Mimosine blocks cell cycle progression by chelating iron in asynchronous human breast cancer cells

We have used severe-combined immunodeficient (SCID) mice to examine the immunoregulatory effects of interleukin (IL)-10 on innate resistance to infection with Listeria monocytogenes. Addition of heat killed Listeria to spleen cells from naive SCID mice resulted in secretion of interferon (IFN)-gamma from natural killer cells in vitro. This response was enhanced up to 15-fold in the presence of exogenous IL-2, but was completely ablated by addition of IL-10 with IC50 of less than 0.5 U/ml. Infection of SCID mice with viable Listeria in vivo resulted in a prolonged course of infection eventually causing death by 12-14 days, whereas daily administration of IL-10 increased bacterial replication in the liver and spleen by up to 1000-fold resulting in death by day 4 post-infection. The immunosuppressive actions of IL-10 in vivo were also observed in immunocompetent BALB/c mice, whereas doses as low as 100 U/day converted a sublethal infection to 100% mortality. To study the events controlling expression of endogenous IL-10, peritoneal macrophage monolayers were challenged with Listeria after preincubation with a panel of recombinant cytokines. IFN-gamma primed macrophages for enhanced tumor necrosis factor (TNF) secretion, but inhibited IL-10 production, whereas granulocyte/macrophage colony-stimulating factor (CSF), macrophage CSF and also IL-4 enhanced macrophage IL-10 responses after ingestion of Listeria in vitro. Finally, monoclonal antibody neutralization of IFN-gamma during infection of SCID mice with Listeria inhibited TNF-alpha mRNA, but augmented expression of IL-10 mRNA in infected tissues. These results demonstrate that exogenous Il-10 is a potent immunosuppressive cytokine in the context of infection with an intracellular bacterium and that expression of endogenous IL-10 versus TNF is differentially regulated by the cytokine environment of the macrophage.     

Neutralization of IL-12 decreases resistance to Listeria in SCID and C.B-17 mice. Reversal by IFN-gamma

Interleukin-12 (IL-12) is necessary for the production of IFN-gamma by NK cells during the generation of innate immunity and by T cells for the development of the Th1 response during specific cell-mediated immunity. Here we demonstrate that the endogenous production of IL-12 is critical to the survival of both immunocompromised SCID mice and normal C.B-17 control mice during a primary infection with Listeria monocytogenes. When IL-12 is neutralized in vivo, both strains of mice die at a normally sublethal dose of Listeria. Anti-IL-12 antibody-treated mice showed a decrease in macrophage I-Ad expression and an increase Listeria burden in the spleen. Furthermore, as has been demonstrated in vitro, these effects of IL-12 in vivo were predominantly regulated through the production of IFN-gamma. Administration of IFN-gamma simultaneously with neutralizing antibodies to IL-12 restored macrophage I-Ad expression, limited the spread of the infection, and resulted in the survival of SCID mice. Thus, IL-12 is critical for resistance to infection with Listeria monocytogenes, and this resistance is mediated through stimulation by IL-12 of IFN-gamma production. Concomitant experiments confirmed that anti-TNF antibodies also resulted in uncontrolled infection and a decrease in macrophage I-Ad expression. However, administration of IFN-gamma restored the levels of I-Ad in macrophages but did not limit Listeria growth.     

