The comparative effects of actinomycin D, imuren and hydrocortisone on interferon and antibody formation in mice]

A single dose of actinomycin-D (2.5 mg/kg body wt) reduced the interferon formation 16-fold in comparison with control; as to the antibody production - it was completely abolished. Hydrocortiosone (100 mg/kg) also diminished the interferon production 16-fold, but had no effect on the production of antibodies against the virus. The effect of hydrocortisone on the antiviral immunity was similar to that of the antilymphocytic serum, whereas the action of actinomycin-D resembled the effect of the X-ray irradiation or imuran. The data obtained were consistent with the suggestion on the heterogeneity of cells, participating in the interferon production in the organism.     

Regulatory effects of macrophage-secreted factors on T-lymphocyte colony growth.

Human fibroblast and leukocyte interferons were found to suppress lymphocyte mitogenesis induced by optimal doses of phytohemagglutinin and concanavalin A. In certain situations (low doses of mitogen and/or low doses of interferon), however, interferon significantly enhanced mitogenesis. In experiments using varying concentrations of interferon, dose-response curves with different slopes were obtained for fibroblast and leukocyte interferons. The effect of interferon was apparently exerted during early stages of the lymphocyte cell cycle. There was no inhibitory effect of interferon if the lymphocytes were washed with medium before being exposed to mitogen. Interferon increased the binding of radiolabeled mitogens to cells. The results suggest that the immunological effects of interferon are consequences of actions on lymphoid cells. Fibroblast and leukocyte interferons seem to have different modes of action, or to bind differently to target cells. Possible mechanisms for the suppressive and enhancing effects of interferons on lymphoid cells are discussed.     

A mouse model of the pathogenesis and postexposure prophylaxis of rabies

A mouse model for the study of postexposure prophylaxis of rabies was established. Mice injected intramuscularly with a street strain of rabies virus were significantly protected from death by five daily 0.2-ml doses of inactivated rabies vaccine of chick embryo cell culture origin initiated immediately or 3 hr after infection. In these mice, a large amount of circulating interferon was induced as early as 1 hr after the first dose of vaccine and lasted until at least 12 hr but no such amount of interferon was induced by additional doses of vaccine. Serum antibody was first detected in the mice on day 6. It was noted that some of the surviving mice manifested an ataxia or paralysis of the legs. Increasing mortality rates were shown in mice treated with decreasing doses of the vaccine. Passive protection tests using concentrated IgG and IgM antibodies with equivalent neutralization titers showed that IgG antibody gave total protection when given 24 hr before the infection, while it was almost totally ineffective in reducing the mortality when given 2 days or more after infection. IgM antibody did not protect the mice even when given 24 hr before infection. These results suggest that interferon production is more important than antibody production in the initial stages of protection by postexposure vaccination. However, the mechanisms of postexposure prophylaxis in this model could not be explained only by the interferon produced by the vaccine and the possible contributions of additional mechanisms were suggested.     

Hepatitis C virus: current understanding and prospects for future therapies.

Hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide and the leading indication for liver transplantation. The hallmark of the disease is its propensity to evolve into chronicity, probably because viral heterogeneity allows the virus to escape immune-mediated neutralization. Treatment with interferon alpha (IFN-alpha) has been disappointing, but higher and more frequent doses, and combination therapies, including nucleoside analogs, might lead to improved suppression of HCV RNA levels. Molecular analysis of HCV before and during treatment has indicated that high viral RNA levels and the presence of HCV genotype 1 are independent predictors of poor treatment outcome. New antiviral agents in development include inhibitors of HCV replicative enzymes, such as protease, helicase and polymerase, as well as several genetic approaches, such as ribozymes and antisense oligonucleotides. The main hindrance to drug development for hepatitis C is the lack of a small animal model or a productive tissue culture system for assessing drug action.     

Suppression of interferon synthesis during the "graft-versus-host" reaction in F1(CBAXC57BL/6) mice]

The development of the graft-versus-host reaction (GVHR) in the F1(1CBA X C57BL/6 hybrid mice after the transplantation of spleen cells from the C57BL/6 parent donor resulted in a strong inhibition of the serum interferon production induced by the intraperitoneal injection of the Newcastle disease virus. In vitro with the mouse bone marrow cells during the development of the GVHR the interferon response was first reduced and then disappeared completely. The described phenomenon could therefore serve as an index of the development of the GVHR.     

