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.

Performance of the Los Alamos National Laboratory spallation-driven solid-deuterium ultra-cold neutron source.

In this paper, we describe the performance of the Los Alamos spallation-driven solid-deuterium ultra-cold neutron (UCN) source. Measurements of the cold neutron flux, the very low energy neutron production rate, and the UCN rates and density at the exit from the biological shield are presented and compared to Monte Carlo predictions. The cold neutron rates compare well with predictions from the Monte Carlo code MCNPX and the UCN rates agree with our custom UCN Monte Carlo code. The source is shown to perform as modeled. The maximum delivered UCN density at the exit from the biological shield is 52(9) UCN/cc with a solid deuterium volume of ~1500 cm(3).

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.

Regulation of autogenous biologicals in the United States.

The U.S. Department of Agriculture's Center for Veterinary Biologics (CVB) is charged with protecting American agriculture. As part of this effort, CVB licenses manufacturers to produce special biologics for use in individual flocks or livestock herds afflicted by a particular microorganism. These special products, autogenous biologicals, are inactivated vaccines used by or under the direction of a veterinarian or USDA-approved specialist and are produced under a variety of special licensing requirements.

Regulation of conditionally licensed biologicals in the United States.

The Virus-Serum-Toxin Act of 1913 (21 US Code 151-159) provides the legal basis for the regulation of veterinary biologicals in the United States and the United States Department of Agriculture's Center for Veterinary Biologics (CVB) has the regulatory authority for the issuing of licences and permits for such products. The law was intended to establish standards and control the importation of products into the United States and the distribution of products interstate assuring the purity, safety, potency, and efficacy of veterinary biological products. Under certain specific circumstances, product licences may be issued in the United States under expedited procedures which assure purity, safety, and a "reasonable expectation" of efficacy. These conditional licences may be authorized to meet an emergency condition, limited market, local situation, or other special circumstances, and provide valuable tools for veterinarians and consumers in improving animal health.

Regulatory considerations for emergency use of non-USDA licensed vaccines in the United States.

The Virus-Serum-Toxin Act of 1913 (21 US Code 151-159) provides the legal basis for the regulation of veterinary biologicals in the United States; the United States Department of Agriculture's Center for Veterinary Biologicals (CVB) has the regulatory authority for the issue of licences and permits for such products. The law was intended to establish standards and control the importation of products into the United States and the distribution of products interstate assuring the purity, safety, potency, and efficacy of veterinary biological products. Administrative regulations and standards appear in the Title 9, Code of Federal Regulations, Parts 101-118, with additional programme guidance found in CVB Notices, Veterinary Services Memoranda, General Licensing Considerations, and other guidance documents. Pre-licensing data evaluation procedures are designed to assess the purity, safety, potency, and effectiveness of each product and support all product label claims. To fulfil these criteria, data from all phases of product development are evaluated against these key elements. Under the standard licensing process, this spectrum of evaluation includes complete characterization and identification of seed material and ingredients, laboratory and host animal safety and efficacy studies, stability studies, and post-licensing monitoring of field performance. This comprehensive evaluation may not be possible during the emergence of a new animal disease. While there are no specific regulations addressing the licensing standards of products for an emerging animal disease, there are mechanisms that allow for the availability of products in an emergency animal health situation. These mechanisms include autogenous biologicals, conditional licences, experimental and emergency use authorizations, and the importation of products in use elsewhere in the world. Pre-approved vaccine banks provide an additional mechanism.

A quantitative analysis of perineurial cell basement membrane collagen IV, laminin and fibronectin in diabetic and non-diabetic human sural nerve.

The thickness of the perineurial cell basement membrane was examined in diabetic and non-diabetic human sural nerve. A significant increase in thickness was found in the diabetic group. The nature of this thickening was investigated using immunohistochemistry and image analysis in order to semi-quantify three of the major intrinsic components of the perineurial cell basement membrane: collagen IV, laminin and fibronectin. Amounts of all three components were shown to be increased in the diabetic group, but not significantly so. However, significant linear correlations between fascicle size and perineurial collagen IV, laminin and fibronectin were identified in both diabetic and non-diabetic nerve.

Measurements of ultracold-neutron lifetimes in solid deuterium.

We present the first measurements of the survival time of ultracold neutrons (UCNs) in solid deuterium (SD2). This critical parameter provides a fundamental limitation to the effectiveness of superthermal UCN sources that utilize solid ortho-deuterium as the source material. These measurements are performed utilizing a SD2 source coupled to a spallation source of neutrons, providing a demonstration of UCN production in this geometry and permitting systematic studies of the influence of thermal up-scatter and contamination with para-deuterium on the UCN survival time.