Role of gamma interferon in natural clearance of Bordetella pertussis infection

Using a mouse model of Bordetella pertussis infection, we have analyzed the role of gamma interferon (IFN-gamma) in bacterial clearance from the respiratory tract. Adult BALB/c mice began to clear a respiratory infection within 3 weeks postinfection, with complete resolution of infection 6 to 8 weeks postinfection. In contrast, neither adult SCID mice (which lack mature B and T lymphocytes) nor adult nude mice (which lack mature T lymphocytes) controlled B. pertussis infection, and both strains died within 3 to 5 weeks postinfection. Short-term T-cell lines generated from the draining lymph nodes of the lungs of infected BALB/c mice were found to be CD4+ and produced IFN-gamma but no detectable interleukin-4. Analyses of IFN-gamma mRNA induction in the lungs of mice following B. pertussis infection showed that in both BALB/c and C57BL/6 mice, IFN-gamma mRNA levels increased sharply by 1 week postinfection and then subsequently declined. Further exploration of a potential role for IFN-gamma demonstrated that infection of adult BALB/c mice depleted of IFN-gamma in vivo with anti-IFN-gamma monoclonal antibodies resulted in greater numbers of bacteria recovered from the lungs than in infected, control BALB/c mice, although IFN-gamma-depleted mice could subsequently clear the infection. Infection of mice which have a disrupted IFN-gamma gene resulted in bacterial clearance with a time course similar to those seen with IFN-gamma-depleted mice. These results indicate that IFN-gamma plays a role in controlling B. pertussis infection.     

The swine as a model for studying exercise-induced changes in lipid metabolism

The function of cytokines produced during Hymenolepis nana egg infection in mice in protective immunity against re-infection was examined. Treatment of mice with monoclonal antibody (MAb) against mouse interferon (IFN)-gamma caused suppression of protective immunity against H. nana re-infection when the MAb was injected intraperitoneally at a daily dose of 40.0 mg kg-1 during the effector phase of protective immunity. Although high levels of IFN-gamma, tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta were released into the intestinal tracts of the parasitised mice at challenge infection, there was almost no release of these cytokines in mice treated with the MAb. Daily administration of rolipram failed to suppress the protective immunity, even when 400 micrograms kg-1 of the agent was administered into mice during the effector phase of immunity. Treatment of mice with rolipram completely suppressed both TNF-alpha and IL-1 beta production in intestinal tracts, induced by H. nana challenge infection. However, endogenous IFN-gamma production in the intestine was scarcely affected by rolipram. These results strongly suggest that IFN-gamma is the most important (or essential) cytokine in protective immunity to H. nana re-infection, rather than TNF-alpha and IL-1 beta.     

Features of Schistosoma mansoni infection in SCID mice

Features of Schistosoma mansoni infection in SCID mice, which lack functional T- and B-lymphocytes, were investigated. The retarded development of parasites as well as reduction of liver egg recovery in SCID mice was significantly lower than those in congenic counterpart C.B-17 mice. Furthermore, the rate of parasite recovery from SCID mice with primary infection was always lower than that from C.B-17 mice by 20%, showing the innate resistance to S. mansoni infection. SCID mice vaccinated with UV-attenuated S. mansoni cercariae did not show protective immunity against a homologous challenge infection. The present innate resistance exhibited in SCID mice is discussed in relation to cell mediated immunity of macrophage activation by IFN-gamma which would not involve T-lymphocytes but is initiated by IL-12 and TNF-alpha cytokines. SCID mice may provide novel information on the host-parasite relationship in schistosome infections.     

IL-4 protects adult C57BL/6 mice from prolonged Cryptosporidium parvum infection: analysis of CD4+alpha beta+IFN-gamma+ and CD4+alpha beta+IL-4+ lymphocytes in gut-associated lymphoid tissue during resolution of infection

