A human immunodeficiency virus-transgenic mouse model for assessing interventions that block microbial-induced proviral expression.

A human immunodeficiency virus (HIV) type 1-transgenic mouse line (166) that previously showed up-regulated expression of viral proteins and infectious particles after infection with pathogenic agents was tested as a model for screening the in vitro and in vivo efficacy of inhibitors of HIV-1 immune activation. Two types of interventions were assessed: use of either the immunosuppressive drug prednisolone or an HIV-1 envelope-targeted toxin (sCD4-PE40). Both agents inhibited lipopolysaccharide-induced p24 expression by splenocytes in vitro and, when administered to transgenic mice, suppressed the induction of plasma p24, as well as the ex vivo production of p24 and infectious virus stimulated by in vivo infection with Mycobacterium avium. Moreover, HIV-1 mRNA levels in the spleen were greatly reduced in mice treated with either agent. Because HIV-1 expression cannot be induced in T lymphocytes from line 166 mice, this model may be of particular advantage for testing interventions that target virus production by non-T cell virus reservoirs.

CD46 is a cellular receptor for human herpesvirus 6.

Human herpesvirus 6 (HHV-6) is the etiologic agent of exanthema subitum, causes opportunistic infections in immunocompromised patients, and has been implicated in multiple sclerosis and in the progression of AIDS. Here, we show that the two major HHV-6 subgroups (A and B) use human CD46 as a cellular receptor. Downregulation of surface CD46 was documented during the course of HHV-6 infection. Both acute infection and cell fusion mediated by HHV-6 were specifically inhibited by a monoclonal antibody to CD46; fusion was also blocked by soluble CD46. Nonhuman cells that were resistant to HHV-6 fusion and entry became susceptible upon expression of recombinant human CD46. The use of a ubiquitous immunoregulatory receptor opens novel perspectives for understanding the tropism and pathogenicity of HHV-6.

Specific killing of HIV-infected lymphocytes by a recombinant immunotoxin directed against the HIV-1 envelope glycoprotein.

BACKGROUND: 3B3 is a high-affinity anti-gp120 antibody that neutralizes a wide range of primary and laboratory isolates of HIV-1. The parental antibody was isolated from a combinatorial phage display library constructed from bone marrow RNA of an HIV-infected individual. We have generated a highly active immunotoxin using the 3B3 single-chain Fv (scFv) which can specifically kill lymphocytes infected by HIV-1. MATERIALS AND METHODS: We used recombinant DNA technology to clone the Fv fragment of 3B3 and produce a single-chain Fv (scFv). 3B3 scFv was then fused to a truncated version of Pseudomonas exotoxin A (PE38), giving rise to a recombinant immunotoxin 3B3(Fv)-PE38 that was expressed in E. coli and purified to near homogeneity. RESULTS: 3B3(Fv)-PE38 binds with the same affinity as the parental Fab antibody to the MN strain of gp120. The immunotoxin specifically kills a gp120-expressing transfected cell line and a chronically HIV-infected lymphocytic cell line. The immunotoxin is very stable at 37 degrees C, retaining 80% of its original activity after 24 hr. CONCLUSIONS: Potent immunotoxins such as 3B3(Fv)-PE38 could be utilized in combination with multidrug cocktails that limit viral replication to help reduce viral reservoirs in patients with AIDS.

HIV-1 coreceptor activity of CCR5 and its inhibition by chemokines: independence from G protein signaling and importance of coreceptor downmodulation.

