Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs.

We describe a strategy for producing human monoclonal antibodies in mice by introducing large segments of the human heavy and kappa light chain loci contained on yeast artificial chromosomes into the mouse germline. Such mice produce a diverse repertoire of human heavy and light chains, and upon immunization with tetanus toxin have been used to derive antigen-specific, fully human monoclonal antibodies. Breeding such animals with mice engineered by gene targeting to be deficient in mouse immunoglobulin (Ig) production has led to a mouse strain in which high levels of antibodies are produced, mostly comprised of both human heavy and light chains. These strains should provide insight into the adoptive human antibody response and permit the development of fully human monoclonal antibodies with therapeutic potential.

Effects of recombinant neutral endopeptidase (EC 3.4.24.11) on the growth of lung cancer cell lines in vitro and in vivo.

Many lung cancers are stimulated by an autocrine/paracrine system of neuroendocrine peptide hormones. Attempts to block this autocrine growth pathway by interactions with specific ligand-receptor binding using monoclonal antibodies and peptide-specific antagonists have been largely unsuccessful because of the heterogeneity of hormone production and receptor expression. In the normal lung, neutral endopeptidase (NEP; CD10, CALLA, enkephalinase, and EC 3.4.24.11) plays a physiological role in degrading biologically active peptides, including all peptides implicated in autocrine growth stimulation of lung cancer. Cigarette smoke decreases the activity of NEP, indicating that the lack of NEP contributes to the dysregulation of the peptide autocrine system. The cloning of the human NEP gene allowed for production of sufficient quantities of recombinant NEP (rNEP) to evaluate its role in inhibiting the growth of lung cancer cells. In this study, we evaluated the ability of rNEP to inactivate the peptides involved in lung cancer signal transduction and to inhibit the growth of lung cancer cells as well as normal lung cells in vitro and in vivo in athymic nude mice. We showed that the growth inhibition of lung cancer cells by rNEP was related to the dose and schedule. Continuous exposure to high doses was required for growth inhibition. These studies confirm the importance of NEP in this autocrine pathway.

Rapid two-stage polymerase chain reaction amplification of chromosomal DNA segments in lysates made from monolayer cultures attached to microcarrier beads.

We describe a method for the rapid two-stage amplification and detection by ethidium bromide staining of chromosomal nucleotide (nt) sequences in lysates made directly from anchorage-dependent cells attached to microcarrier beads. The procedure circumvents the need for cell detachment steps prior to analysis, facilitates the collection, transfer, and manipulation of the cells being studied, and makes unnecessary the use of Southern-blot hybridization for identification of specific nt sequences present in a small fraction of cells within a heterogeneous population.

Chloramphenicol acetyltransferase gene of staphylococcal plasmid pC221. Nucleotide sequence analysis and expression studies.

The nucleotide sequence of the inducible chloramphenicol acetyltransferase gene (cat) of Staphylococcus aureus plasmid pC221 has been determined. The deduced primary structure for the 215 residue polypeptide (25.9 kDa) is in agreement with partial amino acid sequence data on the purified protein, previously designated as the type C variant of CAT. In common with the inducible cat elements of pC194 and B. pumilus, the 5' non-coding region of the cat of pC221 contains an inverted complementary repeat ('stem-loop' or 'hairpin') which may sequester the predicted ribosome bonding site of the mRNA. The likely transcription initiation site has been determined in vitro using purified B. subtilis RNA polymerase. Recombinant plasmids carrying the cat of pC221 on a 1156 bp TaqI fragment are expressed inefficiently in Escherichia coli, wherein induction is both poor and orientation-specific.

Production of antigen-specific human antibodies from mice engineered with human heavy and light chain YACs.

