Factors modifying host resistance to viral infection. 3. Effect of whole body x-irradiation on experimental encephalomyocarditis virus infection in mice.

The resistance of mice to encephalomyocarditis (EMC) virus was markedly decreased by prior exposure to whole body X-irradiation. In contrast to non-irradiated controls, the course of EMC virus infection in X-irradiated animals was characterized by (a) an enhanced mortality, (b) shortening of the incubation period, (c) higher levels of virus in the blood during the viremic phase and persistence of the viremia until death, (d) failure to develop detectable serum levels of neutralizing antibody, and (e) the earlier appearance and higher levels of virus in brain and heart tissue. The level of interferon in the serum during the course of infection was similar in both groups. The administration of relatively small quantities of anti-EMC virus neutralizing antibody to X-irradiated mice during the early phases of the infection with EMC virus restored their resistance to levels comparable to nonirradiated animals. An alteration of local organ defense mechanisms in the central nervous system could not be demonstrated. It is proposed that (a) the inability of the X-irradiated animal to elaborate specific neutralizing antibody was a critical determinant in their failure to clear the viremia, (b) this increase in the level and duration of the viremic phase resulted in the exposure of target organs to a greater inoculum of virus, and (c) the enhanced mortality observed in irradiated mice reflected this greater target organ involvement. The experimental model presented, therefore, suggests that the immunologic response is a critical determinant of host resistance during this primary systemic virus infection.

Effect of cytochalasin B on the adhesion of mouse peritoneal macrophages.

The adhesion of normal mouse macrophages to glass surfaces was reduced by nontoxic levels (1-50 mug/ml) of cytochalasin B in combination with a centrifugal force (1,000-8,000 g). Macrophages nonspecifically activated by Corynebacterium acnes were also detached by this treatment, but less effectively. The effects of cytochalasin B treatment on these cells were shown to be reversible. After detachment, the cells reattached to glass, appeared morphologically normal, and behaved like untreated cells as judged by adhesion, acid phosphatase levels, and phagocytosis. The effect of cytochalasin B on several parameters of phagocytosis by normal macrophages was also examined. The results demonstrate that cytochalasin B can be used to detach macrophages from surfaces and suggest a functional relationship between phagocytosis and macrophage adhesion to surfaces. Furthermore, the effect of cytochalasin B on adhesion of phagocytic cells provides a probe for further investigation of the adhesion of cells to surfaces.

Transfer of interferon-producing macrophages: new approach to viral chemotherapy.

Mice were protected from infection with Semliki Forest virus and encephalomyocarditis virus by the transfer of peritoneal macrophages that were stimulated to produce interferon in vitro by exposure to a nonreplicating virus. This method of therapy was also utilized in animals infected with encephalomy-ocarditis virus after onset of clinical signs. Of these animals 40 percent recovered, but only 9 percent of the control group recovered.

Fetal response to viral infection: interferon production in sheep.

When virus was inoculated intravenously during the third trimester, the gestating ewe produced only low amounts of serum interferon, whereas the fetal lamb had the capacity to produce extremely high amounts. There was no evidence of transplacental transfer of interferon between mother and fetus in either direction.

Effect of interferon administration on pulmonary osteogenic sarcomas in an experimental murine model.

The iv inoculation of a suspension of osteogenic sarcoma cells induced metastatic tumor nodules in the lungs of C57BL/6 mice. The administration of 50,000-100,000 U of interferon daily for 7 days strikingly reduced the tumor mass in the lung and the number of tumor nodules present in histopathologic sections when the interferon treatment was initiated immediately after tumor cell inoculation. In some animals the development of any detectable metastatic lesion was completely prevented. Extending the therapy form 7 days to 21 days failed to improve the protective effect. Interferon therapy delayed until 7 days after tumor cell inoculation had no effect. These findings indicate the effectiveness of exogenous interferon in this murine osteogenic sarcoma model when interferon treatment is initiated within 1 hour of tumor cell inoculation, but not when it is delayed until tumor nodules are established in the lungs.

Antitumor activity of interferon against murine osteogenic sarcoma cells in vitro.

