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.

Considerations for the design and construction of a synthetic platform cell for biotechnological applications.

The design and construction of an artificial bacterial cell could revolutionize biotechnological processes and technologies. A functional platform cell that can be easily customized for a pre-defined task would be useful for applications from producing therapeutics to decontaminating waste streams. The platform cell must be robust and highly efficient. A biotechnological platform cell is related to the concept of a minimal cell, but several factors beyond those necessary for a minimal cell must be considered for a synthetic organism designed for biotechnological applications. Namely, a platform cell must exhibit robust cell reproduction, decreased genetic drift, a physically robust cell envelope, efficient and simplified transcription and translation controls, and predictable metabolic interactions. Achieving a biotechnological platform cell will benefit from insights acquired from a minimal cell, but an approach of minimizing an existing organism's genome may be a more practical experimental approach. Escherichia coli possess many of the desired characteristics of a platform cell and could serve as a useful model organism for the design and construction of a synthetic platform organism. In this article we review briefly the current state of research in this field and outline specific characteristics that will be important for a biotechnologically relevant synthetic cell that has a minimized genome and efficient regulatory structure.

Regulations for vaccines against emerging infections and agrobioterrorism in the United States of America.

The Virus-Serum-Toxin Act of 1913, as amended in 1985, provides the legal basis for the regulation of veterinary vaccines and related biological products in the United States of America (USA). The regulatory authority for the issuance of licences and permits that allow the shipment or importation of pure, safe, potent, and effective veterinary biological products lies with the Center for Veterinary Biologics (CVB), an agency of the United States Department of Agriculture (USDA). Under the standard licensing or permitting process, a manufacturer must develop and completely characterise and evaluate a product prior to licensure, and the CVB must review and evaluate the submitted information, audit and inspect the manufacturing facilities and methods of production and testing, and confirm key product test results through independent testing of product. This complete and comprehensive evaluation may not be possible in emergency situations, so processes and mechanisms are in place that allow for the more rapid availability of veterinary vaccines. Next generation vaccine development against foreign animal diseases such as foot and mouth disease is actively in progress in the USA and the authorities must ensure that there is an adequate supply of these vaccines in the National Veterinary Stockpile.

Reversal of subarachnoid hemorrhage-induced vasoconstriction with an endothelin receptor antagonist.

Increased concentrations of the vasoconstrictor endothelin have recently been demonstrated in the cerebrospinal fluid after subarachnoid hemorrhage (SAH). This observation is consistent with the hypothesis that SAH-induced vasopasm is mediated in part by enhanced constriction due to endothelin. To investigate the issue, an endothelin receptor antagonist (ETant), cyclo(D-Asp-L-Pro-D-Val-L-Leu-D-Trp), was tested for its ability to reverse vasoconstriction after SAH. A transclival surgical approach to the basilar artery in rabbits was used, and the arterial diameter was measured continuously by videomicroscopy. Rabbits were divided randomly into six groups: 1) normal rabbits treated with 40 nmol/L ETant only; 2) normal rabbits treated with 50 mmol/L KCl, then 50 mmol/L KCl + 40 nmol/L ETant; 3) normal rabbits treated with 20 nmol/L endothelin-1 (ET-1), then 20 nmol/L ET-1 + 40 nmol/L ETant; 4) rabbits treated with 20 nmol/L ET-1 only; 5) rabbits subjected to SAH and treated with 40 nmol/L ETant; and 6) rabbits subjected to SAH and treated with artificial cerebrospinal fluid only. In normal (non-SAH) rabbits, ETant: 1) had little or no effect on resting tone; 2) did not reverse potassium-induced constrictions; and 3) substantially reversed endothelin-induced constrictions. The diameter of normal rabbit basilar arteries was 832.1 +/- 20.0 microns (mean +/- standard error). After SAH (double hemorrhage model), the mean diameter was 517.4 +/- 18.3 microns. The addition of ETant reversed this SAH-induced constriction by 70.7%.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of U88999E on experimental cerebral vasospasm in rabbits.

