Production and characterization of recombinant human anti-HBs Fab antibodies.

Recombinant human Fab antibodies were generated with different reactivities against the hepatitis B virus surface (HBs) antigen. To isolate the antibodies, a method was used that combined transformation of human B cells by Epstein-Barr virus (EBV) infection with a primer-vector system developed for isolating DNA fragments of human Ig Fab portions. With this method, monoclonal and oligoclonal cell lines producing anti-HBs antibodies were established and three anti-HBs Fab antibodies were isolated from two of these cell lines. From analysis of affinity characteristics, immunohistochemical activity, and cytolysis activity, these three Fab antibodies were classified into three different groups. The first group had high affinity for HBs, the second had the ability to kill HBV-infected cells, and the third was applicable to immunohistochemical staining with HBV-infected cells. The combined effect of these antibodies was also investigated by complement-dependent cytotoxicity assay.

Association of FcgammaRII with low-density detergent-resistant membranes is important for cross-linking-dependent initiation of the tyrosine phosphorylation pathway and superoxide generation.

IgG immune complexes trigger humoral immune responses by cross-linking of FcRs for IgG (FcgammaRs). In the present study, we investigated role of lipid rafts, glycolipid- and cholesterol-rich membrane microdomains, in the FcgammaR-mediated responses. In retinoic acid-differentiated HL-60 cells, cross-linking of FcgammaRs resulted in a marked increase in the tyrosine phosphorylation of FcgammaRIIa, p58(lyn), and p120(c-cbl), which was inhibited by a specific inhibitor of Src family protein tyrosine kinases. After cross-linking, FcgammaRs and tyrosine-phosphorylated proteins including p120(c-cbl) were found in the low-density detergent-resistant membrane (DRM) fractions isolated by sucrose-density gradient ultracentrifugation. The association of FcgammaRs as well as p120(c-cbl) with DRMs did not depend on the tyrosine phosphorylation. When endogenous cholesterol was reduced with methyl-beta-cyclodextrin, the cross-linking did not induce the association of FcgammaRs as well as p120(c-cbl) with DRMs. In addition, although the physical association between FcgammaRIIa and p58(lyn) was not impaired, the cross-linking did not induce the tyrosine phosphorylation. In human neutrophils, superoxide generation induced by opsonized zymosan or chemoattractant fMLP was not affected or increased, respectively, after the methyl-beta-cyclodextrin treatment, but the superoxide generation induced by the insoluble immune complex via FcgammaRII was markedly reduced. Accordingly, we conclude that the cross-linking-dependent association of FcgammaRII to lipid rafts is important for the activation of FcgammaRII-associated Src family protein tyrosine kinases to initiate the tyrosine phosphorylation cascade leading to superoxide generation.

Cloning and expression of human anti-tumor necrosis factor-alpha monoclonal antibodies from Epstein-Barr virus transformed oligoclonal libraries.

Peripheral blood was obtained from a healthy human volunteer and transformed with Epstein-Barr virus (EBV). This produced an oligoclonal cell library in culture medium that was screened by ELISA for anti-human tumor necrosis factor-alpha (TNFalpha) activity. RNA from two positive clones was applied to RT-PCR using antibody-specific primers, and the light (kappa and lambda) and heavy chain genes (gamma and mu) were cloned into the plasmid vector pFab1-His2. The antibodies produced in Escherichia coli as Fab fragments were assayed for anti-TNFalpha activity utilizing ELISA. Two IgG1/kappa anti-TNFalpha antibodies and two IgM/kappa anti-TNFalpha antibodies were isolated. DNA sequence analysis showed that the VL and VH gene families of IgM and IgG were the same. Both the antibodies showed almost the same activity on ELISA-testing. Ten clones randomly selected from light (kappa and lambda) and heavy (gamma and mu) chain genes in the oligoclonal cell library 1D5 were sequenced, and each gene (kappa, lambda, gamma, and mu) was found to be composed of one to three different genes. These data support the conclusion that the cell clone is oligoclonal at the molecular level.