Murine interferon inhibited the growth of a continuous line of osteogenic sarcoma (OGS) cells in tissue culture. Inhibition of tumor cell growth by interferon was demonstrated by: a) decreased colony formation in soft agar, b) suppression of clone formation in liquid medium, and c) reduction of tumor cell counts in monolayer cultures. This inhibition of cell growth was further documented by suppression of [3H]thymidine uptake by OGS cells exposed to interferon, which suggested inhibition of DNA synthesis of tumor cells. Exposure of tumor cells for 4 hours, 24 hours, and 2,3,4,6, and 8 days demonstrated greater activity with prolonged exposure to interferon. Inhibition of cell growth was significantly greater for OGS cells than for normal mouse embryo fibroblasts. Finally, the antitumor activity of the interferon preparation could be reversed by anti-interferon antibody.

Inhibition of murine osteogenic sarcomas by treatment with type I or type II interferon.

Interferon was used to treat C57BL/6 female mice inoculated with a continuous line of murine osteogenic sarcoma cells. A short 7-day course of 30,000--60,000 U/day of tpe I interferon either completely inhibited or delayed the appearance of tumors in experimental animals. The therapeutic efficacy of type I interferon was compared with murine serum that contained type II interferon as well as other lymphokine activity. Tumor development was strikingly inhibited in animals treated for 7 days with serum containing only 600 U of type II interferon. Inhibition of tumor development was thus achieved with 100-fold less interferon than that required with type I preparation.

Failure of interferon to inhibit plutonium-induced osteosarcomas in mice.

The injection of murine interferon three times weekly in dose levels of 1,250, 5,000, or 20,000 U produced no significant antitumor effect against primary 239Pu-induced osteosarcomas in C57BL/6J mice. The interferon treatment was begun 94 days after the plutonium injection, which is well before the radiographic or microscopic appearance of neoplasia, and was continued until the moribund state or death. The average radiation dose accumulated by the skeleton at the time of first treatment was approximately 300 rad. The largest dose of interferon studied, 20,000 U/injection, was approximately 3 X 10(6) U/m2 of body surface, or 10(6) U/kg body weight.

Alteration of murine mammary tumor metastasis and growth by cytomegalovirus infection.

Host resistance to the development of metastatic lesions is complex and involves both lymphocyte and macrophage functions. Studies in both humans and animals have suggested that cytomegalovirus infection may alter these components of the defense mechanism of the host. In the present study, an experimental model was developed to determine whether cytomegalovirus infection would affect host resistance to the establishment of metastatic tumor nodules in the lungs of C3H mice after i.v. inoculation of a single-cell suspension of mammary tumor cells. The number of tumor nodules in the lungs, the lungs-heart/body weight ratio, and the mean day of death were determined in control animals inoculated i.v. with 10(6) mammary tumor cells and compared with groups of animals also receiving a sublethal i.p. inoculum of murine cytomegalovirus (MCMV) (10(5) plaque-forming units) either 3 days before, on the day of, or 10 or 13 days after tumor cell inoculation. The results suggest a biphasic effect of virus infection on tumor development in the lung. A preexisting or concurrent MCMV infection suppressed tumor growth and prolonged life, while a MCMV infection later in tumorigenesis enhanced tumor growth and shortened survival. These data suggest that MCMV modulates host resistance to the development of metastatic tumor nodules and that this experimental model may be utilized to investigate further the relationship between virus-induced alterations of host defense mechanisms and tumor growth.

Interrelationships of interferon and immunity during viral infections.

Interferon is one determinant of host resistance. The immune responses, cellular or humoral, are other components. Cell-mediated responses appear to be involved in host resistance to certain viral infections, particularly the herpesvirus group and vaccinia virus. It is suggested that immune and interferon responses may complement one another and contribute to host resistance. The relative importance of each component depends upon the virus-host interaction. Finally, evidence has been presented which suggests that production of interferon as a result of antigen-sensitized cell interaction may further link these two components of the host response.

Intrauterine viral infection and the cell-mediated immune response.

In 8 of 23 infants with congenital central nervous system (CNS) anomalies, we observed an increased cell-mediated immune (CMI) response (lymphocyte blastogenesis) to one of six simultaneously tested viral antigens, without detection of concomitant elevation in humoral antibody to that virus. These data suggest possible intrauterine viral exposure in a significant number of anomalous infants and a possible role of that exposure in the pathogenesis of congenital CNS anomalies. The CMI response may offer another approach to the identification of etiologic agents of a significant number of birth defects.