U88999E, 7-[4,(4, 4'-difluorobenzhydryl)piperazino-1-methyl]-4- isopropyl-2-methoxy-2,4,6-cycloheptatrien-1-one hydrochloride, is a recently developed tropolone derivative that inhibits lipid peroxidation and also acts as a calcium antagonist. The effects of U88999E on basilar artery tone were examined in two model systems: 1) an in vitro preparation of arterial rings that measures isometric tension, and 2) an in vivo model of cerebral vasospasm measuring arterial diameter. U88999E elicited dose-dependent relaxation of preconstricted arterial rings maintained in vitro. Ring preparations were preconstricted using elevated potassium (40 mmol/L), uridine triphosphate (10(-3) mol/L), or endothelin-1 (10(-8) mol/L); U88999E reversed these constrictions across a concentration range of 10(-8) to 10(-5) mol/L. The potency of U88999E for relaxing preconstricted vessels was slightly less than that observed for flunarizine or diltiazem. A dose-dependent, relaxing effect of U88999E on potassium-induced contractions was observed in the presence of calcium concentrations ranging from 0.03 to 20 mmol/L. Vasospasm of basilar arteries after subarachnoid hemorrhage was inhibited in a dose-dependent and significant manner by intravenous injections of U88999E. Animals receiving intraperitoneal injections of U88999E also exhibited a tendency for reduced vasospasm; however, this effect did not achieve statistical significance. These findings suggest that U88999E may be useful in the prevention of cerebral vasospasm after subarachnoid hemorrhage.

Cytotoxic effects of bloody cerebrospinal fluid on cerebral endothelial cells in culture.

The release of intracellular products from lysed blood cells is believed to play a critical role in the etiology of vascular pathology following intracerebral hemorrhage. The present studies investigated the effects of a mixture of blood and cerebrospinal fluid (CSF) on bovine intracranial endothelial cells maintained in culture. The incorporation of 3H-leucine into endothelial cells was used as an index of cellular viability. Cerebrospinal fluid alone did not alter the incorporation of 3H-leucine into the cells. In contrast, CSF preincubated with blood for 3 days or longer prior to treatment elicited significant reductions in leucine incorporation. Treatment with CSF preincubated with blood for 5 to 7 days resulted in the rapid deterioration of the culture, with large numbers of cells detaching almost immediately. Concentrations of hemoglobin were elevated profoundly in mixtures of blood and CSF preincubated for periods longer than 3 days. The increases in hemoglobin concentration were related temporally to increases in the cytotoxic impact of the bloody CSF. These findings suggest that factors released during the breakdown of blood exert a deleterious effect on intracranial endothelial cells. The time course of this effect is closely related to the development of vasospasm in humans following subarachnoid hemorrhage. Taken together, these observations are consistent with the hypothesis that intracellular blood products, particularly hemoglobin, contribute to vasospasm by directly compromising endothelial function.

Histidine attenuates cerebral vasospasm in a rabbit model of subarachnoid hemorrhage.

BACKGROUND: Free radical generation following hemolysis of a subarachnoid blood clot is believed to be a key component in the development of cerebral vasospasm. Histidine, an essential amino acid with free radical scavenging characteristics, was examined for its effects on cerebral vasospasm. METHODS: An experimental rabbit model of subarachnoid hemorrhage-induced vasospasm was used in which autologous arterial blood was injected into the cisterna magna. Basilar arteries were removed following perfusion-fixation two days after the injection of blood, and their cross-sectional luminal areas were measured using computerized image analysis. Rabbits received intravenous injections of L-histidine or vehicle starting 30 min prior to induction of subarachnoid hemorrhage (SAH), with additional injections given four times per day for the next 2 days. RESULTS: The luminal area of arteries from animals treated with histidine (50 mg/kg/dose or 100 mg/kg/dose) were significantly larger than those from vehicle-treated animals. Relative to the SAH-only groups (mean cross-sectional area = 106.8 x 10(3) microns 2), vasoconstriction was attenuated by 31% in the low dose treatment group (180.0 x 10(3) microns 2) and by 52% in the high dose treatment group (227.4 x 10(3) microns 2). Mean luminal area of control basilar arteries was 340.5 x 10(3) microns 2. CONCLUSIONS: These findings demonstrate that histidine reduces the amount of cerebral vasospasm occurring subsequent to experimental SAH. It is suggested that the free radical scavenging characteristics of histidine, particularly its ability to scavenge singlet oxygen, may be responsible for the reduction in vasospasm.