A new phosphoglycerolipid, 'phosphatidylglucose', found in human cord red cells by multi-reactive monoclonal anti-i cold agglutinin, mAb GL-1/GL-2.

Cord red cell membranes express many differentiation-related molecules. To study such molecules, we have established human cell lines, termed GL-1 and GL-2, by the Epstein-Barr virus transformation method, both of which produce monoclonal anti-i cold agglutinin [Y. Nagatsuka et al., Immunol. Lett. 46 (1995) 93-100]. Thin layer chromatography immunoblotting analysis revealed that these antibodies had broad specificities reacting with a variety of glycolipid antigens. Of the immunoreactive lipid antigens, a new phosphoglycerolipid containing glucose from human cord red cells was found. The isolated lipid was unstable to alkaline hydrolysis and contained glucose as a sole sugar. Secondary ion mass spectrum-collision-induced dissociation mass spectrometric analysis of this lipid gave the main molecular ion peak at m/z 885 corresponding to phosphatidylhexose. This antigen was susceptible to phospholipases A2, C and D but resistant to phosphatidylinositol-specific phospholipase C. Two-dimensional nuclear magnetic resonance spectroscopy confirmed that glucose is linked to the sn-glycerol 3-phosphate residue with a beta-anomeric configuration. Based upon these combined results, we identified this lipid as phosphatidyl-beta-D-glucose. This is the first report showing the presence of the glucosylated glycerophospholipid in mammalian sources.

A new case of alpha-N-acetylgalactosaminidase deficiency with angiokeratoma corporis diffusum, with Ménière's syndrome and without mental retardation.

alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency is a rare hereditary lysosomal storage disease, and only three alpha-NAGA-deficient patients with angiokeratoma corporis diffusum (Kanzaki) have been described. We report a further case in a 47-year-old Japanese woman, the product of a consanguineous marriage. The remarkable findings in this patient were her normal intelligence, Ménière's syndrome, disturbance of peripheral sensory nerves, hearing loss and cardiac hypertrophy. alpha-NAGA enzyme activity in her plasma was 0.77% of the normal value. Other enzyme activities, such as alpha-galactosidase, beta-galactosidase, alpha-L-fucosidase, beta-mannosidase and aspartylglucosaminidase, were within normal limits. A large quantity of amino acid O-glycans was detected in her urine. Gene analysis revealed a novel point mutation (G-->A transition) at nucleotide 11018 (986 in the cDNA) resulting in an Arg-329-Gln substitution. Kanzaki disease has the same enzyme defect as Schindler disease, but the manifestations are quite different.

Complex gangliosides as cell surface inhibitors for the ecto-NAD+ glycohydrolase of CD38.

Leukocyte cell surface antigen CD38 is a single-transmembrane protein whose extracellular domain has catalytic activity for NAD(+) glycohydrolase (NADase). We previously reported that b-series gangliosides inhibit the NADase activity of the extracellular domain of CD38 expressed as a fusion protein [Hara-Yokoyama, M., Kukimoto, I., Nishina, H., Kontani, K., Hirabayashi, Y., Irie, F., Sugiya, H., Furuyama, S., and Katada, T. (1996) J. Biol. Chem. 271, 12951-12955]. In the present study, we examined the effect of exogenous gangliosides on the NADase activity of CD38 on the surface of retinoic acid-treated human leukemic HL60 cells and CD38-transfected THP-1 cells. After incubation of the cells with G(T1b), inhibition of NADase activity was observed. The time course of inhibition was slower than that of the incorporation of G(T1b) into the cells, suggesting that incorporation into the cell membranes is a prerequisite for inhibition. Inhibition occurred efficiently when G(T1b) and CD38 were present on the same cells (cis interaction) rather than on different cells (trans interaction). Although gangliosides may affect localization of cell surface proteins, indirect immunofluorescence intensity due to CD38 was not affected after G(T1b) treatment. Comparison of the effect of G(T1b) and G(D1a) indicates that the tandem sialic acid residues linked to the internal galactose residue of the gangliotetraose core are crucial to the inhibition. These results suggest a novel role of complex gangliosides for the first time as cell surface inhibitors of CD38 through specific and cis interaction between the oligosaccharide moiety and the extracellular domain.

