Action of quail and chicken interferons on a quail cell line, QT35.

Production of interferon (IFN) in quail cells (QT35) and the activity of quail IFN and heterologous avian IFN (chicken) on QT35 cells were examined. Quail cells produced IFN after induction by bluetongue virus serotype 10; chicken and quail IFN conferred antiviral resistance on the quail cells. Both chicken and quail IFN induced increased levels of 2',5'-oligoadenylate synthetase (2',5'-OAS) in QT35 cells and reduced levels of intracellular Salmonella typhimurium postchallenge. These results indicate that the quail cells produce IFN and respond to homologous and heterologous avian IFN as evidenced by enhanced (1) resistance to viral infection, (2) production of 2',5'-OAS, and (3) resistance to invasion by bacteria. QT35 quail cell monolayer cultures offer an alternative to primary chicken embryo fibroblasts cultures used for avian IFN studies.

Uterine cellular changes in 2',5'-oligoadenylate synthetase during the bovine estrous cycle and early pregnancy.

The signal for maternal recognition of pregnancy in cattle is bovine trophoblast protein-1 (bTP-1), a Type I trophoblast interferon. One of the many functions of interferons is the induction of the 2',5'-oligoadenylate (2-5[A]) system, which is involved in cell division and selective degradation of mRNA. The present study focused on the cellular changes of 2-5(A) synthetase in bovine endometrium during the estrous cycle and early pregnancy. Ability of recombinant bTP-1 to stimulate activity of the enzyme in uterine cells throughout the estrous cycle was also evaluated in vitro. Surface epithelium, glandular epithelium, and stroma were enzymatically separated in cows on Day 5, 10, 15, or 18 postestrus and on Day 15 or 18 of pregnancy. Cell samples were lysed and frozen for determination of the endogenous cellular content of 2-5(A) synthetase. Additional cells obtained during the estrous cycle were cultured and treated with increasing doses of recombinant bovine interferon alpha (rbIFN-alpha) or recombinant bTP-1 (rbTP-1). During the estrous cycle, 2-5(A) synthetase was greatest on Day 5 and declined approximately 10-fold by Day 15 (p < 0.01). Cellular content of 2-5(A) synthetase was similar among all three endometrial cell types. In the gravid horn of pregnant animals, presence of a conceptus significantly increased (p < 0.01) 2-5(A) synthetase in all endometrial cell types compared to levels on Days 15 and 18 of the estrous cycle. On Day 18, levels of 2-5(A) synthetase were 30-fold greater (p < 0.01) in epithelium (surface and glandular) from pregnant cows compared to that from cyclic cows.(ABSTRACT TRUNCATED AT 250 WORDS)

Porcine conceptus proteins: antiviral activity and effect on 2',5'-oligoadenylate synthetase.

Interferon-like proteins synthesized by conceptuses of domestic ruminants inhibit luteolysis during early pregnancy. Although pig conceptuses secrete trophoblast interferons during the period of CL maintenance, estrogen is involved with maintenance of the CL. The principal purposes of this work were to confirm production of trophoblast interferons by porcine conceptuses and to compare the effect of trophoblast interferons on endometrium of pigs and cattle. When measured using Madin-Darby bovine kidney (MDBK) cells challenged with vesicular stomatitis virus, antiviral activity in uterine flushings from cyclic gilts was not detectable throughout the estrous cycle; however, in pregnant gilts, antiviral activity increased from undetectable amounts to 4-11 x 10(3) U on Days 14, 16, and 18. Porcine embryos in culture produced 1,100 U/embryo/ml/24 h. Porcine conceptus secretory proteins induced 2',5'-oligo(A) synthetase in MDBK cells and in endometrial explants of cows but had no measurable effect on 2',5'-oligo(A) synthetase activity of endometrial explants of pigs. Similarly, endometrial 2',5'-oligo(A) synthetase of pregnant pigs was unaffected in vivo during the period of maximal synthesis of conceptus secretory proteins. Porcine conceptus secretory proteins produced no detectable increase in serum antiviral activity or 2',5'-oligo(A) synthetase activity of blood mononuclear leukocytes in utero-ovarian venous blood. These results suggest that conceptus interferons of pigs play different roles in the establishment of pregnancy compared to their roles in ruminants.

Endocrine events associated with endometrial function and conceptus development in cattle.

