Toxoplasma Gondii Replication Research Articles

Molecular analysis and characterization of a protein involved in the replication of intracellular Toxoplasma gondii

Previous studies in our laboratory have identified a cytoplasmic protein (p97) of T. gondii that is involved in the process of intracellular parasite replication. Monoclonal antibody inhibits parasite replication in vitro and recognizes a protein of approximate 97 kDa by Western blot analysis. Using biotinylation, we demonstrate that p97 is not expressed on the surface of the tachyzoite. Polyclonal sera raised against the purified native protein was used to isolate a cDNA of 3.3 kb from a library. The product of this gene expresses a protein of approximate M r 97 kDa that is reactive to the antibody (1B8) raised against the native antigen. The protein sequence of this product suggests that it is within the cytoplasm as suggested by the lack of a signal sequence or hydrophobic trans-membrane domain. This protein fails to dissociate into a monomer in the presence of non-ionic detergents as shown by gel filtration and density gradient. Southern blot analysis demonstrates a homologous gene sequence in two closely related Apicomplexa, Neospora caninum and Besnoitia jellisoni suggesting this protein is conserved among certain species of the Sarcocystidae.

Mechanisms of interferon-induced inhibition of Toxoplasma gondii replication in human retinal pigment epithelial cells

Inflammation associated with retinochoroiditis is a major complication of ocular toxoplasmosis in infants and immunocompetent individuals. Moreover, Toxoplasma gondii-induced retinal disease causes serious complications in patients with AIDS and transplant patients. The retinal pigment epithelial (RPE) cell is an important regulatory cell within the retina and is one of the cells infected with T. gondii in in vivo. We have developed a human RPE (HRPE) cell in vitro model system to evaluate T. gondii replication and the regulation of this replication by cytokines. T. gondii replication was quantitated by counting the foci of infection (plaque formation) and the numbers of tachyzoites released into the supernatant fluids. Pretreatment of cultures with recombinant human tumor necrosis factor alpha, alpha interferon (IFN-alpha), IFN-beta, or IFN-gamma for 24 h prior to inoculation inhibited T. gondii replication in a dose-dependent manner. Of these cytokines, IFN-gamma was the most potent, and T. gondii replication was completely inhibited at a concentration of 100 U/ml. The anti-toxoplasmotic activity of IFN-gamma was significantly blocked by monoclonal antibody to IFN-gamma. Treatment of the cultures with IFN-gamma from day 1 or 2 postinoculation with T. gondii also offered protection against the parasite. The anti-toxoplasmotic activity of tumor necrosis factor alpha or IFN-alpha, -beta, or -gamma in these cultures was found to be independent of the nitric oxide (NO) pathway, since NO production was not found in HRPE cells treated with these cytokines. However, addition of tryptophan to IFN-gamma-treated cells significantly reversed the inhibitory effects of IFN-gamma, suggesting that IFN-gamma acts by depleting cellular tryptophan. This effect was further confirmed by reverse transcription-PCR and Northern (RNA) blot analysis, which indicated induction of indoleamine 2,3-dioxygenase (IDO), an enzyme that converts tryptophan to kynurenine. These results indicated that interferons inhibited T. gondii replication in HRPE by NO-independent but IDO-dependent mechanisms. This in vitro model of T. gondii replication in HRPE may be useful in evaluating the effects of cytokines and drugs on T. gondii replication within the retina.

Influence of antimicrobial agents on replication and stage conversion of Toxoplasma gondii.

