Prophylaxis in cardiac surgery. A controlled randomized comparison between cefazolin and cefuroxime.

In a prospective randomized two center trial, short-term prophylaxis with cefuroxime (CFX) in 189 patients was compared with cefazolin (CFZ) in 196 patients submitted to elective cardiac surgery. A total of 3 g was administered over 24 h in both groups. One major adverse reaction with CFX was noted. Patients were prospectively screened by infectious disease nurses for surgical wound and secondary infections. Sternal wound infections occurred in eight patients treated with CFX and all were minor. One patient from this group eventually died of infectious causes. In the CFZ-treated patients two major and six minor wound infections occurred requiring extensive debridement in two. Secondary infections occurred less frequently in the CFX group (13.2 per 100) than in the CFZ group (16.8 per 100) with two infection-related deaths in the CFX and one in the CFZ group. The most commonly identified organisms were Staphylococcus aureus and a variety of gram-negative organisms. No major differences were observed between the CFX and CFZ groups. Short-term administration of 3 g CFZ or CFX in this study could not demonstrate the advantage of one of the antibiotics used over the other in terms of clinical outcome, incidence or site of infection or organisms identified. The 24 h administration of 3 g CFZ or CFX provided suboptimal prophylaxis for wound infection or secondary infections in patients undergoing elective open heart surgery.

The inhibition of endotoxin-induced local inflammation by LDH virus or LDH virus-infected tumors is mediated by interferon.

The footpad swelling reaction induced by local injection of S. marcescens lipopolysaccharide was found to be inhibited in mice given a transplantable tumor (TA3) or cell-free ascitic fluid from tumor-bearing mice. The tumor was shown to contain LDH virus, which is known to cause inapparent persistent infections in mice. Monoclonal antibodies directed against protein VP3 of the LDH virus could partially abrogate the anti-inflammatory effect of the TA3-ascitic fluid, and, conversely, the anti-inflammatory effect could be obtained by LDH virus isolated from the tumor and reproduced by serial passage of cell-free fluids. Inhibition of the footpad reaction was seen in the acute but not in the chronic phase of LDH virus infection, suggesting that the anti-inflammatory effect might be due to endogenous interferon (IFN) which, similarly, was only detectable in the acute phase. Newcastle disease virus, another potent interferon inducer, had a similar inhibitory effect on the footpad reactivity. Moreover, the inhibitory effect of LDH virus infection could partially be abrogated by administration of a polyclonal antibody directed against murine IFN-alpha,beta. Finally, passively administered natural murine IFN-alpha,beta or recombinant murine IFN-alpha 1 (but not recombinant murine IFN-beta) was found to cause inhibition of the footpad reaction. Since Gram-negative bacteria and their lipopolysaccharides have the ability to induce a systemic interferon response, our findings suggest that this interferon may play a modulatory role in local inflammation caused by these bacteria. Our findings also open a new perspective for interferon therapy of certain inflammatory reactions to bacterial infections.

Influence of carbohydrate side chains on activity of tissue-type plasminogen activator.

When messenger RNA (mRNA) from both untreated and phorbol ester-treated melanoma cells is translated in simple reticulocyte lysates, tissue-type plasminogen activator can be immunoprecipitated by an affinity-purified antibody as a approximately 52,000 mol wt protein, with no detectable biological (plasminogen activating) activity. When the reticulocyte lysate system is supplemented with a preparation of microsomal membranes, biological activity becomes detectable and a 63,000 mol wt protein can be immunoprecipitated with the same antibody. Furthermore, when natural tissue-type plasminogen activator (mol wt approximately equal to 70,000) is incubated with different glycosidases, distinct alterations in the electrophoretic mobility of the molecules are observed, together with alterations in the level of biological activity. While treatment with neuraminidase and beta-galactosidase caused decreases in activity, alpha-mannosidase caused an increase. These results suggest that the carbohydrate part of the molecule can influence its biological behavior.

Cloning of cDNA coding for human tissue-type plasminogen activator and its expression in Escherichia coli.

cDNA clones of the mRNA coding for tissue-type plasminogen activator (t-PA) have been obtained and their nucleotide sequences compared to those reported previously. A gene coding for t-PA has been reconstructed and inserted into vectors for expression in prokaryotic cells. Relatively high levels of t-PA accumulated in inclusion bodies in Escherichia coli containing an optimized expression plasmid, but only a small proportion of the insoluble protein was recovered as active enzyme using a variety of solubilization procedures.