Our paper describes the introduction of large fragments of both the human heavy and light chain Ig genes into the mouse germline to create a mouse strain capable of producing a broad repertoire of antigen-specific, fully human antibodies. The human immunoglobulin gene sequences were functional in the context of the mouse machinery for antibody recombination and expression, either in the presence or absence of functional endogenous genes. This was demonstrated by their ability to undergo diverse rearrangement, to be expressed at significant levels, and to exclude expression of mouse immunoglobulins irrespective of their copy number or site of integration. The decrease in susceptibility to influence by adjacent genomic sequences may reflect the greater size, variable gene content, or structural integrity of the human Ig YACs and/or the presence of unidentified but important regulatory elements needed for optimal expression of the human immunoglobulin genes and their correct regulation. Our results show that mouse B cells coexpressing human heavy and kappa chains, upon immunization, can produce antigen-specific, fully human antibodies. Furthermore, the human heavy and kappa chain YACs induced differentiation and maturation of the growth-arrested B-cell lineage in mice with inactivated endogenous Ig genes, leading to the production of a diverse repertoire of fully human antibodies at levels approaching those in normal serum. These results suggest the potential value of these mice as a source of fully human antibodies for human therapy. Furthermore, it is expected that such mice would lack immunological tolerance to and thus readily yield antibodies to human proteins, which may constitute an important class of targets for monoclonal antibody therapy. Our findings suggest that the introduction of even larger portions of the human heavy and light chain loci, which should be achievable with the ES cell-yeast spheroplast fusion technology described, will result in strains of mice ultimately capable of recapitulating the full antibody repertoire characteristic of the human humoral response to infection and immunization. The present and future mouse strains may prove to be valuable tools for studying the molecular mechanisms and regulatory sequences influencing the programmed assembly and expression of human antibodies in the normal immune response, as well as the abnormal response characteristic of autoimmune disease and other disorders. The strategy we have described for the introduction of large segments of the human genome into mice in conjunction with the inactivation of the corresponding mouse loci may also have broad applicability to the investigation of other complex or uncharacterized loci.

Comparison of febantel tablets and Vercom paste against gastrointestinal nematodes of dogs.

Thirty adult dogs with naturally acquired gastrointestinal nematode infections were assigned at random to ten replicates and treated daily for 3 days with either a combination febantel/praziquantel (Vercom) paste, febantel tablets or placebo tablets. Numbers of hookworm and whipworm eggs after treatment were reduced similarly for both drug formulations when compared with pretreatment egg counts, whereas these counts increased in the controls. Vercom paste reduced the hookworm egg count by 99.9% and the whipworm egg count by 99.6%. The febantel tablet decreased the hookworm egg count by 99.9% and the whipworm egg count by 100%. As determined at necropsy, the controlled test efficacy against adult hookworms and whipworms was similar for the Vercom paste and the febantel tablets. The controlled test efficacies of Vercom paste against Ancylostoma caninum, Ancylostoma braziliense, and Trichuris vulpis were, respectively, 99.7%, 100% and 95.8% and those of febantel tablets were 98.2%, 100% and 99.7%. These results indicate that the nematocidal efficacy of febantel against these nematodes remains unchanged in these two formulations. No adverse reactions to either febantel tablets or to Vercom paste were observed.

Penicillanic acid sulfone: nature of irreversible inactivation of RTEM beta-lactamase from Escherichia coli.

When penicillanic acid sulfone in large molar excess is incubated with the RTEM beta-lactamase, the enzyme becomes inactivated irreversibly. From studies of the consequential spectroscopic changes, from the use of specifically tritiated penicillanic acid sulfone, and from comparison by isoelectric focusing of the enzyme after inactivation by the sulfone and by clavulanic acid, the inactivated enzyme appears to be cross-linked by a beta-aminoacrylate fragment deriving from C-5, C-6, and C-7 of the original beta-lactam. Model studies on the behavior of alcoholic solutions of penicillanic acid sulfone in the presence of amines are entirely consistent with this interpretation.

6-(Methoxymethylene)penicillanic acid: inactivator of RTEM beta-lactamase from Escherichia coli.

The Z and E isomers of 6-(methoxymethylene)-penicillanic acid have been synthesized, and their interaction with the RTEM beta-lactamase has been studied. The Z isomer is an inhibitor and an inactivator of the enzyme, and there is some similarity between its behavior and that of other mechanism-based inactivators such as clavulanic acid and the penam sulfones. Kinetic analysis of the interaction of the enzyme with the Z isomer has allowed a detailed evaluation of the factors that are important in the design of anti-beta-lactamase agents. In contrast to the Z compound, the E isomer of 6-(methoxymethylene)penicillanic acid is not a substrate, an inhibitor, or an inactivator of the enzyme.