As yet, only a few drugs are available that are frequently used for the treatment of human toxoplasmosis. Pyrimethamine and sulfadiazine are regularly used in combination and are the first choice for most clinical conditions. However, due to severe side effects that are observed especially with sulfadiazine, other drugs, such as clindamycin, have successfully been used as alternative treatment. Since pyrimethamine might be teratogenic when used in the first weeks of pregnancy, spiramycin has been introduced as first-choice treatment during the first 16–20 weeks of pregnancy. In addition to these four well-established drugs, others such as tetracycline or cotrimoxazole have been studied as therapeutics against toxoplasmosis. Although their effectiveness has been demonstrated to some extent, they have not been accepted as general treatment (Joss 1992). None of these drugs has been shown to completely eliminate the parasite in the human host. Since persistent infection with cysts is the source of reactivated toxoplasmosis, new drugs have been developed that might even be more effective against the cyst stage. The hydroxynaphthoquinone atovaquone and macrolides such as azithromycin, clarithromycin, and roxithromycin are thought to inhibit the tachyzoite and the cyst stage as well, as has been shown in vitro or/and in vivo (Araujo et al. 1991,1992a,1992b; Blais et al. 1993a, Huskinson-Mark et al. 1991).

Enhancement of intracellular replication of Toxoplasma gondii by IL-6. Interactions with IFN-gamma and TNF-alpha.

Toxoplasma gondii is a major pathogen of immunocompromised hosts, and one defense mechanism against the parasite is activation of macrophages (M phi) for toxoplasmacidal activity by IFN-gamma after triggering by TNF-alpha. IL-6, IFN-gamma, and TNF-alpha are cytokines involved in inflammatory responses and are induced by T. gondii infection. We studied the interaction of these three cytokines using an in vitro model of T. gondii infection. Pretreatment (but not post-treatment) of unelicited murine peritoneal M phi with IL-6 enhanced T. gondii replication in a dose-dependent manner. Pretreatment with IFN-gamma resulted in active killing of parasites whereas the addition of IL-6 to IFN-gamma pretreatment resulted in a reversal of IFN-gamma-mediated toxoplasmacidal activity. Combining TNF-alpha with IL-6 and IFN-gamma pretreatment resulted in restoration of toxoplasmacidal activity. Addition of a polyclonal anti-TNF-alpha Ab to IL-6 and IFN-gamma pretreatment resulted in enhancement in the IL-6-mediated impairment of IFN-gamma function. These data taken together suggest that IL-6 enhances intracellular replication of T. gondii and reverses IFN-gamma mediated activation of murine peritoneal M phi, and that certain of the interactions between these two cytokines may be at the level of TNF-alpha triggering.

Reduced replication of Toxoplasma gondii is necessary for induction of bradyzoite-specific antigens: a possible role for nitric oxide in triggering stage conversion.

Stage conversion between tachyzoites and bradyzoites of Toxoplasma gondii was investigated in vitro by using murine bone marrow-derived macrophages (BMMs) as host cells. Following infection of untreated BMMs with tachyzoites, spontaneous expression of bradyzoite-specific antigens (Bsa) occurred at low frequency with Toxoplasma strain-dependent ratios from 0.03 to 2%. As previously described for peritoneal macrophages, activation of tachyzoite-infected BMMs with gamma interferon (IFN-gamma) or lipopolysaccharide resulted in the induction of Bsa. When bradyzoites were used for infection, prolonged expression of Bsa could be observed in IFN-gamma-activated BMMs. The induction of Bsa expression seemed to be closely linked to parasite multiplication and increased to maximal values of 50 to 70% in intermediately activated macrophages with nitric oxide (NO) levels that allowed reduced parasite replication. Identical results in stage conversion were obtained when sodium nitroprusside was used as a source of exogenous NO, indicating that NO might be a molecular trigger of stage conversion. NO is reactive with iron-sulfur centers in proteins, thereby inhibiting proteins involved in the mitochondrial respiratory chain. Using oligomycin and antimycin A as inhibitors of mitochondrial function, growth inhibition of parasites and induction of Bsa were obtained. Since microglia are the functional correlates of macrophages in the central nervous system and inhibit T. gondii upon activation with IFN-gamma, a similar mechanism might be involved during cyst development in the brain.

Effect of clindamycin on intracellular replication, protein synthesis, and infectivity of Toxoplasma gondii.

