Cerebral edema and cerebral hemorrhages in interleukin-10-deficient mice infected with Plasmodium chabaudi

During a Plasmodium chabaudi infection in interleukin-10 (IL-10) knockout mice, there is greater parasite sequestration, more severe cerebral edema, and a high frequency of cerebral hemorrhage compared with infection of C57BL/6 mice. Anti-tumor necrosis factor alpha treatment ameliorated both cerebral edema and hemorrhages, suggesting that proinflammatory responses contributed to cerebral complications in infected IL-10(-/-) mice.     

Human recombinant tumor necrosis factor alpha protects susceptible A/J mice against lethal Plasmodium chabaudi AS infection.

The effect of intravenous treatment with human recombinant tumor necrosis factor alpha (rTNF-alpha) on infection of susceptible A/J and resistant C57BL/6 mice with Plasmodium chabaudi AS was examined. Treatment of A/J mice with 10(3) or 10(5) U of rTNF-alpha on days 0, 3, 5, 7, and 9 after intraperitoneal infection with 10(6) parasitized erythrocytes resulted in 80% survival and a significant decrease in the peak parasitemia level. Treatment of susceptible A/J hosts with 10(5) but not 10(3) U of rTNF-alpha resulted in increased survival but did not alter the peak parasitemia level following infection with 10(7) parasitized erythrocytes. Moreover, all surviving A/J mice completely eliminated the parasite by approximately 4 weeks and were fully protected against a secondary infection. Except at a dose of 5 x 10(5) U of rTNF-alpha, which resulted in 100% mortality of infected animals, rTNF-alpha did not alter the course or outcome of infection with P. chabaudi AS in resistant C57BL/6 mice.     

Impaired macrophage responses may contribute to exacerbation of blood-stage Plasmodium chabaudi chabaudi malaria in interleukin-12-deficient mice.

Aiming to clarify the role of endogenous interleukin-12 (IL-12) in protective immunity against blood stages of Plasmodium chabaudi chabaudi (AS), we evaluated the course of infection in IL-12p40 gene knockout (IL-12p40KO) and wild-type (WT) C57BL/6 mice, focusing (1) on the ability of T cells to develop adequate type 1 responses and (2) on the potentiality of macrophages to respond to parasites, interferon-gamma (IFN-gamma), or both. We observed that IL-12p40KO mice develop significantly higher parasitemias during the acute infection, although mice from both groups clear the parasites within a month and similarly eliminate a secondary challenge. Thus, fully protective immunity to P. c. chabaudi can be generated in the absence of IL-12. However, this cytokine may promote parasite control during the early phase of infection. The increased acute parasitemia of IL-12p40KO mice was associated with both impaired IFN-gamma and nitric oxide (NO) response by spleen cells. Because stimulation with recombinant IFN-gamma (rIFN-gamma) failed to improve the NO response in IL-12p40KO macrophages, we investigated whether these cells have an intrinsic defect. Analysis of peritoneal macrophages revealed that IL-12p40KO cells produce higher levels of transforming growth factor-beta1 (TGF-beta1) compared with WT cells and respond to infected erythrocytes or rIFN-gamma by releasing little NO. Moreover, IL-12p40KO macrophages had a severely impaired ability to internalize opsonized infected erythrocytes, suggesting that the low effector profile assumed by these cells may compromise antibody-mediated immunity. Taken together, our results support the idea that the absence of IL-12p40 not only affects IFN-gamma production but also has deep consequences in macrophage effector functions that may contribute to exacerbation of the early phase of P. c. chabaudi malaria.     

Transforming growth factor beta-induced failure of resistance to infection with blood-stage Plasmodium chabaudi in mice

The role of transforming growth factor beta (TGF-beta) in infection with Plasmodium chabaudi was investigated with resistant and susceptible mouse models. C57BL/10 mice produced gamma interferon (IFN-gamma) and nitric oxide (NO) shortly after infection and cleared the parasite spontaneously. In contrast, BALB/c mice showed a transient enhancement of TGF-beta production, followed by a relative lack of IFN-gamma and NO production, and succumbed to the infection. However, there was no correlation between levels of serum TGF-beta and splenic TGF-beta mRNA in both mouse strains before and after infection. Administration of recombinant TGF-beta (rTGF-beta) rendered resistant mice susceptible because of suppression of subsequent production of IFN-gamma and NO. Administration of anti-TGF-beta antibody to the infected BALB/c mice resulted in remarkable increases in serum IFN-gamma and NO, and the mice resisted the infection. Splenic CD4(+) T and CD11b+ cells of C57BL/10 mice were significantly activated after infection, but this was completely abrogated by administration of rTGF-beta. These results suggested that, in the P. chabaudi-susceptible but not resistant mice, production of TGF-beta was promoted, and subsequent failure of IFN-gamma- and NO-dependent resistance to the parasite was induced. This study is the first to indicate that TGF-beta production was the key event in failure of resistance to mouse malaria.     

