Translating research into licensed vaccines and validated and licensed diagnostic tests.

The USDA Center for Veterinary Biologics (CVB) has the regulatory authority to issue licenses and permits that allow the marketing of pure, safe, potent, and effective veterinary biological products. Under the standard licensing or permitting process, a manufacturer develops, characterizes, and evaluates a product prior to licensure. The CVB evaluates the submitted information, inspects the manufacturing facilities and methods of production and testing, and confirms key product test results through independent testing. This complete and comprehensive evaluation may not be possible during the emergence of a new animal disease or in response to an introduction of a significant transboundary animal disease agent. Processes are in place in the US that allow for more rapid availability of veterinary products in an emerging or emergency animal health situation. But, it can be advantageous to attain preapproval of products prior to their anticipated need. In this article, issues associated with obtaining approval for use of a biological product under emerging or emergency conditions are discussed.

Human homologs of a Drosophila Enhancer of split gene product define a novel family of nuclear proteins.

Notch and the m9/10 gene (groucho) of the Enhancer of split (E(spI)) complex are members of the "Notch group" of genes, which is required for a variety of cell fate choices in Drosophila. We have characterized human cDNA clones encoding a family of proteins, designated TLE, that are homologous to the E(spI) m9/10 gene product, as well as a novel Notch-related protein. The TLE genes are differentially expressed and encode nuclear proteins, consistent with the presence of sequence motifs associated with nuclear functions. The structural redundancy implied by the existence of more than one TLE and Notch-homologous gene may be a feature of the human counterparts of the developmentally important Drosophila Notch group genes.

Molecular genetics of the Pax gene family.

Three members of the Pax gene family are now known to be responsible for the established mouse developmental phenotypes Splotch, Small eye and undulated; two of these genes are implicated in the human congenital diseases Waardenburg's syndrome and aniridia. The mouse mutants will act as model systems for these human disorders and, in addition, will provide insights into the processes of vertebrate development.

Regulation of major acute-phase plasma proteins by hepatocyte-stimulating factors of human squamous carcinoma cells.

Human squamous carcinoma (COLO-16) cells release factors which specifically stimulate the synthesis of major acute-phase plasma proteins in human and rodent hepatic cells. Anion exchange, hydroxyapatite, lectin, and gel chromatography of conditioned medium of COLO-16 cells result in separation into three distinct forms of hepatocyte-stimulating factors (designated HSF-I, HSF-II, and HSF-III) with apparent molecular weights of 30,000, 50,000 and 70,000, respectively. None of the preparations contains detectable amounts of thymocyte-stimulating activity. Each of the three HSF forms stimulates the accumulation of mRNA for alpha 1-antichymotrypsin in the human hepatoma cell line, HepG2. When the same factors were added to primary cultures of adult rat hepatocytes, the expression of the same set of plasma proteins was modulated as by nonfractionated medium. The hormonally induced accumulation of mRNA for acute phase proteins is qualitatively comparable to that occurring in the liver of inflamed rats. Unlike in human cells, in rat liver cells dexamethasone acts additively and synergistically with HSFs. The only functional difference between the three HSF forms lies in the level of maximal stimulation. HSF-I represents the predominant form produced by normal human keratinocytes and closely resembles in molecular size and isoelectric point the activity produced by activated peripheral blood monocytes while the larger molecular weight forms are more prevalent in human as well as mouse squamous carcinoma cells. The observation that HSFs from different sources elicit essentially the same pleiotropic response in hepatic cells led to the hypothesis that the species-specific reaction of adult liver cells to inflammatory stimuli is pre-programmed and that the function of any HSF is to trigger and tune the execution of this fixed cellular process.

Biosynthesis of vitamin B6: incorporation of three-carbon units.

Pyridoxol, one of the forms of vitamin B(6), is derived from three glycerol units. One of these is incorporated by way of pyruvate as a two-carbon fragment at the oxidation level of acetaldehyde. The other two glycerol units are incorporated intact, possibly by way of triose phosphate.

