Incorporation of a calixarene-based glucose functionalised bolaamphiphile into lipid bilayers for multivalent lectin recognition.

The synthesis, characterization and inclusion in liposomes of a glucosylated bolaamphiphile built on a calix[4]arene scaffold are described. The new glucocalixarene bolaamphiphile destabilizes bilayers of saturated lipids whereas it rigidifies those of unsaturated lipids, thus reducing leakage of calcein from the liposome internal aqueous compartment. Moreover, from fluorescence and turbidimetry experiments it was found that the glucose units of bolaamphiphile 1 functionalised liposomes allow a specific multivalent interaction with the tetrameric glucose binding protein Concanavalin A. These results therefore represent a novel strategy to functionalise liposomes with saccharides, exploiting multivalent glycosylated ligands to be used in the preparation of drug delivery systems potentially able to target specific lectins.

Cell-dependent efficiency of reiterated nuclear signals in a mutant simian virus 40 oncoprotein targeted to the nucleus.

We investigated the requisites for, and functional consequences of, the relocation to the nucleus of a transforming nonkaryophilic mutant of the simian virus 40 large T antigen (a natural deletion mutant lacking an internal large-T-antigen domain that includes the signal for nuclear transport). Synthetic oligonucleotides were used to obtain gene variants with one or more copies of the signal-specifying sequence inserted near the gene 3' end, in a region dispensable for the main large-T-antigen functions. The analysis of stable transfectant populations showed that mouse NIH 3T3 cells, rat embryo fibroblasts, and simian CS cells (a subclone of CV1 cells) differed considerably in their ability to localize some variant molecules into the nucleus. CS cells were always the most efficient, and NIH 3T3 cells were the least efficient. The nuclear localization improved either with reiteration of the signal or with a left-flank modification of the signal amino acid context. Three signals appeared to be necessary and sufficient, even in NIH 3T3 cells, to obtain a nuclear accumulation comparable to that of wild-type simian virus 40 large T antigen; other signal-cell combinations caused a large variability in subcellular localization among cells of the same population, as if the nuclear uptake of some molecules depended on individual cell states. The effect of the modified location on the competence of the protein to alter cell growth was examined by comparing the activity of variants containing either the normal signal or a signal with a mutation (corresponding to large-T-antigen amino acid 128) that prevented nuclear transport. It was found that the nuclear variant was slightly more active than the cytoplasmic variants in rat embryo fibroblasts and NIH 3T3 cells and was notably less active in CS cells.

Murine cell complementation of a cold-sensitive defect for simian virus 40 replication in simian cells.

Mouse 3T3 fibroblasts were found to complement, in simian cell variants semipermissive to simian virus 40, a cold-sensitive defect of an early function, but not a nonconditional defect of viral uncoating. The variant simian cells could rescue simian virus 40 from 3T3 transformants, and this capacity was not temperature dependent.

Limits of transforming competence of SV40 nuclear and cytoplasmic large T mutants with altered Rb binding sequences.

Multiple amino acid substitutions were introduced into the SV40 large T region that harbors the retinoblastoma protein (Rb) binding site and the nuclear transport signal, changing either one or both of these determinants. Mutant activities were examined in a set of assays allowing different levels of transforming potential to be distinguished; phenotypic changes in established and pre-crisis rat embryo fibroblasts (REFs) were detected under isogenic cell conditions, and comparisons made with other established rodent cells. The limit of the transforming ability of mutants with important substitutions in the Rb binding site fell between two transformation levels of the same established rat cells. Such cells could be induced to form dense foci but not agar colonies (their parental pre-crises REFs, as expected, were untransformed either way). Nonetheless, agar colony induction was possible in other cell lines, such as mouse NIH3T3 and (for one of the mutants) rat F2408. All these mutants efficiently immortalized pre-crisis REFs. The transforming ability of cytoplasmic mutants appeared to depend on the integrity of the Rb-binding sequence to approximately the same extent as that of the wild-type large T, although evidence of in vivo Rb-cytoplasmic large T complexes was not found. The presence or absence of small t was critical when the transforming task of mutants was near the limit of their abilities.

Induction of cytokines by HIV-1 and its gp120 protein in human peripheral blood monocyte/macrophages and modulation of cytokine response during differentiation.