Regulation and timing of luteolysis during the bovine oestrous cycle is controlled by the initiation and length of progesterone stimulation. Results have demonstrated that early administration of progesterone shortens the interoestrous interval in the ewe and cow, and removal of progesterone stimulation through a progesterone receptor antagonist delays luteolysis in sheep. Current data suggest that down-regulation of progesterone receptors in the uterine epithelium may initiate events involved in the synthesis and release of prostaglandin F2 alpha (PGF2 alpha) for luteolysis. Progesterone is also involved in the stimulation of the uterine secretions that regulate conceptus growth and the release of the bovine trophoblast protein-1 (bTP-1) necessary for inhibiting endometrial PGF2 alpha release. Conceptus secretion of bTP-1, a Type I trophoblast interferon, increases the concentration of the cellular enzyme 2',5'-oligoadenylate synthetase within the endometrium. The biological role of 2',5'-oligoadenylate synthetase in the establishment of pregnancy is discussed.

Stimulation of 2',5'-oligoadenylate synthetase activity in sheep endometrium during pregnancy, by intrauterine infusion of ovine trophoblast protein-1, and by intramuscular administration of recombinant bovine interferon-alpha I1.

In Expt 1, activity of 2',5'-oligoadenylate (2',5'-A) synthetase in endometrium collected on Day 16 (oestrus is Day 0) from the uterine horn ipsilateral to the corpus luteum was greater (P less than 0.001) for pregnant (135.5 +/- 1.72 nmol/mg protein/h) than for cyclic ewes (58.5 +/- 0.99 nmol/mg protein/h). In pregnant ewes, activity of 2',5'-A synthetase in endometrium collected from the contralateral uterine horn (119.5 +/- 1.72 nmol/mg protein/h) did not differ from that of the ipsilateral horn. In Expt 2, three ovariectomized ewes were treated with progesterone for 10 days and then with oestrogen for 2 days. Activity of 2',5'-A synthetase on Day 13 was 18% greater (P less than 0.10) in endometrium collected from the uterine horn receiving infusions of 30 micrograms ovine trophoblast protein-1 (oTP-1) twice a day on Days 10, 11 and 12(57.7 +/- 0.22 nmol/mg protein/h) than from the uterine horn receiving control infusions of serum protein (SP; 48.8 +/- 0.22 nmol/mg protein/h). In Expt 3, activity of 2',5'-A synthetase on Day 15 was not significantly greater in endometrium collected from the uterine horn of cyclic ewes receiving infusions of 30 micrograms oTP-1 twice a day on Days 12, 13 and 14 (46.5 +/- 0.37 nmol/mg protein/h) than in endometrium from the uterine horn receiving infusions of SP (38.2 +/- 0.37 nmol/mg protein/h). When results of Expt 2 and Expt 3 were combined, intrauterine infusion of oTP-1 increased (P less than 0.05) activity of 2',5'-A synthetase in endometrium by 20%.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro and in vivo 2',5'-oligoadenylate synthetase activity induced by recombinant DNA-derived bovine interferon alpha I1 in bovine alveolar macrophages and blood mononuclear cells.

Biological responses to recombinant DNA-derived bovine interferon alpha (rBoIFN-alpha I1) by bovine alveolar macrophages were examined by measuring viral yield reduction and 2',5'-oligoadenylate synthetase (2',5'-OAS) production by IFN-treated cells. In vitro IFN pretreatment of alveolar macrophages reduced viral yield in cultures challenged exposed with parainfluenza-3 virus, compared with control cultures. In vitro treatment of alveolar macrophages with IFN also resulted in increased 2',5'-OAS activity. The 2',5'-OAS activity was measured in alveolar macrophages and blood mononuclear leukocytes of calves injected IM with 3.6 x 10(6) U of rBoIFN-alpha I1/kg of body weight. The IFN action was monitored by measuring 2',5'-OAS activity of blood mononuclear leukocytes beginning 6 days before and ending 24 hours after IFN treatment. The 2',5'-OAS activity in the blood mononuclear leukocytes sharply increased 24 hours after IFN treatment, indicating response to IFN. The alveolar macrophages collected from the same calves 24 hours after IFN administration also had increased 2',5'-OAS activity, compared with alveolar macrophages from the same calves collected 6 days before treatment. Increased 2',5'-OAS activity indicates: a possible mechanism of IFN action in cattle that may be responsible for viral yield reduction; potential use of high enzyme activity as a marker for IFN induction; and potential use of 2',5'-OAS activity as a marker for determining effects of IFN on bovine macrophages and other cells of the bovine immune system.

Endometrial surface and secretory alterations associated with embryonic mortality in gilts administered estradiol valerate on days 9 and 10 of gestation.