Bacterial expression of a human recombinant monoclonal antibody fab fragment against hepatitis B surface antigen.

The Fab fragment was cloned from the monoclonal cell line TAPC301-CL4, which was produced using the Epstein-Barr virus (EBV) transformation method. This cell line produces a human monoclonal antibody (CL4MAb) against the hepatitis B surface antigen (HBsAg). This MAb was shown to have hepatitis B virus (HBV) neutralizing activity in chimpanzees. The Fab fragment was produced by subjecting the heavy and light chain antibody genes of the TAPC301-CL4 cell line to reverse transcription-polymerase chain reaction, cloning the products in the plasmid vector pFab1-His2 and introducing the plasmid into bacteria. Sequence analyses of the CL4Fab fragment revealed that the light and heavy chains belong to the Vk3a and VH3 groups of the immunoglobulin (Ig) family, respectively. An enzyme-linked immunosorbent assay confirmed that specificity of the recombinant CL4Fab antibody against HBsAg was the same as that of the parental MAb. Flow cytometric analysis using PLC/PRF/5 (Alexander) cells, which express HBsAg, showed the reactivities of the CL4MAb and CL4Fab antibody were the same. These results suggest that the recombinant CL4Fab antibody produced by Escherichia coli using the new vector-primer system developed for human IgG Fab fragments has a very high affinity for the HBsAg and may be useful clinically. A source for generation of human MAb for human therapy with very stable and specific expression was thus produced by isolating antibodies from EBV-transformed cell lines.

The phylogeny of the cactophilic yeasts based on the 18S ribosomal RNA gene sequences: the proposals of Phaffomyces antillensis and Starmera caribaea, new combinations.

The complete sequences of the 18S rRNA gene fragments of the type strains of the cactophilic yeast species, Pichia antillensis, Pichia caribaea, Phaffomyces opuntiae, Phaffomyces thermotolerans, Starmera amethionina var. amethionina, and Starmera amethionina var. pachycereana were determined and compared. The type strain of Phaffomyces opuntiae had two kinds of the 18S rRNA gene sequences of which base differences were counted to be 15 and of which the percent similarity was calculated to be 99.1. The type strains of P. antillensis, P. caribaea, and Starmera amethionina var. pachycereana had the Q-7 system. The phylogenetic analyses showed that the genera Phaffomyces and Starmera were monophyletic and distant from each other and from the other species examined of the ascogenous teleomorphic genera, and that P. antillensis and P. caribaea were included within the clusters of the genera Phaffomyces and Starmera, respectively. The two Pichia species were transferred to the genera Phaffomyces and Starmera as the new combinations, Phaffomyces antillensis and Starmera caribaea. The new family Phaffomycetaceae was proposed as the type genus Phaffomyces.

Establishment and characterization of an Epstein-Barr virus-transformed B cell line, KM/C8, from a patient with infantile sialic acid storage disease.