Determination of tissue-type plasminogen-activator mRNA in human and non-human cell lines by dot-blot hybridization.

A labelled cDNA clone was used in DNA-RNA hybridization on nitrocellulose filter paper (dot-blot technique) to detect and quantify mRNA for endogenous tissue plasminogen activator (PA) in cell extracts and samples of RNA purification runs. Although, for detection purposes, the assay was less sensitive than translation in Xenopus oocytes, it was at least as reliable and much more convenient for the purpose of quantitative determination. In particular, the technique was used to study the kinetics of PA mRNA formation in a human melanoma cell line (Bowes) after exposure to the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). Incubation of the cells with TPA resulted in a 15-20-fold increase in cellular PA mRNA content. The effect was time- and dose-dependent: the increase in PA-specific mRNA was clearly visible as early as 4 h after initiation of TPA treatment in Bowes cells. It was blocked completely by pretreatment of the cells with actinomycin D, indicating that TPA caused enhancement of synthesis of PA mRNA rather than inhibition of PA mRNA degradation. The use of the nitrocellulose dot-blot technique also revealed that two non-human cell lines produce mRNAs which cross-react with the human PA mRNA, namely the mouse melanoma cell line B16 and the rat brain-tumour cell line, RT4-71-1. TPA was found to exert similar stimulatory effects on the synthesis of mRNAs in these cell lines as in Bowes cells.

Stimulation of fibroblast interferon production by a 22K protein from human leukocytes.

We have studied the appearance of human interferon-beta (HuIFN-beta) as well as its mRNA in cells treated with a protein, 22K factor, isolated from the culture supernatant of mitogen-stimulated human peripheral blood leukocytes. By itself 22K was found to be unable to induce production of significant amounts of HuIFN-beta protein. However, when aided by treatment with cycloheximide or cycloheximide and actinomycin D (superinduction), 22K caused increases in production ranging from 3- to 20-fold, depending on the cells (diploid or MG-63 osteosarcoma) and the induction schedule. Cells treated with 22K alone produced small amounts of HuIFN-beta mRNA, which was only detectable with a highly sensitive method. In combination with cycloheximide, 22K induced levels of mRNA detectable with less sensitive methods as well. These experiments provide further support for the concept that the antiviral activity of 22K is mediated by its ability to stimulate transcription of the HuIFN-beta gene in cells.

Homogeneous interferon-inducing 22K factor is related to endogenous pyrogen and interleukin-1.

In vitro stimulation of mononuclear cells from human peripheral blood with mitogens causes the release of factors (monokines and lymphokines) which possess distinct biological activities. One such factor, termed 22K, can induce production of human beta-interferon (HuIFN-beta) in cultured human fibroblasts, thereby rendering these cells resistant to virus infection. Here we report the complete purification and partial sequencing (39 N-terminal amino acids) of this factor, whose relative molecular mass was estimated by SDS-polyacrylamide gel electrophoresis to be 17,000 (17K). In addition to an antiviral effect, the pure protein exhibits several other biological activities. Most significantly, intravenous (i.v.) injection of the factor in rabbits caused fever and granulopenia at doses of 0.1-1 microgram per kg, effects which we attribute to a monokine called endogenous pyrogen (EP). In vitro, the protein was scored as positive in a LAF (lymphocyte-activating factor) assay at 0.1-1 ng ml-1. LAF and EP are considered to be members of one family of monokines, called interleukin-1 (IL-1). For this reason, and also because the amino-acid sequence of the 22K factor is at least partially homologous to a complementary DNA-derived IL-1 sequence, we postulate that the 22K factor also belongs to the IL-1 family.

Molecular cloning of murine interferon gamma (MuIFN-gamma) cDNA and its expression in heterologous mammalian cells.