Impaired interferon status in children with acute respiratory infection and its correction with bifidumbacterin-forte

The state of interferon status was studied in 46 hospitalized children: 33 patients with complicated forms of acute respiratory virus infection (ARVI), such as pneumonia, bronchitis, etc., and 13 patients with vegetovascular dystonia (used as a comparison group). The study revealed that in patients with acute infections of the upper and lower respiratory tract considerable changes in their interferon system were registered. Children with ARVI were treated with Bifidumbacterin forte, a probiotic preparation, in large doses. Bifidumbacterin forte was found to produce a regulatory effect on the interferon system by enhancing the induction of alpha- and gamma-interferon and decreasing the production of serum interferon. The experience of using Bifidumbacterin forte in large doses proved that the preparation was well tolerated and could be used for the correction of interferon status.     

Effect of prodigiozan, levamisole and methyluracil on delayed hypersensitivity against a background of immunosuppressor use

Immunostimulants, such as prodigiosan, levamisol and methyluracil, as well as their combinations with imuran, prednisolone or cyclophosphamide were studied for their effect on the delayed type hypersensitivity (DTH) induced by dinitrofluorobenzene alcohol solutions applied in challenge doses to the floor of the auricle of mice. It was shown that the immunostimulants did not affect the DTH in intact mice. In mice treated with imuran the DTH was significantly increased only by prodigiosan. Prednisolone used for a prolonged period before and after the sensitization and cyclophosphamide administered 24 hours before the sensitization increased the DTH. According to the literature data it was connected with T-suppressor inhibition. With the use of cyclophosphamide it was also connected with B-suppressor inhibition. Under such conditions the DTH was decreased to the control level by prodigiosan after the use of prednisolone or by levamisol after the use of cyclophosphamide. This was probably associated with increasing of the suppressor effect of the macrophages and activation of the T-suppressor effect by levamisol. Methyluracil had no effect on the DTH.     

The ppp(A2'p)nA and protein kinase systems in wild-type and interferon-resistant Daudi cells

Daudi cells, a human lymphoblastoid line, are exceptionally sensitive to the growth inhibitory effects of interferon, 1 unit/ml being sufficient to inhibit cell growth. In addition, interferon treatment of these cells severely inhibits the incorporation of exogenous thymidine into DNA and causes cells to accumulate in the G1(G0) at the expense of the S phase of the cell cycle. The possible involvement of ppp(A2'p)nA(n = 2 to less than or equal to 4) in these effects has been investigated. No (less than 1 nM) ppp(A2'p)nA or (A2'p)nA or alternative products of the ppp(A2'p)nA synthetase [e.g. NAD (2'pA)2] were detected in interferon-treated cells. In addition no evidence was obtained for the occurrence of ppp(A2'p)nA-mediated ribosomal RNA cleavage in these cells even after several days of treatment with relatively high doses of interferon. A line of Daudi cells which is resistant to all three of the above effects of interferon was selected. The wild type and resistant lines were compared with respect to the ppp(A2'p)nA and interferon and double-stranded RNA (dsRNA)-mediated protein kinase systems. The resistant line was not receptor-negative as it responded to interferon by the production of elevated levels of the ppp(A2'p)nA synthetase similar to those observed in extracts from wild-type cells. There was no detectable difference between the lines in the levels of the (2'-5')phosphodiesterase responsible for the degradation of ppp(A2'p)nA. There was, however, about a twofold increase in the ppp(A2'p)nA-dependent endoribonuclease activity in response to interferon with extracts from the wild-type but not the resistant cells. In addition, although the dsRNA-dependent protein kinase activity increased in both types of cell there was a striking reduction in the level of protein phosphorylation in general in response to interferon with material from the wild-type but not the resistant cells.     

