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Biomedical subjects

G Cavallo

Publications and source records attributed to G Cavallo.

At least 19 recordsLinked to original sources
PubMed1992

Impaired transcription of the poly rI:rC- and interferon-activatable 202 gene in mice and cell lines from the C57BL/6 strain.

Activation of 202 and (2'-5')(A)n synthetase genes after injection of interferon (IFN)-inducing, double-stranded, poly rI:rC was compared in various mouse strains. The 202 mRNA level increased 4.5- to 10-fold in DBA/2, BALB/c, and C3H/HeJ mice, whereas in C57BL/6 mice it rose only to about that in untreated DBA/2, BALB/c, and C3H/HeJ mice. To determine whether this low level was due to a reduced transcription rate, a nuclear "run-on" assay was performed with NIH 3T3 cells or BLK cells derived from C57BL/6 mice. IFN-alpha increased the 202 mRNA transcription severalfold in NIH 3T3 cells only, and that of a (2'-5')(A)n synthetase gene in both cell lines. The possibility that an alteration in transacting factors could be responsible for this difference was examined. For this purpose the 5' terminal flanking region (called the b segment, about 0.8 kb) of the 202 gene was linked to a heterologous reporter gene--chloramphenicolacetyl-transferase (CAT) and transfected into normal or transformed NIH 3T3 cells and into various C57BL/6-derived cell lines. IFN-alpha induced strong CAT activity in transfected normal or transformed NIH 3T3 cells, but a much lower activity in those from C57BL/6 mice. The b segment contains an IFN-responsive element (ISRE) (35 bp) homologous to that present in several other IFN-inducible genes. Three tandem copies of the 202 ISRE were linked to an enhancerless SV40 early promoter driving an influenza virus hemagglutinin (HA) cDNA segment. No increase in HA mRNA expression was detected in the transfected BLK cell line derived from C57BL/6 mice following IFN treatment, whereas in the NIH 3T3 cell line, the IFN treatment resulted in a 2.5-fold increase. These and other results suggest that C57BL/6 mice and cell lines derived from them might carry defective transacting factors impairing the ability of IFN-alpha to activate the 202 gene without impairing its ability to activate a (2'-5')(A)n synthetase gene.

2',5'-Oligoadenylate Synthetase↗
PubMed1992

Demonstration of the formation of hydroxyl radicals in acute myocardial infarction in man using salicylate as probe.

Dihydroxybenzoic acid (DHBA) derivatives of acetylsalicylic acid (ASA) are formed in vivo by the action of the hydroxyl radical (OH.). In order to evaluate the possible formation of OH(.) in acute myocardial infarction (AMI) in man, 9 consecutive patients with a first episode of AMI (8 males, 1 female, mean age 50.3 years), treated with rt-PA, and 8 healthy volunteers (7 males, 1 female, mean age 29.8 years) were studied. All subjects received 100 mg ASA p.o. daily; venous blood samples were taken 30 min after the first dose (time 0) and then at 3-, 6-, 12-, 24- and 48 h and 5 days. Serum was analyzed by HPLC and electrochemical detection for 2,3- and 2,5-DHBA contents. 2,3-DHBA was present in all subjects with AMI and undetectable in healthy volunteers at all time points studied. Serum levels of 2,5-DHBA did not show statistically significant differences between AMI patients and healthy volunteers. These data support the hypothesis that hydroxyl radicals are formed during AMI in man.

Aspirin↗
PubMed1992

Effects of interferon alpha on murine cytomegalovirus replication.

The present study examined the protective effect of IFN-alpha against mouse cytomegalovirus (MCMV) infection in embryo fibroblasts (MEF) of genetically resistant (C3H/HeJ) and susceptible (C57BL/6) mouse strains. At a M.O.I. of 1 IFN-alpha was protective in C3H/HeJ-MEF but not in C57BL/6-MEF. Dot-blot analysis during MCMV replication in C3H/HeJ-MEF showed that IFN-alpha pretreatment reduced the steady state level of immediate early and late mRNAs but partially reduced early gene expression.

Animals↗
PubMed1991

Activation of interferon-inducible genes in mice by poly rI:rC or alloantigens.

