Host defenses in murine malaria: induction of a protracted state of immunity with a formalin-killed Plasmodium berghei blood parasite vaccine.

Random-bred mice were immunized with a nonliving antigen prepared from mixed-blood forms of Plasmodium berghei, strain NYU-2, in combination with Corynebacterium parvum and/or living BCG. A high proportion of intravenously immunized mice survived virulent challenge, but subcutaneous vaccination was less effective. Vaccinated mice developed a patent infection after challenge similar to that observed in normal controls. However, between days 12 to 20 postchallenge, infections in some vaccinated mice became subpatent, whereas infections in all normal controls progressed until death. The incidence of recrudescent infection was low and, eventually, a state of sterile immunity was established. The capacity of vaccinated mice to withstand P. berghei challenge was sustained at a fairly stable level for the 6-month period of observation. Mice that had survived a primary infection with P. berghei almost completely suppressed a second and larger challenge with the same organism.     

Host defenses in murine malaria: analysis of the mechanisms of immunity to Plasmodium berghei generated in response to immunization with formalin-killed blood-stage parasites.

Syngeneic B6D2F1 (C57Bl/6 x DBA/2) mice were immunized with a nonliving antigen prepared from mixed blood forms of Plasmodium berghei strain NYU-2. Consistently greater than 80% of the vaccinated mice survived virulent challenge, and protective immunity was demonstrable from 1 week through at least 4 months after immunization. However, vaccination did not prevent the development of patient infection after challenge. Instead, infections in vaccinated mice progressed to about 10% parasitemia and were then subsequently cleared. In contrast, infections initiated in nonvaccinated mice progressed beyond 10% parasitemia and were uniformly fatal within 4 weeks. Sera collected from normal mice, nonvaccinated mice infected with P. berghei, or vaccinated mice before challenge failed to passively protect recipients against virulent infection. On the other hand, sera collected from vaccinated mice after recovery from a challenge infection conferred upon passively immunized recipients protection from homologous virulent challenge, which was manifest as a delay in the onset of overt infection. It was concluded, therefore, that vaccination altered the immunological potential of the host in such a way as to allow the production of a protective humoral factor, probably specific antibody, in response to infection with the virulent parasites.     

Endotoxin-induced serum factor kills malarial parasites in vitro.

We investigated the possibility that malarial parasites may be killed by nonspecific soluble mediators, such as those in tumor necrosis serum, that are obtained from mice given macrophage-activating agents like Corynebacterium parvum or Mycobacterium bovis BCG, followed by endotoxin. Such sera killed parasites in vitro after overnight incubation; killing was measured directly by using an in vivo infectivity assay. Parasite infectivity was not decreased by incubation in sera from mice given C. parvum or BCG alone (no endotoxin) or by incubation in sera from normal mice given endotoxin. Plasmodium yoelii, its lethal variant, and Plasmodium berghei were equally susceptible to inactivation. Sera obtained from mice given endotoxin during the course of infection with these parasites also contained parasite-killing factor. The activity of this factor appeared to be proportional to parasitemia in that it was higher in the sera from mice infected with the lethal parasites than in the sera from mice with infections which resolved either spontaneously or after vaccination.     

Host defenses in murine malaria: characteristics of protracted states of immunity to Plasmodium berghei.

Vaccination of 8-week-old B6D2 (C57BL/6 x DBA/2) mice with Formalin-killed asexual erythrocytic stages of Plasmodium berghei provided a capacity to survive challenge with lethal P. berghei for more than 542 days. Although long-lived, this immunity did not provide a capacity to immediately neutralize parasites in a challenge; significant levels of erythrocytic infection occurred transiently after each challenge. The quality of long-lived immunity was not enhanced by an injection of live parasites 2 weeks after the last injection of killed antigen. The results suggest that it will be difficult to design a vaccination regimen which will provide long-lived protection from overt erythrocytic-stage infection.     