Nakano et al. have recently reported a Japanese case of infantile sialic acid storage disease [C. Nakano, Y. Hirabayashi, K. Ohno, T. Yano, T. Mito, M. Sakurai, Brain Dev., 18 (1996) 153-156]. For further etiological analysis of this disease, we prepared the Epstein-Barr virus (EBV)-transformed cell line (LCL) from the peripheral lymphocytes of this patient and performed initial characterization of the cells. Electron microscopy of the cells showed that the cells contained many vacuoles and swelled lysosomes. Cytochemical staining with sialic acid-specific lectin, Limax flavus agglutinin (LFA), showed strong staining on membranes and subcellular organelles on the patient-derived cells, whereas LCL from a normal person was only weakly stained. The cells from the patient contained 5.5-7.3 nmol/107 cells of free N-acetyl neuraminic acid, whereas three strains of LCLs derived from normal persons contained 1 nmol/107 cells. The culture supernatant of LCL from the patient contained 144 nmol/ml of free N-acetyl neuraminic acid, whereas the LCL culture supernatant from normal persons contained 57-73 nmol/ml of free sialic acid, which was the same or only at a slightly higher level than the fresh medium. In addition, cellular acidic sialidase measured as 4-methylumbelliferyl sialidase was elevated (107 nmol 4-methylumbelliferon released/mg cellular protein/60 min). The EBV-LCL from an ISSD patient is considered to remain as the abnormality of the cell donor.

The Epstein-Barr virus (EBV) alters the B cell glycolipid which is recognized by the human monoclonal antibody to i-blood group antigen.

To analyze the differentiation-related glycolipids, we have recently developed human monoclonal anti-i antibodies (mAbs) using a combination of EBV-transformation and bovine i-active glycolipid (NeuAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc beta 1-->1Cer)-containing liposome immune lysis assay (LILA). Using complement cytolysis with these mAbs, we found the occurrence of a surface antigen (Ag) in the EBV-negative but not in the EBV-positive cell lines. The fresh EBV infection reveals that the suppressed expression of the antigen was a result of the EBV infection. The Ag recognized with these mAbs appeared to have a very low density on the B cell lines. Unexpectedly, thin layer chromatogram (TLC)-immunostaining using the mAbs revealed that the major immunoreactive substance in the EBV-negative B cell lines was an extremely minor glycolipid that was distinct from the i-active glycolipid however, this was not the case in the EBV-positive B cell lines.

Repression of cell cycle progression by antisense HMG2 RNA.

The expression level of the HMG2 gene during the cell cycle of rat fibroblast cell lines was analyzed. Northern analysis demonstrated that the level of HMG2 mRNA was markedly enhanced in the post-S phase and reached a maximum at G2 phase, suggesting that expression of the HMG2 gene is not coupled with DNA synthesis. Progression of the cell cycle of COS-1 cells was repressed during G1 to S phase by expression of the antisense RNA for HMG2, resulting in a decrease of cell growth. These results suggest that the fluctuation of the HMG2 message during the cell cycle is not a consequence of but a prerequisite for cell proliferation.

Production of human monoclonal antibodies to i blood group by EBV-induced transformation: possible presence of a new glycolipid in cord red cell membranes and human hematopoietic cell lines.

To study the differentiation-associated glycolipid two anti-i mAb producers, GL-1 and GL-2, were established from the combination of EBV-induced transformation of normal PBL and immune lysis of fluorescent dye-trapped liposome-containing bovine i-active glycolipid. The mAb GL-1 reacted with both sialosylparagloboside and pentahexosyl ceramide and the bovine i-active glycolipid whereas mAb GL-2 reacted only with the bovine i-active glycolipid in LILA. Both mAbs cold-agglutinate human cord red cells but not adult red cells. However, unexpectedly, the majority of the reactivity of these mAbs in human cord red cells on TLC was not identical to the i-active glycolipid. The GL-1 antigenic substance is considered to be a glycolipid distinct from the i-active glycolipid because the immunoreactivity was canceled with endoglycoceramidase which cleaves a linkage between the oligosaccharide and ceramide. Based on complement cytolysis with the mAb, 15 hematopoietic cell lines and normal peripheral lymphocytes were screened for susceptibility to the mAbs. A Burkitt lymphoma cell line, Ramos, was most sensitive among those tested, and BJA-B, Daudi, Namalwa in the B cell lines, TALL-1, Jurkatt in the T-cell lines and HL-60 in the non-lymphoid cell lines were sensitive whereas normal lymphocytes or other 8 cell lines were not. An immunoreactive spot with the same Rf with cord red cells was also detected in sensitive cell lines. The possible presence of a new glycolipid antigen determined from the mAb and related to the differentiation of hematopoietic cells was speculated.