We studied the effects of clindamycin and a combination of clindamycin and pyrimethamine on the proliferation of Toxoplasma gondii in cultured mammalian cells and the effect of clindamycin on the parasite's RNA and protein syntheses. Infected macrophages were treated for 48 h with clindamycin or a combination of clindamycin and pyrimethamine, and the 50% inhibitory concentrations for parasite growth were 32.50 +/- 1.30 and 10.78 +/- 0.56 micrograms/ml, respectively. A modified susceptibility assay was also used to measure the effect of low concentrations of clindamycin on T. gondii. Macrophages and bovine turbinate cells were infected with low numbers of tachyzoites and were exposed to low concentrations of clindamycin for 5 days. In these systems, a concentration of 10 ng of clindamycin per ml inhibited 50% of the growth of the parasite in macrophages, while it completely prohibited the growth of the parasite in epithelial cells. When free tachyzoites were preexposed to clindamycin for 4 h, the reduction of parasite infectivity was proportional to the amount of drug; 100 ng of clindamycin per ml reduced the infectivity of T. gondii to 46.5% +/- 8.5% of that of the untreated control. A concentration of 40 micrograms of clindamycin per ml reduced protein synthesis by 56.2% +/- 6.0% but had no effect on RNA synthesis after a 4-h exposure of free tachyzoites of T. gondii to the drug. Our results show that long-term exposure to low concentrations of clindamycin reduces the level of replication of T. gondii, that clindamycin affects the protein synthesis of free parasites, and that clindamycin impairs the ability of tachyzoites to infect host cells.

The inhibitory effect of ovine recombinant interferon‐gamma on intracellular replication of Toxoplasma gondii

A model for the in vitro infection of ovine cells with Toxoplasma gondii tachyzoites has been developed and used to investigate the effect of treatment with ovine recombinant interferon-gamma (ov.rIFN gamma) on parasite replication. Treatment of both alveolar macrophages and fibroblast cells either 24 h pre-infection or 2 h post-infection with ov.rIFN gamma inhibited replication of T. gondii and was quantified by suppression of 3H uracil uptake by the parasite. Replication of T. gondii in the fibroblast cells was significantly inhibited by treatment with 200-300 U/ml ov.rIFN gamma, whereas concentrations as low as 1 U/ml suppressed parasite replication in the alveolar macrophages.

Interferon-gamma protects endothelial cells from damage by Candida albicans.

Endothelial cells activated with interferon-gamma (IFN-gamma) have been shown to inhibit the replication of Toxoplasma gondii. To determine if this cytokine protects endothelial cells from damage by Candida albicans, human umbilical vein endothelial cells were pretreated with IFN-gamma and infected with C. albicans; endothelial cell damage was measured by the release of 51Cr. Pretreatment with IFN-gamma decreased the extent of endothelial cell injury caused by C. albicans by up to 100% +/- 8.2%. This diminution of endothelial cell damage was confirmed by scanning electron microscopy. The degree of protection was dependent on the concentration of IFN-gamma, with maximum protection occurring at 13 units/mL. Higher concentrations of IFN-gamma were toxic to the endothelial cells. Pretreating the endothelial cells with this cytokine had no effect on candidal germination and growth, suggesting that IFN-gamma stimulates endothelial cells to become resistant to or inhibit the action of candidal virulence factors.

Interaction between human immunodeficiency virus and Toxoplasma gondii replication in dually infected monocytoid cells.

THP-1 monocytoid cells, either not infected or chronically infected with human immunodeficiency virus type 1 (HIV-1), were challenged with Toxoplasma gondii. Parasitic growth, as assessed by trophozoite counting and measurement of supernatant p30 membrane antigen, was similar in HIV-infected and noninfected THP-1 cells. Also, T. gondii did not affect HIV replication. These experiments therefore failed to demonstrate any interaction between HIV-1 and T. gondii replication in concurrently infected monocytoid cells.

Coexistence of heterogeneous populations of Toxoplasma gondii parasites within parasitophorous vacuoles of murine macrophages as revealed by a bradyzoite-specific monoclonal antibody.