The use of synthetic oligonucleotides with universal templates for rapid DNA sequencing: results with staphylococcal replicon pC221.

A rapid sequencing strategy has been devised and applied to determine the complete nucleotide sequence (4555 bp) of Staphylococcus aureus plasmid pC221. The entire replicon was cloned into phage M13mp8 in both orientations to provide 'universal templates' for primed DNA synthesis from internally-sited oligonucleotide primers. The latter were synthesized by a modification of a recently described paper disc method which employs phosphotriester chemistry. Less than 4 weeks was required for the synthesis of the required primers and for the sequencing experiments. Plasmid pC221 bears a substrate-inducible chloramphenicol acetyltransferase (CAT) gene that shares much homology with its counterparts in pC194 (S. aureus) and the chromosomal cat-86 gene of Bacillus pumilus, both in coding regions and upstream sequences believed to be involved in the induction phenomenon. A second plasmid-specified protein, REP D, has an 81% identity in the REP C polypeptide that has been shown to be essential for the replication of staphylococcal plasmid pT181. The 5' flanking region of rep D shows striking similarities with its counterpart in rep C that determines copy number and incompatibility. The nucleotide sequence reveals two additional and overlapping open reading frames that may specify proteins that play roles in plasmid relaxation and transfer.

Penicillanic acid sulfone: an unexpected isotope effect in the interaction of 6 alpha- and 6 beta-monodeuterio and of 6,6-dideuterio derivatives with RTEM beta-lactamase from Escherichia coli.

Penicillanic acid sulfone (1) is both a substrate and an inactivator of the RTEM beta-lactamase. About 7000 hydrolytic events occur before enzyme inactivation. The 6,6-dideuterio sulfone shows a 3-fold acceleration of both the hydrolysis reaction and the enzyme inactivation. The kinetic and spectroscopic results are nicely accommodated by a scheme in which a transiently stable intermediate is formed in an isotopically sensitive step. The deuterated material partitions less readily toward this transiently stable intermediate by virtue of a primary isotope effect, and more enzyme is then available for the hydrolysis and inactivation pathways. Use of the stereospecifically monodeuterated sulfones shows that the 6 beta hydrogen is preferentially abstracted in the formation of the transiently stable intermediate and allows a detailed picture of the interaction of the sulfone and the beta-lactamase to be drawn. The crystal structures of both the labeled and unlabeled compounds are reported.

Regulation of antibiotic resistance in bacteria: the chloramphenicol acetyltransferase system.

The evaluations of antibiotic resistance has been a subject of interest to workers in several disciplines. Our current understanding of the molecular biology of plasmids, phages, and transposable elements provides a basis for appreciating the range of mechanisms likely to be involved in the horizontal spread of resistance determinants through microbial ecosystems. Rather less can be imagined with confidence about the origins of the genes or the constraints and selection pressures operating at the level of protein structure. The CAT system illustrates the extent of variation possible for an accessory gene product which is required infrequently and which is encoded by multicopy and promiscuous vectors which can cross taxonomic boundaries. Still less is known with certainty about the evolution of genetic control of the expression of antibiotic resistance. While there are sound reasons for looking in detail at prokaryotic antibiotic-producing organisms such as Streptomyces to find the progenitors of present resistance mechanisms (44, 45), it seems likely that controls of expression have been acquired during the "passage" of selectable markers through more distant bacterial genera. The CAT system is illustrative of the variety we may expect to find in control strategies used by microbial systems generally. It might indeed be a surprise to find an expression mechanism operating in the CAT system (or for any other family of resistance genes) which was not illustrative of a general strategy exploited by essential genes specifying biosynthetic or degradative functions. There may be some truth in referring to the cat structural gene as a "cartridge" for the isolation and manipulation of promoter functions. It would seem that nature has been at it for some time.

Analysis of mammalian cell genetic regulation in situ by using retrovirus-derived "portable exons" carrying the Escherichia coli lacZ gene.