Post-Transcriptional Control of Interferon Synthesis

Low to moderate doses of cycloheximide had a stimulatory effect on interferon production in rabbit kidney cell cultures treated with double-stranded polyinosinate-polycytidylate (poly I:poly C). A very marked stimulation occurred in the presence of a dose of cycloheximide inhibiting amino acid incorporation into total cellular protein by about 75%. Higher doses of cycloheximide caused a shift in interferon release towards later intervals and a gradual decrease in the overall degree of stimulation. An even greater increase in the amount of interferon produced was observed if cells were treated with cycloheximide for only 3 to 4 hr immediately after their exposure to poly I:poly C. Under the latter conditions, a rapid burst of interferon production occurred after the reversal of cycloheximide action. Treatment with a high dose of actinomycin D before the reversal of cycloheximide action caused a further increase and a marked prolongation of interferon production. It is postulated that inhibitors of protein synthesis suppress the accumulation of a cellular regulatory protein (repressor) which interacts with the interferon messenger ribonucleic acid mRNA and thereby prevents its translation. Therefore, active interferon mRNA can apparently accumulate in rabbit kidney cells which, after exposure to poly I:poly C, are kept in the presence of an inhibitor of protein synthesis. Some of this accumulated interferon mRNA can be translated during a partial block of cellular protein synthesis, but its most efficient translation occurs after the reversal of the action of the protein synthesis inhibitor.     

Failure of interferon alfa and tribavirin in rabies encephalitis.

OBJECTIVE--To test the effect of interferon alfa and tribavirin (ribavirin) in patients with rabies encephalitis. DESIGN--An open trial of chemotherapy and intensive care in patients with early rabies. SETTING--The intensive care unit of a Bangkok hospital. PATIENTS--Four conscious men with clinical rabies encephalitis. INTERVENTIONS--Rapid virological diagnosis of rabies. Treatment with intravenous and intraventricular injections of high doses of lymphoblastoid interferon alfa in three patients and tribavirin in one patient. Intensive care was given throughout. MAIN OUTCOME MEASURES--Rabies infection confirmed by antigen detection and virus isolation. Rabies neutralising antibody and specific IgM sought in serum and cerebrospinal fluid. Interferon concentrations monitored before and during treatment in three patients. RESULTS--Interferon alfa treatment produced high concentrations in serum and cerebrospinal fluid. All four patients died after 5 1/2 to 12 1/2 days of treatment with no evidence of virostatic or clinically beneficial effects from either treatment. CONCLUSION--Interferon alfa treatment is not effective in rabies encephalitis. The use of tribavirin warrants further study, possibly combined with new therapeutic methods.     

Inhibition of Mengo virus by interferon

Gauntt, Charles J. (The University of Texas, Austin), and Royce Z. Lockart, Jr. Inhibition of Mengo virus by interferon. J. Bacteriol. 91:176-182. 1966.-The inhibition of Mengo virus replication in L cells resulting from interferon was studied quantitatively. Interferon was titrated on L cells with Western equine encephalomyelitis (WEE) virus as the challenge virus. One protective unit (PU) of interferon is the least amount of interferon which prevents cytopathic effects when a large multiplicity of WEE virus is added subsequent to overnight incubation with interferon. Ten PU of interferon reduced the yields of Mengo virus by about 90%. Larger doses of interferon, up to 220 PU, caused no further reduction in the amount of virus produced. Plaque formation by Mengo virus was also reduced in number by about 85 to 90%, but could not be further reduced. The plaques which formed on interferon-treated cells were reduced in size. We were unable to obtain a virus population with increased resistance to interferon action by use of five successive growth cycles in interferon-treated cultures. Analysis of the cell population for the proportion of cells able to act as infectious centers revealed that incubation of cells with 10 PU of interferon decreased the proportion of virus-yielding cells by 80%. The yield of virus per virus-producing cell was decreased by 40 to 60%. Despite the reduction in yields, plaques, and infectious centers resulting from interferon, all doses of interferon failed to prevent the complete destruction of the cells. Experiments with puromycin indicated that the cytopathic effects observed in L cells infected with Mengo virus required that a virus-directed protein be synthesized between 4 and 5 hr postinfection. The evidence suggested, therefore, that the Mengo virus genome was able to code for new protein synthesis in the absence of the production of infectious virus.     