The expression of bradyzoite-specific antigens (Bsa) of Toxoplasma gondii was studied in murine bone-marrow-derived macrophages that had previously been infected with tachyzoites. Growth conditions that allowed only restricted replication of Toxoplasma gondii resulted in heterogeneous populations; (1) Bsa-positive and Bsa-negative parasites could be observed within one parasitophorous vacuole (heterogeneous vacuole community), and (2) homogeneous Bsa-positive and homogeneous Bsa-negative vacuole communities coexisted within one macrophage host cell. These observations suggest that stage conversion does not seem to be a synchronous event for a vacuole community.

Human endothelial cells are activated by IFN-gamma to inhibit Toxoplasma gondii replication. Inhibition is due to a different mechanism from that existing in mouse macrophages and human fibroblasts.

Toxoplasma gondii invaded and proliferated in cultured human umbilical vein endothelial cells. Preincubation of the human umbilical vein endothelial cells with human rIFN-gamma induced a high degree of inhibition of T. gondii replication, with the effect being dose dependent. In order to try to elucidate the inhibitory mechanism, we tested the presence of several factors that are known to operate against intracellular parasites in other cell types. We found, by means of a competitive inhibitor, that L-arginine-dependent production of reactive nitrogen intermediates was not the cause of inhibition of T. gondii proliferation, thus contrasting with the inhibitory mechanism found in activated mouse macrophages. Furthermore, the inhibition of replication was not overcome by oxygen scavengers or by saturation of the system with tryptophan, suggesting that neither reactive oxygen intermediates nor the induction of tryptophan starvation was responsible.

Beta interferon inhibits Toxoplasma gondii growth in human monocyte-derived macrophages

Gamma interferon (IFN-gamma) previously has been shown to block the replication of Toxoplasma gondii in fibroblasts by the induction of indoleamine 2,3-dioxygenase (IDO) activity. IFN-beta also is known to induce IDO activity in monocyte-derived macrophages, but its ability to block the growth of T. gondii has not been demonstrated. We found not only that the combination of IFN-beta and lipopolysaccharide induced greater IDO activity in monocyte-derived macrophages than did IFN-beta alone but that this combination also was effective in inhibiting the growth of T. gondii. In addition, the inhibition was reversed by the addition of exogenous tryptophan, thus demonstrating that a mechanism by which IFN-beta inhibited T. gondii replication was by the induction of IDO.

Inability of recombinant interferon-gamma to activate the antibacterial activity of mouse peritoneal macrophages against Listeria monocytogenes and Salmonella typhimurium.

The effect of recombinant murine interferon-gamma (rIFN-gamma) as single stimulus for the activation of antibacterial activity of macrophages was investigated on the basis of the rate of intracellular killing of Listeria monocytogenes and Salmonella typhimurium by normal and rIFN gamma-activated peritoneal macrophages of CBA and C57BL/10 mice, which differ in natural resistance to infection by these bacteria. Eighteen hours after i.p. injection of 10 to 1 X 10(4) U rIFN-gamma, resident and exudate peritoneal macrophages which had phagocytosed L. monocytogenes or S. typhimurium in vivo, killed both species in vitro just as efficiently as did resident macrophages of normal mice. Similar results were obtained after 18 hr of in vitro incubation of resident or exudate peritoneal macrophages with 0.1 to 1 X 10(4) U/ml rIFN-gamma. Consistent with the in vitro findings, two i.v. injections of 5 X 10(4) U rIFN-gamma did not affect the rate of in vivo proliferation of L. monocytogenes or S. typhimurium in the spleens of mice during the first 2 days after i.v. injection of the bacteria. Compared with the effect on the controls, two i.p. injections of 5 X 10(2) to 5 X 10(4) U rIFN-gamma did not decrease the numbers of viable S. typhimurium in either the peritoneal cell suspension or the spleen 24 hr after i.p. injection of the bacteria. Checking the state of activation of rIFN-gamma-activated macrophages on the basis of two commonly used criteria for macrophage activation showed that rIFN-gamma-activated macrophages inhibited the intracellular replication of Toxoplasma gondii and displayed enhanced O2 consumption and H2O2 release after stimulation with phorbol myristate acetate compared with macrophages from normal CBA and C57BL/10 mice. The present findings show that as single activating stimulus, rIFN-gamma is not capable of activating the antibacterial effector functions of peritoneal macrophages against facultative intracellular pathogens such as L. monocytogenes and S. typhimurium.