Initiation zone of DNA replication at the aldolase B locus encompasses transcription promoter region.

Aldolase A (AldB) gene is one of the liver-specific genes, which is activated in the fetal stage. As a first step to investigate the functional relationship between transcription and DNA replication, we intended to determine the initiation zone of replication nearest to the AldB gene region. BrdU-labeled nascent DNA was obtained from G1/S arrested hepatoma cells at various times after entering S phase. Hybridization of the newly synthesized, BrdU-labeled DNA with probes corresponding to regions spanning about 26 Kb, revealed that replication zone locates within the AldB gene region. This result, together with the result of hybridization of nascent DNA obtained by alkaline sucrose density-gradient centrifugation, suggested that the initiation zone is located within a more defined region (about 1.0 Kb) containing AldB promoter. In the predicted initiation zone, a purine-rich element which shows high homology to known mammalian origin sequences and other replication components are found. Further, autonomously replicating activity of this initiation zone was examined by DNA transfection. The results showed that the predicted initiation zone confers replication initiation in Cos-1 cells.

Anti-bacterial antibodies in Epstein-Barr virus (EBV)-transformed oligoclonal B-cell lines established from normal persons and autoimmune disease patients.

We have established 950 and 430 oligoclonal B-lymphoblastoid cell lines (LCL) from two normal persons and eight autoimmune disease patients, respectively by using Epstein-Barr virus (EBV)-induced transformation. To re-evaluate the EBV technique for production of human monoclonal antibodies (mAb) related to infectious disease, we screened these oligoclonal LCLs for antibodies against 31 bacterial strains systematically. A total of 74 cultures out of 1380 were reactive to a total of 18 strains out of 31. Among these, eight cultures showed 10(-3) antibody (Ab) titers to Pseudomonas aeruginosa serotypes C, E, F and I, Staphylococcus aureus, Serratia marcescens and Bacillus cereus. Ten cultures showed 10(-2) Ab titers to Ps. aeruginosa serotypes D, E, F and I, Ps. maltophilia, Staph. epidermidis, Klebsiella ozaenae, Ser. marcescens and B. subtilis. The results reveal the further possibilities for the EBV technique to produce various infectious disease-related human mAbs.

Anti-glycolipid antibodies produced by Epstein-Barr virus (EBV)-transformed oligoclonal B cell lines obtained from normal persons and autoimmune disease patients.

Oligoclonal B-lymphoblastoid cell lines (LCL) were blindly established by nonimmunized protocol from natural populations of two normal persons and 8 autoimmune disease patients using Epstein-Barr virus (EBV)-induced transformation. We systematically screened these LCLs on antibodies against a panel of glycolipids using liposome immune lysis assay (LILA). Eventually we found antibodies to 12 out of 15 compounds containing CTH, globoside, Forssman, paragloboside, CPH, sulfatide, NAGM3, NGGM3, i active glycolipid, GM1, GA1 and GA2 in 81 out of 950 LCLs from normal PBL, and antibodies to 2 out of 15 compounds containing sulfatide and CPH were also detected in 11 out of 430 LCLs from autoimmune disease patients. Unexpectedly, the antibody repertoire of LCLs from autoimmune disease patients was impoverished. The possibility of the EBV technique for production of various kinds of human monoclonal antibodies (MAbs) to glycolipid was shown.

Differential roles of the caudate nucleus and putamen in motor behavior of the cat as investigated by local injection of GABA antagonists.