We have examined the effects of synthetic dsRNA (poly rI:rC) treatment or of immunization with irradiated allogeneic cells on the expression in vivo of several interferon (IFN)-inducible genes. For this purpose, DBA/2 mice were injected i.p. once with poly rI:rC, or once and then again 3 weeks later, with irradiated C3H/He spleen cells and the effect of these treatments on the levels of the following mRNAs was determined: 202, 2',5'-oligoadenylate synthetase (2-5A synthetase), class I and class II major histocompatibility antigens, and beta-actin. After poly rl:rC treatment, the levels of the 202 and 2-5A synthetase mRNAs in the spleen and in the bone marrow peaked between 12 and 24 h and decreased thereafter. The class I mRNA levels started to increase at 12 h, peaked at 24 h, and declined thereafter. No increase in class II mRNA expression was observed after poly rl:rC injection, whereas beta-actin levels remained unchanged. Pretreatment of DBA/2 mice with sheep anti-murine IFN-alpha/beta antibodies before poly rI:rC injection strongly diminished the induction of 202 mRNA, indicating that IFN-alpha/beta mediated this induction. When irradiated C3H/He spleen cells were injected into DBA/2 mice, the class I and class II mRNAs in the spleen, but not in the bone marrow, started to increase at 12 h, peaked between 48 and 96 h, and decreased thereafter. No increase in the levels of 202 and 2-5A synthetase mRNAs was detected, whereas beta-actin levels remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

2',5'-Oligoadenylate Synthetase↗
PubMed1991

Interferon action: binding of viral RNA to the 40-kilodalton 2'-5'-oligoadenylate synthetase in interferon-treated HeLa cells infected with encephalomyocarditis virus.

The 40-kDa 2'-5'-oligoadenylate [(2'-5') (A)n] synthetase isoenzyme was proven to be a mediator of the inhibition of encephalomyocarditis virus (EMCV) replication by interferon (IFN). When activated by double-stranded RNA, this enzyme converts ATP into 2'-5'-oligoadenylate [(2'-5') (A)n], and (2'-5') (A)n was found to accumulate in IFN-treated, EMCV-infected cells. The only known function of (2'-5') (A)n is the activation of RNase L, a latent RNase, and this was also implicated in the inhibition of EMCV replication. Intermediates or side products in EMCV RNA replication, presumed to be partially double stranded, were shown to activate (2'-5') (A)n synthetase in vitro. These findings served as the basis of the long-standing hypothesis that the activator of (2'-5') (A)n synthetase in IFN-treated, EMCV-infected cells is the viral RNA. To test this hypothesis, we have generated a polyclonal rabbit antiserum to the human 40-kDa (2'-5') (A)n synthetase. The antiserum immunoprecipitated, from IFN-treated HeLa cells that had been infected with EMCV, the 40-kDa (2'-5') (A)n synthetase protein in complex with both strands of EMCV RNA. The immunoprecipitate was active in (2'-5') (A)n synthesis even without addition of double-stranded RNA, whereas the immunoprecipitate from IFN-treated, uninfected cells was not. These and other results demonstrate that in IFN-treated, EMCV-infected cells, viral RNA is bound to the (2'-5') (A)n synthetase and suggest that the agent activating the (2'-5') (A)n synthetase is the bound viral RNA.

2',5'-Oligoadenylate Synthetase↗
PubMed1991

Activation of interferon-inducible genes in vivo by synthetic double-stranded RNA, poly rI:rC.

We analyzed the effects of synthetic dsRNA (poly rI:rC) treatment on the expression in vivo of two interferon (IFN)-inducible genes. DBA/2 mice were injected i.p. with poly rI:rC and its effect on the levels of the following mRNAs was determined; 202, 2'-5' oligoadenylate synthetase (2-5A synthetase) and beta-actin. After poly rI:rC treatment the levels of the 202 and 2-5A synthetase mRNAs in the spleen and in bone marrow peaked between 12 and 24 h and decreased thereafter, whereas beta-actin levels remained unchained unchanged. Pretreatment of DBA/2 mice with sheep anti-murine IFN-alpha/beta antibodies before rI:rC injection strongly diminished the induction of 202 mRNA indicating that IFN-alpha/beta mediated this induction.