Role of tumor necrosis factor alpha in Helicobacter pylori gastritis in tumor necrosis factor receptor 1-deficient mice

Increased gastric production of interleukin 8 and tumor necrosis factor alpha (TNF-alpha) has been implicated in the pathogenesis of Helicobacter pylori-associated gastroduodenal disease. In the present study we used a mouse model to demonstrate whether loss of the tumor necrosis factor receptor 1 (TNF-R1) function leads to differences in gastric inflammation or the systemic immune response in H. pylori infection. Six different clinical isolates of H. pylori (three cytotoxin-positive and three cytotoxin-negative strains) were adapted to C57BL/6 mice. TNF-R1-deficient (TNF-R1(-/-)) mice (n = 19) and isogenetic controls (n = 24) were infected and sacrificed after 4 weeks of infection. Inflammation of the stomach and the humoral immune response to H. pylori were evaluated by histological, immunohistochemical, and serological methods. There was no detectable difference in the grade or activity of gastritis in TNF-R1(-/-) mice when they were compared with wild-type mice, but the number of lymphoid aggregates was slightly reduced in the gastric mucosa of TNF-R1(-/-) mice. Interestingly, total immunoglobulin G (IgG), as well as IgG1, IgG2b, and IgG3, H. pylori-specific antibody titers were significantly higher in wild-type mice. As revealed by immunoblot analysis, the difference in reactivity against H. pylori antigens was not based on a failure to recognize single H. pylori antigens in TNF-R1(-/-) mice. We therefore suggest that TNF-R1-mediated TNF-alpha signals might support a systemic humoral immune response against H. pylori and that the gastric inflammatory response to H. pylori infection seems to be independent of TNF-R1-mediated signals.     

Reversal of lipopolysaccharide-analog-induced antibody suppression by anti-transforming growth factor beta and indomethacin.

Monophosphoryl lipid A and a synthetic, nontoxic monosaccharide analog of lipid A, termed GLA 60, both strongly suppressed antibody production when administered 1 to 2 days prior to antigen. Evidence is presented that this suppression was mediated by two cytokines, prostaglandin E and transforming growth factor beta, because it was reversed by multiple injections of the cyclooxygenase inhibitor indomethacin and by in vitro addition of antibody to transforming growth factor beta.     

Porphyromonas gingivalis infection during pregnancy increases maternal tumor necrosis factor alpha,suppresses maternal interleukin-10, and enhances fetal growth restriction and resorption in mice

Epidemiological studies have shown a potential association between maternal periodontitis and pregnancy complications. We used a pregnant murine model to study the effect of infection with the periodontal pathogen Porphyromonas gingivalis on pregnancy outcomes. Female BALB/c mice were inoculated with heat-killed P. gingivalis (10(9) CFU) in a subcutaneous chamber and mated 2 weeks later. At gestation day (GD) 7.5, mice were challenged with live P. gingivalis (10(7) CFU) (n = 20) or broth (control; n = 8) and sacrificed at GD 16.5. Fetal growth restriction (FGR, <0.46 g) was defined as fetuses with weights 2 standard deviations (SD) smaller than controls (0.56 +/- 0.05 g [mean +/- SD]). Among the 20 challenged mice, 8 had both normal-weight (0.51 +/- 0.11 g) and FGR (0.34 +/- 0.1 g) fetuses within the same litter. All other challenged dams had normal-weight fetuses (0.57 +/- 0.04 g). Maternal liver, uterus, and spleen samples were examined for P. gingivalis DNA using a PCR technique. Of the eight challenged mice with FGR fetuses, three had PCR signals for P. gingivalis in liver and uterus, but not in the spleen. Liver, uterus, and spleen were negative for P. gingivalis DNA among all other challenged and control mice. In serum of dams with FGR fetuses, tumor necrosis factor alpha levels were elevated significantly, while interleukin-10 levels were significantly reduced compared to levels in dams with normal fetuses. P. gingivalis-specific serum immunoglobulin G levels were significantly elevated in dams with FGR fetuses compared to dams without any FGR fetuses. These data demonstrate that P. gingivalis-induced murine FGR is associated with systemic dissemination of the organism and activated maternal immune and inflammatory responses.     

