Protection of rhesus macaques against disease progression from pathogenic SHIV-89.6PD by vaccination with phage-displayed HIV-1 epitopes.

The antigenic polymorphism of HIV-1 is a major obstacle in developing an effective vaccine. Accordingly, we screened random peptide libraries (RPLs) displayed on phage with antibodies from HIV-infected individuals and identified an array of HIV-specific epitopes that behave as antigenic mimics of conformational epitopes of gp120 and gp41 proteins. We report that the selected epitopes are shared by a collection of HIV-1 isolates of clades A-F. The phage-borne epitopes are immunogenic in rhesus macaques, where they elicit envelope-specific antibody responses. Upon intravenous challenge with 60 MID50 of pathogenic SHIV-89.6PD, all monkeys became infected; however, in contrast to the naive and mock-immunized monkeys, four of five mimotope-immunized monkeys experienced lower levels of peak viremia, followed by viral set points of undetectable or transient levels of viremia and a mild decline of CD4+ T cells, and were protected from progression to AIDS-like illness. These results provide a new approach to the design of broadly protective HIV-1 vaccines.

The expression of the interleukin 6 gene is induced by the human immunodeficiency virus 1 TAT protein.

Human immunodeficiency virus 1 (HIV1) infection is associated with severe psoriasis, B cell lymphoma, and Kaposi's sarcoma. A deregulated production of interleukin 6 (IL-6) has been implicated in the pathogenesis of these diseases. The molecular mechanisms underlying the abnormal IL-6 secretion of HIV1-infected cells may include transactivation of the IL-6 gene by HIV1. To test this hypothesis, we used the pIL6Pr-chloramphenicol acetyltransferase (CAT) plasmid, an IL-6 promoter-CAT construct, as a target of the transactivating function of the HIV1 TAT protein. By cotransfecting the pIL6Pr-CAT and the tat-expressing pSVT8 plasmid in MC3 B-lymphoblastoid or in HeLa epithelial cells, we observed that TAT transactivates the human IL-6 promoter. These results were confirmed when pIL6Pr-CAT was transfected in MC3 or HeLa cells that constitutively expressed the tat gene in a sense (pSVT8 cells) or antisense (pSVT10 cells) orientation. 5' deletion plasmids of pIL6Pr-CAT, in which regions at -658, -287, and -172 were inserted 5' to the cat gene, were transiently transfected in pSVT10 and pSVT8 cells and showed that TAT-induced activation of the IL-6 promoter required a minimal region located between -287 and -54 bp. Moreover, experiments with plasmids carrying the -658, -287, and -172 bp regions of the IL-6 promoter inserted downstream to a TAR-deleted HIV1-LTR identified the sequence of -172 to -54 as the minimal region of the IL-6 promoter required for TAT to transactivate the TAR-deleted HIV1-LTR. By DNA-protein binding experiments, tat-transfected cells expressed a consistent increase in kappa B and nuclear factor (NF)-IL-6 binding activity. Accordingly, the pDRCAT and IL-1REK9CAT, carrying tandem repeats of NF-kappa B or NF-IL6 binding motifs, respectively, were activated in TAT-expressing cells. The biological relevance of the TAT-induced IL-6 secretion was addressed by generating 7TD1 cells, an IL-6-dependent mouse cell line, stably expressing the tat gene. These tat-positive cells expressed the endogenous IL-6 gene, secreted high amounts of murine IL-6, and grew efficiently in the absence of exogenous IL-6. Moreover, the tat-positive 7TD1 cells sustained the growth of parental 7TD1 cells and showed a dramatic increase in their tumorigenic potency. These results suggest that TAT protein may play a role in the pathogenesis of some HIV1-associated diseases by modulating the expression of host cellular genes.

Expression of an exogenous interleukin 6 gene in human Epstein Barr virus B cells confers growth advantage and in vivo tumorigenicity.

The biological role of interleukin 6 (IL-6) molecules in human B cell tumorigenesis was studied by using an episomal expression vector, pHEBoSV-IL6, to introduce stably the human IL-6 gene into human Epstein Barr virus (EBV)-transformed B lymphoblasts. The gene was present in the IL-6-transfected cells in a high copy number and was efficiently expressed, resulting in the secretion of consistent levels of IL-6 molecules. The constitutive expression of the IL-6 gene led to an altered pattern of growth and to a malignant phenotype, as shown by clonogenicity in to an altered pattern of growth and to a malignant phenotype, as shown by clonogenicity in soft agar cultures and tumorigenicity in nude mice. These data suggest that the combined action of EBV, which exerts an immortalizing function, and of the growth-promoting activity of IL-6 molecules, can give rise to fully transformed B cell tumors in immunodeficient subjects.