Comparative development. Seeing eye to eye.

Comparative studies of homologous developmental genes in mouse and Drosophila are suggesting that organs in these species may have closer evolutionary relationships than was hitherto suspected.

Colipase and maximally activated pancreatic lipase in normal subjects and patients with steatorrhea.

Human pancreatic lipase in duodenal secretions was studied under conditions of maximal activation by porcine colipase and maximal inhibition by sodium taurodeoxycholate. In almost all samples, total lipase activity in 4 mM sodium taurodeoxycholate was activated by the addition of porcine colipase. Activation was linear until saturation by cofactor was reached, and maximum activity was greater than that obtained in the absence of bile salts. At pH 8.0 in 4 mM sodium taurodeoxycholate, lipase activity was due to pancreatic lipase in samples from normal and steatorrheic individuals and was proportional to the concentration of endogenous colipase in samples that could be activated by exogenous colipase. In these samples, therefore, colipase activity could be conveniently assayed as the lipase activity at pH 0.8 in 4 mM sodium taurodeoxycholate. Colipase to total pancreatic lipase ratios varied widely from individual to individual and on average were significantly lower in steatorrheic patients. In individual samples, colipase secretion was stimulated by pancreozymin and secretin roughly in parallel with total pancreatic lipase, but some variation in the ratio of the two was often seen in successive collection periods. Because pancreatic lipase is usually unsaturated with respect to cofactor, lipolytic activity in duodenal secretions may be finely controlled by modulation of colipase secretion.

A genetic locus closely linked to a protease inhibitor gene complex controls the level of multiple RNA transcripts.

The two major protease inhibitors in mouse plasma are alpha 1-protease inhibitor (alpha 1-PI), putative inhibitor of neutrophil elastase, and contrapsin, an inhibitor in vitro of trypsinlike proteases. We have shown by nucleotide sequence analysis that these two inhibitors are related (R. E. Hill, P. H. Shaw, P. A. Boyd, H. Baumann, and N. D. Hastie, Nature (London) 311:175-177, 1984). Here, we show that the contrapsin and alpha 1-PI genes are members of two different multigene families, each containing at least three genes in mice and rats. We established the chromosomal locations of these genes by analyzing the segregation of restriction fragment length polymorphisms in recombinant inbred mouse strains. These experiments show that the multiple genes in each family are clustered and that the two gene families are closely linked on chromosome 12. Thus the genes for contrapsin and alpha 1-PI are likely to have evolved by duplication of a common ancestral gene. The contrapsin multigene family codes for multiple mRNA transcripts in the liver. There is a genetic difference among inbred mouse strains in the regulation of two of these transcripts. In some inbred strains the transcripts are synthesized constitutively; in others they are induced by inflammation. We mapped in recombinant inbred strains the regulatory locus responsible for this genetic variation and found it is linked to the contrapsin multigene family, which suggests a cis-acting regulatory element. We also found that the contrapsin and the alpha 1-PI multigene families have acquired very different regulatory responses since the time of the gene duplication event.

Most highly repeated dispersed DNA families in the mouse genome.

The construction of a small library of mouse repetitive DNA has been previously reported (Pietras et al., Nucleic Acids Res. 11:6965-6983, 1983). Here we report that the 35 plasmids in this library corresponding to highly repeated (greater than 30,000 copies per genome) dispersed DNA sequences can be grouped into no more than 5 distinct families. These families together comprise 8 to 10% of the mouse genome. They include the previously described small elements B1, B2, and R and the large MIF-1 element. Twelve of the 35 clones contain evolutionarily conserved (EC) sequences. One EC clone in our library mostly consists of alternating dCdT residues; another consists of tandem repeats of the sequence CCTCT. The majority of B1s and B2s in the genome appear to be homogeneous, whereas R sequences, ECs, and MIF-1s are heterogeneous. Two earlier reports showed highly repeated mammalian DNA sequences in the herpesvirus genome (Peden et al., Cell 31:71-80, 1982; Puga et al., Cell 31:81-87, 1982). We show that sequences homologous to our EC clones are present in the herpesvirus genome, although these polypyrimidine stretches are not detected in poxvirus, adenovirus, and simian virus 40 genomes. We detect transcripts containing homology to all of these sequences in a nuclear transcription assay. Also, we show that small, polyadenylated RNA molecules homologous to B2 sequences are expressed in undifferentiated embryonal carcinoma cells but not in their differentiated derivatives. The significance of these findings is discussed.