Resistance of adult C57BL/6 mice to severe Cryptosporidium parvum infection is dependent on CD4+alpha beta+ TCR lymphocytes. In this study, we demonstrated that treatment with anti-IFN-gamma mAb extended oocyst excretion 18 days longer, and anti-IL-4 mAb extended oocyst excretion at least 11 days longer than isotype control mAb treatment. Analysis of the specific activity of anti-IFN-gamma mAb present in treated mouse sera suggested that IFN-gamma may have a limited role in the resolution phase of infection. Changes were also documented in numbers of CD4+alpha beta+IFN-gamma+ and CD4+alpha beta+IL-4+ lymphocytes in Peyer's patches and intraepithelium of adult C57BL/6 mice during resolution of C. parvum infection. Resistance to initial severe infection was associated with CD4+alpha beta+IFN-gamma+ lymphocytes, and eventual resolution of infection was associated with CD4+alpha beta+IL-4+ lymphocytes. Analysis of cytokine expression following in vitro stimulation with C. parvum Ags during resolution of infection demonstrated consistent increases in CD4+alpha beta+IL-4+ lymphocytes, but not CD4+alpha beta+IFN-gamma+ lymphocytes. The relevance of CD4+alpha beta+IL-4+ lymphocytes in protection against C. parvum was then evaluated in C57BL/6 IL-4 gene knockout mice (IL-4(-/-)). Adult IL-4(-/-) mice excreted oocysts in feces approximately 23 days longer than IL-4(+/+) mice. Further, anti-IFN-gamma mAb treatment increased the severity and the duration of infection in IL-4(-/-) mice compared with those in IL-4(+/+) mice. Together, the data demonstrated that IFN-gamma was important in the control of severity of infection, and either IFN-gamma or IL-4 accelerated termination of infection. However, neither IL-4 nor IFN-gamma was required for the final clearance of infection from the intestinal tract of adult mice.     

Protective effect of a single interleukin-12 (IL-12) predose against the toxicity of subsequent chronic IL-12 in mice: role of cytokines and glucocorticoids

The mechanisms of interleukin-12 (IL-12) toxicity were studied in mice using a schedule (murine rIL-12, 400 ng/mouse, intraperitoneally [IP] once daily for 5 days) that markedly reduced body weight and food intake. On day 5, IL-12-treated mice had elevated serum and spleen IFN-gamma and tumor necrosis factor (TNF). Serum sTNFR-P75 and corticosterone (CS) were also elevated. IL-12 toxicity was partially prevented by anti-IFN-gamma antibodies or dexamethasone (DEX). A pre-dose of IL-12 (200 ng/mouse on day -14) completely prevented the toxicity of subsequent IL-12. The IL-12 predose also inhibited IL-12-induced IFN-gamma levels, but did not modify IL-12-induced CS, TNF or sTNFR-P75. A protective effect was observed with a predose of lipopolysaccharide (LPS) or murine recombinant (r)IL-10. The protective effect of the IL-12 predose was reduced by coadministration of anti-IFN-gamma, but a predose of murine rIFN-gamma was not protective, suggesting that IFN-gamma is necessary but not sufficient for the protective effect of IL-12. The IL-12 predose specifically protected against IL-12 toxicity and did not modify LPS toxicity. These data indicate that IL-12 can induce tolerance to its own toxicity, probably through a downregulation of IL-12-induced IFN-gamma but independently of endogenous glucocorticoids. IFN-gamma, and possibly IL-10, might be important in this tolerance.     

High sensitivity of transgenic mice expressing soluble TNFR1 fusion protein to mycobacterial infections: synergistic action of TNF and IFN-gamma in the differentiation of protective granulomas

To investigate the role of tumor necrosis factor (TNF) in protective immune responses to Mycobacterium tuberculosis and M. bovis Bacillus Calmette Guérin (BCG), we have used transgenic mice unable to use TNF because of the expression of high amounts of a soluble TNF receptor (R) type I (sTNFR1) fusion protein, and studied resistance of these mice to infection by lethality assays, evaluation of bacterial recovery and histologic examination. These mice showed a strongly increased sensitivity to M. tuberculosis and BCG infections, with bacterial overgrowth and marked inhibition of macrophage differentiation within granulomas; after M. tuberculosis infection, this resulted in extensive lesions of caseous necrosis in the lung. To explore the respective roles of TNF and interferon (IFN)-gamma in resistance to BCG and granuloma differentiation, controls and sTNFR1-transgenic mice were compared to IFN-gammaR mutant mice and mice double defective in TNF and IFN-gamma activity (obtained by crossing transgenic and mutant mice). The three groups of deficient mice showed a strongly enhanced susceptibility to BCG infection, with the following decreasing order of sensitivity between groups: TNF + IFN-gamma --> TNF --> IFN-gamma-deficient mice. The hepatic granulomas of IFN-gammaR mutant mice were small and contained eosinophils but few differentiated macrophages; compared to those of sTNFR1-transgenic mice, acid-fast bacilli were less numerous within the macrophages. Granulomas of double-deficient mice were strikingly different by their very large size and cellular content, made up large numbers of polymorphonuclears, eosinophils, and cells undergoing apoptosis, but without detectable differentiated macrophages; acid-fast bacilli were spread in the lesions. These studies show the essential role of both TNF and IFN-gamma in the development, during mycobacterial infections, of protective granulomas containing highly differentiated macrophages capable of destroying ingested bacteria, and emphasize that these two cytokines act synergistically in granuloma formation.     

