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

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

In Vivo Toxicity, Pharmacokinetics, and Anti-Human Immunodeficiency Virus Activity of Stavudine-5′-(p-Bromophenyl Methoxyalaninyl Phosphate) (Stampidine) in Mice

We have evaluated the clinical potential of stavudine-5′-(p-bromophenyl methoxyalaninyl phosphate(stampidine [STAMP]), a novel aryl phosphate derivative of stavudine, as a new anti-human immunodeficiency virus (anti-HIV) agent, by examining its acute, subacute, and chronic toxicity profile in mice as well as by testing its antiviral activity in a surrogate human peripheral blood lymphocyte (Hu-PBL)-SCID mouse model of human AIDS. STAMP was very well tolerated in BALB/c and CD-1 mice, without any detectable acute or subacute toxicity at single intraperitoneal or oral bolus doses as high as 500 mg/kg of body weight. Notably, daily administration of STAMP intraperitoneally or orally for up to 8 consecutive weeks was not associated with any detectable toxicity at cumulative dose levels as high as 6.4 g/kg. Micromolar concentrations of the active STAMP metabolite in plasma were rapidly achieved and maintained for more than 4 h after parenteral as well as oral administration of a nontoxic 100-mg/kg bolus dose of STAMP. In accordance with its favorable pharmacokinetic profile and in vitro potency, STAMP exhibited dose-dependent and potent in vivo anti-HIV activity in Hu-PBL-SCID mice against a genotypically and phenotypically nucleoside analog reverse transcriptase inhibitor (NRTI)-resistant clinical HIV type 1 (HIV-1) isolate (BR/92/019; D67N, L214F, T215D, K219Q) at nontoxic dose levels. The remarkable in vivo safety and potency of STAMP warrants the further development of this promising new antiretroviral agent for possible clinical use in patients harboring NRTI-resistant HIV-1

In Vivo Antiretroviral Activity of Stampidine in Chronically Feline Immunodeficiency Virus-Infected Cats

Here we report the antiretroviral activity of the experimental nucleoside reverse transcriptase inhibitor (NRTI) compound stampidine in cats chronically infected with feline immunodeficiency virus (FIV). Notably, a single oral bolus dose of 50 or 100 mg of stampidine per kg resulted in a transient ≥1-log decrease in the FIV load of circulating peripheral blood mononuclear cells in five of six FIV-infected cats and no side effects. A 4-week stampidine treatment course with twice-daily administration of hard gelatin capsules containing 25 to 100 mg of stampidine per kg was also very well tolerated by cats at cumulative dose levels as high as 8.4 g/kg and exhibited a dose-dependent antiretroviral effect. One of three cats treated at the 25-mg/kg dose level, three of three cats treated at the 50-mg/kg dose level, and three of three cats treated at the 100-mg/kg dose level (but none of three control cats treated with placebo pills) showed a therapeutic response, as evidenced by a ≥1-log reduction in the FIV load in peripheral blood mononuclear cells within 2 weeks. The previously documented in vitro and in vivo antiretroviral activity of stampidine against primary clinical human immunodeficiency virus type 1 isolates with genotypic and/or phenotypic NRTI resistance, together with its favorable animal toxicity profile, pharmacokinetics, and in vivo antiretroviral activity in FIV-infected cats, warrants further development of this promising new NRTI compound