Evaluation of fentanyl transdermal patches in rabbits: blood concentrations and physiologic response.

In the study reported here, we sought to evaluate transdermal fentanyl patches for their ability to achieve detectable plasma concentrations with minimal adverse effects in New Zealand White rabbits. Fentanyl patches were applied to the dorsum after removing hair either by clipping or by application of a depilatory agent. Blood samples were collected every 12 h for a total of 96 h (24 h after patch removal) for determination of plasma fentanyl concentration. At those times, rabbits were assessed for changes in body temperature, heart rate, respiratory rate, and body weight. In rabbits with clipped hair, where rapid hair re-growth was not a mitigating factor, mean plasma fentanyl concentration reached a mean (+/- SEM) peak of 1.11 +/- 0.32 ng/ml at 24 h, decreased to 0.77 +/- 0.21 ng/ml at 72 h, and was negligible at 96 h. In rabbits with depilated hair, peak concentration was obtained at 12 h (6.7 +/- 0.57 ng/ml) and decreased gradually to 0.27 +/- 0.06 ng/ml at 72 h. In a second group of fentanyl-treated rabbits in which hair started growing back within 24 h, plasma fentanyl concentration was not detectable. Control and fentanyl-treated rabbits with clipped hair had no effect from the experimental manipulations other than slight loss in body weight. In the depilatory group, two rabbits appeared moderately sedated during the initial 12-h period, and had decreased respiratory rate for 24 h. In conclusion, rabbits tolerate the transdermal fentanyl patch well. Hair regrowth in rabbits may present a complicating factor that impedes dermal absorption of fentanyl. The application of a depilatory agent lead to early and rapid absorption of fentanyl causing undue sedation in some rabbits and lack of sustained plasma concentrations for the desired three-day period.

Attenuation of vasospasm and hemoglobin-induced constriction in the rabbit basilar artery by a novel protease inhibitor.

Calcium-activated proteolysis mediated by the protease inhibitor, calpain, has recently been implicated in the pathogenesis of cerebral vasospasm. The effect of one inhibitor of calcium-activated proteolysis, z-Leu-Phe-CONH-morpholene (zLF), on cerebrovascular constriction was examined in two experimental paradigms. In the first paradigm, the rabbit basilar artery (BA) was visualized via a transclival exposure, and its diameter was monitored using videomicroscopy. In the second experimental paradigm two intracisternal injections of autologous blood were administered to mimic a subarachnoid hemorrhage (SAH). The BA was visualized via the transclival exposure, and its luminal diameter was measured. Topical application of oxyhemoglobin (OxyHb), a known pathogenic agent in cerebral vasospasm, elicited vasoconstriction in normal animals, reducing arterial diameter to approximately 75% of resting levels. Pretreatment with zLF (100, 200, or 300 microM) attenuated vasoconstriction induced by OxyHb. In an experimental model of SAH, the diameter of the BA was reduced after the first injection of blood to approximately 67% of normal resting levels when measured 3 to 4 days later. This vasospastic response was reversed significantly by topical application of zLF (100 microM); vascular diameter was increased to approximately 84% of normal resting levels. These findings demonstrate that both acute OxyHb-induced constriction and blood-induced vasospasm are sensitive to an inhibitor of the proteolytic enzyme, calpain. Together, these observations indicate an important role for calcium-activated proteolysis in the development and maintenance of vasospasm after SAH. In addition, it may be inferred from the data that inhibitors of calcium-activated proteolysis may be useful therapeutic agents for treating this form of cerebrovascular disease.

