Search

We have examined the mechanisms by which interferon (IFN)-gamma and IFN-alpha regulate the expression of 2'-5'-oligoadenylate synthetase (2-5A synthetase) and class I major histocompatibility complex antigens in murine T cells and in cell types of other histological origin. When treated with IFN-alpha both fibroblasts and T cell lines displayed a marked increase of the 2-5A synthetase activity and of the corresponding mRNA. The augmentation of the enzyme activity in T cells was induced by IFN-alpha at the transcriptional level, as determined by nuclear run-on analysis. In contrast IFN-gamma was capable of increasing 2-5A synthetase activity only in fibroblasts, but not in T cells. Nuclear run-on assays revealed that the 2-5A synthetase gene in T cells is not transcriptionally activated by IFN-gamma. After IFN-alpha and -gamma treatment we also observed a significant increase in class I gene expression in fibroblasts and T cell lines as measured both on the cell surface and by cytoplasmic RNA accumulation. In the case of the T cell line, DO1110, the observed increase in the steady-state levels of class I transcripts was a consequence of a high rate of H-2 gene transcription as demonstrated by run-on analysis. However, the molecular mechanisms involved in this IFN-dependent H-2 gene transcriptional activation are different between IFN-alpha and IFN-gamma. When the T cell lines DO1110, L12-R4 and EL4 were transfected with a plasmid containing a reporter gene (chloramphenicol acetyltransferase) under the control of a regulatory IFN-responsive DNA element of 237 bp or 1.4 kb, IFN-alpha was able to activate the transcription of these constructs. In contrast, IFN-gamma did not recognize the IFN-responsive element which, by itself, activated transcription of the reporter gene in response to IFN-gamma in other cellular types of non-T cell origin. Therefore, in the T cell lines examined, IFN-gamma increases the H-2 gene expression by acting on DNA elements located upstream of the regulatory segment used in this study or downstream of the cap site. This suggests a possible cell specificity in the activation of an IFN-responsive element, that in turn may regulate the IFN-inducible gene expression in a cell-specific fashion. Thus, the differential biological activities of IFN-gamma on T cells could be generated by a differential gene activation at the transcriptional level.

A 20-amino acid peptide corresponding to a common part of the 40 and 46 kD forms of human 2'-5' oligoadenylate synthetase was coupled to keyole lymphet haemocyanin (KLH) and used as immunogen in sheep. After a cycle of four immunizations, immunoglobulins able to recognize the 20-amino acid peptide as evaluated in ELISA assays were purified by an immunoadsorbent with the peptide immobilized on Sepharose CL-4B and used in Western blot employing a secondary anti-sheep antibodies and iodinated protein A as indicator system. Results obtained using extracts from human and mouse cells treated for 15 hr with IFN-alpha as antigen demonstrated that the anti-peptide antibodies recognize several forms of the 2'-5' oligoadenylate synthetase enzyme complex. These antibodies therefore represent a useful tool for monitoring the induction of the above enzymes.

IFNs are a family of proteins produced by various cells following stimulation by biological or synthetic inducers. The interaction with the membrane receptor is followed by the activation of the expression of particular genes that are responsible for their antiviral, antiproliferative and immunomodulatory properties. Initiation of transcription is an early and critical event in the control of eukaryotic gene expression. In this review we will briefly discuss the mechanisms exploited by IFNs to control at transcriptional level the expression of inducible genes. In particular we will focus on some characteristics if cis-acting DNA elements that are located upstream from the initiation site for RNA transcription and of nuclear trans-acting factors that are required for modulation of gene expression by IFNs.

The central role of Streptococcus mutans in the pathogenesis of caries is universally recognized. In this article we discuss bacterial metabolism relevant to plaque formation and enamel destruction, and review current methods and new prospects in caries prophylaxis: the use of fluorine, possible substitutes for sucrose, active and passive immunoprophylaxis, teeth colonization with non-cariogenic strains of S. mutans. No one of these methods can be considered a definitive solution for every caries problem, but short term major achievements can be reasonably attained by ongoing field research.

Laser irradiation of tissues treated in vivo with haematoporphyrin (Hpr) is known to result in a cytocidal effect, reportedly more pronounced in tumour tissues. To ascertain whether this cytocidal phenomenon can occur not only in eukaryotic but also in prokaryotic cells, the authors devised a model system in vitro consisting of bacterial cultures in liquid and in solid media. Bacteria photosensitized with Hpr were subsequently exposed to laser beams and to daylight; then normal microbiological techniques were used to determine whether any bactericidal effect occurred. Satisfactory results were achieved, particularly against Gram-positive microorganisms; a partial inhibition of Gram-negative microorganisms was also observed.

The incidence of methicillin-resistant Strains is increasing in these last years. This increase is particularly evident in Strains isolated from hospital environments. In this work we evaluated the methicillin-resistance of 58 Staphylococcus Strains, isolated from a surgical intensive care ward, in various experimental conditions, which changing in: culture medium, inoculum, incubation period and antibiotic concentration. Besides this we took in consideration various factors which can influence the frequency of methicillin-resistance and can also lead to errors in the evaluation itself. From our study we can conclude that the factors are: survey "in vitro" of the resistance or of the sensitivity, exclusion or inclusion of staphylococci different from Staphylococcus aureus, double count of the same Strains isolated in different times.