Multiple sclerosis and elevation of cerebrospinal fluid vaccinia virus antibody.

Thirty percent of 33 patients with a clinical diagnosis of multiple sclerosis and an elevation of cerebrospinal fluid (CSF) immunoglobulin G/albumin ratio greater than 2 standard deviations above the mean had elevation of the CSF vaccinia neutralizing antibody titer. Elevated titers were found in 1 of 23 patients with non-multiple sclerosis neurologic diseases and in none of the normal control patients. No linear correlation was found between the magnitude of the relative or absolute CSF immunoglobulin G concentration and the magnitude of the CSF vaccinia neutralizing antibody titers.

Evaluation of central nervous system vaccinia antibody synthesis in multiple sclerosis patients.

A serum/cerebrospinal fluid (CSF) ratio method was used to determine whether elevated CSF vaccinia antibody titers in some patients with multiple sclerosis are the result of central nervous system antibody synthesis or of a leak in the blood-brain barrier. Nine of 20 multiple sclerosis patients were noted to have a depressed serum/CSF vaccinia antibody ratio and a normal ratio for poliovirus-I, an agent thought not to be involved in the pathogenesis of multiple sclerosis. These data suggest central nervous system synthesis of vaccinia neutralizing antibody. Vaccinia virus antigens may play an important direct or indirect role in the pathogenesis of multiple sclerosis.

Effect of acyclovir treatment on acute and chronic murine cytomegalovirus infection.

Murine cytomegalovirus (MCMV) is inhibited in vitro by 1 to 2 microM acyclovir. Therapy of a systemic MCMV infection in weanling mice with acyclovir was only minimally effective when drug was administered intraperitoneally, while oral administration by addition of acyclovir to the drinking water was highly efficacious in mice with disseminated MCMV. Effective therapy was characterized by reduction of virus titers in lung, liver, spleen, and kidney. In mice chronically infected with MCMV, treatment for 30 days with oral acyclovir eliminated or reduced virus titers in all target organs except the salivary gland. Therapeutic efficacy in this model infection using oral administration of acyclovir could be correlated with the achievement of acyclovir levels in the plasma of experimental animals two to 10 times greater than the mean inhibitory concentration for MCMV in vitro throughout treatment. The lack of efficacy observed when drug was administered intraperitoneally was associated with acyclovir levels exceeding 1 microM for one to three hours after each dose.

Optimal treatment of herpes simplex virus encephalitis in mice with oral acyclovir.

The effect of oral or intraperitoneal administration of acyclovir was evaluated in four experimental models of herpes simplex virus (HSV) encephalitis in mice. Mice were inoculated with HSV-1 or HSV-2 intracerebrally or with HSV-2 intranasally, intraperitoneally, or intravaginally. With all four routes of inoculation, oral acyclovir therapy significantly reduced mortality when started as late as 72 to 96 hours after viral challenge. Intraperitoneal acyclovir was not effective in protecting mice inoculated intravaginally, but was effective if given 24 to 48 hours after intracerebral or intranasal challenge and as late as 96 hours after intraperitoneal infection. Oral acyclovir was more active than intraperitoneal treatment in all four model infections. Levels of acyclovir inhibitory for HSV in cell culture were maintained in plasma and brain tissue throughout oral treatment but lasted only three to six hours after each intraperitoneal treatment. These results suggest that acyclovir may be useful in treating serious HSV infections in humans.

Potentiation of Clostridium botulinum toxin aminoglycoside antibiotics: clinical and laboratory observations.

A child with infant botulism became apneic and died while receiving ampicillin and gentamicin therapy. As aminoglycosides have been implicated in the induction and/or exacerbation of neuromuscular blockade, we used an animal model to test the hypothesis that aminoglycosides potentiate neuromuscular blockade of botulinum toxin. In the range of aminoglycoside doses utilized in these experiments, both gentamicin and tobramycin enhance neuromuscular impairment and death of botulinum toxin-exposed mice. These results support recently published clinical observations that aminoglycosides may potentiate neuromuscular weakness caused by botulinum toxin, and suggest that these antibiotics should be used with caution in suspected cases of infant botulism.

Infant botulism: clinical spectrum and epidemiology.