Interleukin-18 protects mice against acute herpes simplex virus type 1 infection

We examined the effects of interleukin-18 (IL-18) in a mouse model of acute intraperitoneal infection with herpes simplex virus type 1 (HSV-1). Four days of treatment with IL-18 (from 2 days before infection to 1 day after infection) improved the survival rate of BALB/c, BALB/c nude, and BALB/c SCID mice, suggesting innate immunity. One day after infection, HSV-1 titers were higher in the peritoneal washing fluid of control BALB/c mice than in that of IL-18-treated mice. A genetic deficiency of gamma interferon (IFN-gamma), however, diminished the survival rate and the inhibition of HSV-1 growth at the injection site in the mice. Anti-asialo GM1 treatment had no influence on the protective effect of IL-18 in infected mice. IL-18 augmented IFN-gamma release in vitro by peritoneal cells from uninfected mice, while no appreciable IFN-gamma production was found in uninfected mice administered IL-18. Although IFN-gamma has the ability to induce nitric oxide (NO) production by various types of cells, administration of the NO synthase inhibitor NG-monomethyl-L-arginine resulted in superficial loss of the improved survival, but there was no influence on the inhibition of HSV-1 replication at the injection site in IL-18-treated mice. Based on these results, we propose that IFN-gamma produced before HSV-1 infection plays a key role as one of the IL-18-promoted protection mechanisms and that neither NK cells nor NO plays this role.     

Control of IL-12 and IFN-gamma production in response to live or dead bacteria by TNF and other factors

When mice were infected i.v. with either Listeria monocytogenes or Brucella abortus, bioactive IL-12 was briefly detected in serum and supernatants of spleen homogenates immediately ex vivo. Although the time scale was more prolonged for the more slowly growing B. abortus, in both instances IL-12 production ceased while bacteria still persisted in high numbers. Production of IL-12, detected in serum and spleen, was neither increased nor prolonged by injecting Abs to IL-10 or IL-4. In contrast with live organisms, heat-killed bacteria did not induce detectable IL-12 in vivo and were less efficient when added in vitro to resident peritoneal cells or spleen cells. Mice lacking the receptors for TNF (TNFR-/- mice) were severely deficient in IL-12 production, suggesting a controlling role for TNF, which we have previously shown to be triggered by live, rather than dead, bacteria. Infection in the TNFR-/- mice was exacerbated, although in the Brucella-infected mice splenomegaly, the main indicator of immunopathology, was reduced. Production of NO by macrophages was deficient, but the TNFR-/- mice were not deficient in IFN-gamma production. In addition to being poor inducers of IL-12, killed bacteria actively suppressed IL-12 production in response to live bacteria, by mechanism(s) unknown. The implications of these findings are discussed in light of the fact that only live bacteria satisfactorily induce cell-mediated immunity to infection.     

