Aryl hydrocarbon (Ah) receptor-independent induction of Cyp1a2 gene expression by acenaphthylene and related compounds in B6C3F1 mice.

Treatment of B6C3F1 mice with acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene and dibenzofuran resulted in induction of hepatic microsomal methoxyresorufin O-deethylase (MROD) activity. Acenaphthylene was the most potent inducer of MROD, a Cyp1a2-dependent activity, and was utilized as a prototypical inducer for this group of tricyclic hydrocarbons. Acenaphthylene (300 mg/kg) caused a > 80-fold induction of hepatic microsomal MROD activity; no induction was observed in kidney or lung. Analysis of induced hepatic microsomes with antibodies to Cyp1a1 and Cyp1a2 showed that acenaphthylene induced immunoreactive Cyp1a2 but not Cyp1a1 proteins and subsequent mRNA analysis confirmed with a cDNA probe for Cyp1a1 and Cyp1a2 that acenaphthylene induced Cyp1a2 but not Cyp1a1 mRNA. Results from nuclear run-on experiments using hepatic nuclei showed that acenaphthylene caused an approximately 4-fold increase in the rate of Cyp1a2 gene transcription in B6C3F1 mice. Results of competitive binding studies indicated that the tricyclic hydrocarbons did not competitively displace [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin or [3H]benzo[a]pyrene from the mouse hepatic cytosolic aryl hydrocarbon (Ah) receptor or 4S carcinogen binding protein respectively. The data indicate that acenaphthylene and related tricyclic hydrocarbons induce Cyp1a2 gene expression in B6C3F1 mice via an Ah receptor-independent pathway. Thus, tricyclic hydrocarbons induce Cyp1a2 without the co-induction of Cyp1a1 and therefore these relatively non-toxic compounds can be used to further probe the role of Cyp1a2 in the metabolism and metabolic activation of diverse chemical carcinogens.

Enzyme-kinetic and immunochemical characteristics of mouse cDNA-expressed, microsomal, and purified CYP1A1 and CYP1A2.

Kinetics of benzo[alpha]pyrene hydroxylase (AHH), 7-methoxyresorufin o-demethylase (MROD), and 7-ethoxyresorufin o-deethylase (EROD) were estimated in microsomes of Hep G2 cells infected with a recombinant vaccinia virus bearing mouse CYP1A1 or CYP1A2 cDNAs. The kcat and Km values obtained were compared with those of liver microsomal and purified mouse CYP1A1 and CYP1A2. In the matter of AHH activity, the kcat CYP1A1/CYP1A2 ratios were 21.2, 12.3, and 1.5 for expressed, microsomal, and purified CYPs, respectively. As to MROD activity, the kcat CYP1A2/CYP1A1 ratio was 3.0 for both expressed and microsomal CYPs and was 8.0 for purified CYPs. As regards EROD activity, the kcat CYP1A2/CYP1A1 ratios were 1.0, 1.1, and 6.25 for expressed, microsomal, and purified CYPs, respectively. Whereas furafylline displayed an isozyme-specific inhibition of CYP1A2-catalyzing MROD and EROD activities, alpha-naphthoflavone was an equally strong inhibitor of AHH activity of the CYP1A1s and MROD activities of the CYP1A2s. Immunodepleted polyclonal anti-CYP1A1(-A2) and anti-CYP1A2(-A1) showed an isozyme-specific immunoblotting and inhibition of mouse CYP1A1 and CYP1A2 while monoclonal antibody (Mab) 1-7-1 displayed a striking difference between its immunoblotting and inhibitory effects. Western blot/densitometry analysis revealed a 4.8 times lower binding of Mab 1-7-1 to cDNA-expressed CYP1A2 than to CYP1A1. The results demonstrate the reliability of the vaccinia virus expression system for studies on the enzymology of mouse CYP1A1 and CYP1A2.

Complete sequence of the genes encoding the VH and VL regions of low- and high-affinity monoclonal IgM and IgA1 rheumatoid factors produced by CD5+ B cells from a rheumatoid arthritis patient.