Modulating effect of passive immunization with anti-Mycobacterium tuberculosis antibodies on humoral response in BCG-infected mice

Previous work has shown that the passive immunization with rabbit anti-Mycobacterium tuberculosis H37Rv antiserum of Mycobacterium bovis BCG-infected mice promotes the growth of bacilli in their spleen and induces a late production of antimycobacterial antibodies in their serum. The effect of the passive immunization on the early antibody response in infected mice has now been investigated. It was found that passive immunization with H37Rv antiserum of BCG-infected mice depressed the early humoral response as determined by the plaque-forming cell response to BCG extract when compared with BCG-infected mice treated with the antiserum freed from its mycobacterial antibodies as controls. In the BCG-infected mice treated with the rabbit antiserum freed from mycobacterial antibodies or treated with saline, the antibodies were present in the serum as soluble immune complexes which reached a peak 4 days after infection. These immune complexes were formed with mice antimycobacterial antibodies as determined with an antimouse immunoglobulin serum in the double diffusion test. On the other hand, in BCG-infected mice passively immunized with rabbit antimycobacterial serum, the immune complexes detected were mainly composed of transfer rabbit antimycobacterial antibodies as established with an antirabbit immunoglobulin serm. Comparison of the biphasic humoral response in passively-enhanced mycobacterial infection and allotransplanted normal tissue in the host is discussed.     

Effects of BCG, Corynebacterium parvum, and methanol-extration residue in the reduction of mortality from Staphylococcus aureus and Candida albicans infections in immunosuppressed mice.

An immunosuppressed mouse model was devised to test the effects of immunopotentiators on the prevention of bacterial and fungal infections. The effects of BCG and Corynebacterium were tested against Staphylococcus aureus and Candida albicans infection. The effect of methanol-extraction residue (MER-BCG) was tested against S. aureus septicemia. CDF mice were given various doses of BCG, 1.0 mg of C. parvum, or 0.5 mg of MER intraperitoneally at varying intervals before injection of an intravenous bacterial challenge. Four days before challenge, 300 mg of cyclophosphamide per ml was given intraperitoneally. BCG (106 colony-forming units) reduced mortality due to S.aureus at pretreatment intervals of 3, 7, 14, and 28 days. Isonicotinic acid hydrazide treatment elimated the protective effect of the live BCG. C. parvum was as effective as BCG against S. aureus septicemia when given 3 days before infection, but lost most of its protective effect after that time. MER protected at doses as small as 0.25 mg when given 25 days prior to challenge. Both BCG and C. parvum exerted a protective effect against Candida albicans infection.     

BCG, in contrast to C. parvum, does not induce increased sensitivity to the toxic effects of indomethacin in mice

Treatment of mice with killed Corynebacterium parvum (also designated Propionibacterium acnes, P. acnes) or Bacille Calmette Guerin (BCG) leads to modification of several of the same host systems. However, BCG, in contrast to C. parvum, did not induce increased sensitivity to the toxic effects of indomethacin in BALB/c or C57B1/6 mice. In addition, treatment of mice with BCG did not interfere with the induction of sensitivity by C. parvum. Therefore C. parvum must uniquely induce changes in host systems which alter the sensitivity to this anti-inflammatory drug. Additional experiments with splenectomized animals revealed that the presence of this organ, which undergoes hypertrophy following C. parvum treatment, was not necessary for the induction of indomethacin sensitivity. Presentation of C. parvum via the subcutaneous route versus the intraperitoneal route revealed that the two routes were equally efficient in inducing sensitivity in C57B1/6 mice but the former route was less effective (50% deaths) than the intraperitoneal route (95% deaths) in BALB/c mice. These results indicate that host related factors (genetic) may be important in the generation of enhanced sensitivity to the toxic effects of indomethacin.     

Differential susceptibility of rodent malaria parasites to nonspecific immunity

Nonspecific immunity to rodent malaria parasites can be produced by immunising mice with heterologous parasites or pretreating them with BCG, Corynebacterium parvum or Brucella abortus. Nonspecific immunity is easily produced against Plasmodium vinckei but not P. berghei. This is due to the difference between the cells occupied by the parasites, young red blood cells in the case of P. berghei and mature red blood cells in the case of P. vinckei.     