International harmonisation of regulatory requirements.

The International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) was formed in April 1996 and is a programme of collaboration between regulatory authorities and the animal health industries of three world regions: the European Union, Japan and the United States of America. Two other regions, Canada and Australia/New Zealand, have observer status. The principal goal of VICH is to harmonise technical data requirements of participating regulatory authorities before granting marketing authorisation or registration. VICH has finalised six guidelines on the technical requirements for marketing authorisation/registration of biological products. These guidelines have been fully implemented in the regions. Three more technical guidelines are under development by two expert working groups. VICH has also finalised a guideline which specifically deals with pharmacovigilance and veterinary medicinal products, including biological products. A further four guidelines relating to pharmacovigilance are under development by an expert working group.

Two enhancer domains control early aspects of the complex expression pattern of Msx1.

To understand how the complex embryonic expression pattern of the Msx1 gene is produced a transgenic analysis of 13 kb of DNA around the Msx1 locus was carried out. Most of the extensive expression pattern of the Msx1 gene was reproduced in transgenics using the LacZ gene fused to 5 kb of Msx1 5' flanking DNA. Two enhancer domains were identified which produced this pattern. The distal element produced expression in the first arch and the nasal epithelium and was restricted to 240 bp. However, the proximal element which produced expression in superficial nasal epithelium, dorsal and ventral myotome, limb mesenchyme, eye, ear, roof plate, second arch, genital ridge and epiphysis, was contained in only 78 bp.

Disruption of PAX6 function in mice homozygous for the Pax6Sey-1Neu mutation produces abnormalities in the early development and regionalization of the diencephalon.

Pax6 expression in the diencephalon of the mouse embryo is restricted both antero-posteriorly and dorso-ventrally, with changes in level occurring at prosomere boundaries. Small eye (Pax6Sey-1Neu) mice homozygous for Pax6 mutations have multiple defects in early forebrain development. In the diencephalon of Pax6Sey-1Neu/Pax6Sey-1Neu mice there is an apparent enlargement of the zona limitans (the boundary region between prosomeres p2 and p3), and a blurring of the p1-p2 boundary. PAX6 function is also required for the normal development of the posterior commissure at the midbrain-p1 boundary. In the posterior diencephalon PAX6 appears to regulate its own transcription, and that of Wnt7b. In p2 and p3, ventral markers are expressed more dorsally than normal, and this is accompanied in p3 by a reduction in the size of the zona incerta. Thus, PAX6 is essential for the normal development and regionalization of the diencephalon.

Antagonistic effects of FGF4 on BMP induction of apoptosis and chondrogenesis in the chick limb bud.

In an effort to define the roles of bone morphogenic proteins (BMPs) and fibroblast growth factors (FGFs) during chick limb development more closely, we have implanted beads impregnated with these growth factors into chick limb buds between stages 20 and 26. Embryos were sacrificed at the time the bone chondrocyte condensations first appear (stages 27-28). Implantation of beads containing BMPs at the earlier stages (20-22) caused apoptosis to occur, in the most severe cases leading to complete limb degeneration. Application of FGF4, either in the same, or in a different bead, prevented the BMP-induced apoptosis. We argue that the apoptosis observed on removal of the AER prior to stage 23 of development could be brought about by BMPs. The action of epithelial FGF in preventing BMP-mediated apoptosis in the mesenchyme would define a novel aspect of epithelial-mesenchymal interactions. Implanting the BMP4 beads into the core of the limb bud a day later (stages 25-26) caused intense chondrogenesis rather than apoptosis. FGF4 could again nullify this effect and by itself caused a reduction in bone size. This is the reverse of the functional relationship these growth factors have in mouse tooth specification (where it is BMP4 that inhibits the FGF8 function), and suggests that the balance between the effects of FGFs and BMPs could control the size of the chondrocyte precursor cell pool. In this way members of these two growth factor families could control the size of appendages when they are initially formed.