Increased mortality and dysregulated cytokine production in tumor necrosis factor receptor 1-deficient mice following systemic Klebsiella pneumoniae infection

A significant clinical complication of pulmonary infections with Klebsiella pneumoniae is peripheral blood dissemination, resulting in a systemic infection concurrent with the localized pulmonary infection. In this context, little is known about the role of tumor necrosis factor receptor 1 (TNFR1)-mediated innate immune responses during systemic Klebsiella infections. Mice lacking TNFR1 were significantly more susceptible to Klebsiella-induced mortality following intravenous inoculation. Bacterial clearance was impaired in TNFR1-deficient mice at early times following infection. Unexpectedly, bacterial burdens at the onset of mortality (days 2 to 3 postinfection) were not higher in mice lacking TNFR1. However, elevated production of liver-associated proinflammatory cytokines (interleukin-12, tumor necrosis factor alpha [TNF-alpha[, and gamma interferon [IFN-gamma]) and chemokines (MIP-1 alpha, MIP-2, and MCP-1) was observed within the first 24 h of infection. Additionally, excessive plasma-associated IFN-gamma was also observed late in the course of infection (day 3). Spleen cells from day-3 infected TNFR1-deficient mice secreted markedly enhanced levels of IFN-gamma when cultured in vitro. Additionally, there was a marked increase in the total number of activated lymphocyte subsets as indicated by CD69 upregulation. A notable exception was the sharp decrease in the frequency of splenic NK T cells in infected TNFR1 knockout (KO) mice. Anti-TNF-alpha therapy in TNFR1 KO mice significantly reduced chemokine production and liver injury. Combined, these data indicate a dysregulated antibacterial host response following intravenous Klebsiella infection in the absence of TNFR1 signaling, resulting in heightened cytokine production and hyperactivation of specific splenic lymphocyte subsets.     

High serum interleukin-10 and tumor necrosis factor alpha levels in chronic paracoccidioidomycosis

In patients with chronic paracoccidioidomycosis (n = 10), levels of tumor necrosis factor alpha, interleukin-10, and interleukin-2 in serum, measured by enzyme-linked immunosorbent assay (in picograms per milliliter, as mean +/- standard error of the mean), were higher than in normal controls (n = 8): 186 +/- 40 versus 40 +/- 7 (P < 0.05), 203 +/- 95 versus 20 +/- 8 (P = 0.001), and 96.3 +/- 78.57 versus 1.19 +/- 1.19 (P = 0.045), respectively. Gamma interferon and interleukin-4 levels were similar in patients and controls.     

Cytokine appearance and effects of anti-tumor necrosis factor alpha antibodies in a neonatal rat model of group B streptococcal infection.

Cytokines are suspected of playing an important role in the pathophysiology of septic shock. This study was undertaken to determine whether tumor necrosis factor alpha (TNF-alpha) induces the production of other cytokines and mediates mortality in a neonatal rat model of sepsis caused by group B streptococci (GBS). We have measured TNF-alpha, interleukin-1 alpha (IL-1 alpha), interleukin-6 (IL-6), and gamma interferon (IFN-gamma) levels in neonatal rats infected with different strains (H738, 259, and 90) and doses (1 50% lethal dose [LD50] and 5 90% lethal doses [LD90]) of type III GBS. TNF-alpha and IL-6 were detected by the L929 cytotoxicity and the B9 proliferation assays, respectively, in serial plasma samples. IL-1 alpha and IFN-gamma were measured in spleen homogenates by enzyme-linked immunosorbent assay kits by using antibodies raised against the corresponding mouse cytokines. Plasma TNF-alpha levels significantly rose above baseline values within 12 h after intraperitoneal challenge with 5 LD90 of GBS strain H738, corresponding to 3 x 10(3) CFU. A mean peak TNF-alpha concentration of 232 +/- 124 U/ml was reached at 20 h. Peak IL-1 alpha and IL-6 levels of 766 +/- 404 U/g and 1,033 +/- 520 U/ml, respectively, were reached at 24 h after bacterial challenge. Maximal spleen concentrations of IFN-gamma (449 +/- 283 U/g) were measured at 36 h. Concentrations of TNF-alpha, but not other cytokines, remained significantly elevated at 72 h, a time when mortality approached 100%. Significant correlations were found between concentrations of each of the cytokines tested and the logs of CFU concentrations in the blood. In order to ascertain whether TNF-alpha influenced the production of other cytokines, rat pups received two injections of anti-murine TNF-alpha or normal rabbit serum at 2 h before and at 26 h after challenge with live GBS. Plasma TNF-alpha bioactivity was undetectable in anti-TNF-alpha-treated animals, while IL-6 and IFN-gamma, but not IL-1 alpha, levels were significantly reduced, compared with normal serum controls. Rat pups pretreated with anti-TNF-alpha serum and infected with 1 and 5 LD90 of strains H738 and 259 showed enhanced early (48 to 72 h) survival. However, by 96 h this protection was no longer apparent.     