Animals↗
PubMed1990

Cell and type specificity of interferon action. Unusual characteristics of the transcriptional control of gene expression by interferon-gamma in T cells.

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.

2',5'-Oligoadenylate Synthetase↗
PubMed1990

Augmentation of mouse natural killer (NK) activity by GM-1/P, a processed form of monosialoganglioside GM-1.

We describe the immunomodulatory activity of GM-1/P a processed form of GM-1 (monosialoganglioside) extracted from ox brain, purified and physically modified. We examined the effect of in vivo and in vitro treatment of GM-1/P on natural (NK) activity and its ability to induce the production of interleukin-2 (IL-2) in the mouse. In vivo treatment with GM-1/P (1 mg/Kg, i.v., day-1) resulted in a marked increase and in a change of distribution of NK activity, which was associated with lower density Percoll fractions. Marked increase was already observed at 18 hrs and then declined by day 4. In vitro treatment with GM-1/P (2 micrograms/ml) enhanced NK activity of B6 spleen cells, already after 6 hours of incubation, remaining at plateau levels within 18 hours. A role of IL-2 in this enhancement was suggested by the ability of an anti-IL-2 rabbit antiserum to abolish in vitro increased cytotoxicity. The presence of IL-2 in the supernatants of splenocytes from GM-1/P (1mg/Kg, i.v., ,day-1) treated mice stimulated with Con A or Con A plus TPA for 48 hrs was evaluated by proliferation of an IL-2 dependent CTLL cell line. GM-1/P by itself was unable to stimulate IL-2 production; however it markedly increased IL-2 production induced by Con A or Con A plus TPA.

Adjuvants, Immunologic↗
PubMed1990

Enhancement of lymphocyte proliferation and IL-2 receptor expression by a processed form (GM-1/P) of monosialoganglioside GM-1.

In this study we investigated the ability of GM-1/P, a calcium mediated processed form of monosialoganglioside GM-1, of in vivo augmenting mouse T and B-lymphocyte blastogenesis induced by mitogens. We have also determined its effect on IL-2 responsiveness by analyzing the induction of the expression of IL-2 receptor (IL-2r) on mouse spleen cells. Lymphocyte blastogenesis was evaluated by 3H-TdR incorporation of spleen cells from untreated or GM-1/P (1mg/Kg, i.v., day-1) treated mice cultured in the presence of T (PHA, ConA) B (LPS) cell specific mitogens. The stimulatory effects appeared to be due to a direct action on T and B lymphocytes, since proliferative response was not abolished by removal of macrophages. Splenocytes from GM-1/P treated mice showed increased proliferation in response to various concentrations of HrIL-2; moreover under these conditions an increased generation of LAK activity was found. A direct evidence for enhanced expression of IL-2r was obtained by immunofluorescence and FACS analysis using a monoclonal antibody (PC.61) directed against the p55 subunit of murine IL-2r. 29% PC.61+ cells were found in IL-2 cultures from treated spleen cells.

Animals↗
PubMed1990

The role of interferons in the resistance to murine cytomegalovirus.

The protective effect of IFN alpha/beta or IFN-gamma against MCMV infection in mouse embryo fibroblasts (MEF) of genetically resistant and susceptible strains have been examined. For this purpose, MEF derived from Balb/c, C3H, DBA2, C57BL6 mouse strains were used. Cells were pretreated for 24 hrs with either IFN alpha/beta or IFN-gamma and subsequently infected with MCMV at different M.O.I. No difference in susceptibility to MCMV was observed between untreated Balb/c, C3H, C57 and DBA2-MEF when infected at a M.O.I. of 5 whereas at a M.O.I. of 1 or 0.5 untreated DBA2-MEF displayed the highest susceptibility to viral infection. Pretreatment of MEF with IFN alpha/beta showed that the degree of protection to MCMV was dependent on the M.O.I. used. At a M.O.I. of 5, IFN alpha/beta did not inhibit viral replication in any MEF tested, whereas at a M.O.I. of 1 or 0.5 was protective in C3H-MEF and to a lesser extent in Balb/c-MEF. No protection was observed in DBA2 and C57-MEF. Different results were observed when MEF were pretreated with IFN-gamma. C57-MEF were protected when infected at a M.O.I. of 5 or 1; no protection was observed at a M.O.I. of 0.5. Balb/c-MEF were protected only at a M.O.I. of 1. No protection was observed in C3H and DBA2-MEF.