Partial purification and MALDI-TOF MS analysis of UN1, a tumor antigen membrane glycoprotein.

UN1 is a membrane glycoprotein that is expressed in immature human thymocytes, a subpopulation of peripheral T lymphocytes, the HPB acute lymphoblastic leukemia (ALL) T-cell line and fetal thymus. We previously reported the isolation of a monoclonal antibody (UN1 mAb) recognizing the UN1 protein that was classified as "unclustered" at the 5th and 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens. UN1 was highly expressed in breast cancer tissues and was undetected in non-proliferative lesions and in normal breast tissues, indicating a role for UN1 in the development of a tumorigenic phenotype of breast cancer cells. In this study, we report a partial purification of the UN1 protein from HPB-ALL T cells by anion-exchange chromatography followed by immunoprecipitation with the UN1 mAb and MALDI-TOF MS analysis. This analysis should assist in identifying the amino acid sequence of UN1.

Mutagenic epoxide impurities discovered in two new beta-adrenergic blocking agents.

Preparations of two new beta-adrenergic blocking drugs, Zami 1305 [1-(2-nitro-3-methyl-phenoxy)-3-tert-butylaminopropan-2-ol] and Zami 1327 [1-(6-nitro-3-methyl-phenoxy)-3-tert-butylaminopropan-2-ol], were found to be contaminated by expoxides which are direct acting mutagens on TA 100 and TA 1535 in the Salmonella/microsome mutagenicity test. Because of the suggested correlation between mutagenicity and carcinogenicity of a chemical [10,11], beta-adrenergic blocking agents contaminated by mutagenic expoxide impurities may be a health hazard.

Carcinogenic potency in rodents versus genotoxic potency in E. coli: a correlation analysis for bifunctional alkylating agents.

The mutagenic (M), recombinagenic (R) and SOS inducing (I) potencies of 6 bifunctional directly acting alkylating agents (mitomycin C, thiotepa, chlorambucil, nitrogen mustard, bis(2-chloroethyl)ether and bis(2-chloroethyl)nitrosourea) were measured in an E. coli test system (E. coli multitest) as the integral under the yield-dose curve obtained for each event. This potency corresponds to the cumulative yield of the affected cell population over the entire effective dose range of the chemical treatment. A weak mutagenic activity was detected only for mitomycin C and thiotepa. Except for bis(2-chloroethyl)ether, all agents were recombinagenic and SOS inducing. When the 3 genotoxic potencies (M, R and I) of these bifunctional alkylating agents were correlated, separately or in combination, with the respective carcinogenic potencies in rodents, a highly significant correlation was obtained with both the recombinagenic and SOS inducing potencies.

The E. coli multitest: a set of strains to characterize diverse genotoxic effects.

A set of E. coli strains was developed by Toman et al. (1985) to study the effects of chemical and physical agents on forward mutation, homologous recombination and induction of the SOS system. New tester strains have been constructed to improve this test system in order to explore quantitative genotoxicity spectra. Through the use of these strains: (i) SOS induction can be specifically detected without interference from mutagenesis; (ii) SOS-dependent and SOS-independent mutational events can be distinguished; (iii) the sensitivity of the recombination system has been considerably increased.

Evaluation of Propineb, a dithiocarbamate pesticide, in the mouse-sperm morphology assay.

Propineb, a dithiocarbamate fungicide, was studied by using the sperm morphology assay in (C57BL6 male X C3H female) F1 mice. At all dose levels, no statistically significant increase in the percentage of sperm abnormalities was observed. Methyl methanesulfonate (MMS) and 2-acetylaminofluorene (2-AAF), which were tested as positive controls, induced a dose-effect-related increase in teratospermia.

Genotoxic potency of monofunctional alkylating agents in E. coli: comparison with carcinogenic potency in rodents.

A quantitative correlation between carcinogenicity and genotoxicity was investigated by a comparison between the carcinogenic potency in rodents and the mutagenic (M), recombinogenic (R) and SOS-inducing (I) potencies in a bacterial test (E. coli multitest) for 9 monofunctional alkylating agents: N-nitroso-N-methylurethane, N-nitroso-N-ethylurea, epichlorohydrin, N-nitroso-N-methylurea, N-nitroso-N-methyl-N'-nitroguanidine, methyl methanesulfonate, diethylsulfate, dimethylsulfate, ethyl methanesulfonate. A significant positive correlation between the carcinogenic potency and the product of the mutagenic and recombinogenic potencies was found for all tested compounds. Thus, the E. coli multitest may be used as a simple test to search for correlations between carcinogenicity and genotoxicity of DNA-damaging agents.