Treatment with anti-interleukin-10 monoclonal antibody enhances early resistance to but impairs complete clearance of Listeria monocytogenes infection in mice

Mice that received an anti-interleukin-10 (anti-IL-10) neutralizing monoclonal antibody (MAb) (SXC-1) prior to infection with Listeria monocytogenes initially demonstrated resistance to the infection, as indicated by reduced recovery of L. monocytogenes from their spleens and livers during the first 5 days after challenge. Anti-IL-10 MAb-treated mice then demonstrated reduced resistance during the later stage of infection, as indicated by persistent infection with L. monocytogenes in their livers 11 days after challenge. Aspartate aminotransferase (AST) levels (a measure of liver damage) in the sera of control mice increased between 1 and 5 days after challenge, while anti-IL-10 MAb-treated mice maintained lower AST levels. At 7 days after challenge, AST levels in the sera of control mice decreased as the numbers of organisms declined. In contrast, AST levels increased as the infections persisted in anti-IL-10 MAb-treated mice. The AST levels in serum reflected liver histopathology as anti-IL-10 MAb-treated mice exhibited fewer granulomatous lesions and less necrosis of liver tissue than the control mice during the first 5 days after challenge. Anti-IL-10 MAb treatment altered the expression of inflammatory cytokine mRNAs during L. monocytogenes infection. Control MAb-treated mice exhibited increased expression of tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor mRNA in their lives during L. monocytogenes infection, but this increase did not occur in anti-IL-10 MAb-treated mice. Gamma interferon mRNA expression in the livers of the control MAb-treated mice was increased between 1 and 5 days after L. monocytogenes challenge and then decreased at 7 days after challenge. In contrast, gamma interferon mRNA expression in the livers of anti-IL-10 MAb-treated mice was not decreased until 7 days after challenge. These results indicate that endogenous IL-10 has both beneficial and detrimental effects on the host response to L. monocytogenes infection in mice.     

Attenuation and vaccine potential of aroQ mutants of Corynebacterium pseudotuberculosis

Corynebacterium pseudotuberculosis, a gram-positive intracellular bacterial pathogen, is the etiological agent of the disease caseous lymphadenitis (CLA) in both sheep and goats. Attenuated mutants of C. pseudotuberculosis have the potential to act as novel live veterinary vaccine vectors. We have cloned and sequenced the aroB and aroQ genes from C. pseudotuberculosis C231. By allelic exchange, aroQ mutants of both C231, designated CS100, and a pld mutant strain TB521, designated CS200, were constructed. Infection of BALB/c mice indicated that introduction of the aroQ mutation into C231 and TB521 attenuated both strains. In sublethally infected BALB/c mice, both CS100 and CS200 were cleared from spleens and livers by day 8 postinfection. The in vivo persistence of these strains was increased when the intact aroQ gene was supplied on a plasmid in trans. Mice infected with TB521 harbored bacteria in organs at least till day 8 postinfection without ill effect. When used as a vaccine, only the maximum tolerated dose of CS100 had the capacity to protect mice from homologous challenge. Vaccination with TB521 also elicited protective immunity, and this was associated with gamma interferon (IFN-gamma) production from splenocytes stimulated 7 days postvaccination. The role of IFN-gamma in controlling primary infections with C. pseudotuberculosis was examined in mice deficient for the IFN-gamma receptor (IFN-gammaR(-/-) mice). IFN-gammaR(-/-) mice cleared an infection with CS100 but were significantly more susceptible than control littermates to infection with C231 or TB521. These studies support an important role for IFN-gamma in control of primary C. pseudotuberculosis infections and indicate that aroQ mutants remain attenuated even in immunocompromised animals. This is the first report of an aroQ mutant of a bacterial pathogen, and the results may have implications for the construction of aromatic mutants of Mycobacterium tuberculosis for use as vaccines.     

Endogenous IL-12 is required for control of Th2 cytokine responses capable of exacerbating leishmaniasis in normally resistant mice