A complete cDNA clone of murine interferon-gamma (MuIFN-gamma) was obtained by recombining two appropriate segments from partial cDNA clones originally identified by colony hybridization with rat IFN-gamma chromosomal gene fragments as probes. An expression vector was constructed in which the cDNA was placed under control of the SV40 early promoter. Transient expression of MuIFN-gamma was obtained by transformation of COS-1 cells. Subsequently, this interferon expression unit was linked to a vector containing a dihydrofolate reductase (DHFR) modular gene and used to transform DHFR(-)-CHO cells. Cell clones were selected that constitutively produce an interferon activity which by several criteria was found to be indistinguishable from natural, splenocyte-derived MuIFN-gamma.

An interferon-beta-like or interferon-inducing protein released by mitogen-stimulated human leukocytes.

Human peripheral blood leukocytes were treated with concanavalin A (Con A) to produce interferon gamma (HuIFN-gamma). On gel filtration this interferon eluted as a protein with a molecular weight of 45000. In addition to this, the culture supernatant contained an interferon-like protein of apparent molecular weight 22000 (22K factor). The antiviral activity of this protein was neutralizable by a highly specific antibody to HuIFN-beta. Yet, the 22K factor differed from classical HuIFN-beta in several characteristics: lack of activity on certain homologous and heterologous cells which are sensitive to HuIFN-beta; lack of affinity for zinc-chelate and Con A-Sepharose columns; failure to bind to an anti-HuIFN-beta antibody column. Moreover, a specific antiserum raised against the 22K factor did not neutralize HuIFN-beta. Two alternative explanations of these findings are proposed: (i) the 22K factor is an interferon whose molecular structure resembles that of the known HuIFN-beta but it is not identical to it, or (ii) the 22K factor is not an interferon but a protein that can induce the production of HuIFN-beta in certain lines of fibroblastoid cells.

Effects of 12-O-tetradecanoylphorbol 13-acetate on the production of mRNAs for human tissue-type plasminogen activator.

The mRNA for human (tissue type) plasminogen activator from a human melanoma cell line (Bowes) was investigated in different translation systems. After translation of poly(A)-rich RNA in Xenopus oocytes a biologically active plasminogen activator was obtained. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of the secreted translation products revealed a protein band precipitable with affinospecific antibody and migrating at the same position (apparent molecular mass of approximately 70000 Da) as the native melanoma cell product. Translation in rabbit reticulocyte lysate yielded an immunoprecipitable band migrating at position corresponding to a molecular mass of 52000 Da. Addition of 12-O-tetradecanoylphorbol 13-acetate to the cell cultures resulted in increased production of plasminogen activator. Concomittantly more poly(A)-rich RNA could be extracted from the cells and this RNA was more effectively translated by oocytes into biologically active plasminogen activator. Translation of poly(A)-rich RNA from phorbol-ester-treated cells in the reticulocyte lysate system yielded the 52000-Da protein also seen with RNA from untreated cells. However, in addition a prominent protein band of apparent molecular mass of 48000 Da was detectable. Its intensity increased with increasing doses of tetradecanoylphorbol acetate. This phorbol-ester-induced protein was not precipitable with the affinospecific antibody against plasminogen activator.

Messenger RNA for human tissue plasminogen activator.

A human melanoma cell line (Bowes), which secretes extrinsic (tissue-type) plasminogen activator, was used as a source for the preparation of mRNA for extrinsic plasminogen activator. The cells were lysed and total RNA was extracted with the phenol method. A poly(A)-rich RNA fraction was isolated by affinity chromatography on oligo(dT)-cellulose. This preparation was translated by oocytes into (a) protein(s) that had biological activity in the assay for extrinsic plasminogen activator. On sucrose gradient centrifugation the translatable fraction of the RNA migrated with a sedimentation coefficient of approximately 19 S, a value compatible with the molecular weight of extrinsic plasminogen activator (70 000). The translation product was characterized as being similar to or identical with authentic extrinsic plasminogen activator by the following criteria: (a) serological cross-reactivity with purified extrinsic plasminogen activator in neutralization and immunoprecipitation reactions; (b) plasminogen dependency of fibrinolytic activity and (c) apparent molecular weight of 70 000 on sodium dodecyl sulphate/polyacrylamide gel electrophoresis.

Interferon induced in human leukocytes by concanavalin A: isolation and characterization of gamma- and beta-type components.

