Enhancement of Pasteurella Haemolytica A1 Leukotoxin Activity by Bovine Serum Albumin�

Growth of Pasteurella haemolytica A 1 in media containing fetal bovine serum has been observed to enhance leukotoxin (LKT) activity, but the mechanism of this increase is not understood. We found that bovine serum albumin in the absence of other serum components also enhances LKT activity. During the logarithmic growth phase, LKT activity was 30,700 � 12,900 Toxic Units (TU)/ml for P. haemolytica grown in RPM I medium containing 0. 5% bovine serum albumin (BSA) (BSA-LKT), compared to 120 � 40 TU/ml in medium containing RPMI alone (RPMILKT). In other experiments, addition of 0.5% BSA to RPMI-LKT culture filtrate(RPMI-LKT/BSA) resulted in LKT activity intermediate ( 13,000 � 1,600 TU/ml) between that of BSA-LKT (55,600 � 11,500 TU/ml) and RPMI-LKT (2,200 � 900 TU/ml). The activity of RPMI-LKT, BSA-LKT and RPMI-LKT /BSA decreased when the toxin preparations were incubated at room temperature (25�C) for 2 hours; however, the decrease in RPMI-LKT activity was more pronounced. Concentrated toxin from logarithmic growth phase RPMI-LKT culture supernatants contained a single LKT activity peak (Peak I) on Sephacryl HR-400 which had a K of 0.01 and estimated av molecular weight > > 669,000. In contrast, gel filtration of concentrated toxin from logarithmic growth phase BSA-LKT culture supernatants had three activity peaks with K values of 0.04 (Peak 1), 0.66 (Peak II), and av 0.87 (Peak Ill). Gel filtration of RPMI-LKT/BSA also consisted of Peaks I, II, and Ill. Peaks I, II, and Ill contain a prominent 97000 band on SDS-PAGE which was identified as LKT by Western blot analysis with an monoclonal antibody (MAB) to LKT. The identity of Peaks I, II, and Ill were further confirmed as LKT by target cell specificity (lysed bovine lymphoma cells and not equine leukocytes) and neutralization with a MAB toP. haemolytica LKT. It is concluded that LKT produced in RPMI medium alone is a large aggregate which undergoes conformational changes in the presence of BSA resulting in at least two additional LKT forms which may be partially disaggregated forms of the large aggregate LKT produced in RPMI.Veterinary Patholog

CNS activity of Pokeweed Anti-viral Protein (PAP) in mice infected with Lymphocytic Choriomeningitis Virus (LCMV)

BACKGROUND: Others and we have previously described the potent in vivo and in vitro activity of the broad-spectrum antiviral agent PAP (Pokeweed antiviral protein) against a wide range of viruses. The purpose of the present study was to further elucidate the anti-viral spectrum of PAP by examining its effects on the survival of mice challenged with lymphocytic choriomeningitis virus (LCMV). METHODS: We examined the therapeutic effect of PAP in CBA mice inoculated with intracerebral injections of the WE54 strain of LCMV at a 1000 PFU dose level that is lethal to 100% of mice within 7–9 days. Mice were treated either with vehicle or PAP administered intraperitoneally 24 hours prior to, 1 hour prior to and 24 hours, 48 hours 72 hours and 96 hours after virus inoculation. RESULTS: PAP exhibits significant in vivo anti- LCMV activity in mice challenged intracerebrally with an otherwise invariably fatal dose of LCMV. At non-toxic dose levels, PAP significantly prolonged survival in the absence of the majority of disease-associated symptoms. The median survival time of PAP-treated mice was >21 days as opposed to 7 days median survival for the control (p = 0.0069). CONCLUSION: Our results presented herein provide unprecedented experimental evidence that PAP exhibits antiviral activity in the CNS of LCMV-infected mice

Virtual Reality and 3D Imaging to Support Collaborative Decision Making for Adaptation of Long-Life Assets