Frameshift mutagenicity in Salmonella typhimurium of furocoumarins in the dark.

The dark mutagenicity of 4,5',8-trimethylpsoralen (4,5',8-TMP), 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), 3-carbethoxypsoralen (3-CPs) and two new pyridopsoralens (PyPs and MePyPs) was tested using the Ames Salmonella plating assay in the absence of metabolic activation. 4,5',8-TMP, 8-MOP and the two pyridopsoralens were found to be weak frameshift mutagens in strain TA1537 whereas 5-MOP and 3-CPs did not demonstrate any significant mutagenic activity. These findings support the notion that the genetic risks of these psoralens in the dark may be considered to be negligible.

Effect of DNOC, Ferbam and Imidan exposure on mouse sperm morphology.

DNOC, Ferbam and Imidan were tested in (C3H X C57BL/6) F1 mice to assess their potential testicular toxicity. Chemicals were administered i.p. and per os at different doses for 5 consecutive days. After 35 days the testicular was toxicity was evaluated by measuring the testicular weights, the sperm counts and the percentage of abnormal sperm. DNOC and Imidan failed to induce teratospermia in mice treated by both routes of administration. Conversely Ferbam induced a statistically significant increase in teratospermia only following per os administration to mice at a dose of 1000 mg/kg b.w./day. These data indicate that per os administration of Ferbam succeeded in producing active metabolites able to interfere with the differentiation process of spermatogenic cells.

Induction of sperm abnormalities in mice by ifosfamide and trofosfamide.

The antitumor drugs ifosfamide (IF) and trofosfamide (TF) were evaluated for their capability to induce sperm abnormalities in (C3H X C57BL/6)F1 mice. A statistically significant increase in teratospermia was observed at the 35th day after 5 daily consecutive intraperitoneal injections of the drugs at doses of 25, 50, 100 mg/kg b.w. of TF and 100 mg/kg b.w. of IF. Thus, IF and TF are able to interfere with the differentiation process of spermatogenic cells.

Evolutionary screening and adsorption behavior of engineered M13 bacteriophage and derived dodecapeptide for selective decoration of gold interfaces.

There is a growing interest in identifying biomacromolecules such as proteins and peptides to functionalize metallic surfaces through noncovalent binding. One method for functionalizing materials without fundamentally changing their inherent structure is using biorecognition moieties. Here, we proved a general route to select a biomolecule adhesive motif for surface functionalization by comprehensively screening phage displayed peptides. In particular, we selected a genetically engineered M13 bacteriophage and a linear dodecapeptide derived from its pIII domain for recognizing gold surfaces in a specific and selective manner. In the phage context, we demonstrated the adhesive motif was capable to adsorb on gold in a preferential way with a morphological and viscoelastic signature of the adsorbed layer as evidenced by QCM-D and AFM investigations. Out of the phage context, the linear dodecapeptide is reproducibly found to adhere to the gold surface, and by quantitative SPR measurements, high affinity constants (K(eq)~10(6)M(-1), binding energy ~-8 kcal/mol) were determined. We proved that the interactions occurring at gold interface were mainly hydrophobic as a consequence of high frequency of hydrophobic residues in the peptide sequence. Moreover, by CD, molecular dynamics and steered molecular dynamics, we demonstrated that the molecular flexibility only played a minor role in the peptide adsorption. Such noncovalent but specific modification of inorganic surfaces through high affinity biomolecule adsorption represents a general strategy to modulate the functionality of multipurpose metallic surfaces.

[Mutagenicity of alkylnitrites in the Salmonella test]. / Mutagenicita' dei nitriti alchilici nel test della Salmonella.

It has been shown that five alkylnitrites are mutagens by the Salmonella/microsome assay. n-Propyl-, n-butyl, iso-butyl and amyl-nitrite are direct mutagens on TA 1535; sec-butyl-nitrite is mutagen on TA 1535 only following metabolic activation by Aroclor-induced rat liver homogenate. Because of the known correlation between mutagenicity and carcinogenicity, we believe that amyl-nitrite and iso-butyl-nitrite, which are used as human drugs, should be tested for carcinogenicity in animals; in the meanwhile, their use should be allowed only in emergencies.

