Edward A. Havell

Production of High-Titered Interferon in Cultures of Human Diploid Cells

The effect of incubation with interferon prior to the stimulation of interferon production (priming) and of sequential treatment with cycloheximide and actinomycin D (superinduction) on the interferon yield from polyinosinic-polycytidylic acid (poly I·poly C)-induced diploid human foreskin cell cultures (FS-3 strain) was studied. Suitable priming with interferon produced, on the average, about an eightfold increase over the control yield, with a greater increase noted on some occasions when the control interferon yield was very low. Under the optimal conditions carefully defined in our experiments, superinduction produced about a 100-fold increase over the average control yield, resulting in interferon yields of about 10,000 reference units from cultures containing about 106 cells. Combined superinduction and priming did not produce yields markedly higher than obtainable by superinduction alone. Essentially similar results were obtained in cultures of human embryonic kidney cells and in FS-3 cells stimulated with other double-stranded polynucleotide inducers. However, stimulation of cells with certain concentrations of a mixture of diethylaminoethyl-dextran and poly I·poly C altered the interferon response; the yield was considerably higher than in cells stimulated with poly I·poly C alone, but it could not be markedly increased further by superinduction.

Synthesis of two distinct interferons by human fibroblasts

Abstract Human interferon species Le and F can be distinguished on the basis of their distinct antigenic, biological, and physicochemical characteristics. Until now, Le interferon was thought to be produced only in cells of lymphoid origin. This study demonstrates that cultures of human fibroblast cell strains GM-258 and FS-4 produce both F and Le interferons. Le interferon constituted up to about 20% of the total interferon activity produced in fibroblast cultures after stimulation with Newcastle disease virus or vesicular stomatitis virus. In contrast, only F interferon was detected in preparations obtained after induction with polyinosinate-polycytidylate. These results indicate that the Le interferon gene is inducible in cells of nonlymphoid origin and that the expression of this gene depends on the nature of the inducing agent.

Characteristics of human lymphoblastoid (Namalva) interferon.

Interferon derived from the human lymphoblastoid cell line, Namalva, was fractionated by antibody affinity chromatography into two antigenically distinct interferon subspecies. At least 13% of the total Namalva interferon activity possessed the F antigenic determinant found on human interferon derived from fibroblast cultures, while the bulk of the Namalva interferon activity had the Le antigenic determinant characteristic for human leukocyte interferon. The separated Le and F subspecies of Namalva interferon differed in the degree of their heterospecific activities on bovine cells. The Le moiety resembled crude leukocyte interferon in that it was highly active in bovine cells. The F component of Namalva interferon showed a lower degree of activity in bovine cells, thus resembling crude fibroblast interferon. When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and by isoelectric focusing, crude Namalva interferon qualitatively resembled crude leukocyte interferon.

Characterization of Escherichia coli Type 1 Pilus Mutants with Altered Binding Specificities

PCR mutagenesis and a unique enrichment scheme were used to obtain two mutants, each with a single lesion in fimH, the chromosomal gene that encodes the adhesin protein (FimH) of Escherichia coli type 1 pili. These mutants were noteworthy in part because both were altered in the normal range of cell types bound by FimH. One mutation altered an amino acid at a site previously shown to be involved in temperature-dependent binding, and the other altered an amino acid lining the predicted FimH binding pocket.

ANTIGENIC, PHYSICOCHEMICAL, AND BIOLOGIC CHARACTERIZATION OF HUMAN INTERFERONS *

It has long, been known that interferons exert some degree of species specificity and that s t ruchral differences exist among interferons of different species. Antisera raised against interferons from one animal species generally failed to neutralize the activity of interferons made in cells of other species.’ Only much later did it become known that antigenic differences could also exist among interferons produced in different cell types of the same species. The distinct antigenic specificity of two mouse interferons was first demonstrated by Youngner and Salvin2 who found that an interferon present in the serum of sensitized mice after injection with tuberculin was not neutralized by antibodies raised against virus-induced mouse L-cell interferon. The authors named the interferon made by immune stimulation “Type 11” interferon, as opposed to conventional or “Type I” mouse interferon. More recently it was shown that human interferons made in culturesof buffy coat cells (“leukocyte” interferon) and in diploid cell strains (“fibroblast” interferon) are dissimilar in many respects. First of all, sera made against fibroblast interferon, while neutralizing the activity of homologous interferon, completely failed to neutralize leukocyte interfer~n.~.‘ Furthermore, the interferons present in the two types of preparations were found to differ in a number of physicochemical characterist i c ~ . ~ . ~

Suppression of human interferon production by inhibitors of glycosylation

Abstract Treatment of diploid human foreskin cell cultures with either 2-deoxy- d -glucose or d -glucosamine resulted in dose-dependent inhibition of interferon production induced with polyinosinate-polycytidylate [poly(I) · poly(C)]. This inhibitory action was readily demonstrable even if the addition of the inhibitor was delayed until 1.5 hr after induction. The appearance of intracellular interferon activity was also inhibited, suggesting that synthesis of biologically active interferon, and not its secretion from the cell, was the main target of the inhibitory action. The inhibitory action of 2-deoxy- d -glucose on interferon production was completely reversible by d -mannose while the action of d -glucosamine was only slightly affected by the addition of mannose. Interferon produced in the presence of a partially inhibitory concentration of 2-deoxy- d -glucose was more heat-labile than control interferon. Residual active interferon made in the presence of d -glucosamine was less efficiently neutralized by anti-interferon antibody than control interferon. The results are compatible with the idea that the two inhibitors suppress the production of biologically active interferon by virtue of their interfering with the proper glycosylation of the molecule.