European companies of today are involved in many stages of the product life cycle. There is a trend towards the view of their business as a complex industrial product-service system (IPSS). This trend shifts the business focus from a traditional product oriented one to a function oriented one. With the function in focus, the seller shares the responsibility of for example maintenance of the product with the buyer. As such IPSS has been praised for supporting sustainable practices. This shift in focus also promotes longevity of products and promotes life extending work on the products such as adaptation and upgrades. Staying competitive requires continuous improvement of manufacturing and services to make them more flexible and adaptive to external changes. The adaptation itself needs to be performed efficiently without disrupting ongoing operations and needs to result in an acceptable after state. Virtual planning models are a key technology to enable planning and design of the future operations in parallel with ongoing operations. This chapter presents an approach to combine digitalization and virtual reality (VR) technologies to create the next generation of virtual planning environments. Through incorporating digitalization techniques such as 3D imaging, the models will reach a new level of fidelity and realism which in turn makes them accessible to a broader group of users and stakeholders. Increased accessibility facilitates a collaborative decision making process that invites and includes cross functional teams. Through such involvement, a broader range of experts, their skills, operational and tacit knowledge can be leveraged towards better planning of the upgrade process. This promises to shorte

Antiretroviral Spermicide WHI-07 Prevents Vaginal and Rectal Transmission of Feline Immunodeficiency Virus in Domestic Cats

WHI-07 [5-bromo-6-methoxy-5,6-dihydro-3′-azidothymidine-5′-(p-bromophenyl)-methoxy alaninyl phosphate] is a novel dual-function aryl phosphate derivative of zidovudine with potent anti-human immunodeficiency virus (HIV) and spermicidal activities. WHI-07 was active against the feline immunodeficiency virus (FIV). This study evaluated whether topical application of WHI-07 as a single agent and in combination with an organometallic vanadium complex, vanadocene dithiocarbamate (VDDTC), via a nontoxic gel microemulsion can block vaginal as well as rectal transmission of feline AIDS (FAIDS) by chronically FIV-infected feline T cells in the natural host model. Genital transmission of FIV was monitored in recipient cats by the appearance of viral antibodies to FIV Gag proteins and by virus isolation of blood leukocytes as measured by FIV reverse transcriptase activity and FIV-specific PCR. Microbicidal activity was considered effective when the treated cats did not show evidence of FIV infection for up to 18 weeks postchallenge. An aggregate analysis of 46 specific-pathogen-free cats revealed that a single dose of the infected cell inoculum efficiently transmitted FIV infection when delivered into the vagina (100%) or rectum (66%). Pretreatment of the vagina or rectum with 2% WHI-07 alone or in combination with 0.25% VDDTC significantly (P = 0.004) protected cats from genital transmission by the highly infectious inoculum (7 million FIV(Bangston)-infected feline T cells). Collectively, using the vaginal and rectal transmucosal model for FAIDS, our studies demonstrated that WHI-07 either alone or in combination with a vanadocene has clinical potential for the development of a dual-function anti-HIV microbicide for sexually active women

Structure-Based Design and Engineering of a Nontoxic Recombinant Pokeweed Antiviral Protein with Potent Anti-Human Immunodeficiency Virus Activity

A molecular model of pokeweed antiviral protein (PAP)-RNA interactions was used to rationally engineer FLP-102((151)AA(152)) and FLP-105((191)AA(192)) as nontoxic PAPs with potent anti-human immunodeficiency virus (anti-HIV) activities. FLP-102 and FLP-105 have been produced in Escherichia coli and tested both in vitro and in vivo. These proteins depurinate HIV type 1 (HIV-1) RNA much better than rRNA and are more potent anti-HIV agents than native PAP or recombinant wild-type PAP. They are substantially less toxic than native PAP in BALB/c mice and exhibit potent in vivo activities against genotypically and phenotypically nucleoside reverse transcriptase inhibitor-resistant HIV-1 in a surrogate human peripheral blood lymphocyte (Hu-PBL) SCID mouse model of human AIDS. Rationally engineered nontoxic recombinant PAPs such as FLP-102 and FLP-105 may provide the basis for effective salvage therapies for patients harboring highly drug-resistant strains of HIV-1. The documented in vitro potencies of FLP-102 and FLP-105, their in vivo antiretroviral activities in the HIV-infected Hu-PBL SCID mouse model, and their favorable toxicity profiles in BALB/c mice warrant the further development of these promising new biotherapeutic agents