HIV-1 infection requires the presence of specific chemokine receptors on CD4+ target cells to enable the fusion reactions involved in virus entry. CCR5 is a major fusion coreceptor for macrophage-tropic HIV-1 isolates. HIV-1 entry and fusion are mediated by the viral envelope glycoprotein (Env) and are inhibited by CCR5 ligands, but the mechanisms are unknown. Here, we test the role of G protein signaling and CCR5 surface downmodulation by two separate approaches: direct inactivation of CCR5 signaling by mutagenesis and inactivation of G(i)-type G proteins with pertussis toxin. A CCR5 mutant lacking the last 45 amino acids of the cytoplasmic C-terminus (CCR5306) was created that was expressed on transfected cells at levels comparable to cells expressing CCR5 and displayed normal chemokine binding affinity. CCR5 ligands induced calcium flux and receptor downmodulation in cells expressing CCR5, but not in cells expressing CCR5306. Nevertheless, CCR5 or CCR5306, when coexpressed with CD4, supported comparable HIV-1 Env-mediated cell fusion. Consistent with this, treatment of CCR5-expressing cells with pertussis toxin completely blocked ligand-induced transient calcium flux, but did not affect Env-mediated cell fusion or HIV-1 infection. Also, pertussis toxin did not block chemokine inhibition of Env-mediated cell fusion or HIV-1 infection. However, chemokines inhibited Env-mediated cell fusion less efficiently for CCR5306 than for CCR5. We conclude that the C-terminal domain of CCR5 is critical for G protein signaling and receptor downmodulation from the surface, but that neither function is required for CCR5 fusion coreceptor activity. The contrasting phenotypes of CCR5 and CCR5306 suggest that coreceptor downmodulation and direct blockage of Env interaction sites both contribute to chemokine inhibition of HIV-1 infection.

Inherited resistance to HIV-1 conferred by an inactivating mutation in CC chemokine receptor 5: studies in populations with contrasting clinical phenotypes, defined racial background, and quantified risk.

BACKGROUND: CC chemokine receptor 5 (CCR5) is a cell entry cofactor for macrophage-tropic isolates of human immunodeficiency virus-1 (HIV-1). Recently, an inactive CCR5 allele (designated here as CCR5-2) was identified that confers resistance to HIV-1 infection in homozygotes and slows the rate of progression to AIDS in heterozygotes. The reports conflict on the effect of heterozygous CCR5-2 on HIV-1 susceptibility, and race and risk levels have not yet been fully analyzed. Here we report our independent identification of CCR5-2 and test its effects on HIV-1 pathogenesis in individuals with contrasting clinical outcomes, defined race, and quantified risk. MATERIALS AND METHODS: Mutant CCR5 alleles were sought by directed heteroduplex analysis of genomic DNA from random blood donors. Genotypic frequencies were then determined in (1) random blood donors from North America, Asia, and Africa; (2) HIV-1+ individuals; and (3) highly exposed-seronegative homosexuals with quantified risk. RESULTS: CCR5-2 was the only mutant allele found. It was common in Caucasians, less common in other North American racial groups, and not detected in West Africans or Tamil Indians. Homozygous CCR5-2 frequencies differed reciprocally in highly exposed-seronegative (4.5%, n = 111) and HIV-1-seropositive (0%, n = 614) Caucasians relative to Caucasian random blood donors (0.8%, n = 387). This difference was highly significant (p < 0.0001). By contrast, heterozygous CCR5-2 frequencies did not differ significantly in the same three groups (21.6, 22.6, and 21.7%, respectively). A 55% increase in the frequency of heterozygous CCR5-2 was observed in both of two cohorts of Caucasian homosexual male, long-term nonprogressors compared with other HIV-1+ Caucasian homosexuals (p = 0.006) and compared with Caucasian random blood donors. Moreover, Kaplan-Meier estimates indicated that CCR5-2 heterozygous seroconvertors had a 52.6% lower risk of developing AIDS than homozygous wild-type seroconvertors. CONCLUSIONS: The data suggest that homozygous CCR5-2 is an HIV-1 resistance factor in Caucasians with complete penetrance, and that heterozygous CCR5-2 slows the rate of disease progression in infected Caucasian homosexuals. Since the majority (approximately 96%) of highly exposed-seronegative individuals tested are not homozygous for CCR5-2, other resistance factors must exist. Since CCR5-2 homozygotes have no obvious clinical problems, CCR5 may be a good target for the development of novel antiretroviral therapy.

CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1.