The aim of this study was to determine the role of mezlocillin, a new acylureidopenicillin, in otorhinolaryngology. One hundred and forty-four bacterial strains were isolated from 100 patients suffering from infections of the oropharyngeal tract. Among the isolates only 10 (6.9%) resulted in being mezlocillin-resistant by the agar diffusion method. Minimum inhibitory and minimum bactericidal concentrations were also determined. Pharmacokinetic parameters e.g., diffusion in tonsillar tissue and serum concentrations, were studied. Mean mezlocillin tonsillar levels of 3.5 micrograms/g and 1.8 micrograms/g and serum concentrations of 60.2 micrograms/ml and 10.6 micrograms/ml, 1 h and 4 h respectively after administration, were found.

The degree of toxicity of the antibiotic Cefonicid on the cellular reactivity of the immune system was evaluated. The effects on some lymphokine (IL-2 and IFN-gamma) production and the degree of proliferation of splenic lymphocytes following mitogen stimulation have been considered. Our results show that Cefonicid does not impair the immune response, except at very high doses (500 micrograms/ml).

These studies were designed to investigate the characteristics of the intracellular messengers induced by interferons (IFN-alpha/beta and IFN-gamma) after receptor binding. Pretreatment of target cells with V. cholerae toxin, which is known to activate a membrane GTP-binding stimulatory protein (Gs), potentiated the action of IFN-gamma, but not of IFN-alpha/beta. By contrast, B. pertussis toxin, which is known to activate the GTP-binding inhibitory protein (Gi), had no effects on the action of both IFN-alpha/beta and IFN-gamma. Further support to the involvement of G proteins in IFN-gamma transduction signal came from the finding that a non-hydrolizable GTP analog, GTP-gamma-S, enhanced in the presence of phorbol esters (PMA) the antiviral and antiproliferative activity of IFN-gamma, but not of IFN-alpha/beta. On the other hand, forskolin or PGE1, known to increase the intracellular cAMP levels by different metabolic pathways, when added together with IFN-gamma, significantly potentiated its antiviral and antiproliferative activity. Pretreatment of the cultures with the above drugs completely prevented IFN-gamma activity. No effects were observed when forskolin or PGE1 were used with IFN-alpha/beta. Finally, the modulation of IFN-gamma activity by the above drugs was not a consequence of changes in the expression of the specific surface receptors, since [125I]IFN-gamma binding by pretreated target cells was comparable to that of untreated cultures. Altogether these results demonstrate that the IFN-gamma, but not the IFN-alpha/beta, transduction signal is mediated after receptor binding by a G protein with functional characteristics similar to those of the known Gs proteins. Activation of the adenylate cyclase system could be one of the subsequent steps involved in IFN-gamma action.

Using radioiodinated murine interferon-gamma (IFN-gamma), various cell lines were analyzed for their capacity to bind IFN-gamma and to respond to its biological activity as measured by activation of the 2',5'-oligoadenylate (2-5A) synthetase, induction of the antiviral activity, and inhibition of [3H]thymidine uptake. A T-cell lymphoma, EL-4, was found to express relatively high number of specific membrane receptors for IFN-gamma (60,000/cell) with a dissociation constant of around 9 X 10(-10) M. By contrast, a fibroblast cell line, K-BALB, transformed by the Kirstein murine sarcoma virus, was found negative for binding of radioiodinated IFN-gamma, even in the presence of high ligand concentrations. Other cell lines displayed numbers of receptors varying between 650 to 20,000 binding sites/cell with KD ranging between 5 X 10(-9) to 9 X 10(-10) M. When incubated with EL-4 expressing high levels of specific receptors, IFN-gamma caused stimulation of cell growth, but not resistance to viral (vesicular stomatitis virus) infection and expression of 2-5A synthetase. By contrast, when L1210 and TS/A cells, expressing intermediate levels of specific receptors, were incubated with IFN-gamma, inhibition of cell growth, induction of antiviral resistance and activation of 2-5A synthetase was observed. Finally, K-BALB cells, lacking specific receptors for IFN-gamma, were completely unsensitive to its biological activity. The present system could represent a useful model for the characterization of the interaction of IFN-gamma with target cells at molecular levels.

Teicoplanin is a new antibiotic isolated from the fermentation broth of Actinoplanes teichomyceticus nov. sp. It's a glycopeptide antibiotic belonging to the same family as vancomycin, active against Gram-positive bacteria. The antimicrobial activity of this drug was evaluated in 32 patients suffering from different infections due to Gram-positive bacteria. Among the 36 Gram-positive microorganisms isolated, 15 staphylococci were methicillin resistant, 10 were sensitive, 1 was intermediate and for another one the methicillin susceptibility was not evaluated. After the treatment with teicoplanin, cure and improvement occurred in 26 cases (81%), in 2 cases the therapy failed (6%), in 2 cases there was a relapse and 2 cases were not evaluable. From this clinical study Teicoplanin results safe and effective in the therapy of Gram-positive infections.