卡介苗接种对小鼠伯氏疟原虫感染早期树突状细胞活化状态的影响

为观察疟原虫感染早期卡介苗（BCG）接种对小鼠树突状细胞（DC）活化状态的影响,用Plasmodium berghei ANKA感染C57BL/6小鼠,3天后接种BCG（P.b-3-B实验组）,同时以未接种BCG的感染小鼠为对照.通过动态观察两组感染鼠的原虫血症水平和生存率,流式细胞术检测感染后0、5和8天脾细胞中CD11c＋CD11b＋DC、CD11c＋B220＋DC、CD11c＋CD80＋DC和CD11c＋MHCⅡ＋ DC百分率.结果显示,P.b-3-B实验组的原虫血症从感染后第8天开始逐渐增高,66.7%的小鼠存活至第20天死亡,而对照组小鼠多于感染后8～10天死于脑型疟疾;感染后第5和8天,P.b-3-B实验组小鼠CD11c＋CD11b＋DC、CD11c＋CD80＋DC和CD11c＋MHCⅡ＋DC百分率均显著低于对照组（P〈0.05或P〈0.01）,感染后第8天,CD11c＋B220＋DC百分率也明显低于对照组（P〈0.01）.这些结果表明,疟原虫感染早期接种BCG可下调DC亚群百分率和表面分子表达水平,并以此影响疟疾的感染进程.     

The role of IgA immunoglobulins in the passive transfer of protection to Taenia taeniaeformis in the mouse.

Normal mice were protected against infection with metacestodes of Taenia taeniaeformis when administered intestinal, colostral or serum immunoglobulins obtained from adult mice previously orally infected with the parasitie. The protective capacity of these preparations was found to be associated mainly with IgA of colostrum and intestinal secretions and IgG of serum. The removal of IgA and IgG from immune colostrum and serum, respectively, abolished the protective effect. Neonatal mice were protected against infection with T. taeniaeformis when fed purified colostral IgA and serum IgG from immune donors. The intraduodenal injection of intestinal IgA from immune donors into 4-week-old mice passively protected the recipients against infection with T. taeniaeformis, but intestinal IgG from immune donors had no protective effect when given in this manner. The protective capacity of IgA and IgG was largely eliminated by prior absorption with T. taeniaeformis antigen or hatched, activated oncospheres of T. taeniaeformis.     

Recombinant interleukin-2 limits the replication of Mycobacterium lepraemurium and Mycobacterium bovis BCG in mice.

BALB/c mice were infected with Mycobacterium lepraemurium in the footpad or with Mycobacterium bovis BCG intravenously with 5 x 10(7) bacilli. Recombinant interleukin-2 (IL-2) was injected intraperitoneally as a single dose (20,000 U), as a single course of five injections (400 U each), or as a 6-month course starting 3 days after the M. lepraemurium infection. BCG-infected mice received a single dose (1,000 U) or five daily injections of 100 or 1,000 U each. IL-2 significantly reduced the total bacterial counts in the footpad, lymph nodes, and liver of M. lepraemurium-infected mice (50 to 85%) by 6 months and viable counts in the spleen (30 to 50%) by 60 days after BCG infection. The courses of IL-2 started at 60 days were more effective than those started at 3 days after M. lepraemurium infection (P less than 0.05 to 0.001), and for BCG, 100 U of IL-2 was better than 1,000 U (P less than 0.05 to 0.01). These results indicate that IL-2 limits mycobacterial infections in mice and raise the question of its possible use in humans.     

Differential sensitivity in vivo of lethal and nonlethal malarial parasites to endotoxin-induced serum factor.