We previously reported that in vitro culture of human peripheral blood monocytes resulted in a time-dependent differentiation into macrophages and in an enhanced capacity for producing certain cytokines [i.e., tumor necrosis factor alpha, interleukin-6 (IL-6), and interferon-beta (IFN-beta)] in response to bacterial lipopolysaccharide (LPS). HIV-1 infection or gp120 treatment of monocyte/macrophages resulted in the induction of low levels of IFN-beta, which were very effective in restricting viral replication in 7-day cultured macrophages but not in freshly isolated cells. This enhanced response of macrophages was due to a higher sensitivity of these cells to the antiviral effect of IFN-beta. Consistent with this finding, 7-day cultured macrophages exhibited higher levels of type I IFN receptors than 1-day cultured monocytes. Treatment of monocyte/macrophages with gp120 also caused a marked increase in IL-10 secretion, regardless of the differentiation state. No IL-12 secretion was detected in monocyte/macrophage cultures treated with gp120 alone. However, consistent IL-12 secretion was found in 7-day cultured macrophages primed with IFN-beta and subsequently stimulated with gp120. Macrophages responded more efficiently than monocytes to the priming effect of IFN-beta for IL-12 production. This was consistent with a stronger antiviral response against vesicular stomatitis virus by these cells as well as with a higher expression of IFN-beta receptors. The finding that the acquisition of the macrophage phenotype is associated with an increased capacity to respond to environmental signals (such as type I and type II IFNs) underlines the importance of the differentiation process for the selection of a certain repertoire of responses that may allow these cells to have important functions in vivo.

Role of endogenous interferon-beta in the restriction of HIV replication in human monocyte/macrophages.

In vitro culture of human monocytes results in a time-dependent differentiation into macrophages. Monocyte/macrophages were infected with HIV-1Ba-L at different times after isolation and subsequent culture. When 7-day macrophages were infected in the presence of antibodies to interferon-beta (IFN-beta), a significant increase in HIV-1 p24 release was observed. This effect was not detected in 1-day monocytes. Treatment of 7-day cultured macrophages with HIV-1 rgp120 resulted in resistance to vesicular stomatitis virus infection. This rgp120-induced antiviral state was neutralized in the presence of antibodies to IFN-beta. The overall results indicate that the infection of monocyte/macrophages with HIV-1 results in the induction of IFN-beta, which, in turn, inhibits HIV-1 expression in macrophages. The finding that HIV-1 itself (possibly through its gp120) can induce a potent antiviral factor (IFN-beta) in macrophages underlines the complex physiological function of these cells in maintaining normal homeostasis in vivo in response to virus infection.

IFN-alpha and IL-18 exert opposite regulatory effects on the IL-12 receptor expression and IL-12-induced IFN-gamma production in mouse macrophages: novel pathways in the regulation of the inflammatory response of macrophages.

We characterized the IL-12 response of mouse macrophages in terms of modulation of IFN-gamma production by cytokines (IFN-alpha and IL-18) and regulation of IL-12 receptor expression. Beta1 and beta2 IL-12R chain mRNA expression increased with time in culture in the absence of exogenous stimulation, with concomitant acquisition of responsiveness to IL-12 for IFN-gamma production. Expression of the IL-12R beta1 chain mRNA was increased further following IL-12 treatment as a consequence of IFN-gamma expression. IL-12 response was regulated differentially by IFN-alpha and IL-18. Neutralization of endogenous type I IFN increased IFN-gamma secretion, whereas exogenous IFN-alpha reduced it. In contrast, IL-18 enhanced IFN-gamma mRNA accumulation and IFN-gamma secretion in IL-12-stimulated, but not -untreated, cultures. The opposite effects exerted by IFN-alpha and IL-18 mirror their mutual capacity of regulating-in a negative or positive manner, respectively-the expression of the IL-12R beta1 chain. We suggest that differential regulation of IL-12 response by IFN-alpha and IL-18 can represent previously unrecognized regulatory mechanisms for maintaining suitable levels of differentiation/activation in macrophages.

Regulation of chemokine/cytokine network during in vitro differentiation and HIV-1 infection of human monocytes: possible importance in the pathogenesis of AIDS.

The monocyte/macrophage lineage represents heterogeneous cell populations characterized by major differences in the phenotype and functional activities. These cells are a major source of soluble factors, such as cytokines and chemokines, which can both affect HIV replication and AIDS pathogenesis. Although monocytes/macrophages are unanimously considered important targets of HIV-1 infection, the HIV-induced alterations in their physiological functions at different stages of differentiation are still matter of debate. In this article, we review our data on the regulation of chemokine/cytokine network with regard to macrophage differentiation and HIV-1 infection, in comparison with studies from other groups. The ensemble of the results emphasizes that: 1) macrophages markedly differ with respect to monocytes for a variety of responses potentially important in the pathogenesis of HIV infection; and 2) the experimental conditions can influence the HIVmonocyte/macrophage interactions, reflecting the possible in vivo existence of a spectrum of responses among macrophage populations.

