatitis B surface antigen were investigated. HBV genotypes were determined by two methods: one based on restriction fragment length polymorphism (RFLP) targeting to S region, and another on enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies to pre-S2 region. Seven (13%) and 47 (87%) of the 54 subjects were classified into genotypes A and D, respectively. Dual infection of two viral populations of the same genotype was observed in one subject. No significant difference of ALT level (203.3 +/- 244.7 vs. 190.6 +/- 39.5) and HBeAg (42.9% vs. 42.6%) were found between genotypes A and D. In this study, the validity of the genotyping systems in this region was confirmed.