Human immunodeficiency virus-type 1 (HIV-1) entry requires fusion cofactors on the CD4+ target cell. Fusin, a heterotrimeric GTP-binding protein (G protein)-coupled receptor, serves as a cofactor for T cell line-tropic isolates. The chemokines RANTES, MIP-1alpha, and MIP-1beta, which suppress infection by macrophage-tropic isolates, selectively inhibited cell fusion mediated by the corresponding envelope glycoproteins (Envs). Recombinant CC CKR5, a G protein-coupled receptor for these chemokines, rendered CD4-expressing nonhuman cells fusion-competent preferentially with macrophage-tropic Envs. CC CKR5 messenger RNA was detected selectively in cell types susceptible to macrophage-tropic isolates. CC CKR5 is thus a fusion cofactor for macrophage-tropic HIV-1 strains.

HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor.

A cofactor for HIV-1 (human immunodeficiency virus-type 1) fusion and entry was identified with the use of a novel functional complementary DNA (cDNA) cloning strategy. This protein, designated "fusin," is a putative G protein-coupled receptor with seven transmembrane segments. Recombinant fusin enabled CD4-expressing nonhuman cell types to support HIV-1 Env-mediated cell fusion and HIV-1 infection. Antibodies to fusin blocked cell fusion and infection with normal CD4-positive human target cells. Fusin messenger RNA levels correlated with HIV-1 permissiveness in diverse human cell types. Fusin acted preferentially for T cell line-tropic isolates, in comparison to its activity with macrophagetropic HIV-1 isolates.

Expression of foreign genes in cultured human primary macrophages using recombinant vaccinia virus vectors.

Recombinant vaccinia viruses (re-VVs) provide an extremely versatile method for the expression of foreign genes in a wide range of cultured cell types of different lineages and species. In the present report, we examine the utility of re-VV vectors for re-protein production in cultured human primary macrophages obtained through in vitro differentiation of peripheral blood monocytes. Primary macrophages supported early stages of the VV infection cycle, including morphologic cytopathic effect, shut-off of host protein synthesis and activation of early viral protein synthesis; however, late stages of infection were blocked, including synthesis of late viral proteins, replication of viral DNA, and production of infectious progeny virions. Abortive infection was observed with several independent VV strains. Using re-VVs containing Escherichia coli lacZ as a reporter gene, we assayed the activities of different classes of VV promoters. Consistent with the results noted above, human primary macrophages supported reporter gene expression driven by an early or intermediate VV promoter, but not by a late promoter; expression was obtained with synthetic bifunctional promoters containing early and/or intermediate components. Primary macrophages also supported the VV/bacteriophage T7 RNA polymerase hybrid gene expression system. The utility of re-VV vectors for production of proteins of biological interest in human primary macrophages was demonstrated using re-VVs encoding human CD4 and the human immunodeficiency virus type-1 envelope glycoprotein.

Primary HIV-1 isolates refractory to neutralization by soluble CD4 are potently inhibited by CD4-Pseudomonas exotoxin.

Despite the ability of soluble forms of CD4 (sCD4) and related CD4 derivatives to neutralize human immunodeficiency type 1 (HIV-1) infectivity in vitro, these agents have shown little evidence of efficacy in clinical trials with infected individuals. These disappointing findings may be related to recent observations that much higher concentrations of sCD4 are required for in vitro neutralization of primary HIV-1 isolates compared to laboratory-adapted strains. An alternative CD4-based therapeutic strategy exploits CD4 as a targeting agent to direct cytotoxic molecules to selectively kill HIV-infected cells. In this report we demonstrate that CD4-Pseudomonas exotoxin inhibits spreading infection by primary HIV-1 isolates known to be highly refractory to neutralization by soluble CD4; the observed potency is at least as great as for a prototypic sCD4-sensitive, laboratory-adapted HIV-1 strain. Thus, the in vitro efficacy of a CD4-based agent, which acts by targeted killing of infected cells, appears not to be compromised by features which render primary HIV-1 isolates refractory to neutralization by sCD4 derivatives. These results have important conceptual and practical implications for CD4-based therapeutic strategies.

Evaluation of two over-the-counter natural thyroid hormone preparations in human volunteers.