Mammalian and Drosophila dachshund genes are related to the Ski proto-oncogene and are expressed in eye and limb.

We have isolated mammalian homologues of the Drosophila dachshund gene. Two domains of high conservation, one of which contains an alpha-helical, coiled-coil motif, show similarity to the Ski family of genes. We therefore propose that Dachshund belongs to a superfamily including these genes. Mouse Dachshund (Dach) is expressed in the eye and limb, structures affected by the Drosophila loss-of-function mutant, and rib primordia, CNS and genital eminence. Pax6 and Dach show overlapping but non-identical expression patterns. Dach expression is unaffected in smalleye mouse brain, indicating that Pax6 is not directly activating Dach. In Drosophila eye development dachshund is a component of an interacting network of proteins. Genes homologous to many of these exist in mammals; Dach joins this expanding group.

Expression of the chicken homeobox-containing gene GHox-8 during embryonic chick limb development.

Homeobox-containing genes are thought to be involved in the regulation of pattern formation and specification of positional information during vertebrate limb development. Because of its accessibility to microsurgical manipulation, the developing chick limb bud provides a powerful system for investigating the role of homeobox-containing genes in patterning events. We report the isolation from a chick limb bud cDNA library of a chicken homeobox-containing cDNA, which on the basis of its nucleotide and deduced amino acid sequences has been identified as the chicken cognate of mouse Hox-8. The gene encoding this chicken (Gallus) homeobox-containing cDNA has been designated GHox-8, and is a member of a family of vertebrate homeobox-containing genes that are highly similar in sequence to the Drosophila msh gene. GHox-8 encodes an mRNA transcript of about 3 kb that is expressed at several early stages of chick limb development. In situ and dot-blot hybridization analyses have revealed that GHox-8 is expressed in limb bud mesoderm in a temporal and spatial fashion consistent with its involvement in specifying anterior positional identity. At early stages (stages 20-21) of chick limb development when positional values along the anterior-posterior (A-P) axis are being specified, GHox-8 is expressed in high amounts in the anterior mesoderm of the wing bud. Little expression of the gene is detectable in the middle region of the wing bud mesoderm or in the posterior mesoderm that contains the zone of polarizing activity, which is thought to be the source of a diffusible morphogen, possibly retinoic acid, that specifies the A-P positional values of the skeletal elements of the limb according to its local concentration. Similarly, at later stages of development (stages 23-25), high expression of GHox-8 is localized to the proximal anterior periphery of the wing bud, with no detectable expression in the proximal dorsal and ventral (myogenic) regions, or in the chondrogenic central core. In the proximal posterior periphery of the wing bud at these later stages of development, expression of GHox-8 is limited to a small region in the mid-proximal periphery corresponding to the posterior necrotic zone in which programmed cell death is occurring. The possible involvement of GHox-8 in programmed cell death during limb development is also suggested by the fact that it is expressed in the necrotic interdigital mesenchyme in 6-7 day (stage 31-32) wing buds.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of maximal daily doses of acetaminophen on the liver of alcoholic patients: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Retrospective reports suggest that therapeutic doses of acetaminophen may be associated with fulminant hepatic failure and death in alcoholic patients. Millions of patients use acetaminophen; the prevalence of alcoholism in the United States is 5% to 10%. OBJECTIVE: To determine if hepatic injury was associated with maximal therapeutic dosing of acetaminophen to chronic alcohol abuse patients immediately following cessation of alcohol intake (the presumed time of maximal vulnerability). METHODS: Patients entering an alcohol detoxification center were enrolled in a randomized, double-blind, placebo-controlled trial. Exclusion criteria were baseline values of aspartate or alanine aminotransferase greater than 120 U/L, international normalized ratio greater than 1.5, serum acetaminophen level greater than 20 mg/L, or a history of ingesting more than 4 g/d of acetaminophen. Acetaminophen, 1000 mg, or placebo was administered orally 4 times daily for 2 consecutive days and liver test results were monitored for 2 more days. Acetaminophen was not administered until the alcohol had been eliminated. RESULTS: There were 102 patients in the acetaminophen-treated group and 99 patients in the placebo-treated (control) group. Demographic data, alcohol history, and baseline blood test results were similar in both groups. The mean (SD) aspartate aminotransferase level on day 4 was 38.0 +/- 26.7 U/L in the acetaminophen-treated group and 37.5 +/- 27.6 U/L in the placebo-treated group. There were 4 patients in the acetaminophen-treated group and 5 in the placebo-treated group who developed an increase in their serum aspartate aminotransferase level to greater than 120 U/L; it did not exceed 200 U/L in any patient. The mean (SD) international normalized ratio on day 4 was 0.96 +/- 0.09 in the acetaminophen-treated group and 0.98 +/- 0.11 in the placebo-treated group. CONCLUSION: Repeated administration of the maximum recommended daily doses of acetaminophen to long-term alcoholic patients was not associated with evidence of liver injury.