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.

Heteropolymer-mediated clearance of immune complexes via erythrocyte CR1: mechanisms and applications.

Opsonization of particulate pathogens by antibodies and complement can lead to their binding to the complement receptor (CR1), specific for C3b, on primate erythrocytes (E). This process of immune adherence may play a role in immunologic defense by immobilizing bacteria and viruses, thus preventing them from leaving the bloodstream to invade susceptible tissue and organs. Immune adherence of C3b-opsonized and immune complexed pathogens to E may also facilitate their transfer to, and destruction by, fixed tissue macrophages. We have used mAbs specific for CR1 crosslinked with pathogen specific mAbs to generate heteropolymers (HP) which can bind a wide range of substrates to primate erythrocytes. Both prototype and bonafide pathogens bound to primate E via HP are handled in the circulation of non-human primates in a manner which appears to be virtually identical to the mechanism by which C3b-opsonized substrates bound to E CR1 are cleared. In this process of focused phagocytosis, Fc receptors on the phagocytic cell engage the E-bound complex, CR1 is removed by proteolysis, and the entire immune complex and CR1 are internalized while sparing the E. It may be possible to use HP to target pathogens in the bloodstream in a wide range of therapeutic applications.

A recombinant vector derived from the host range-restricted and highly attenuated MVA strain of vaccinia virus stimulates protective immunity in mice to influenza virus.

The immunogenicity of a recombinant virus derived from modified vaccinia virus Ankara (MVA), a host range-restricted, highly attenuated and safety-tested strain, was investigated. Plasmid transfer vectors that provide strong synthetic early/late promoters for the simultaneous expression of two genes as well as a transient or stable selectable marker and flanking sequences for homologous recombination with the MVA genome were constructed. A recombinant MVA containing influenza virus haemagglutinin and nucleoprotein genes was isolated in avian cells and shown to express both proteins efficiently upon infection of human or mouse cells in which abortive replication occurs. Mice, inoculated by various routes with recombinant MVA, produced antibody and cytotoxic T-lymphocyte responses to influenza virus proteins and were protected against a lethal influenza virus challenge as effectively as mice immunized with a recombinant derived from the replication-competent WR strain of vaccinia virus.

Hemoglobin penetration in the wall of the rabbit basilar artery after subarachnoid hemorrhage and intracisternal hemoglobin injection.

The ability of hemoglobin (Hb) to penetrate the basilar arterial wall in vivo after experimental subarachnoid hemorrhage was examined using immunohistochemistry. The distribution of anti-Hb antibodies in rabbit basilar artery was studied following the injection of autologous blood in the cisterna magna. Vessels removed two or four days after subarachnoid hemorrhage exhibited varying degrees of vasospasm, and exhibited Hb immuno-fluorescence throughout the vessel wall. Hemoglobin immunofluorescence was most conspicuous in the adventitia but was also seen in the smooth muscle and endothelial cell layers in 7 of 10 animals. The degree of vasoconstriction correlated with the total amount of Hb-fluorescence present in the vessel wall. When Hb solution alone was injected into the subarachnoid space, vasoconstriction was evident but penetration into the vascular layers was not as extensive as that observed after injection of autologous blood. These findings demonstrate that Hb is able to penetrate through the arterial wall after subarachnoid hemorrhage. The results provide direct support for the hypothesis that Hb-induced changes in smooth muscle and/or endothelial function can contribute to the pathogenesis of vasospasm.

Oxyhemoglobin-induced cytotoxicity and arachidonic acid release in cultured bovine endothelial cells.