Sera from mice infected with Plasmodium yoelii or Plasmodium berghei and given endotoxin contained nonspecific mediators which killed both species of parasite and tumor cells in vitro. The sera resembled tumor necrosis sera obtained from mice given macrophage-activating agents such as Propionibacterium acnes (formerly designated Corynebacterium acnes) or Mycobacterium bovis BCG and then endotoxin. Cytotoxicity developed parallel to parasite killing activity and indicated that macrophages were activated. Activation occurred sooner with P. berghei, which is lethal, and serum activity remained on a plateau until the mice died. In nonlethal P. yoelii infections, activation was related to the course of parasitemia. Endotoxin given to mice infected with P. yoelii caused an immediate decrease in parasitemia, presumably through the release of parasite killing factors. The extent of the decrease depended upon the time of administration. No immediate drop in the parasitemia caused by P. berghei was observed at any time. Early administration of endotoxin prolonged survival; late administration accelerated death. Passive transfer of rabbit tumor necrosis serum to infected mice decreased the parasitemia caused by P. yoelii but not that caused by P. berghei. Other components of the immune response appeared to act together with these soluble mediators to eliminate P. yoelii; they may be absent or suppressed in infections with P. berghei.     

Neutralization of gamma interferon and tumor necrosis factor alpha blocks in vivo synthesis of nitrogen oxides from L-arginine and protection against Francisella tularensis infection in Mycobacterium bovis BCG-treated mice.

Peritoneal cells from Mycobacterium bovis BCG-infected C3H/HeN mice produced nitrite (NO2-, an oxidative end product of nitric oxide [NO] synthesis) and inhibited the growth of Francisella tularensis, a facultative intracellular bacterium. Both NO2- production and inhibition of bacterial growth were suppressed by NG-monomethyl-L-arginine, a substrate inhibitor of nitrogen oxidation of L-arginine, and monoclonal antibodies (MAbs) to gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). Intraperitoneal injection of mice with BCG increased urinary nitrate (NO3-) excretion coincident with development of activated macrophages capable of secreting nitrogen oxides and inhibiting F. tularensis growth in vitro. Eight days after BCG inoculation, mice survived a normally lethal intraperitoneal challenge with F. tularensis. Treatment of these BCG-infected mice with MAbs to IFN-gamma or TNF-alpha at the time of BCG inoculation reduced urinary NO3- levels to those found in normal uninfected mice for up to 14 days. The same anticytokine antibody treatment abolished BCG-mediated protection against F. tularensis: mice died within 4 to 6 days. Intraperitoneal administration of anti-IFN-gamma or anti-TNF-alpha antibody 8 days after BCG infection also reduced urinary NO3- and abolished protection against F. tularensis. Isotype control (immunoglobulin G) or anti-interleukin 4 MAbs had little effect on these parameters at any time of treatment. IFN-gamma and TNF-alpha were clearly involved in the regulation of macrophage activation by BCG in vivo. Protection against F. tularensis challenge by BCG depended upon the physiological generation of reactive nitrogen oxides induced by these cytokines.     

Strain differences in mouse cellular responses to Mycobacterium lepraemurium and BCG subcutaneous infections. I. Analysis of cell surface phenotype in local granulomas.

C57BL/6, BALB/c and CBA mice were subcutaneously infected with either Mycobacterium lepraemurium (MLM) or BCG, and studied for bacillary growth, granuloma size of infected footpads and draining lymph nodes (DLN), and DLN cell surface phenotype. Whereas, BCG-infected mice controlled the infection and developed early and large granulomas, MLM-infected mice exhibited major strain variations in their resistance to the infection, as well as in the granuloma size and kinetics. C57BL/6 mice, highly resistant, displayed early and regressive granulomas; BALB/c mice showed lower resistance and early granulomas that grew continuously; CBA mice, highly susceptible, developed late, soft, phagocyte-rich granulomas. Important strain differences in lymph node lymphocyte subset distribution could be observed prior to any infection: C57BL/6 mice displayed higher B cell percentages than both of the other strains and BALB/c mice showed the highest CD4/CD8 ratios, followed by CBA and C57BL/6 mice. BCG and MLM infections both induced similar changes of these parameters in all three strains: that is a decrease of the B cell percentage and a decrease of the CD4/CD8 ratio, and the strain differences observed in uninfected mice persisted. On the other hand, DLN cells stimulated by the infecting bacillus and interleukin 2 also displayed an increase of the CD8 T cell percentage as compared with normal lymph node cells, but this phenomenon was much less pronounced in BALB/c mice, whether infected by MLM or BCG, and in MLM-infected CBA mice, than in BCG- or MLM-infected C57BL/6 (B6) mice. Thus the ability of C57BL/6 mice to generate an early and persistent CD8 T cell response to mycobacteria may contribute to their resistance to MLM.     