OBJECTIVE: To determine the pharmacologic activity of over-the-counter (OTC) thyroid preparations. DESIGN: In vitro analysis and a prospective, crossover study in vivo. SETTING: Tertiary care center. PARTICIPANTS: Two healthy adult volunteers. INTERVENTION: Three OTC preparations (Thyrotrophin PMG [bovine thyroid PMG extract], Thyro Forte [thyroid lymphogland concentrate with synergistic complex], and Thyro Complex [thyroid lyophilized gland concentrate with synergistic complex]) were analyzed in vitro. Volunteers were administered two times the manufacturer's maximum recommended daily dose of either Thyrotrophin PMG or Thyro Forte for one week, washed out for four to five weeks, and crossed over to receive the opposite tablet preparation for an additional week. MAIN OUTCOME MEASURES: The triiodothyronine (T3) and thyroxine (T4) contents of OTC preparations were measured by HPLC. Vital signs, serum total and free T4, total T3, thyroid stimulating hormone, thyroxine binding globulin, thyroglobulin, and general chemistry tests (including glucose and cholesterol) were monitored before, during, and between administration of the products. RESULTS: HPLC analysis of the three OTC preparations showed no T4 but did show possible T3 in two of these products. We found no definite clinical or laboratory evidence of thyroid hormone excess with either product. CONCLUSIONS: Healthcare professionals should advise against the use of these scientifically unsound and relatively expensive OTC thyroid preparations, of which the therapeutic efficacy is unknown.

Isolation of HIV-1 from plasma of infected individuals: an analysis of experimental conditions affecting successful virus propagation.

Experimental conditions affecting the successful propagation of HIV-1 from the plasma of seropositive individuals were examined. It was determined that whole blood samples collected with lithium heparin as the anticoagulant, immediate plasma separation, and immediate culturing were best suited for obtaining viable virus from plasma. Virus was isolated by infecting fresh phytohemagglutinin-stimulated normal donor peripheral blood mononuclear cells (PBMCs) with plasma followed by weekly cocultivation with new target cells. The plasma virus isolation rate was the greatest and HIV-1 titers were the highest for those individuals with less than 200 CD4+ cells/mm3 and decreased as the level of CD4+ cells approached normal values. We were able to obtain positive cultures from 29.5% of those patients with CD4+ counts greater than 500/mm3. HIV-1 titers in plasma also correlated with high serum p24 antigen levels when serum was treated with glycine to dissociate antigen-antibody complexes.

Stability of zidovudine in 5% dextrose injection and 0.9% sodium chloride injection.

The stability of zidovudine at a concentration of 4 mg/mL in 5% dextrose injection and 0.9% sodium chloride injection in polyvinyl chloride infusion bags stored at room and refrigerated temperatures for up to eight days was studied. Zidovudine was diluted in 5% dextrose injection and in 0.9% sodium chloride injection to a concentration of 4 mg/mL. Six admixtures were prepared with each diluent; three were stored at room temperature (25 +/- 1 degree C) and three were refrigerated (4 +/- 1 degree C). At 0, 3, 6, 24, 48, 72, and 192 hours, 2-mL aliquots were removed. One milliliter of each aliquot was diluted to a zidovudine concentration of approximately 40 micrograms/mL and assayed in duplicate by a stability-indicating high-performance liquid chromatographic method. Visual inspection was performed at each sampling time for precipitation, turbidity, color change, and gas formation. Sample pH was recorded at 0 and 192 hours. In all admixtures, more than 97% of the initial zidovudine concentration remained throughout the study period. No visual or pH changes were observed. Zidovudine 4 mg/mL in admixtures with 5% dextrose injection or 0.9% sodium chloride injection stored in polyvinyl chloride infusion bags was stable for up to 192 hours (eight days) at room temperature and under refrigeration.

Stability of amphotericin B in 5% dextrose injection at concentrations used for administration through a central venous line.

The stability of amphotericin B in 5% dextrose injection was studied at concentrations used for administration through a central venous line. Amphotericin B 60, 80, and 100 mg was diluted in 50 mL of 5% dextrose injection; final mean +/- S.D. concentrations after adjustment for total volume were 0.92 +/- 0.01, 1.20 +/- 0.03, and 1.40 +/- 0.03 mg/mL, respectively. For each concentration, six admixtures were prepared; of these, three were stored at 25 degrees C and three at 6 degrees C. Amphotericin B concentration was tested at 0, 4, 12, 24, and 36 hours by stability-indicating high-performance liquid chromatography. The admixtures were inspected visually at each time point for precipitation, turbidity, gas formation, and color change, and the pH was measured. Concentrations of amphotericin B remained within 3% of initial concentrations at each time point at both storage temperatures. No precipitation, turbidity, gas formation, or color change was observed, and no changes in pH were measured. Amphotericin B in 5% dextrose injection was stable at concentrations of 0.92, 1.20, and 1.40 mg/mL when stored at 6 and 25 degrees C for up to 36 hours.