Isolation of two cDNAs encoding novel alpha 1-antichymotrypsin-like proteins in a murine chondrocytic cell line.

We have isolated two novel serpin-encoding sequences from EB22, a chondrocytic cell line derived from a mouse teratocarcinoma. Both sequences fall within the Spi-2 sub-family, and are related to the gene encoding human alpha 1-antichymotrypsin (ACT), a major acute-phase reactant. Considerable amplification of the Spi-2 gene family in the mouse has occurred, hindering the identification of a functional equivalent of the human gene. However, one of the sequences described here, EB22/4, exhibits several features which indicate that it may represent the physiological rodent equivalent of ACT. The sequence is expressed in the liver, as expected, and is induced several-fold during the acute-phase response. The P1 amino acid residue, which is primarily responsible for inhibitor specificity, is Met rather than the human Leu, most probably a functionally conservative substitution. Analysis of the orthologous sequence in related rodents demonstrates conservation of the predicted reactive centre-encoded specificity. The second isolated cDNA, EB22/3, encodes an unexpected Cys residue at the P1 position in the reactive centre, and represents a novel sub-class of the Spi-2 serine proteinase inhibitor (serpin)-encoding gene family. At least one of the sequences appears to be expressed at sites of skeletal deposition during the later stages of mouse foetal development, indicating a role for serpins during development.

Growth response to 4-hydroxy-L-threonine of Escherichia coli mutants blocked in vitamin B6 biosynthesis.

Mutants of Escherichia coli (pdx B and pdx C) which are blocked in the biosynthesis of pyridoxol (vitamin B6) showed a growth response to 4-hydroxy-L-threonine. This observation constitutes the first direct evidence in support of the view that 4-hydroxy-L-threonine is implicated in the biosynthesis of vitamin B6. 1-Aminopropan-2,3-diol, the decarboxylation product of 4-hydroxy-L-threonine, does not support the growth of these mutants. Deuterium from deuterium-labelled 1-aminopropan-2,3-diol was not incorporated into pyridoxol.

Smith-Lemli-Opitz syndrome: phenotypic extreme with minimal clinical findings.

Smith-Lemli-Opitz syndrome (SLO) is caused by inherited enzymatic deficiency of 7-dehydrocholesterol-delta7-reductase and resultant cholesterol deficiency. It comprises a characteristic combination of facial features, malformations, and mental retardation. We report on three related patients (two brothers and their first cousin) with mental retardation and minimal physical signs in whom the diagnosis of SLO was delayed for a number of years. The presence of a third-degree relative in the absence of consanguinity in this family supports the proposed high population carrier frequency. Our report suggests that cases of mild SLO remain undiagnosed and untreated, and that awareness of this common cause of mental retardation is low.