Immunological consequences of innate resistance and susceptibility to BCG

The immunological consequences of genetically controlled innate resistance and susceptibility to Mycobacterium bovis (BCG) infection in mice were investigated. The susceptible (BcgS) BALB/c and the congenic resistant BALB/c.Bcgr mouse strains were employed to test for differences in the specific immune response to BCG. Antigen-specific lymphocyte proliferation and production of interleukin 2 (IL-2) to purified protein derivative (PPD) in vitro were measured following infection of congenic mice with low (10(4)) and high (10(6)) doses of BCG. The lymphocyte subsets were identified by testing the ability of spleen cells to respond after separation of T and B cells and after cytotoxic depletion of T cell subsets. In addition, fluorescence activated cell sorter (FACS) analysis with anti-T and -B cell monoclonal reagents was used to enumerate lymphocyte populations in BCG-infected spleens. The results indicated that the innately resistant mice displayed antigen-specific T helper cell function three weeks following the injection of BCG. The susceptible animals were found to be T cell-unresponsive since they lacked both proliferative and IL-2 secreting specific T cells. No evidence for suppression of IL-2 production or proliferation was detected in BALB/c (susceptible) spleen cultures in cell mixing experiments. The results provide evidence for a regulatory role of the Bcg gene on the generation of lymphocyte responses to BCG.     

Mycobacterium bovis BCG-induced protection against cutaneous and systemic Leishmania major infections of mice.

We examined the protective effects of Mycobacterium bovis bacillus Calmette-Guérin (BCG) administration on Leishmania major infections of BALB/c and P/J mice. There were two treatment protocols. In the first, the footpads of naive animals were inoculated with mixtures of L. major and BCG (viable or heat killed) or the soluble mycobacterial antigen, purified protein derivative. Viable BCG, but not heat-killed BCG or purified protein derivative, inoculated with L. major amastigotes into the footpads of naive BALB/c or P/J mice protected these animals from the metastatic spread of parasites to the viscera and from ensuing lethal systemic infection. This treatment also induced cures of the cutaneous lesions of P/J mice but not of BALB/c mice. In the second protocol, we induced an immune response to BCG before inoculation of L. major. BCG given intraperitoneally 10 days before infection of footpads with leishmania offered protection against the metastatic spread of amastigotes in both P/J and BALB/c mice, regardless of intralesional treatment, and modulated the severity of cutaneous infection by 30 to 50%. Inoculation of a mixture of viable BCG and L. major amastigotes into BCG-immune mice completely protected both BALB/c and P/J strains from cutaneous disease; we recovered no parasites from the inoculated footpads of these animals. Furthermore, each of the nonspecifically protected mice of both the BALB/c and P/J strains developed immunity to rechallenge with viable L. major. Injection of amastigotes at a site remote from the original lesion, the contralateral footpad, resulted in the complete clearance of parasites in the inoculum with no evidence of either cutaneous or systemic disease over an extended observation period.     

Interferon Production by Corynebacterium Parvum and BCG-Activated Murine Spleen Macrophages

Macrophages were identified to be a major source of interferon produced in murine spleen cell cultures after intravenous injection of Corynebacterium parvum (C. parvum), strain CN 6134 or Bacille Calmette Guérin (BCG). Another strain of C. parvum, CN 5888, which lacks RES stimulating activity and adjuvant activity in vivo, was not effective when injected intravenously. Protein synthesis was required for interferon activity to be produced and protein synthesis was also required for the antiviral state to be expressed. The antiviral activity was relatively stable to pH 2 and neutralized by an antiserum against virus-induced fibroblast interferon, thus exhibiting some properties of type I interferon. In vitro only CN 6134, the biologically active strain, could induce small amounts of interferon in spleen macrophage cultures. Macrophages from CN 6134 or BCG-infected athymic nu/nu mice produced similar interferon titers as their controls. It is concluded that infection with certain immunomodulators can activate splenic macrophages via a predominantly T-cell independent mechanism. Interferon in turn may operate locally as a mediator of immunoregulation.     