We have characterized the VH and VL genes of three low-affinity polyreactive and two high-affinity monoreactive IgM and IgA1 rheumatoid factor (RF) mAb generated using circulating CD5+ B cells from a single rheumatoid arthritis patient. We found that four and one RF mAb utilized genes of the VHIV and VHIII families, respectively. The VHIV gene usage by these RF mAb differs from the preferential VHIII, VHI, and, to a lesser extent, VHII gene usage by the IgM with RF activity found in patients with mixed cryoglobulinemia, Waldenstrom's macroglobulinemia, and other monoclonal gammopathies. In addition, in contrast to the preponderant kappa L chain usage by the RF in these patients, a lambda L chain was utilized by all RF mAb from our rheumatoid arthritis patient. Two RF mAbs utilized V lambda I, two V lambda IV, and one V lambda III L chains. The VH genes of the two low-affinity polyreactive IgM RF mAb were in germline configuration. When compared with the deduced amino acid sequence of the putatively corresponding genomic segment, the VH gene of the high-affinity monoreactive IgM RF mAb displayed five amino acid differences, all of which are in the complementarity determining regions (CDR), possibly the result of a process of somatic point mutation and clonal selection driven by Ag. The unavailability of the corresponding genomic VH segment sequences made it impossible to infer whether the VH genes utilized by the two IgA1 RF were in a germline or somatically mutated configuration. Sequencing of the genes encoding the H chain CDR3 (D segments) revealed that all three low-affinity polyreactive RF mAb displayed a much longer D segment (36-45 bases) than their high-affinity monoreactive counterparts (15-24 bases), raising the possibility that a long D segment may be one of the factors involved in antibody polyreactivity.

Model for studying virus attachment. II. Binding of biotinylated human T cell leukemia virus type I to human blood mononuclear cells potential targets for human T cell leukemia virus type I infection.

Purified human T cell leukemia virus type I (HTLV-I) was biotinylated and used to study its attachment to human PBMC. The use of biotinylated HTLV-I (biot-HTLV-I) in conjunction with mouse mAb specific for selected cell-surface molecules and flow cytometric analysis allowed us to positively identify virus-binding cells among a heterogeneous blood mononuclear cell population. Biot-HTLV-I efficiently bound not only to T cells, but also to B cells and monocytes. Preincubation of monocytes with excess of unlabeled HTLV-I significantly reduced the attachment of biot-HTLV-I. HTLV-I not only bound to, but also infected, B cells, as suggested by: i) in situ hybridization of a 35S-labeled full length HTLV-I DNA probe with EBV-transformed B cells, previously cocultured with HTLV-I-producing (G11MJ) T cells, and ii) hybridization of the same nick-translated 32P-labeled DNA probe with blotted DNA from similar HTLV-I-infected EBV-transformed B cells. HTLV-I infection did not affect the ability of B cells to secrete IgG. These findings suggest that HTLV-I cannot only infect cells of the T lineage, but can also infect B cells.

Clonal analysis of a human antibody response. Quantitation of precursors of antibody-producing cells and generation and characterization of monoclonal IgM, IgG, and IgA to rabies virus.

We quantitated and characterized the changes in the human B cell repertoire, at the clonal level, before and after immunization with rabies virus. Moreover, we generated 10 monoclonal cell lines producing IgM, IgG, and IgA antibodies to the virus. We found that in healthy subjects, not previously exposed to the virus, nearly 2% of the circulating B lymphocytes were committed to the production of antibodies that bound the virus. These B cells expressed the surface CD5 molecule. The antibodies they produced were polyreactive IgM that displayed a relatively low affinity for the virus components (Kd, 1.0-2.4 x 10(-6) g/microliters). After immunization, different anti-virus (IgG and IgA) antibody-producing cells consistently appeared in the circulation and increased from less than 0.005% to greater than 10% of the total B cells committed to the production of IgG and IgA, respectively. Most of such B cells do not express CD5 and produce monoreactive antibodies of high affinity for rabies virus (Kd, 6.5 x 10(-9) to 1.2 x 10(-10) g/microliters). One of these IgG mAbs efficiently neutralized rabies virus in vitro and in vivo, as detailed elsewhere (Dietzschold, B., P. Casali, Y. Ueki, M. Gore, C. E. Rupprecht, A. L. Notkins, and H. Koprowski, manuscript submitted for publication). Hybridization experiments using probes specific for the different human V gene segment families revealed that cell precursors producing low affinity IgM binding to rabies virus utilized a restricted number of VH gene segments (i.e., only members of the VHIIIb subfamily), whereas cell precursors producing high affinity IgG and IgA to rabies virus utilized an assortment of different VH gene segments (i.e., members of the VHI, VHIII, VHIV, and VHVI families and VHIIIb subfamily). In conclusion, our studies show that EBV transformation in conjunction with limiting dilution technology and somatic cell hybridization techniques are useful methods for quantitating, at the B cell clonal level, the human antibody response to foreign Ags and for generating human mAbs of predetermined specificity and high affinity.