In order to reveal a functional difference between the two distinctly separate nuclei of the striatum, i.e. the caudate nucleus (Cd) and the putamen (Put), we studied the effects of local bicuculline (BIC) and picrotoxin (PTX) injection into these nuclei on the motor behavior in the cat. The extent of diffusion of the injected BIC could be approximated by determining the extent of spreading of the dye fast green FCF (FCF) mixed with BIC solution since the extent of diffusion of radioactive [3H]BIC mixed with BIC and FCF solution was almost the same as that of FCF.BIC and PTX are GABA antagonists and are assumed to activate efferent neurons of the Cd and Put by removing the action of GABA-ergic inhibitory synapses on them. A total of 28 BIC and 22 PTX injections was made in 20 adult cats, 22 aimed at the Cd and 28 at the Put. Injection of BIC or PTX to either the head or body of the Cd unilaterally induced locomotor hyperactivity without any postural asymmetry or circling tendency. When BIC or PTX was injected into the Put, dystonic movements (dystonia) towards the contralateral side appeared frequently in the neck and trunk. Even though the injected BIC or PTX often spread to the external segment of the globus pallidus, claustrum, or anterior sylvian gyrus, none of these areas was consistently associated with dystonia. These results demonstrate that the Cd and Put are differentially associated with locomotor and postural functions.

Disturbance of two simultaneous motor acts in patients with parkinsonism and cerebellar ataxia.

Parkinsonian patients showed great difficulty in performing two motor acts in space and time although each single motor act was relatively well performed. In contrast, patients with cerebellar ataxia had no more difficulty in performing two motor acts than did normal subjects although they performed each motor act clumsily. The results suggest that parkinsonian patients use special strategies of motor performance, requiring higher levels of brain function such as attention, strong effort, and feedback, and that abnormality of basal ganglia uses higher levels of brain function for motor performance.

Seroepidemiological studies on a non-A, non-B hepatitis specific antigen/antibody system (SO-antigen/anti-SO).

The patients and staff members of a haemodialysis unit were examined for their serological responses to SO-antigen, which was isolated from the urine of epidemic type non-A, non-B hepatitis patients at Tohoku University Hospital. To understand how SO-antigen or SO-antigen-related aetiology can be incriminated for the hepatitis found in the haemodialysis unit, the prevalence of SO-antigen/anti-SO system and hepatitis A and B virus-related antibodies was compared in the sera of patients and staff members. Although the SO-antigen was rarely detected in the serum, anti-SO antibody was frequently detected in the sera of patients and staff. A significantly higher prevalence was found in the serum of patients (15%, 54 out of 361) than staff members (7.1%, 13 out of 184) and volunteer blood donors (1%, 3 out of 305). The same prevalence percentages of HBV-related antibodies (either positive for anti-HBs or anti-HBc) and anti-HAV were observed among the patients, staff, and volunteer blood donors, irrespective of whether the sera were anti-SO positive or negative. Among the staff, anti-SO antibody was more frequently found in those with a history of acute hepatitis (16.7%, 3 out of 18) than in those without (6%, 10 out of 166). These prevalence ratios conformed with those of HBV-related antibodies, but the same prevalence ratios of antibody to HAV were observed between the staff with and without a history of acute hepatitis. These results indicate that the SO-antigen/anti-SO system or entity related to this immune system is distinct from HBV or HAV, and this immune system was found widely in the haemodialysis unit where type B and non-A, non-B hepatitis were also found frequently.

Long-term recording of extracellular unitary spikes of the basal ganglia neurons in moving cats.

A technique for long-term recording of unitary spikes of the basal ganglia neurons in moving cats has been introduced. For recording of extracellular unitary spikes teflon-coated platinum/iridium wires of 25 microns diameter (A-M Systems, WA, U.S.A.) are used. A suitable preamplifier using small diodes and FETS is made. A crucial point is to make the head of the preamplifier as small as possible and to connect it to the main part of the preamplifier with soft wires. The same amplitude spikes are picked up by a slicer device and the spikes are converted into square pulses of 1 ms duration. To study the relationship between changes of unitary activities and behavior, a video-tape recording system which includes two cameras, mixing device and editing machine is used. A way of identification of sites of recorded cells is described.