We investigated the mechanisms by which treatment with anti-IL-12 Ab prevents cure of infection with Leishmania major in resistant C57BL/6 mice. Consistent with delayed production of IL-12, anti-IL-12 Abs could be administered as late as 2 wk after infection to exacerbate disease. Starting at 2 wk of infection, the cultured lymph node cells from mice treated with either polyclonal or monoclonal anti-IL-12 Abs persistently generated 3- to 10-fold more IL-4 and IL-10 in response to L. major Ag compared with cells from mice receiving preimmune goat IgG. Reciprocal decreases in Ag-specific IFN-gamma production were observed in mice receiving anti-IL-12 Abs. A similar reversal of IFN-gamma and IL-4 production accompanied progressive disease induced by pretreatment with a single dose of anti-IFN-gamma mAb. Although IFN-gamma production was suppressed for up to 4 wk in mice treated with monoclonal anti-IL-12 or anti-IFN-gamma, coadministration of neutralizing anti-IL-4 IgG reversed progressive illness. These findings demonstrate that IL-12 produced in vivo is necessary for both the emergence of IFN-gamma producing cells and the down-regulation of Th2 cell responses during murine leishmaniasis. Furthermore, the uninhibited production of IL-4 was required to sustain progressive infection initiated by the decreased IFN-gamma synthesis observed in anti-IL-12 and anti-IFN-gamma-treated mice.     

Treatment with the antigranulocyte monoclonal antibody RB6-8C5 impairs resistance of mice to gastrointestinal infection with Listeria monocytogenes

Treatment with the antigranulocyte monoclonal antibody (MAb) RB6-8C5 increased the severity of infection in mice intragastrically inoculated with Listeria monocytogenes. Most MAb RB6-8C5-treated mice died when inoculated intragastrically with as few as 4 x 10(4) L. monocytogenes bacteria, whereas most control mice survived intragastric inoculation with 4 x 10(8) L. monocytogenes bacteria. The increased severity of infection in MAb RB6-8C5-treated mice appeared to result from listerial multiplication in the spleen and liver rather than from local proliferation in the intestinal tract or mesenteric lymph nodes.     

Immunity to Cryptosporidium muris infection in mice is expressed through gut CD4+ intraepithelial lymphocytes

The role of gut intraepithelial lymphocytes (IEL) in immunity to cryptosporidial infection was investigated with a murine infection model involving Cryptosporidium muris. Oocyst shedding was monitored in severe combined immunodeficiency (SCID) mice infected with C. muris following intravenous injection of mesenteric lymph node (MLN) cells or intestinal IEL from BALB/c donor mice which were naive or previously infected with C. muris. SCID mice receiving no lymphoid cells developed chronic infections and excreted large numbers of oocysts until the end of the experiment. SCID mice injected with IEL from immune animals, however, were able to overcome the infection, and furthermore, these animals produced fewer oocysts and recovered sooner than ones which received IEL or MLN cells from naive BALB/c donors. Similar levels of protection were obtained in SCID mice injected with either 2 X 10(6) IEL or MLN cells from immune donor mice. Depletion of CD4+ cells from immune IEL, however, abrogated the ability to transfer immunity to SCID mice, while depletion of CD8+ cells only marginally reduced the protective capacity of immune IEL. Finally, control SCID mice which received no lymphocytes had < or = 1% CD4+ cells in the IEL from the small intestine, whereas the IEL from SCID mice recovered from infection, as a result of injection with immune IEL, contained 15% CD4+ cells. Thus, the ability to control C. muris infection correlated with the presence of the protective CD4+ cells in the gut epithelium.     

Th1 response in Salmonella typhimurium-infected mice with a high or low rate of bacterial clearance

Previous studies have shown that the capacity to clear an attenuated strain of Salmonella typhimurium after the second week of infection varies widely among mouse strains. Bacterial clearance is mediated by CD4+ T cells and is regulated in part by the H-2 complex. The aim of the present study was to compare the patterns of cytokine mRNA expression in the spleens of C57BL/6 (H-2b) and CBA (H-2k) mice, which exhibit a low and a high rate of bacterial clearance, respectively. A transient increase in interleukin-12 (IL-12) mRNA levels was found in both mouse strains. Gamma interferon (IFN-gamma) gene expression was higher and more sustained in C57BL/6 than in CBA mice. No increase in IL-4 mRNA was detected. A transient increase in IL-10 mRNA was found in C57BL/6 mice. Separation of spleen cells into CD4+ and CD4- fractions showed that CD4+ T cells produced the bulk of IFN-gamma in both mouse strains and of IL-10 in C57BL/6 mice. Infection of H-2 congenic mice induced a higher level of IFN-gamma mRNA expression by CD4+ T cells in mice with a low rate of clearance (H-2b) than in mice with a high rate of clearance (H-2q). Treatment of infected C57BL/6 mice with anti-IFN-gamma or anti-CD4 monoclonal antibodies indicated that IFN-gamma participates in resistance in the early phase of infection, but not in bacterial clearance, and that CD4+ T cells mediate bacterial clearance during the 3rd week of infection. Taken together, these results suggest that defective bacterial clearance in H-2b mice is not linked to defective IFN-gamma production and that CD4+ T cells mediate bacterial clearance by an IFN-gamma-independent mechanism.     