Self-inactivating derivatives of Moloney murine leukemia retrovirus containing the Escherichia coli lacZ gene were used to detect and study the regulation of transcription initiated at chromosomally located promoters in mouse fibroblasts. The introduction of splice acceptor sites in all three translational reading frames relative to lacZ and the inclusion of an in-frame ATG translation start codon in one construct allowed synthesis of beta-galactosidase fusion proteins upon insertion of retrovirus vectors containing lacZ into introns 3' to either protein-coding or noncoding exons. Selection of lacZ-expressing cells by fluorescence-activated cell sorting and the analysis of beta-galactosidase production after serum deprivation has yielded lines in which lacZ was fused to genes induced by growth arrest in the G0 state.

CD10/neutral endopeptidase 24.11 is phosphorylated by casein kinase II and coassociates with other phosphoproteins including the lyn src-related kinase.

CD10/neutral endopeptidase 24.11 (NEP) regulates peptidemediated proliferation of lymphoid progenitors and certain epithelial cells and is itself regulated by cellular proliferation. To further characterize mechanisms by which cell-surface signaling might regulate CD10/NEP expression, we determined whether CD10/NEP was phosphorylated and whether the enzyme co-associated with additional cellular phosphoproteins. The CD10/NEP cytoplasmic tall contains two consensus recognition sequences for casein kinase II (CKII), a serine and threonine kinase that increases in activity following peptide signaling. In standard in vitro kinase assays, CKII phosphorylated full-length recombinant CD10/NEP but did not phosphorylate a truncated CD10/NEP protein that lacked the transmembrane region and cytoplasmic tail. To determine whether CD10/NEP might interact with additional cellular phosphoproteins, in vitro kinase assays were performed on CD10/NEP immune complexes from Nalm-6 cells. Three additional tyrosine phosphoproteins of approximately 40 kD, approximately 58 kD, and approximately 75 kD were identified in the CD10/NEP immunoprecipitates. The approximately 56-kD CD10/NEP-associated phosphoprotein was immunoprecipitated with an anti-lyn antibody confirming its identity as the lyn src-related kinase. Taken together, these data indicate that CD10/NEP is itself phosphorylated by CKII and that CD10/NEP co-associates with additional tyrosine phosphoproteins including lyn.

Effect of recombinant neutral endopeptidase (EC 3.4.24.11) on neuropeptide-mediated nasal fluid secretion and plasma exudation in the rat.

The nasal mucosa harbors sensory nerves containing neuropeptides such as substance P (SP), which are released by capsaicin. The neuropeptides are degraded by peptidases, e.g., neutral endopeptidase (NEP) that is present in the nasal mucosa. We studied the effect of enzymatically active recombinant NEP (rNEP) on neuropeptide-evoked secretion of nasal fluid and plasma exudation in rats. rNEP administered intranasally (i.n.) reduced the capsaicin-evoked nasal fluid secretion but failed to reduce the secretion evoked by SP (exogenous) under the experimental conditions used. rNEP reduced the increase in nasal plasma exudation evoked by capsaicin (endogenous neuropeptides). Because rNEP reduced neuropeptide-mediated nasal fluid secretion and plasma exudation in the rat, we suggest that peptidase activity in the nasal mucosa will determine the magnitude of the response to locally released neuropeptides.

Analysis of homozygous mutant chimeric mice: deletion of the immunoglobulin heavy-chain joining region blocks B-cell development and antibody production.

Using a recently described method for efficiently deriving homozygous targeted alleles in embryonic stem cells, we produced chimeric mice whose tissues were derived partially from embryonic stem cells bearing homozygous deletion of the mouse immunoglobulin heavy-chain joining (JH) region. Characterization of these chimeric mice indicated that homozygous JH deletion leads to arrest of B-cell development at an early stage, resulting in a total lack of peripheral B cells and serum IgM. These results were confirmed in mice containing the homozygous JH deletion in their germ line. This novel B-cell-deficient mouse strain provides a tool for studying the recombination and expression of exogenous immunoglobulin genes introduced into the mouse germ line.