Restoration of T-cell responsiveness by thymosin: expression of anti-tuberculous immunity in mouse lungs.

Specific pathogen-free, adult thymectomized, irradiated, and bone marrow-reconstituted (THXB) B6D2 mice were infected aerogenically with 1 X 10(3) to 5 X 10(3) live BCG Pasteur. Seven days later a group of the mice was placed on a 14-day regimen of 20 mg of calf thymosin per kg per day, and the growth of the BCG in the lungs, spleen, inguinal lymph node, bone marrow, and blood was determined for up to 90 days. The thymosin treatment was followed by a decline in the BCG counts for the lungs and spleens of the THXB mice, whereas the saline-treated controls showed no such decline with time. The thymosin-treated mice did not develop progressive BCG infections in the test lymph nodes or in the bone marrow, both of which became positive in the THXB mice. Spleen cells were harvested from thymosin-treated THXB donors, filtered through nylon wool, and infused three times into BCG-infected THXB recipients. The lung BCG counts declined approximately 10-fold by day 90 compared with THXB mice which received THXB spleen cells. The transferred immune response was only slightly smaller numerically than that seen in THXB mice infused with BCG-immune lymphocytes from normal donors.     

Correlation of Increased Metabolic Activity, Resistance to Infection, Enhanced Phagocytosis, and Inhibition of Bacterial Growth by Macrophages from Listeria- and BCG-Infected Mice

Macrophages from mice infected with facultative intracellular organisms such as Listeria monocytogenes and BCG have been shown to resist infection by antigenically unrelated intracellular bacterial parasites. This study compares phagocytosis, bacterial growth inhibition, and oxidation of glucose by macrophages from normal mice, mice infected with listeria or BCG, or mice immunized with killed listeria in incomplete Freund's adjuvant. Macrophages from listeria- and BCG-infected mice ingested more listeria; 67 and 57%, respectively, had three or more cell-associated bacteria versus 22% of controls (P < 0.001). Peritoneal macrophages from listeria- and BCG-infected animals significantly (P < 0.001 covariance analysis) inhibited growth of listeria in suspension, whereas control macrophages had no such inhibitory effect. The rate of oxidation of glucose-1-(14)C was higher in macrophages from listeria- and BCG-infected mice than from either uninfected animals or those immunized with killed listeria. During phagocytosis of killed or live bacteria, or latex particles, the rate of glucose oxidation was increased (P < 0.01). These data suggest that the cellular immunity after infection by an intracellular organism is associated with an increase in metabolic activity of macrophages, namely, an increase in the rate of glucose oxidation resulting in enhancement of phagocytosis and killing.     

Spleen cell transfer induces T cell-dependent granulomas in tuberculous nude mice

After IV infection with an attenuated strain of Mycobacterium bovis BCG, athymic nude mice produced a few hepatic granulomas; most of the other foci of bacterial parasitization lacked obvious cellular reactions. Nude mice BCG-infected four weeks earlier received spleen cells from normal euthymic littermates. After transient splenomegaly which peaked at about two weeks, numerous hepatic granulomas appeared by four weeks and the number of viable BCG decreased. The development of hepatic granulomas required viable spleen cells and depended on the spleen cell dose and corresponded with positive footpad reactions. The effective population seemed to be T cells, since they were sensitive to treatment with anti-brain associated theta antigen serum (antiBAT) plus complement, and were found in nylon-wool passed fractions. After transfer with sensitized spleen cells, adoptive immunity was observed, provided that the recipient BCG-infected nude mice had been splenectomized before transfer. AntiBAT plus complement treatment of the sensitized spleen cells decreased the granuloma-producing as well as antimycobacterial capacity. When BCG-infected nude mice were irradiated with 500 r of gamma ray, granuloma production was decreased after immune spleen cell transfer. Co-transfer of immune spleen cells with normal nude mouse bone marrow cells to BCG-infected irradiated nude recipients restored the granuloma-forming ability.