Between 1977 and 1979, 12 cases of infant botulism were diagnosed in Utah, and 87 control patients (normal, nonbotulism neurologic disease, and nonbotulism systemic disease) were evaluated. Observations from these patients suggest an expanded clinical spectrum of infant botulism including asymptomatic carriers of organism; mild hypotonia and failure to thrive; typical cases with constipation, bulbar weakness, and hypotonia; and children with a picture compatible with sudden infant death syndrome. Clostridium botulinum was isolated from the stools of three normal control infants and nine control infants who had neurologic diseases that were clearly not infant botulism. These infants were termed "asymptomatic carriers" of the organism. The occurrence of the asymptomatic carrier state suggests that a diagnosis of infant botulism cannot be made on a basis of culture results alone, but must rest in historical documentation and physical confirmation of progressive bulbar and extremity weakness with ultimate complete resolution of symptoms and findings over a period of several months. A common set of environmental features characterizes the home environment of children with infant botulism and "asymptomatic carriers" and includes: nearby constructional or agricultural soil disruption, dusty and windy conditions, a high water table, and alkaline soil conditions.

Effect of interferon on cyclic adenosine monophosphate levels in cells.

Treatment of murine and human cells with homologous interferon (IFN) resulted in an elevation of the cellular cyclic adenosine monophosphate (cAMP) levels in a time- and dose-dependent manner. A similar elevation of cAMP levels could be produced by treatment of cells with isoproterenol, prostaglandin or methylxanthine. However, these agents did not produce an antiviral state. Pretreatment with an inhibitor of adenyl cyclase, N-ethylmaleimide, prevented the effect of IFN on cAMP levels, but did not influence tis antiviral activity.

A comparison of phosphonoacetic acid and phosphonoformic acid activity in genital herpes simplex virus type 1 and type 2 infections of mice.

The activity of phosphonoacetic acid (PAA) and phosphonoformic acid (PFA) against four strains of herpes simplex virus type 1 (HSV-1) and four strains of HSV-2 were compared in tissue culture and in a murine model of genital herpes. In mouse embryo fibroblast cells, both drugs were three-fold more active against the HSV-1 strains than against the HSV-2 strains. In contrast, in the animal model infections, PAA appeared to be more active against the HSV-2 strains, while PFA was equally effective against both HSV types. In mice infected intravaginally with HSV-2 and treated with intravaginal 5% PAA, none of the treated mice became infected, replication of virus in the genital tract was completely inhibited, none of the infected mice died from encephalitis, and latent infection in lumbosacral ganglia of surviving animals was completely prevented. In HSV-1 genital infection treated with PAA, 20-60% of mice became infected, replication of virus in the genital tract was strikingly reduced, none of the infected mice died, and latent infection was completely prevented. In both HSV-2 and HSV-1 genital infections, 20-70% of animals treated with 8% PFA became infected, growth of virus in the genital tract was reduced significantly but not completely suppressed, mortality was variably altered, and there was a trend towards reduction in the frequently of latent infection. These results indicate that HSV-1 strains are more sensitive to PAA and PFA in tissue culture, but the HSV-2 strains are generally more amenable to therapy in the murine model of genital herpes. Although PAA appeared to be more active that PFA in the genital infection, both drugs significantly altered the course of the infection. Since dermal toxicity associated with PAA precludes its use in humans and since PFA is already undergoing trials in patients with recurrent herpes labialis, the current results suggest that topical PFA deserved further evaluation in the treatment of mucocutaneous HSV infections, including genital herpes.

Acyclovir treatment of experimental genital herpes simplex virus infections. I. Topical therapy of type 2 and type 1 infections of mice.

Intravaginal inoculation of mice with herpes simplex virus (HSV) provides a model infection of genital herpes to determine the effectiveness of potential antiviral agents. topical (intravaginal) treatment with 1% or 5% acyclovir (ACV) in an ointment of gel vehicle initiated 3, 6 or 24 h after inoculation with HSV type 2, significantly inhibited viral replication in the genital tract and usually reduced final mortality. Treatment with 5% ACV initiated 48 or 72 h after infection also reduced vaginal virus titers but did not alter final mortality. When mice were inoculated with HSV type 1 treatment with 5% ACV significantly reduced viral replication in the genital tract when begun as late as 72 h. In HSV-2 infected mice, treatment initiated 3 h but not 24 h after infection prevented the establishment of latent infection in sacral ganglie. These results suggest that topical ACV may be effective antiviral agent for primary genital herpes in humans.