Direct cardiac effects in isolated perfused rat hearts measured at increasing concentrations of morphine, alfentanil, fentanyl, ketamine, etomidate, thiopentone, midazolam and propofol

The development of distant metastases to multiple organs is a critical problem in the treatment of human lung cancer. In this study, we evaluated the therapeutic efficacy of a mouse-human chimeric anti-ganglioside GM2 (GM2) monoclonal antibody (MAb), KM966 against metastasis formation of GM2-positive human lung cancer cells inoculated intravenously (i.v.) into natural killer (NK) cell-depleted severe combined immunodeficient (SCID) mice. GM2-positive human small cell lung cancer (SCLC), SBC-3 cells (1 x 10(6)), injected through a tail vein into NK cell-depleted SCID mice, formed large number of metastatic colonies in the liver, kidneys and lymph nodes by 42 days after inoculation (day 42). KM966, but not control MAb, given on days 2 and 7, almost completely inhibited metastasis formation of SBC-3 cells in the liver, kidneys and lymph nodes in a dose-dependent fashion. Moreover, treatment with KM966 at advanced stages of metastasis (even from day 28) significantly suppressed multiple organ metastases of SBC-3 cells. The anti-metastatic effect of KM966 in vivo was mainly due to an antibody-dependent cell-mediated cytotoxicity (ADCC) reaction mediated by macrophages of the SCID mice. Our findings suggest that the mouse-human chimeric anti-GM2 MAb, KM966 may be useful for eradicating multiple organ micrometastases of lung cancer in humans.     

Induction of protective T cells against Listeria monocytogenes in mice by immunization with a listeriolysin O-negative avirulent strain of bacteria and liposome-encapsulated listeriolysin O

Only listeriolysin O (LLO)-producing strains of Listeria monocytogenes generate protective immunity in mice. Based on the findings that endogenous gamma interferon (IFN-gamma) production was induced only by such strains and that purified LLO could induce IFN-gamma from NK cells, we have postulated that LLO may play a pivotal role in the induction of Th1-type protective T cells, which are highly dependent on IFN-gamma. In this study, mice were immunized with L. monocytogenes ATCC 15313, an LLO-nonproducing avirulent strain, along with LLO encapsulated in liposome (LLO-liposome). LLO-liposome was highly potent in the induction of various cytokines, including IFN-gamma. Immunization of mice with either LLO-liposome or the viable strain ATCC 15313 alone did not induce protection against challenge infection. In contrast, the combination of LLO-nonproducing bacteria plus LLO-liposome induced a significant level of protective immunity mediated mainly by Th1-type cells capable of producing a large amount of IFN-gamma in an antigen-specific manner. The protection afforded by the combination was not dependent on LLO-specific cytotoxic T cells. These results support the idea that the inability of an LLO-nonproducing avirulent strain or killed bacteria to induce the generation of protective T cells is due not to the lack of a central T-cell epitope(s) but to the lack of ability to induce the production of endogenous cytokine during the early stage of immunization; the results also suggest that an appropriate use of LLO at least in an animal model may be effective in the induction of antigen-specific Th1-dependent protective immunity to various kinds of intracellular parasitic bacteria.     