Host and Bacterial Factors Involved in the Innate Ability of Mouse Macrophages To Eliminate Internalized Unopsonized Escherichia coli

ABSTRACT In an effort to better understand genetic and cellular factors that influence innate immunity, we examined host and bacterial factors involved in the nonopsonic phagocytosis and killing ofEscherichia coli K-12 by mouse macrophages. Unelicited (resident) peritoneal macrophages from five different mouse strains, BALB/c, C57BL/6, CD-1, C3H/HeJ, and C3H/HeN, were employed. Additional macrophage populations were obtained from CD-1 mice (bone marrow-derived macrophages). Also, for BALB/c and C57BL/6 mice, peritoneal macrophages elicited with either thioglycolate or proteose peptone, bone marrow-derived macrophages, and macrophage-like cell lines derived from the two strains were employed. Two E. coli K-12 strains that differed specifically in their abilities to produce type 1 pili containing the adhesive protein FimH were examined. The parameters used to assess macrophage bacteriocidal activity were (i) the killing of internalized (gentamicin-protected)E. coli during the approximately 4-h assay and (ii) the initial rate at which internalized E. coli were eliminated. Data on these parameters allowed the following conclusions: (i) unelicited or proteose peptone-elicited peritoneal macrophages were significantly better at eliminating internalized bacteria than thioglycolate-elicited peritoneal macrophages, bone marrow-derived macrophages, or macrophage cell lines; (ii) the host genetic background had no significant effect upon the ability of unelicited peritoneal macrophages to kill E. coli (even though the mouse strains differ widely in their in vivo susceptibilities to bacterial infection); and (iii) the FimH phenotype had no significant effect uponE. coli survival once the bacterium was inside a macrophage. Additionally, there was no correlation between the bacteriocidal effectiveness of a macrophage population and the number of bacteria bound per macrophage. However, macrophage populations that were the least bacteriocidal tended to bind higher ratios of FimH+ to FimH−E. coli. The effect of gamma interferon, fetal calf serum, and the recombination proficiency of E. coli were examined as factors predicted to influence intracellular bacterial killing. These had no effect upon the rate of E. coli elimination by unelicited peritoneal macrophages.

Influence of extracellular bactericidal agents on bacteria within macrophages.

ABSTRACT We employed gentamicin-sensitive and -resistant derivatives of Escherichia coli in a macrophage phagocytosis assay that compared λ bacteriophage and gentamicin as extracellular bactericidal agents. Colony counts and direct microscopic examination of phagocytized E. coli supported the conclusion that gentamicin entered macrophages, even at low concentrations, and contributed to their bactericidal activity. Also, two E. coli strains differing in the ability to express the adhesin of type 1 pili (FimH) were distinguishably different in intracellular survival when λ was used as the extracellular killing agent but were indistinguishable when gentamicin was employed.

The Mucosal Phase of Listeria Infection

Listeria monocytogenes is an enteroinvasive bacterial pathogen of man and animals. Listeriae have been shown capable of infecting the host by translocating from the intestinal lumen through Peyers Patches (PP), however, results of experiments now indicate that these facultative intracellular parasites may also translocate through PP-independent routes. With regards to this, on occasion we observed that listeriae were absent from the PP of mice inoculated intragastrically with L. monocytogenes, but were present in the mesenteric lymph nodes of these same mice. These observations suggested that PP were not necessary for listerial translocation from the intestinal lumen. Two experimental approaches were used to determine whether luminal listeriae could indeed infect the host through PP-independent routes. First, since it is known that: 1) following the intragastric inoculation of L. monocytogenes, listeriae rapidly transit the length of the gastrointestinal tract and reside in the colonic lumen for up to a week, 2) the colon lacks PP, and 3) the descending colon and rectum are drained exclusively by the caudal lymph node (CLN), it was determined whether colonic listeriae could access the CLN. Inoculation of listeriae into the rectum of mice resulted in the infection of the CLN which indicated that PP were not required for listerial translocation. Second, since germfree SCID mice lack PP, it was determined whether listeriae could translocate from the intestinal lumen and infect these immunoincompetent mice. Shortly after the intragastric inoculation of L. monocytogenes into germfree SCID mice, listeriae were found in the mesenteries, livers and spleens. These results also indicate that PP are not required for listerial translocation from the intestinal lumen. One possible route of translocation from the intestinal lumen might occur by listeriae entering enterocytes. Results were obtained showing that listeriae were capable of entering cultured mouse small intestine enterocytes. Internalized listeriae were observed to multiply and spread intracellularly between enterocytes.

Characterization of a subspecies of mouse interferon cross-reactive on human cells and antigenically related to human leukocyte interferon

Abstract Mouse interferon preparations from various virus-induced cell cultures exhibited some antiviral activity (about 0.1% of homologous cell titers) on human cells. When analyzed by SDS-polyacrylamide gel electrophoresis the activity of mouse interferon on mouse cells migrated as two bands with activity peaks at 38,000 and 22,000 daltons, while the activity on human cells was detectable only in the faster migrating portion of the lower molecular weight band, at about 21,000 daltons. When the mouse interferon preparation was chromatographed on a column of immobilized anti-human leukocyte interferon antibodies, the specifically retained interferon was found to migrate in SDS-polyacrylamide gels as a 21,000-dalton species which exhibited similar degrees of activity on both mouse and human cells. Thus, mouse interferon preparations contain a low molecular weight interferon species which is both active on mouse cells and is responsible for all the heterologous antiviral activity on human cells and is antigenically related to human leukocyte interferon. These data suggest significant structural similarities between the active cores of certain interferons from phylogenetically diverse animal species.