Molecular cloning and restriction mapping of a simian virus 40 deletion mutant derived from simian transformants expressing a non-karyophilic T antigen.

A replication-defective Simian virus 40 genome, with a deletion of about 120 nucleotides in the region encoding the N-terminal fourth of the large T antigen, has been isolated from the DNA of Simian cells transformed by SV40. Both the original transformants, and the murine transformants obtained by transfection with this cloned mutant DNA, produced a large T antigen displaying in immunofluorescence an exclusively cytoplasmic localization. The protein apparent molecular mass (83 kDa) was about 6% smaller than that of normal karyophilic large T. Restriction analysis showed that the deletion eliminated two close HinfI sites, at nucleotides 4459 and 4376 (map unit 0.50).

IFN-beta stimulates the production of beta-chemokines in human peripheral blood monocytes. Importance of macrophage differentiation.

We investigated the effect of IFN-beta on beta-chemokine expression in differentiating human peripheral blood monocytes. MCP-1, MIP-1alpha and MIP-1beta were constitutively expressed in 1 day-cultured monocytes, and their secretion increased with time in culture despite any change in mRNA accumulation. IFN-beta treatment of differentiating monocytes resulted in a marked and dose-dependent increase of beta-chemokine secretion, which was regulated differently with respect to the differentiation stage. In particular, IFN-beta upregulated MCP-1 secretion in monocytes at all stages of differentiation although its effect was significantly higher in 1-day cultured monocytes as compared to monocyte-derived macrophages (MDM). In contrast, MIP-1alpha and MIP-1beta secretion was up-regulated by IFN-beta only in MDM. Although MCP-1, MIP-1alpha and MIP-1beta mRNA expression was up-regulated by IFN-beta in both 1 day-cultured monocytes and MDM, no correlation was found between mRNA level and protein secretion. These results suggest that the regulation of beta-chemokine secretion in monocytes/macrophages by IFN-beta occurred through different mechanisms, involving both a direct effect of this cytokine on chemokine gene expression and translational/post-translational steps of regulation more likely linked to the differentiation process. This finding reveals a novel role for this cytokine in the recruitment of specific cell types during the immune response, which may be relevant in the control of viral infections in vivo.

[Modification of the Pack method for the microdetermination of diacetyl in mixed cultures]. / Modificación del método de Pack para la microdeterminación de diacetilo en cultivos mixtos.

Prill and Hammer's method (4) for microdetermination of diacetyl was modified by several authors (1-3, 7), but retaining the same principle: diacetyl is converted into dimethylglyoxime by reaction with hydroxylamine; the oxime is subsequently converted into a pink ammonoferrous glyoximate and its colour is measured by absorbance at 530 nm. In the present communication a procedure based on the method of Pack et al. (3) is proposed with the following modifications: 1. Omission of carboy and suction flask; 2. Elongation of the connecting tubing between sample and trap tubes and relocation of the trap tubes to a higher level. 3. Replacement of rubber tubing by pvc connection and of rubber stoppers by neoprene ones. 4. A more accurate regulation of the nitrogen flow. 5. Protection of the Fe SO4 against oxidation. 6. Omission of the rinse of the trap tips with K2 HPO4 solution. 7. Use of diacetyl as a standard instead of dimethylglyoxime.

[Leuconostocs in raw milk: isolation and identification of species rapidly utilizing lactose]. / Leuconostocs en leche cruda: aislamiento e identificación de especies de utilización rápida de lactosa.

Fast lactose fermenting Leuconostoc species and subspecies were isolated from raw milk. Samples were obtained from dairy farms of the surroundings of Buenos Aires city. A lactose, non selective, isolation medium was employed (YCL). Differentiation of leuconostocs from Lactobacillus viridescens and L. confusus was avoided on account of the use of this medium. 801 typical colonies of lactic acid bacteria were selected from YCL agar; 710 of them were identified as lactic acid bacteria from which 114 strains belonged to the genus Leuconostoc. These last strains were then tested for species and subspecies differentiation by dextran production and sugar fermentation. Leuconostoc mesenteroides subsp. dextranicum and L. lactis were identified. Four strains identified as Leuconostoc spp do not belong to any known species.

HIV-1 gp120 stimulates the production of beta-chemokines in human peripheral blood monocytes through a CD4-independent mechanism.