Down-regulation of the interferon receptor

The binding of 125I-labeled alpha A interferon to human lymphoblastoid Daudi cells decreased when these cells were incubated with unlabeled alpha or beta interferon. This decrease could not be accounted for by the occupancy of interferon receptors with unlabeled interferon and it apparently resulted from the loss or down-regulation of receptors. The binding activity gradually increased when Daudi cells were incubated in fresh medium after a treatment with interferon, but inhibition of protein synthesis with cycloheximide prevented this recovery. Treatment of Daudi cells with this inhibitor resulted in the loss of half the interferon binding activity within 5 h. These findings suggested that the interferon receptors turn over at a basal rate in interferon-free medium and at an increased rate in cells incubated with interferon. The dose-response for the down-regulation was investigated by treating Daudi cells with different concentrations of alpha interferon. Down-regulation was observed in cells treated with relatively low doses of interferon, sufficient to elicit a biological response. The synthesis of the enzyme (2',5')oligo(A) polymerase was induced at the lowest interferon concentrations tested which caused receptor down-regulation.     

Electrodissociation of high molecular weight complexes of interferon alpha during recycling isoelectric focusing

Natural human interferon alpha has been separated by selective ultrafiltration into low molecular weight components and the molecules exceeding 100K daltons. Interferon associated with a higher molecular weight fraction showed partial pH sensitivity and resisted dissociation after treatment with urea, mercaptoethanol, sodium chloride or significant changes in pH. However, interferon activity was released from high molecular weight components during recycling isoelectric focusing. Electrodissociation was carried out in 1% ampholytes for 574 watt-hours. The interferon activity was concentrated in a pH range of 6-6.5, whereas, the majority of proteins were generally found in a more acidic position. The dissociated interferon was neutralized by polyclonal antibody to human interferon alpha (IFN alpha) and showed no presence of pH labile form. A pH sensitivity of high molecular weight interferon (HMW-IFN) may reflect an aggregation phenomenon rather than intrinsic structural differences.     

Effect of interferon on growth and division cycle of Friend erythroleukemic murine cells in vitro

The administration of appropriate doses of interferon to cultures of Friend leukemia cells causes a pronounced inhibition of cell growth. Several lines of evidence indicate that this effect is due to interferon itself, rather than to unknown contaminants of interferon preparations. Autoradiograph analysis of growth parameters of Friend leukemia cells during treatment with interferon demonstrates that the rate of entry into the S phase, the percent decline of unlabeled mitoses, and the mitotic indexes are significantly lower in interferon- treated cell cultures than in control untreated cultures when tritiated thymidine was added 12 h after the administration of interferon. These data indicate that fractions of interferon-treated cell population are delayed in both G1 and in G2 phases of the cell cycle. This was confirmed by exact measurements of the length of the various phases of the cycle. The interferon-induced inhibition of growth of Friend leukemia cells is reversible after removal of the compound. Autoradiograph data obtained from control cultures and from cultures previously treated with interferon that had been washed free of interferon and reseeded in interferon-free medium, demonstrate that during the first 12 h after removal of interferon, a large majority of the cells previously treated with interferon had a deranged flow into the S phase, a high number of unlabeled mitoses, and a low mitotic index. These data provide further evidence for the above-mentioned prolongations of G1 and G2 phases of the cell cycle. All growth parameters tested reverted to normal values within 12 h after washing out interferon.     

Effect of estrogen deprivation on the reproductive physiology of male and female primates

The availability of CGS 16949A, CGS 20267 and CGP 47645, a series of aromatase inhibitors (AIs) having high specific activity and specificity, made possible this study wherein the need for estrogen (E) for regulating (a) follicular maturation/ovulation, luteal function and pregnancy establishment, and (b) testicular function of the bonnet monkey (Macaca radiata) has been examined. Generally these compounds, used in the range of 500 μg to 2.5 mg/day did not inhibit follicular maturation although they did reduce E levels. Although low doses had no effect on ovulation it appears that relatively high doses of CGS 20267 and CGP 47645 could be inhibiting it. Three oral doses of letrozole (CGS 20267, each dose of 2 mg) during the follicular phase resulted in the formation of multiple follicles in cycling females, and these could be ovulated by exogenous hCG (1000 IU) treatment. Although administration of AI during the early luteal phase had no effect on progesterone (P) production, it prevented pregnancy establishment. Whereas AI administration in the female had no significant effect on luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels (except at high drug dosages), it significantly increased serum testosterone (T) levels in the male. Sustained high levels of T (30–50 ng/ml) could be maintained for 100 days by administering 2.5 mg of CGP 47465 orally once in 5 days. Blockade of E synthesis in the male led to the disruption of testicular germ cell transformation, which in turn resulted in a significant reduction in sperm production. These studies with aromatase inhibitors in the monkey suggest that these compounds have a potential for use as fertility regulating agents in both the male and female primate.     