Interferon (IFN) was induced in suspensions of fresh human leukocytes by incubation with concanavalin A (Con A). The crude supernatant was concentrated and partially purified by adsorption to silicic acid and elution with an ethylene glycol (EG)-containing buffer. Th EG eluate was shown to contain two antivirally active components (tentatively called 22K), separable from each other by gel filtration. The 45K component was strictly species specific, relatively sensitive to acid (pH 2) and poorly neutralized by anti-human IFN-beta (HuIFN-beta) antibody. In all probability, it predominantly contained (a) molecular variant(s) of human gamma-type IFN (HuIFN-gamma ). The 22K component was acid resistant and serologically indistinguishable from HuIFN-beta prepared from fibroblasts. However, in contrast to the latter, it had little activity on bovine kidney cells and no activity on human HEp-2 and monkey Vero cells. A preparation with the same properties was obtained by substituting glycine buffer (pH 2) for EG in the elution of Con A-induced IFN from silicic acid. This IFN failed to adsorb to a zinc chelate column, while HuIFN-beta from fibroblasts can easily be purified by affinity chromatography on this type of column. It was concluded that the Con A-induced IFN contained an acid-resistant component of apparent molecular weight 22000 serologically related to classical HuIFN-beta but differing by certain physicochemical and biological criteria.

The preparation of antibodies directed against human immune interferon.

Antisera against human immune interferon (HuIFN-gamma) were prepared by immunization of rabbits and a goat with antigens of different degrees of purity: a) crude supernatant from concanavalin A-stimulated human leukocytes; b) a preparation partially purified by adsorption to controlled pore glass; and c) pooled fractions (molecular weight - 45,000) obtained by gel filtration and corresponding to the peak of HuIFN-gamma. Antisera with a relatively high titer were obtained in animals immunized with the latter two antigens. The sera were specific for HuIFN-gamma in that they failed to neutralize preparations of HuIFN-alpha and and HuIFN-beta. From the time course of the antibody titers in each animal it seemed that frequent boosters within a short time interval led to a decrease rather than an increase in neutralizing activity of the sera.

Purification of human fibroblast interferon by zinc chelate chromatography.

Human interferon was prepared by superinduction of cultures of either diploid embryonic skin and muscle cells or of the osteosarcoma cell line MG-63. The interferon so obtained was concentrated and partially purified by adsorption to controlled pore glass (CPG) beads at neutral pH and desorption by glycine-HCl buffer at pH 2. After neutralization, this interferon was applied to a column of zinc chelate which was eluted with buffers of decreasing pH. Most of the proteins eluted ahead of the interferon activity, which itself eluted in two distinct peaks. The first peak occurred in the effluent fractions around pH 5.9, and the second one in fractions around pH 5.2. The interferon found in fractions of pH 5.9 contained 5% of the original contaminating proteins. In contrast, the amount of total protein in the pH 5.2 peak was so small that it could not accurately be assayed by the fluorescamine method. Consequently, the interferon in the peak fraction was estimated to have a specific activity of about 2 x 10(9) units/mg. This material was radiolabelled and analysed by electrophoresis. A major peak of about 22000 mol. wt. with only minor contaminating proteins appeared on the autoradiographs. The total recovery of the zinc chelate chromatographical procedure was nearly 100%, and the interferon recovered from each peak behaved consistently on rechromatography. Fibroblast interferon produced by most diploid cells contained less than 10% of the variant eluting at pH 5.9. MG-63 cells and high-passage cultures of some diploid cell strains produced up to 50% of this variant.

Tissue distribution of human interferons after exogenous administration in rabbits, monkeys, and mice.

Preparations of human leukocyte (alpha) and fibroblast (beta) interferon were given intramuscularly to rabbits and monkeys, and circulating interferon was measured. In rabbits, but not in monkeys, a marked difference between the two interferons was noted in that higher titers of circulating antiviral activity were obtained with leukocyte than with fibroblast interferon. In mice, injected interperitoneally, a similar difference could be noted. However, levels of antiviral activity in homogenates of spleens and lungs did not differ between mice injected with either interferon. Fibroblast interferon that was injected intrathecally in monkeys was found to diffuse throughout the cerebrospinal canal and to reach the serum compartment. Some interferon could also be recovered from the pia mater surrounding the brain hemispheres, but none was found in the deeper layers of the brain.