The present study was designed to evaluate the effect of the HIV-1 envelope glycoprotein gp120 on the expression of beta-chemokines in cultured monocytes/macrophages. Treatment of either freshly isolated 1-day-cultured monocytes or 7-day-cultured monocyte-derived macrophages (MDM) with recombinant gp120-IIIB resulted in a specific and dose-dependent enhancement of secretion of monocyte chemoattractant protein-1, macrophage inflammatory protein-1beta, and RANTES as well as a clear-cut increase in transcript accumulation. The expression of these mRNA was increased, but not superinduced, in the presence of cycloheximide. beta-Chemokine secretion was also induced after exposure of monocyte cultures to gp120-JRFL and aldrithiol-2-inactivated R5 and X4 HIV-1 strains, retaining conformational and functional integrity of envelope proteins. In contrast, no beta-chemokine secretion was triggered by X4 and R5 gp120 or aldrithiol-2-inactivated virus treatment of monocytoid cell lines that were fully responsive to LPS. The gp120-mediated effect was independent of its interaction with CD4, as preincubation with soluble CD4 did not abrogate beta-chemokine induction. Moreover, triggering of CD4 receptor by a specific Ab did not result in any beta-chemokine secretion. Interestingly, engagement of CCR5 and CXCR4 receptors by specific Abs as well as treatment with CCR5 and CXCR4 ligands induced beta-chemokine secretion. On the whole, these results indicate that HIV-1 stimulates monocytes/macrophages to produce beta-chemokines by a specific interaction of gp120 with HIV-1 coreceptors on the cell membrane. The expression of these related polypeptides may represent an important cellular response for regulating both the extent of viral infection and the recruitment of immune cells.

A nonkaryophilic T antigen of SV40 can either immortalize or transform rodent cells, and cooperates better with cytoplasmic than with nuclear oncoproteins.

We investigated the cell growth alterations brought about by a mutant nonkaryophilic large T antigen (NKLT) of SV40, alone and in combination with other oncogenes. NKLT by itself exhibited bivalent functional competence: it induced immortalization in early-passage rat embryo fibroblasts (REFs) and transformation in established NIH3T3 cells, although it was totally unable to transform nonestablished REFs. The absence of a normal small T reduced but did not suppress such effects. Coexpression of NKLT with the nuclear oncoprotein Polyoma large T significantly increased the efficiency of the same activities sustained by NKLT alone, but did not confer the ability to transform nonestablished REFs. Similar results were also observed in cells cotransfected with NKLT and another nuclear oncoprotein, E1A of adenovirus. In contrast, NKLT coexpression with either of the cytoplasmic oncoproteins, Polyoma middle T or activated Ha-ras, produced full transformation of early passage REFs. Thus the NKLT stimulus has the following properties: (i) it straddles the immortalizing/transforming distinction of oncogenic competence, (ii) it potentiates the effects of some nuclear oncoproteins, and (iii) it complements certain cytoplasmic oncoproteins to promote transformation of nonestablished cells.

Deletion of 43 amino acids in the NH2-terminal half of the large tumor antigen of simian virus 40 results in a non-karyophilic protein capable of transforming established cells.

We have characterized a simian virus 40 (SV40) mutant, derived from the viral DNA insertion present in simian cell transformants, which carries a deletion affecting the NH2-terminal region of the SV40 large tumor antigen. This mutant protein is 6% smaller than normal, has lost the typical nuclear localization of the SV40 large tumor antigen, and accumulates in the cytoplasm. The deletion begins at nucleotide position 4490 of the SV40 DNA and ends in-frame at nucleotide position 4362. The missing 43 amino acids begin with proline-110 and end with serine-152 of the predicted sequence; they include a cluster of basic residues, presumably important for the viral origin-DNA binding, and most of the phosphorylation sites present in the NH2-terminal half of the molecule. The protein can still be phosphorylated considerably in vivo. This mutant viral genome is replication-defective but has conserved the competence to transform established cells, such as NIH/3T3 cells. Transfection of cloned mutant DNA into such cells resulted in the production of full transformants. Full transformants were not produced in similar transfections carried out in primary rat embryo fibroblasts, although some primary transfectants expressing the non-karyophilic large tumor antigen might be considered minimally transformed.

Cold-sensitive growth of simian virus 40 in semipermissive variants of CV1 cells.