T-lymphocyte function and sensitivity to interferon in trisomy 21

Previous studies have shown that cells from subjects with trisomy 21 have enhanced sensitivity to the antiviral effects of interferon, presumably because of the location of the gene, IfRec, coding for the species-specific response to interferon on chromosome 21. Interferon is also known to have many other effects including the ability to inhibit the proliferation of many types of cells. To determine whether proliferating trisomic lymphocytes are more sensitive to the antiproliferative effect of interferon we have investigated, using healthy noninstitutionalized subjects with trisomy 21, the ability of interferon to inhibit the proliferation of lymphocytes stimulated with phytohemagglutinin P(PHA), concanavalin A (Con A), and tetanus toxoid. The trisomic subjects had normal numbers of peripheral blood leukocytes, and normal numbers and proportions of T and B lymphocytes. The production of interferon by PHA-stimulated trisomic T lymphocytes was normal. Trisomic lymphocytes also had normal proliferative responses to PHA and Con A. There were no differences between the inhibitory effects of interferon on the proliferation of PHA-stimulated trisomic and normal lymphocytes. However, trisomic lymphocytes stimulated with low doses of Con A did display significantly enhanced sensitivity to the antiproliferative effects of interferon. In contrast to normal lymphocytes, trisomic lymphocytes were not stimulated to proliferate by tetanus toxoid, and exposure to interferon resulted in enhancement, rather than inhibition, of DNA synthesis.     

Electron microscope analysis of the transplacental effect of azathioprine on the pancreas of albino mouse embryos

An electron microscopic investigation has been performed to study the transplantation effect of azathioprine (imuran) on the pancreas of the white mouse embryos. Azathioprine is administered to female mice at various time of pregnancy. The pancreas of 19-20-day-old embryos is studied. Azathioprine produces an unfavourable effect on the embryonal pancreacytes; it is manifested as some disorders in structure of organelles, appearance of the intracellular degeneration foci, disturbed maturation of the zymogen granules, premature secretion of zymogen into the acinar lumen during the prenatal period. In the insulocytes similar changes are observed in their organelles, as well as appearance of unusual in size alpha- and beta-granules. As a whole, the epithelium of the endocrine part of the gland is evidently more resistive to the preparation.     

Effect of T-activin on the production of immune interferon

A study was made of the effect of T-activin on the biosynthesis of immune gamma-interferon. It was shown that in 27% of patients with chronic nonspecific pulmonary diseases, production of gamma-interferon by lymphocytes was substantially reduced during exacerbation of inflammatory process in the lungs. It was discovered that T-activin was not an interferon inductor but enhanced its synthesis in patients with a low capacity of producing immune interferon even at small doses of interferon inductor. The preparation does not produce any effect on this process in normal subjects and in patients showing the normal level of gamma-interferon. Thus T-activin can be used for stimulation of interferonogenesis.     

Inhibition of parathormone-stimulated bone resorption by type I interferon

The effect of Type I interferon on bone resorption was studied by measuring its effect on parathormone-stimulated calcium release from neonatal murine calvaria in vitro. A pure human recombinant leukocyte interferon hybrid of the A and D subtypes was used, which has high antiviral activity on mouse cells. Calcium release was inhibited in a dose dependent fashion with 50% inhibition at about 10(-10) M or 600 U/ml, and the inhibition was reversible. The presence of interferon was required before or during the activation phase of the resorptive response, when the formation of osteoclasts from precursor cells would occur. When added to actively resorbing bone it had no effect. The data suggest that Type I interferon can inhibit the parathormone-regulated development of active osteoclasts, possibly by inhibiting osteoclast precursor differentiation.