Reconstitution of C.B-17 scid mice with BALB/c T cells initiates a T helper type-1 response and renders them capable of healing Leishmania major infection

C.B-17 scid mice, which were found to be very susceptible to infection with Leishmania major, were reconstituted with various doses of T cells, T plus B cells or unfractionated spleen cells from nonhealer BALB/c mice. All reconstitution protocols, except for the transfer of very high numbers of BALB/c spleen cells, led to a spontaneously healing infection and resistance to reinfection, rather than the lethal, nonhealing infection typical of BALB/c mice. These healing responses were associated with a strong T helper 1 (Th1)-like response characterized by delayed-type hypersensitivity (DTH) responsiveness, but no elevation of serum IgE, and by the production of high levels of interferon-gamma (IFN-gamma), but no interleukin-4 (IL-4) by lymph node and spleen cells after restimulation with antigen in vitro. The development of this Th1 response from BALB/c Th cells requires IFN-gamma during the initial infection period. Treatment of scid mice with a single injection of neutralizing anti-IFN-gamma antibody prior to infection and reconstitution prevented healing and permitted the development of a Th-2 like response as indicated by elevated serum IgE, but no DTH, and by the production of IL-4, but very little IFN-gamma, after antigen stimulation in vitro. As few as 10(4) transferred T cells led to a Th1-like response, suggesting that the IFN-gamma is of host rather than donor origin. The transfer of very high numbers (7.5 x 10(7)) of BALB/c spleen cells overcame the effects of the IFN-gamma and led to the nonhealing infection and cytokine pattern characteristic of BALB/c mice. The enrichment or depletion of B cells from the transferred T cells had no measurable effect upon the development of a healing response in reconstituted scid mice.     

Route of infection that induces a high intensity of gamma interferon-secreting T cells in the genital tract produces optimal protection against Chlamydia trachomatis infection in mice

The induction of local T helper type 1 (Th1)-mediated cellular immunity is crucial for resistance of mice to genital infection by the obligate intracellular bacterium Chlamydia trachomatis. We tested the hypothesis that the route of immunization that elicits relatively high numbers of chlamydia-specific, gamma interferon (IFN-gamma)-secreting T lymphocytes (ISTLs) in the genital tract would induce optimal protective immunity against reinfection. Female BALB/c mice were infected intravaginally (i.v.), intranasally (i.n.), orally (p.o.), or subcutaneously (s.c.) with C. trachomatis. At days 7, 14, 21, and 28 postinfection, T cells isolated from the genital tract tissues were restimulated with chlamydial antigen in vitro, and the amounts of IFN-gamma induced were measured by a sandwiched enzyme-linked immunosorbent assay method. At day 7 postinfection, i.n.- and i.v.-immunized mice had high levels of chlamydia-specific ISTLs in their genital tracts (203.58 +/- 68.1 and 225.5 +/- 12.1 pg/ml, respectively). However, there were no detectable ISTLs in the genital tracts of p.o.- or s.c.-infected mice. When preinfected mice were challenged i.v. 70 days later, animals preexposed by the i.n. route were highly resistant to reinfection, with greatly reduced chlamydial burden, and suffered an attenuated infection that resolved by day 6 postchallenge. Animals preexposed by the i.v. route were modestly protected, whereas p.o. and s.c. groups were indistinguishable in this regard from control mice. The resistance of i.n.-immunized mice (and to some extent the i.v.-exposed mice) to reinfection was associated with early appearance (within 24 h) of high levels of genital ISTLs compared with mice preinfected by other routes. Furthermore, although i.n. and i.v.-immunized mice had comparable levels of chlamydia-specific immunoglobulin A (IgA) antibodies in their vaginal washes, the levels of IgG2a were four- sixfold higher in i.n.-immunized mice than in any of the other groups. The results suggested that immunization routes that foster rapid induction of vigorous genital mucosal cell-mediated immune (CMI) effectors (e.g., IFN-gamma), the CMI-associated humoral effector, IgG2a, and to some extent secretory IgA produce protective immunity against chlamydial genital infection. Therefore, i.n. immunization is a potential delivery route of choice in the development of a vaccine against Chlamydia.