Mutagenic effects of psoralens in yeast and V79 Chinese hamster cells.

The mutagenic effects of monofunctional and bifunctional furocoumarins (psoralens) plus 365 nm radiation were analyzed in the yeast Saccharomyces cerevisiae. Per unit dose of 365-nm radiation, bifunctional compounds were more effective than were the monofunctional for the induction of reverse and forward mutations. The same was observed for the induction of 6-thioguanine-resistant mutants in V79 Chinese hamster cells when we compared the activity of 8-methoxypsoralen and 3-carbethoxypsoralen. An analysis of the kinetics of mutation induction indicated that DNA interstrand cross-links induced by bifunctional psoralens are more prone to error than are monoadducts induced by monofunctional psoralens. In yeast, 8-methoxypsoralen and 4,5'-dimethylangelicin were shown to photoinduce mitotic and nondisjunction. The implications of these findings for the use of psoralens in photochemotherapy and cosmetics are discussed.

High efficiency of site-directed mutagenesis mediated by a single PCR product.

We describe a highly efficient procedure for site-specific mutagenesis of double-stranded plasmids. The method relies on a single PCR primer which incorporates both the mutations at the selection site and the desired single base substitutions at the mutant site. This primer is annealed to the denatured plasmid and directs the synthesis of the mutant strand. After digestion with selection enzyme, the plasmid DNA is amplified into Escherichia coli strain BMH71-18 and subjected to a second digestion and amplification into the bacterial strain DH5alpha. A mutagenesis efficiency >80% was consistently achieved in the case of two unrelated plasmids.

Intercellular adhesion molecule-1 mediated interactions and leucocyte infiltration in IgA nephropathy.

BACKGROUND: Mononuclear leucocytes have a role in IgA nephropathy (IgAN). Renal leucocyte recruitment is mediated by adhesive interactions between leucocytes and their ligands on renal cells. METHODS: We have assessed interstitial and glomerular leucocytes by avidin-biotin-peroxidase with monoclonal antibodies (MA) against leucocytes (CD45), beta 2-integrin (CD18), monocyte-macrophages (CD14), T (CD3) and T-cell subsets (CD4, CD8), and intercellular adhesion molecule-1 (ICAM-1) (CD54), and analysed their relation with the abnormal expression of ICAM-1 on proximal tubule epithelium in sequential renal sections from 48 patients with IgAN stratified according to the severity of the interstitial cellular infiltration observed by light microscopy. RESULTS: In IgAN without (n = 15) and with (n = 7) interstitial cellular infiltration of 1+, ICAM-1 expression on vascular endothelium was unchanged with respect to that observed in the normal kidney; the proximal tubule epithelium was negative for this stain. In IgAN with interstitial cellular infiltration of 2+ (n = 10), 3+ (n = 11), and 4+ (n = 5), ICAM-1+ stain was observed on the proximal tubule epithelium, the median value of its quantitative expression being 0.3, 0.1, and 0.2 (P = 0.0008), respectively. The tubular ICAM-1 + stain was significantly associated with the interstitial leucocytes identified by MA, and correlated with CD45+ (r = 0.59, P = 0.02), CD14+ (r = 0.54, P < 0.02), and CD3+ (r = 0.51, P = 0.02) interstitial leucocytes in IgAN with interstitial cellular infiltration. Interstitial ICAM-1+ and CD18+ leucocytes were correlated (r = 0.56, P < 0.001). Correlation was found between the quantitative tubular expression of ICAM-1+ and the number of CD45+ (r = 0.98, P < 0.0001), CD3+ (r = 0.48, P = 0.02), and CD8+ (r = 0.76, P < 0.02) glomerular leucocytes. CONCLUSIONS: Our results suggest that tubular and interstitial ICAM-1+ cells may participate in adhesive interactions with interstitial leucocytes. Interstitial T-cells and macrophages as well as glomerular T-cells bearing predominantly CD8+ phenotype could play a role in the induction of the tubular expression of ICAM-1 in IgAN.

[Mutagenicity of twelve imidazole derivatives with trichomonacide activity]. / Mutagenicità di dodici composti imidazolici ad attività tricomonicida.

The mutagenic action of twelve imidazole derivatives was investigated by Salmonella/microsome assay. All of them were mutagenic on TA 100 without metabolic activation. No direct relationship between antitrichomonad action in vitro and mutagenicity was established. As the compound XII showed much less mutagenic activity compared to the other imidazoles, we propose to perform more mutagenic studies on the chemical analogues of this drug.