Divergent changes in antimicrobial activity after immunologic activation of mouse peritoneal macrophages.

To find out whether activated macrophages display a nonspecific enhancement of antibacterial activity, we determined the intracellular killing of bacteria by peritoneal macrophages from CBA and C57BL/10 mice infected with BCG and challenged with mycobacterial antigens (purified protein derivative (PPD]. After in vivo phagocytosis, the rate of in vitro intracellular killing of Listeria monocytogenes by bacillus Calmette-Guérin (BCG)-PPD-activated macrophages from CBA mice increased by a factor of 1.7 and that of those from C57BL/10 mice by a factor of 2.0, relative to the rate in normal resident macrophages. The increased listericidal activity of BCG-PPD-activated macrophages could not have been due to an increased number of peroxidase-positive macrophages because exudate macrophages obtained after i.p. injection of proteose peptone into BCG-infected mice or PPD into control mice, killed ingested Listeria about as efficiently as normal resident macrophages did. In contrast, BCG-PPD-activated macrophages from both mouse strains killed Salmonella somewhat less efficiently and Escherichia coli and Staphylococcus aureus with the same efficiency as normal resident macrophages did. These cells, however, inhibited the intracellular replication of Toxoplasma gondii. Activated peritoneal macrophages from listeria-infected mice showed a similar increase of the rate of intracellular killing of Listeria and absence of change in rate of intracellular killing of Salmonella. Consistent with the in vitro findings, the number of viable L. monocytogenes in the spleen and liver of BCG-infected CBA and C57BL/10 mice decreased during the first 2 days after i.v. injection, whereas Salmonella typhimurium proliferated in these organs of both mouse strains. Checking the state of activation of BCG-PPD-activated macrophages showed that these cells displayed enhanced O2-consumption and H2O2 release after stimulation with phorbol myristate acetate compared with resident macrophages. The present findings show that the antimicrobial activity of immunologically activated macrophages is not uniformly increased: for certain microorganisms (L. monocytogenes, T. gondii), this effector function is enhanced, whereas for others (S. typhimurium, S. aureus, E. coli), it is not.

Differential activation of resident macrophage subsets with two sources of lymphokine preparations.

Resident murine macrophages were separated into subsets by Percoll density gradient centrifugation before treatment with lipopolysaccharide-stimulated lymphocytes or different lymphokine preparations. The lymphokines used were culture supernatants from lymphocytes obtained from lipopolysaccharide-injected mice or from purified protein derivative-treated lymphocytes from mice bearing an active BCG infection. The macrophage subsets were activated by the stimulated lymphocytes or lymphokine preparations to express C3b receptor-mediated ingestion or to inhibit the intracellular replication of Toxoplasma gondii or both. The results showed that the macrophage subsets were heterogeneous with respect to ingestion and T. gondii inhibition when activated with lipopolysaccharide-stimulated lymphocytes or lipopolysaccharide-derived lymphokines but were all homogeneous when activated with lymphokines from purified protein derivative-stimulated lymphocytes. When the macrophage subsets were allowed to remain in vitro for various times before lymphokine treatment, the relative pattern of subset activation changed when treated with lipopolysaccharide-derived lymphokines. In contrast, the macrophage subsets remained equally activated throughout the in vitro period when treated with the lymphokines from purified protein derivative-stimulated lymphocytes. The results suggested that functional macrophage heterogeneity depends not only on the nature of the activating signal but also on a state of receptivity of that signal by the macrophage population.

Lymphokine stimulated macrophages inhibit intracellular Chlamydia psittaci replication by mechanisms distinct from intracellular inhibition of Toxoplasma gondii replication.