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.

Attenuation of verapamil-induced myocardial toxicity in an ex-vivo rat model using a verapamil-specific ovine immunoglobin.

OBJECTIVE: To determine whether an ovine verapamil-specific immunoglobin G (V-IgG) attenuates verapamil toxicity in an ex-vivo rat left ventricular papillary muscle model. METHODS: The authors dissected left ventricular papillary muscle strips from male Sprague-Dawley rats (350-410 g) and suspended them in an oxygen-perfused Tyrode buffer bath at 37.5 degrees C. Muscle strips equilibrated for 90 minutes under electrical stimulation of 1 Hz. Resting and developed tension (mg) were monitored continuously. A concentration-response trial was performed with verapamil concentrations ranging from 31 to 1,020 nM; 510 nM produced consistent reduction in developed tension. A trial of V-IgG was then conducted by administering the following treatments to papillary muscle strips in a randomized manner: V-IgG + 510 nM verapamil, nonspecific ovine IgG (N-IgG) + 510 nM verapamil (protein control), and 510 nM verapamil alone. Immunoglobin G was administered in equimolar concentrations to verapamil. Attenuation was expressed as inhibition of the verapamil-induced reduction of developed tension. RESULTS: The V-IgG comparative trial indicated the V-IgG + verapamil treatment had a mean reduction in developed tension of 14.1% (SD +/- 12.2) compared with 36.2% (SD +/- 14.9) for N-IgG + verapamil and 34.9% (SD +/- 8.1) for verapamil alone (p < 0.05). There was no difference between the two control groups. CONCLUSION: Verapamil-specific IgG attenuated verapamil-induced reduction of developed tension in an ex-vivo rat model.

Calcium neutralizes fluoride bioavailability in a lethal model of fluoride poisoning.

OBJECTIVES: Acute systemic fluoride poisoning can result in systemic hypocalcemia, cardiac dysrhythmias, and cardiovascular collapse. Topical and intraarterial therapy with calcium or magnesium salts reduces dermal injury from fluoride burns. The mechanism of these therapies is to bind and inactivate the fluoride ion. The purpose of this study is to evaluate the effect of calcium and magnesium to decrease the bioavailability of fluoride in a lethal model of fluoride poisoning. METHODS: In preliminary studies, we determined that fluoride 3.6 mM/kg intraperitoneally in the form of sodium fluoride was uniformly and rapidly fatal in a mouse model. Using this fluoride dose, we performed a controlled, randomized, blinded study of low- and high-dose calcium chloride (1.8 and 3.6 mM/kg intraperitoneally, respectively) and magnesium sulfate (3.6 mM/kg intraperitoneally) to decrease the bioavailability of the fluoride ion. After injection with sodium fluoride, animals were immediately treated with injections of sodium chloride (control), calcium chloride (low- or high-dose), or magnesium sulfate. The major outcome was 6-hour survival using a Cox Proportional Hazard model. RESULTS: All untreated animals died within 60 minutes. Using a Cox Proportional Hazard model, each 1.8 mM/kg dose of calcium chloride administered reduced the risk of death by 33%. Magnesium sulfate treatment was not associated with a hazard reduction. CONCLUSION: Calcium chloride administered simultaneously with sodium fluoride reduces the bioavailability of fluoride poisoning in a mouse model. The equivalent dose of magnesium sulfate does not significantly decrease fluoride bioavailability.

Primary defects in the lens underlie complex anterior segment abnormalities of the Pax6 heterozygous eye.

We describe lens defects in heterozygous small eye mice, and autonomous deficiencies of Pax6(+/-) cells in the developing lens of Pax6(+/+) <--> Pax6(+/-) chimeras. Two separate defects of the lens were identified by analyzing the distribution of heterozygous cells in chimeras: Pax6(+/-) cells are less readily incorporated into the lens placode than wild type, and those that are incorporated into the lens are not maintained efficiently in the proliferating lens epithelium. The lens of chimeric eyes is, therefore, predominantly wild type from embryonic day 16.5 onwards, whereas heterozygous cells contribute normally to all other eye tissues. Eye size and defects of the iris and cornea are corrected in fetal and adult chimeras with up to 80% mutant cells. Therefore, these aspects of the phenotype may be secondary consequences of primary defects in the lens, which has clinical relevance for the human aniridia (PAX6(+/-)) phenotype.