Administration of estrogen to gilts on Days 9 and 10 of pregnancy results in total embryonic loss by Day 18. The present study examined changes in the uterine endometrial surface and secretion during conceptus attachment in control and estrogen-treated (Days 9 and 10) pregnant gilts. Gilts were unilaterally hysterectomized on either Days 12 and 14 or Days 16 and 18 of gestation. Uterine horns were flushed with saline and conceptuses were evaluated. Intact conceptuses were recovered from all control gilts, whereas estrogen-treated gilts contained normal intact conceptuses only on Day 12 of gestation. Antiviral activity, which reflects conceptus viability, was reduced (p less than 0.01) in uterine flushings after Day 14 in estrogen-treated gilts. Culture of endometrial explants with [3H]glucosamine revealed several glycoproteins that are synthesized during the period of conceptus attachment; however, no difference in glycoprotein synthesis between treatment groups was detected by analysis with two-dimensional PAGE and fluorography. Analyses of the uterine epithelium by scanning and transmission electron microscopy demonstrated that estrogen administration caused an alteration in the uterine surface, a thinning of the uterine epithelial glycocalyx, and a reduction of cationic ferritin binding to the microvilli of the uterine epithelium. Results indicate that conceptus mortality after early administration of estrogen is associated with alterations in the uterine endometrial surface during the period of conceptus attachment in the pig.

Expression of antiviral activity and induction of 2',5'-oligoadenylate synthetase by conceptus secretory proteins enriched in bovine trophoblast protein-1.

Establishment of pregnancy in cattle has been proposed to depend on production of a conceptus protein, bovine trophoblast protein-1 (bTP-1), which has a high degree of sequence homology with bovine interferon-alpha (bIFN-alpha), especially the alpha II subfamily. A preparation of bovine conceptus secretory proteins enriched for bTP-1 has antiviral and physico-chemical properties similar to other bIFN-alpha. Antiviral activity is initially detectable in uterine flushings on Day 14 of pregnancy, when the conceptus measures 4-5 mm in length, and increases as the conceptus elongates through Day 18. Day 17 conceptuses produce more than 10(6) U antiviral activity during 24 h of culture. All IFNs induce the enzyme 2',5'-oligoadenylate synthetase, which catalyzes production of 2',5'-oligo(A), which in turn is involved in antiviral and growth inhibitory effects of IFNs. This enzyme activity is induced in Madin-Darby bovine kidney cells by the partially purified bTP-1 preparation similarly to IFN-alpha, -beta, and -gamma. Likewise, the partially purified bTP-1 and bIFN-alpha 1 induce 2',5'-oligo(A) synthetase activity in monolayers of endometrial epithelial and stromal cells. Compared to epithelial cells, stromal cells have higher baseline activity of 2'-5'-oligo(A) synthetase activity (p less than 0.01) and show a greater degree of induction in the presence of either the partially purified bTP-1 or bIFN-alpha 1 (p less than 0.01). Also, 2',5'-oligo(A) synthetase of endometrial stromal cells is induced to a greater degree by our enriched bTP-1 preparation than by bIFN-alpha 1 (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Interferon and 2',5'-oligo(A) synthetase activities in serum and blood mononuclear leukocytes of cattle after injection of bovine interferon-alpha 1.

Cell extracts that were prepared from blood mononuclear leukocytes from 66 samples obtained from 6 clinically normal calves contained mean 2',5'-oligoadenylate (2',5'-oligo[A]) synthetase activity sufficient to synthesize 186 +/- 82 pmol of 2',5'-oligo(A)/h/10(6) cells. Calves had no measurable serum interferon (IFN) activity. Five calves were given IM injections of 10(4), 10(5), 5 x 10(5), 10(6), and 10(7) U of bovine IFN-alpha 1/kg of body weight at 2-week intervals. Five dosing sequences were used with a 5 x 5 Latin square design so that each calf received each dose once. Activity of 2',5'-oligo(A) synthetase increased at 24 hours in response to all dosages of IFN and then declined following first-order kinetics, with an apparent half-life (t1/2) of 2.1 +/- 0.5 days. The area under the concentration-time curve for 2',5'-oligo(A) synthetase increased with dose of IFN more rapidly than did peak response. Serum IFN that was measured at 1-day intervals following administration of IFN was consistently measurable only at dosages above 10(6) U of IFN/kg. The t1/2 for circulating IFN was 12.4 +/- 1.0 hours. Over all dosages, increases in 2',5'-oligo(A) synthetase activity were measurable for 3.5 days longer than were increases in IFN following IM injection of IFN. None of the calves developed detectable anti-IFN antibodies.

Induction and measurement of 2',5'-oligoadenylate synthetase in Madin-Darby bovine kidney cells and in cattle.