Chlamydia and Toxoplasma are obligate intracellular parasites that replicate within macrophages and other eucaryotic host cells. Although chlamydia are procaryotes and Toxoplasma are eucaryotes, these organisms have several features in common during their intracellular growth and development. Each of these parasites is taken into the host cell by an endocytic mechanism that resembles phagocytosis (1,7), and each subverts normal phagocytic function by inhibiting fusion of host cell lysosomes with parasite-containing vacuoles (3,5). The mechanisms involved in this inhibition are not known, although Eissenberg and Wyrick (2) have reported that inhibition of fusion in peritoneal macrophages challenged with C. psittaci and either Saccharomyces ceriviciae or Escherichia coli was restricted to chlamydiae-containing vesicles. Chlamydiae and Toxoplasma remain within membrane bound vesicles during their entire intracellular development. Toxoplasma replicate by a process called endodyogeny. Chlamydiae first differentiate from a metabolically inactive infective form called an elementary body to an intracellular reticulate body, then grow and divide by binary fission forming a microscopically visible inclusion.KeywordsPeritoneal MacrophagePhorbol Myristate AcetateChlamydia TrachomatisPhorbol Myristate AcetateToxoplasma GondiiThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Host defenses to intracellular pathogens

and Historical Perspective.- I. Microbicidal Mechanisms of Leukocytes in Host Defenses.- Oxygen Independent Microbicidal Mechanisms of Human Polymorphonuclear Leukocytes.- Oxidative Metabolism of Leukocytes and Its Relationship to Bactericidal Activity.- Some Paradoxes of Macrophage Function.- Genetic Disorders of Granulocyte Function: What They Tell Us About Normal Mechanisms.- II. The Immune System and Phagocytic Cell Function.- Modulation of Effector Lymphokines.- Stimulation of Host Resistance to Metastatic Tumors by Macrophage Activating Agents Encapsulated in Liposomes.- Effect of Prostaglandins on the Production of Interleukin-2.- Interaction of Mycobacteria with Normal and Immunologically Activated Alveolar Macrophages.- Guinea Pig Alveolar Macrophages Probably Kill M. tuberculosis H37Rv and H37Ra in vivo by Producing Hydrogen Peroxide.- Interferon and Host Defense Systems.- Mediator Interactions Regulating Macrophage Secretion of Interleukin 1 and Interferon.- Macrophage Oxygen-Dependent Killing of Intracellular Parasites: Toxoplasma and Leishmania.- Immunologic Lesions During Toxoplasma gondii Infection.- Immunodepression in BALB/c Mice Infected with Leishmania tropica.- III. Host Defenses to Intracellular Bacteria.- Cellular Mechanisms of Anti-Mycobacterial Immunity.- Host Response to Infection with Mycobacterium bovis (BCG) in Mice: Genetic Study of Natural Resistance.- Cell Mediated Lysis of Lymphocytes Expressing Bacterial Antigens.- Improvement of Abnormal Lymphocyte Responses in Atypical Mycobacteriosis with Indomethacin.- Immunoregulatory Defects in Leprosy.- Cellular Mechanisms of Resistance to Listeria monocytogenes.- Effect of Interferon Inducers and Purified Mouse Interferon on the Susceptibility of Mice to Infection with Listeria monocytogenes.- Natural Resistance to Listeria monocytogenes as a Function of Macrophage Inflammatory Response.- Effect of Acute Nutritional Deprivation on Host Defenses Against Listeria monocytogenes - Macrophage Function.- Pilus-Mediated Clearance of Salmonella typhimurium by the Perfused Mouse Liver.- Immunity to Salmonella Infection.- Strain Dependent Variation of Delayed-Type Hypersensitivity in Salmonella typhimurium Infected Mice.- Monoclonal Antibodies to Salmonella typhimurium and Escherichia coli Lipopolysaccharides.- Characterization of Monoclonal Antibodies Which Recognize Specific Cell Surface Determinants on Salmonella typhimurium.- Monoclonal Antibodies as Probes for Antigens of Mycoplasma pulmonis.- Electron Microscopic Examination of the Inflammatory Response of Guinea Pig Neutrophils and Macrophages to Legionella pneumophila.- IV. Immunity to Rickettsia, Parasites and Fungi.- Activation of Macrophages for Killing of Rickettsiae: Analysis of Macrophage Effector Function After Rickettsial Inoculation of Inbred Mouse Strains.- Parameters of Cellular Immunity in Acute and Chronic Rickettsia tsutsugamushi Infections of Inbred Mice.- Lymphokine Stimulated Macrophages Inhibit Intracellular Chlamydia psittaci Replication by Mechanisms Distinct from Intracellular Inhibition of Toxoplasma gondii Replication.- Natural and Acquired Resistance to Trypanosoma cruzi.- Immunity to Fungal Infections.- Antibody-Independent Mechanisms in the Development of Acquired Immunity to Malaria.- Intracellular Destruction of Leishmania tropica by Macrophages Activated In Vivo with Mycobacterium bovis Strain BCG.- In Vitro Macrophage Antimicrobial Activities and In Vivo Susceptibility to Leishmania tropica Infection.- V. Mechanisms of Antiviral Immunity.- Virus-Immune T Cells and Monoclonal Antibodies in the Mouse Influenza Model.- Escape from Immune Surveillance During Persistent Virus Infection.- Influence of Viruses on Cells of the Immune Response System.- Genetically Controlled Resistance to Viruses.- Macrophage Oxidative Metabolism: A Defense Mechanism Against virus Infection?.- Interferon-Induced Augmentation of Natural Killer Cell Activity by Splenocytes from Leukemia Virus Immunosuppressed Mice.- Immunological Comparison of Ocular Disease Induced by Two Strains of Herpes Virus of Different Virulence.- Cellular Processing of the Large Glycoprotein of Lacrosse Virus (Family Bunyaviridae) Implications for Virion Assembly and Host Defense.- CMV and Renal Allograft Survival.- Martyrs, A Poem by Dr. L. J. Berry.- Contributors.- Author Index.