Neutralization of Latrodectus mactans and L. hesperus venom by redback spider (L. hasseltii) antivenom.

OBJECTIVE: To test the effectiveness of L. hasseltii (redback spider) antivenom in neutralizing the lethal effects of L. hesperus and L. mactans (North American black widow) venoms. METHODS: LD50 values for the L. hesperus and L. mactans venom preparations were determined. A prospective, randomized, double-blind antivenom efficacy experiment was then performed for each venom using a mouse envenomation model. The following treatments were premixed and incubated at 25 degrees C for 1 hour prior to intraperitoneal injection: 1) saline control + protein control, 2) saline control + L. hasseltii antivenom, 3) L. hesperus or L. mactans venom + protein control, and 4) L. hesperus or L. mactans venom + L. hasseltii antivenom. The study endpoints were time elapsed until death and survival at 24 hours. RESULTS: The mouse LD50 values for L. hesperus and L. mactans venoms were 0.64 mg/kg and 0.26 mg/kg, respectively. In the efficacy trial, all mice in group 3 (L. hesperus or L. mactans venom and protein control) died. In both experiments, all mice in group 4 (L. hesperus or L. mactans venom + antivenom) survived (p < 0.0001). CONCLUSION: This is the first study to derive mouse LD50 values for L. hesperus and L. mactans venom obtained by electrical stimulation of live adult spiders. Redback spider antivenom is effective in neutralizing the lethal effects of L. hesperus and L. mactans venoms in a mouse envenomation model. While this study is limited by the optimized premixing of antigen with antibody, it generates the hypothesis that redback antivenom would be effective in the treatment of latrodectism in humans caused by the two clinically relevant species of North American widow spiders.

Time to reconstitution: purified Fab antivenom vs. unpurified IgG antivenom.

We conducted prospective, randomized analytical and observational trials to assess reconstitution times of two lyophilized crotaline snake antivenoms, Antivenin (Crotalidae) Polyvalent [Wyeth-Ayerst] (ACP) and affinity-purified, mixed monospecific crotalid antivenom ovine Fab (CroTAb) (Fab antivenom). The analytical experiment indicated Fab antivenom and ACP reached their maximum protein concentration at 25 and 45min, respectively. In the observational experiment, Fab antivenom (median 40min) had a shorter reconstitution time than ACP (>90min, p<0.008).

Characterization of venom (Duvernoy's secretion) from twelve species of colubrid snakes and partial sequence of four venom proteins.

R.E. Hill and S.P. Mackessy. Characterization of venom (Duvernoy's secretion) from twelve species of colubrid snakes and partial sequence of four venom proteins. Toxicon XX, xx-yy, 2000. - Venomous colubrids, which include more than 700 snake species worldwide, represent a vast potential source of novel biological compounds. The present study characterized venom (Duvernoy's gland secretion) collected from twelve species of opisthoglyphous (rear-fanged) colubrid snakes, an extremely diverse assemblage of non-venomous to highly venomous snakes. Most venoms displayed proteolytic activity (casein), though activity levels varied considerably. Low phosphodiesterase activity was detected in several venoms (Amphiesma stolata, Diadophis punctatus, Heterodon nasicus kennerlyi, H. n. nasicus and Thamnophis elegans vagrans), and acetylcholinesterase was found in Boiga irregularis saliva and venom, but no venoms displayed hyaluronidase, thrombin-like or kallikrein-like activities. High phospholipase A(2) (PLA(2)) activity was found in Trimorphodon biscutatus lambda venom, and moderate levels were detected in Boiga dendrophila and D. p. regalis venoms as well as B. dendrophila and H. n. nasicus salivas. Non-reducing SDS-PAGE revealed 7-20 protein bands (3.5 to over 200 kD, depending on species) for all venoms analyzed, and electrophoretic profiles of venoms were typically quite distinct from saliva profiles. Components from A. stolata, Hydrodynastes gigas, Tantilla nigriceps and T. e. vagrans venoms showed protease activity when run on gelatin zymogram gels. N-terminal protein sequences for three 26 kD venom components of three species (H. gigas, H. torquata, T. biscutatus) and one 3.5 kD component (T. nigriceps) were also obtained, and the 3.5 kD peptide showed apparent sequence homology with human vascular endothelial growth factor; these data represent the first sequences of colubrid venom components. Protease, phosphodiesterase and PLA(2) activities are also common to elapid and viperid snake venoms, but it is apparent that numerous other (as yet undescribed) components make up the majority of colubrid venom proteins. The complex nature of venoms produced by most species surveyed, and the high levels of protease or phospholipase A(2) activity of some venoms, suggest that many colubrids could become an important source of human health concern as encounters with these snakes increase.