The effects of interferon (IFN)-gamma or IFN-alpha/beta on virus yield, (2'-5')oligo(A) synthetase activation, H-2 antigen expression and proliferation of T lymphocytes have been investigated. Under the culture conditions used, vesicular stomatitis virus or Semliki Forest virus replication in T cells was not impaired by the addition of IFN-gamma, whereas it was completely inhibited by the addition of IFN-alpha/beta. In contrast, B cell lines, macrophage-transformed cell lines and fibroblasts were fully protected by both IFN-gamma as well as IFN-alpha/beta following virus infection. The lack of sensitivity of T lymphocytes to the antiviral effects of IFN-gamma was not due to absence of specific membrane receptors, since in saturation binding experiments with 125I-labeled murine IFN-gamma most T cell lines displayed a number of binding sites and a degree of affinity comparable to those found on B cells, which are fully sensitive to IFN-gamma antiviral activity. Analysis of IFN-induced dsRNA-dependent (2'-5')oligo(A) synthetase activity, one of the biochemical markers for cellular responses to IFN, showed that it was not induced in T lymphocytes after IFN-gamma treatment, whereas IFN-alpha/beta induced high levels. Both IFN-gamma and IFN-alpha/beta enhanced H-2 antigen expression on T cells as well as on cells of different histological type. Moreover, when IFN-gamma was tested for its antiproliferative activity on T cells, it was found to consistently potentiate the response of these cells to mitogens or growth factors, rather than inhibit their proliferation. Taken as a whole these results suggest that on T lymphocytes IFN-gamma should not be regarded as an antiviral agent, but rather as a modulator of T cell growth and functional differentiation, transducing intracellular signals dissimilar to those observed with target cells of different origin.

The effects of interferon-gamma (IFN-gamma) on protein synthesis of a pre-B cell leukemia, L1210, have been studied by two-dimensional gel electrophoresis. In total cell extracts, at least ten proteins were induced de novo, or increased in their expression, after an 18-h IFN-gamma treatment, whereas in the nuclear extracts eight proteins were specifically induced. Of these, increased synthesis of a 40-kDa/pI 5.9 cytoplasmic protein was the most prominent and reproducible. Most of these proteins appear to be specific for a defined step of differentiation, since they are not found in other B cell leukemias upon IFN-gamma treatment. Others appear to be tissue specific, since they are not induced in fibroblasts nor in T cells. In addition, synthesis of some of the induced proteins appeared to require rapid transcription of new mRNA, because actinomycin D markedly inhibited their formation when added immediately before IFN-gamma. In keeping with this finding, in vitro translation of mRNA from IFN-gamma-treated L1210 cells into a rabbit reticulocyte lysate system, followed by analysis of the labeled proteins by two-dimensional gel electrophoresis, revealed the appearance of at least seven proteins. Taken as a whole, these results demonstrate that in leukemic pre-B cells IFN-gamma induces the transcriptional activation of genes coding for cytoplasmic and nuclear proteins, some of which could be employed as specific cell activation markers.

In this study, we evaluated the effect of ras oncogene activation on cell response to interferons (IFNs). For this purpose, we treated NIH 3T3 murine fibroblasts transformed by transfection with K-, Ha-, or N-ras oncogenes, either mutated or amplified, for 24 hours with IFN-gamma or IFN-alpha. We evaluated cell response by measuring virus replication, [3H]thymidine incorporation, 2',5'-oligoadenylate synthetase activation, and class I antigen induction. Transformed cells were much less responsive to IFN-gamma antiviral and antiproliferative activities than normal NIH 3T3 cells. Similarly, the induction of 2',5'-oligoadenylate synthetase following IFN-gamma treatment was completely depressed in transformed cells. Only class I antigens, measured at the cell surface and mRNA levels, appeared partially inducible by IFN-gamma in ras-transformed cells. When the same cell lines were treated with IFN-alpha, we observed full response. Because both normal and ras-transformed NIH 3T3 cells were able to bind [125I]IFN-gamma with comparable Kd values (8.3 X 10(-11) M vs. 3 X 10(-11) M, respectively), these findings suggest that ras oncogenes may differentially impair IFN-gamma activities by affecting activation of IFN-inducible genes downstream from the receptor binding event.

These studies were designed to investigate the characteristics of the intracellular second messengers induced by interferons (IFN-alpha/beta and IFN-gamma) after receptor binding. Pretreatment of target cells with V. cholerae toxin, which is Known to activate the GTP-binding stimulatory protein (Gs), potentiated the action of IFN-gamma, but not of IFN-alpha/beta. By contrast, B. pertussis toxin, which is known to act on the GTP-binding inhibitory protein (Gi doesn't affect the action of both IFN) (Gi). Besides this forskolin and PGE1, known to increase intracellular cAMP levels, completely prevented antiviral state induction by IFN-gamma, but had no effects on IFN-alpha/beta. Altogether these results demonstrate that IFN-gamma transduction signal is mediated by a G protein with functional characteristics similar to those of the known Gs protein.