BACKGROUND AND PURPOSE: An impairment of endothelial function is associated with vasospasm after subarachnoid hemorrhage. Oxyhemoglobin is considered to be a critical trigger in the pathogenesis of vasospasm. The present studies examined the direct effects of oxyhemoglobin on cultured endothelial cells from bovine carotid artery. METHODS: Confluent endothelial cells were treated with oxyhemoglobin, and the following were studied: 1) cell morphology, 2) cell density, and 3) the release of radiolabel from [3H]arachidonic acid-treated cells. RESULTS: Endothelial cells exposed to oxyhemoglobin exhibited detachment vacuoles, and cell density was significantly decreased in time- and dose-dependent manners. Superoxide dismutase, a free radical scavenger, provided partial protection against the cytotoxic effects of oxyhemoglobin. The release of radiolabel from [3H]arachidonic acid-treated cells was increased by oxyhemoglobin in time- and dose-dependent manners. Treatment with an inhibitor of phospholipase A2 or a calcium chelator inhibited the effects of oxyhemoglobin on arachidonic acid release and cellular viability. CONCLUSIONS: Oxyhemoglobin exerts a direct cytotoxic effect on cultured endothelial cells, and this effect is associated with increased release from [3H]arachidonic acid-labeled cells. Phospholipase A2 and free radicals appear to participate in the pathogenesis of endothelial cell damage. Oxyhemoglobin-induced compromise of endothelial cells may contribute to cerebrovascular pathology.

Protective roles of nitric oxide and testosterone in endotoxemia: evidence from NOS-2-deficient mice.

Lipopolysaccharide (LPS)-induced septic shock, which triggers nitric oxide (NO) overproduction, multiple organ dysfunction, and death, can be affected by gender and sex hormones. We hypothesized that NO is beneficial during endotoxemia and that this beneficial effect is influenced by sex hormones. C57BL/6 wild-type (WT) mice and congenic inducible NO synthase knockout (KO) mice were injected with LPS, and mortality was recorded for 4 days. After 5 mg/kg LPS, female KO mice had significantly higher mortality than WT. After 12.5 mg/kg LPS, both male and female KO mice had significantly higher mortality than WT. Ovariectomy did not alter mortality, but orchiectomy dramatically increased mortality in KO mice. After 5 mg/kg LPS, exogenous testosterone completely prevented the increased mortality in KO female and orchiectomized KO male mice. WT survival was not affected by exogenous testosterone. After 12.5 mg/kg LPS, exogenous testosterone significantly improved survival of female KO mice. Serum enzymes and organ edema, which may not correlate with mortality, were significantly and similarly increased in both WT and KO endotoxemic mice; however, edema was not observed in KO hearts. Thus, NO plays a protective role in endotoxemia while having differential effects on different organs. Importantly, testosterone is beneficial in endotoxemia when NO production is deficient, and may be therapeutic in certain septic patients.

Targeting of Pseudomonas aeruginosa in the bloodstream with bispecific monoclonal antibodies.

We examined the ability of a bispecific mAb reagent, consisting of a mAb specific for the primate erythrocyte complement receptor cross-linked with an anti-bacterial mAb, to target bacteria in the bloodstream in an acute infusion model in monkeys. In vitro studies demonstrated a variable level of complement-mediated binding (immune adherence) of Pseudomonas aeruginosa (strain PAO1) to primate E in serum. In vivo experiments in animals depleted of complement revealed that binding of bacteria to E was <1% before administration of the bispecific reagent, but within 5 min of its infusion, >99% of the bacteria bound to E. In complement-replete monkeys, a variable fraction of infused bacteria bound to E. This finding may have significant implications in the interpretation of animal models and in the understanding of bacteremias in humans. Treatment of these complement-replete monkeys with the bispecific reagent led to >99% binding of bacteria to E. Twenty-four-hour survival studies were conducted; several clinical parameters, including the degree of lung damage, cytokine levels, and liver enzymes in the circulation, indicate that the bispecific mAb reagent provides a degree of protection against the bacterial challenge.