Stability of concentrated trimethoprim-sulfamethoxazole admixtures.

The stability of trimethoprim-sulfamethoxazole (TMP-SMX) at various concentrations in 5% dextrose injection or 0.9% sodium chloride injection was studied. Appropriate volumes of TMP-SMX formulation (80 mg TMP and 400 mg SMX/5 mL) were mixed with 5% dextrose injection or 0.9% sodium chloride injection to provide dilutions of 1:25 v/v, 1:20 v/v, 1:15 v/v, and 1:10 v/v. Aliquots were removed at 0, 0.5, 1, 2, 4, 8, 14, 24, and 48 hours and filtered. The pH of the samples was determined, and the samples were assayed for trimethoprim and sulfamethoxazole content by high-performance liquid chromatography. Admixtures were visually inspected for precipitate before each sample was removed. The concentration of SMX in all admixtures did not change during the study period. The stability of TMP was dependent on concentration and vehicle. At a 1:25 v/v dilution, TMP was stable for 48 hours in 5% dextrose injection and 0.9% sodium chloride injection. At a 1:20 v/v dilution, TMP was stable for 24 hours in 5% dextrose injection and 14 hours in 0.9% sodium chloride injection. At a 1:15 v/v dilution, TMP was stable for four hours in 5% dextrose injection and two hours in 0.9% sodium chloride injection. At a 1:10 v/v dilution, TMP was stable for one hour in 5% dextrose injection and 0.9% sodium chloride injection. Concentrated solutions of TMP-SMX should be prepared in 5% dextrose injection, infused within one hour of preparation, and visually inspected for precipitation before and during infusion.

Design, synthesis, and testing of potential antisickling agents. 5. Disubstituted benzoic acids designed for the donor site and proline salicylates designed for the acceptor site.

This paper reports the discovery of a new class of potent antigelling agents. The new compounds, disubstituted benzoic acid derivatives, were designed by using molecular modeling experiments. These molecules contain functional groups positioned to interact with several polar amino acid residues near the Val-6 beta mutation site (donor site) in HbS. The compounds also contain a hydrophobic group designed to occupy a nonpolar ara on the surface of the protein created by several hydrophobic amino acids. The synthesis and testing of these new molecules using a standard solubility assay is reported. A structural comparison is made between one of the most active antigelling agents, compound 13, which has little effect on the oxygen affinity of Hb in solution, and bezafibrate, a known antilipidemic drug that is progelling and has a very potent effect on decreasing Hb oxygen affinity (Perutz, M. F.; Poyart, C. Lancet 1983, 2, 881-882). We also report the synthesis and testing of a series of proline-salicylate molecules designed to react covalently at the mutation acceptor area. This class of molecules did not show significant activity.

Design, synthesis, and testing of potential antisickling agents. 4. Structure-activity relationships of benzyloxy and phenoxy acids.

In this paper we further establish the activity of two classes of small molecules, benzyloxy and phenoxy acids, as potent inhibitors of hemoglobin S (HbS) gelation. Structural modifications with a large number of each class confirm our earlier work that the highest activity is observed with compounds that contain dihalogenated aromatic rings with attached polar side chains. We have also found a halogenated aromatic malonic acid derivative to be quite active. Compounds reported in this paper are compared with other antigelling agents studied in our laboratory. Comments are made concerning the antigelling activity and binding sites of four derivatives and their effect on the allosteric mechanism of hemoglobin (Hb) function.

Methylphenylmercury: a novel heavy atom reagent for protein crystallography.

Methylphenylmercury reacts with two normally inaccessible cysteine residues in crystals of carbonmonoxyhaemoglobin, but not with the third, normally reactive one. It may, therefore, be useful in the preparation of new heavy atom derivatives for protein crystallography.