Different roles for Pax6 in the optic vesicle and facial epithelium mediate early morphogenesis of the murine eye.

Chimaeric mice were made by aggregating Pax6(-/-) and wild-type mouse embryos, in order to study the interaction between the optic vesicle and the prospective lens epithelium during early stages of eye development. Histological analysis of the distribution of homozygous mutant cells in the chimaeras showed that the cell-autonomous removal of Pax6(-/-) cells from the lens, shown previously at E12.5, is nearly complete by E9.5. Most mutant cells are eliminated from an area of facial epithelium wider than, but including, the developing lens placode. This result suggests a role for Pax6 in maintaining a region of the facial epithelium that has the tissue competence to undergo lens differentiation. Segregation of wild-type and Pax6(-/-) cells occurs in the optic vesicle at E9.5 and is most likely a result of different adhesive properties of wild-type and mutant cells. Also, proximo-distal specification of the optic vesicle (as assayed by the elimination of Pax6(-/-) cells distally), is disrupted in the presence of a high proportion of mutant cells. This suggests that Pax6 operates during the establishment of patterning along the proximo-distal axis of the vesicle. Examination of chimaeras with a high proportion of mutant cells showed that Pax6 is required in the optic vesicle for maintenance of contact with the overlying lens epithelium. This may explain why Pax6(-/-) optic vesicles are inefficient at inducing a lens placode. Contact is preferentially maintained when the lens epithelium is also wild-type. Together, these results demonstrate requirements for functional Pax6 in both the optic vesicle and surface epithelia in order to mediate the interactions between the two tissues during the earliest stages of eye development.

The dominant hemimelia mutation uncouples epithelial-mesenchymal interactions and disrupts anterior mesenchyme formation in mouse hindlimbs.

Epithelial-mesenchymal interactions are essential for both limb outgrowth and pattern formation in the limb. Molecules capable of communication between these two tissues are known and include the signaling molecules SHH and FGF4, FGF8 and FGF10. Evidence suggests that the pattern and maintenance of expression of these genes are dependent on a number of factors including regulatory loops between genes expressed in the AER and those in the underlying mesenchyme. We show here that the mouse mutation dominant hemimelia (Dh) alters the pattern of gene expression in the AER such that Fgf4, which is normally expressed in a posterior domain, and Fgf8, which is expressed throughout are expressed in anterior patterns. We show that maintenance of Shh expression in the posterior mesenchyme is not dependent on either expression of Fgf4 or normal levels of Fgf8 in the overlying AER. Conversely, AER expression of Fgf4 is not directly dependent on Shh expression. Also the reciprocal regulatory loop proposed for Fgf8 in the AER and Fgf10 in the underlying mesenchyme is also uncoupled by this mutation. Early during the process of limb initiation, Dh is involved in regulating the width of the limb bud, the mutation resulting in selective loss of anterior mesenchyme. The Dh gene functions in the initial stages of limb development and we suggest that these initial roles are linked to mechanisms that pattern gene expression in the AER.

The transcription factor, Pax6, is required for cell proliferation and differentiation in the developing cerebral cortex.

The cerebral cortex develops from the dorsal telencephalon, at the anterior end of the neural tube. Neurons are generated by cell division at the inner surface of the telencephalic wall (in the ventricular zone) and migrate towards its outer surface, where they complete their differentiation. Recent studies have suggested that the transcription factor Pax6 is important for regulation of cell proliferation, migration and differentiation at various sites in the CNS. This gene is widely expressed from neural plate stage in the developing CNS, including the embryonic cerebral cortex, where it is required for radial glial cell development and neuronal migration. We report new findings indicating that, in the absence of Pax6, proliferative rates in the early embryonic cortex are increased and the differentiation of many cortical cells is defective. A major question concerns the degree to which cortical defects in the absence of Pax6 are a direct consequence of losing the gene function from defective cells themselves, rather than being secondary to abnormalities in other cells. Cortical defects in the absence of Pax6 become much more pronounced later in cortical development, and we propose that many result from a compounding of abnormalities in proliferation and differentiation that first appear at the onset of corticogenesis.