Animals↗
PubMed1989

Inhibition of interferon-gamma antiviral and antiproliferative activities by ras oncogene expression.

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.

2',5'-Oligoadenylate Synthetase↗
PubMed1989

Characterization of cytoplasmic and nuclear polypeptides induced by interferon-gamma in a murine pre-B cell leukemia.

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.

Animals↗
PubMed1989

Effects of protein kinase C (PK-C) activators and inhibitors on human large granular lymphocytes (LGL): role of PK-C on natural killer (NK) activity.

The role of protein kinase C (PK-C) in the early metabolic events involved in human natural killer (NK) cell activation has been studied through the action of PK-C-specific activators and inhibitors. Highly purified human large granular lymphocytes (LGL) were treated for 1 hr with the diacylglycerol analog 1-oleoyl-2-acetyl glycerol (OAG) (10(-4)-10(-5) g/ml) or with 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-8)-10(-10) g/ml), both specific activators of PK-C. Both these agents consistently increased NK activity against K562 target cells. Suboptimal doses of either OAG or TPA also synergized with Ca2+ ionophores to augment spontaneous cytotoxic activity. Pretreatment of LGL with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrocloride (H7) (5-40 microM), a potent PK-C inhibitor, greatly reduced NK activity in a time- and dose-dependent fashion. By contrast, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA 1004), a potent cAMP- and cGMP-dependent PK inhibitor with almost no effect on PK-C, marginally reduced NK activity. Moreover, almost complete NK activity inhibition was observed when H7 (10 microM), but not HA 1004 (50 microM), was present in the NK assay. Finally, 48 hr stimulation of LGL with TPA (10(-6) g/ml), a treatment able to inactivate most of the PK-C cellular pool, almost completely abrogated NK activity. This functional evidence was supported by phosphorylation of several endogenous substrates which occurs within 5 min in TPA-treated LGL. Two proteins of 70 and 56 kDa have been identified as major PK-C substrates, together with other phosphorylated proteins with MW ranging from 177 to 43 kDa. H7, but not HA 1004, almost completely inhibited the TPA-induced phosphorylation of all of these proteins in the NK cells. These data strongly suggest that selective activation of PK-C plays an essential role in the mechanisms of NK cell activation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Obligatory role of IFN-gamma in induction of lymphokine-activated and T lymphocyte killer activity, but not in boosting of natural cytotoxicity.

The biological role of the murine IFN-gamma endogenously secreted during cell activation has been probed by the use of a rat mAb (AN18) that specifically neutralizes its activity. When An18 mAb is added to the cultures of BALB/c and C57BL/6 nylon nonadherent spleen cells (naSpc) stimulated for 18 h with 1000 U of IL-2, the normally released IFN-gamma can no longer be detected in the supernatants and the IL-2-induced proliferative response is markedly reduced as compared with control cultures set up in the presence of an unrelated rat mAb. By contrast, the increased natural cytotoxicity is not affected. The 96-h culture of both BALB/c and C57BL/6 naSpc in the presence of 1000 U of IL-2 resulted in marked lymphocyte proliferation and generation of lymphokine-activated killer activity. The presence of An18 mAb strongly inhibited both functions. Similarly, when naSpc were stimulated with mitomycin C-inactivated allogeneic leukocytes in the presence of An18 mAb, the normal proliferative response and specific cytotoxicity were almost abolished, whereas the secretion of IL-2 was in no way affected. Lymphocytes recovered from 3-day cultures stimulated by IL-2 or allogeneic cells in the presence of An18 mAb displayed a decrease of the expression of the p55 chain of IL-2R, as shown by flow cytofluorimetry. Moreover, binding experiments with 125I-labeled IL-2 showed that lymphocytes from allostimulated cultures set up in the presence of An18 mAb display a decreased number of low affinity and almost no high affinity IL-2R as compared with control cultures. These data show that endogenous IFN-gamma plays an obligatory role for the de novo induction of cytolytic activity in lymphokine-activated killer cells and CTL, most probably by affecting the membrane expression of high affinity IL-2R.

Animals↗