Macrophage oxygen-dependent antimicrobial activity. III. Enhanced oxidative metabolism as an expression of macrophage activation.

The capacity of 15 separate populations of mouse peritoneal macrophages to generate and release H2O2 (an index of oxidative metabolism) was compared with their ability to inhibit the intracellular replication of virulent Toxoplasma gondii. Resident macrophages and those elicited by inflammatory agents readily supported toxoplasma multiplication and released 4-20X less H2O2 than macrophages activated in vivo by systemic infection with Bacille Calmette-Guérin or T. gondii, or by immunization with Corynebacterium parvum. Immunologically activated cells consistently displayed both enhanced H2O2 production and antitoxoplasma activity. Exposure to lymphokines generated from cultures of spleen cells from T. gondii immune mice and toxoplasma antigen preserved both the antitoxoplasma activity and the heightened H2O2 release of toxoplasma immune and immune-boosted macrophages, which otherwise were lost after 48-72 h of cultivation. In vitro activation of resident and chemically-elicited cells by 72 h of exposure to mitogen- and antigen-prepared lymphokines, conditions that induce trypanocidal (5) and leishmanicidal activity (14), stimulated O2- and H2O2 release, and enhanced nitroblue tetrazolium reduction in response to toxoplasma ingestion. Such treatment, however, failed to confer any antitoxoplasma activity, indicating that intracellular pathogens may vary in their susceptibility to macrophage microbicidal mechanisms, including specific oxygen intermediates. In contrast, cocultivating normal macrophages with lymphokine plus heart infusion broth for 18H rendered these cells toxoplasmastatic. This in vitro-acquired activity was inhibited by scavengers of O2-, H2O2, OH., and 1O2, demonstrating a role for oxidative metabolites in lymphokine-induced enhancement of macrophage antimicrobial activity. These findings indicate that augmented oxidative metabolism is an consistent marker of macrophage activation, and that oxygen intermediates participate in the resistance of both in vivo- and vitro-activated macrophages toward the intracellular parasite, T. gondii.