Two cell clones were isolated from the simian line CV1, permissive for simian virus 40 (SV40), by selection at low temperature with the tsA239 mutant of SV40. These clones exhibited cold-sensitive semipermissivity to both SV40 virions and SV40 DNA. On the basis of virus yields, their resistance to viral DNA was increased approximately 15 times over that of CV1 cells when the incubation temperature was lowered from 38.5 to 33.5 degrees C. A further 30- to 40-fold resistance increase was exhibited at both temperatures upon infection with SV40 virions. Partial characterization of these clones indicated that the cold sensitivity affected an early function in viral growth, between viral uncoating and the appearance of T-antigen positivity, with a burst-size decrease in all cells at the restricted temperature. This conditional defect appeared to be superimposed upon a temperature-independent uncoating defect, presumably carried in a CV1 subpopulation from which the two clones were ultimately selected.

Loss of CCR2 expression and functional response to monocyte chemotactic protein (MCP-1) during the differentiation of human monocytes: role of secreted MCP-1 in the regulation of the chemotactic response.

Human peripheral blood monocytes differentiate into macrophages when cultured in vitro for a few days. In the present study, we investigated the expression of C-C chemokine and CXCR4 receptors in monocytes at different stages of differentiation. Culturing of monocytes for 7 days resulted in a progressive decrease of the mRNA that encodes for CCR2 and CCR3, whereas the expression of mRNA for other chemokine receptors (CCR1, CCR4, CCR5, and CXCR4) was not substantially affected. The loss of CCR2 mRNA expression in 7-day-cultured macrophages was associated with a strong reduction in the receptor expression at the plasma membrane, as well as in the monocyte chemotactic protein (MCP-1) binding, as compared with freshly isolated monocytes. Furthermore, the biologic response to MCP-1, as measured by intracellular calcium ions increase and chemotactic response, was lost in 7-day-cultured macrophages. Differentiation of monocytes into macrophages also resulted in an increased secretion of MCP-1 that, at least in part, was responsible for the downmodulation of its receptor (CCR2). The loss of CCR2 expression and the parallel increase of MCP-1 secretion triggered by differentiation may represent a feedback mechanism in the regulation of the chemotactic response of monocytes/macrophages.

Induction of beta interferon by human immunodeficiency virus type 1 and its gp120 protein in human monocytes-macrophages: role of beta interferon in restriction of virus replication.

In vitro cultivated human monocytes show a time-dependent differentiation into macrophages, characterized by an increased expression of macrophage-specific antigens. Monocytes-macrophages were infected with human immunodeficiency virus type 1 strain Ba-L (HIV-1Ba-L) at different stages of differentiation. When 7-day cultured macrophages were infected in the presence of antibodies to beta interferon (IFN-beta), a significant increase in HIV-1 p24 release was detected. This effect was not observed in 1-day monocytes. This finding suggests that IFN-beta secreted by the infected macrophages inhibits p24 release. Treatment of cultured macrophages with recombinant gp120 (rgp120) protein resulted in the induction of IFN-beta mRNA and in an antiviral state to vesicular stomatitis virus. This rgp120-induced antiviral state was largely neutralized by antibodies to IFN-beta, whereas anti-IFN-alpha antibodies were ineffective. In cultured macrophages, 0.1 IU of IFN-beta per ml was sufficient to induce a marked inhibition of vesicular stomatitis virus yield, whereas this dose was ineffective in 1-day monocytes. These results indicate that (i) HIV-1 (possibly in part through its gp120 protein) induces low levels of IFN-beta in macrophages and (ii) this IFN-beta is very effective in inducing an antiviral state in differentiated macrophages.

Induction of interleukin-12 (IL-12) by recombinant glycoprotein gp120 of human immunodeficiency virus type 1 in human monocytes/macrophages: requirement of gamma interferon for IL-12 secretion.

We studied the effects of the gp120 glycoprotein of human immunodeficiency virus type 1 on the expression of interleukin-12 (IL-12) in human monocytes and in monocyte-derived macrophages. Induction of the mRNA for both the p35 and p40 subunits of IL-12 was observed in both cell types after gp120 treatment. We then evaluated cytokine secretion by using an enzyme-linked immunosorbent assay which recognizes only the IL-12 heterodimer. No IL-12 was detected in monocytes/macrophages treated with gp120 alone. A consistent IL-12 secretion was found in macrophages primed with gamma interferon (IFN-gamma) and subsequently treated with gp120. Low levels of IL-12 were occasionally observed in IFN-gamma-primed monocytes stimulated with gp120. The greater response of macrophages than of monocytes to the priming effect of IFN-gamma was consistent with the finding that IFN-gamma induced a much stronger antiviral state to vesicular stomatitis virus in macrophages than in monocytes. These data indicate that gp120 is an inducer of IL-12 expression in monocytes/macrophages and that IFN-gamma is an essential cofactor for IL-12 secretion, especially in differentiated macrophages.