2',5'-Oligoadenylate [2',5'-oligo(A)] was separated from 14C-labeled nucleosides produced in the 2',5'-oligo(A) synthetase assay by using 100-microliters columns of Dowex 1. No detectable nucleoside remained on the column after elution with 20 column volumes of water, whereas less than 1% of oligonucleotides were eluted from the column. At least 99% of oligonucleotides were eluted from the column with 1 M NaCl, pH 2. The major product had properties consistent with 2',5'-oligo(A). Exposure to alpha-1 bovine interferon (IFN) caused an increase in cellular 2',5'-oligo(A) synthetase activity which was proportional to the concentration of IFN in the medium up to 10(4) U of IFN per ml and then leveled off at about 15 X control activity. Under the assay conditions used, 2',5'-oligo(A) synthetase activity was directly proportional to the amount of cell extract over a 10-fold range. Cattle inoculated with IBR/BVD/PI-3 modified live virus vaccine showed an increase in 2',5'-oligo(A) synthetase activity in peripheral blood mononuclear leukocytes which persisted for at least 3 days postvaccination. Intramuscular injection of cattle with IFN caused a similar increase in 2',5'-oligo(A) synthetase activity. Changes in 2',5'-oligo(A) synthetase activity should be of value in (i) assessing the response of cattle to experimental viral infections or inoculations with viral vaccines or IFN or (ii) indicating a possible viral etiology in disease.

Studies on the origin of the alpha-haemolysin produced by Escherichia coli.

Synthesis of alpha-haemolysin by Escherichia coli was proportional to the amount of meat-broth factor present in the medium and not to bacterial growth. The meat-broth component required for the synthesis of haemolysin was found to have several physical and chemical properties in common with alpha-haemolysin itself. Both molecules are trypsin-sensitive, acidic substances with similar elution volumes when subjected to gel-filtration chromatography with Sepharose 6B. Isotopic labelling experiments in which the bacteria were grown on 14C-labelled meat broth showed that partially purified haemolysin preparations contained molecules of mammalian origin. Similar experiments in which 14C-glucose was used to label bacterial molecules showed that little or no material of bacterial origin was present in the haemolysin preparation. These data suggest that alpha-haemolysin may be produced by bacterial modification of a molecule present in meat broth rather than by synthesis de novo within the bacterial cell.

Pathogenesis of edema disease in swine: pathologic effects of hemolysin, autolysate, and endotoxin of Escherichia coli (O141).

Hemolysin, cell-free autolysate, and lipopolysaccharide (LPS) prepared from Escherichia coli (O141) were parenterally administered to 113 weaned pigs. Both the hemolysin and the cell-free autolysate were crude preparations which probably contained several biologically active substances. Pigs in all groups which die less than 72 hours after injection had similar gross and microscopic lesions. The pigs which survived (chronically affected pigs) were killed 3 to 12 days after injection. Of the pigs that lived more than 72 hours after injection, those given hemolysin and autolysate had generalized vascular myolysis and fibrinoid necrosis, whereas those given LPS had morphologically normal blood vessels. The vascular changes produced by hemolysin and autolysates of E coli (O141) were the same as the histologic angiopathy of naturally occurring edema disease of pigs. The LPS produced acute lesions of endotoxin shock in the pigs, but did not produce the angiopathy characteristic of edema disease. Typical clinical signs of naturally occurring edema disease were not a consistent observation in any of the treatment groups.

Properties of the Hemolytic Activities of Escherichia coli.

Some properties of the cell-free and cell-associated hemolysins of Escherichia coli were studied. Several strains of E. coli that were isolated from intestines of pigs with edema disease produce large quantities of cell-free hemolysin when grown in the presence of an extract of meat. The component of meat that stimulates production of cell-free hemolysin is not extracted by lipid solvents and is not dialyzable. The cell-free hemolysin is an acidic substance that occurs in two forms. It is inactivated by trypsin but not by lecithinase, lysozyme, ribonuclease, or deoxyribonuclease, shows optimum activity between pH 7 and 8, and requires calcium ion for activity. It does not appear to be an enzyme. The kinetics of the lytic reaction are most consistent with the hypothesis that one molecule of cell-free hemolysin is sufficient to lyse one erythrocyte and that it is inactivated in the lytic reaction. The cell-free hemolysin does not sufficiently damage the cell during the prelytic period to cause lysis after the hemolysin-calcium-erythrocyte complex has been disrupted. The cell-associated hemolysin was not separated from the cell by autolysis, freezing, sonic treatment, or treatment with trypsin or lysozyme. It appears to be closely associated with the metabolic status of the cell. Organisms that are highly hemolytic under usual conditions of assay immediately lose most of their hemolytic capability in the presence of sodium cyanide, streptomycin, nalidixic acid, and rifampin.