新生儿感染性疾病IL-6、IL-8和TNFα的研究

目的分析比较新生儿感染性疾病(败血症与一般感染)血浆中细胞因子直接及诱生水平的变化特征.方法采用双抗体夹心酶联免疫吸附实验法(ELISA)测定白细胞介素6(IL-6)、白细胞介素8(IL-8)和肿瘤坏死因子α(TNFα)的水平 .结果入院时败血症、一般感染患儿血浆IL-6、IL-8、TNFα直接水平显著高于各自恢复期(P<0.05),败血症患儿三者极显著高于健康对照组(P<0.01),一般感染患儿三者显著高于健康对照组(P<0.05).两组恢复期三者与健康对照组无显著性差异(P>0.05),入院时败血症三者显著高于一般感染患儿(P<0.05).结论新生儿血浆中高水平的IL-6、IL-8和TNFα提示机体存在感染,可做为早期诊断的灵敏指标,并与感染轻重程度有关,还可做为判断疗效的指标.     

Role of interferon-gamma and tumor necrosis factor in host resistance to Plasmodium chabaudi AS

The contribution of the T cell- and macrophage-derived cytokines, interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF), respectively, in the cell-mediated mechanisms leading to acquired immunity to blood-stage Plasmodium chabaudi AS was investigated. To examine the contribution of IFN-gamma, resistant C57BL-derived mice were treated during infection with two different neutralizing, anti-murine IFN-gamma mAbs. Such treatment impaired the ability of the host to limit parasite multiplication just before and at the time of the peak parasitemia but did not abrogate the development of acquired immunity resulting in control and elimination of acute infection. The requirement of endogenous IFN-gamma around the time of the peak parasitemia was confirmed by quantification of IFN-gamma production in vitro by spleen cells from infected animals in response to malaria antigen. To investigate the role of TNF, resistant C57BL/6 and susceptible A/J mice were treated with rTNF during P. chabaudi AS infection. Treatment with 10(3) or 10(5) U rTNF resulted in increased resistance in A/J hosts (that is, increased survival and a less severe course of infection); there was no difference between control and treated C57BL/6 mice in the course of infection but there was increased mortality among the animals treated with rTNF. Splenic macrophages harvested from C57BL/6 mice during infection were found to produce high levels of TNF from day 3 to day 28 post-infection. In conclusion, both IFN-gamma and TNF appear to contribute to host resistance to blood-stage infection with P. chabaudi AS.     

Pneumolysin stimulates production of tumor necrosis factor alpha and interleukin-1 beta by human mononuclear phagocytes.

Human peripheral blood monocytes and a human monocyte cell line were exposed to the toxin pneumolysin. Pneumolysin-exposed cells produced significantly larger amounts of tumor necrosis factor alpha and interleukin-1 beta than cells not exposed to the toxin. The viability of cells was not affected by the concentrations of pneumolysin used in the experiments.     

Endotoxin-induced lethality in neonatal mice is counteracted by interleukin-10 (IL-10) and exacerbated by anti-IL-10.

The lethal effects occurring in neonatal (<24-h-old) BALB/c mice after challenge with 25 mg of lipopolysaccharide (LPS) per kg of body weight were significantly counteracted by pretreatment with recombinant interleukin-10 (rIL-10; 25 or 50 ng/mouse). Concordantly, blockage of endogenous IL-10 with the SXC1 monoclonal antibody increased LPS-induced mortality. Both IL-10 and SXC1 modulated the release of tumor necrosis factor alpha (TNF-alpha) so that, relative to controls, peak TNF-alpha values after LPS challenge were decreased by rIL-10 and increased by anti-IL-10.     

Involvement of tumor necrosis factor alpha and interleukin-1beta in enhancement of pentylenetetrazole-induced seizures caused by Shigella dysenteriae

Neurologic manifestations, mainly convulsions, are the most frequent extraintestinal complications of shigellosis. We used an animal model to study the roles of tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) in Shigella-related seizures. Administration of Shigella dysenteriae 60R sonicate enhanced the sensitivity of mice to the proconvulsant pentylenetetrazole (PTZ) within 7 h. This was indicated by a significantly higher mean convulsion score and an increased number of mice responding with clonic-tonic seizures in the Shigella-pretreated group. Preinjection of mice with anti-murine TNF-alpha (anti-mTNF-alpha) or anti-murine IL-1beta (anti-mIL-1beta) 30 min prior to administration of Shigella sonicate abolished their enhanced response to PTZ at 7 h. Mean convulsion scores were reduced by anti-mTNF-alpha from 1.2 to 0.8 (P = 0.017) and by anti-mIL-1beta from 1.3 to 0.7 (P = 0.008). Preinjection of anti-mTNF-alpha also reduced the percentage of mice responding with clonic-tonic seizures, from 48 to 29% (P = 0.002), and preinjection of anti-mIL-1beta reduced it from 53 to 21% (P = 0. 012). Neutralization of TNF-alpha or IL-1beta did not protect the mice from death due to S. dysenteriae 60R. These findings indicate that TNF-alpha and IL-1beta play a role in the very early sensitization of the central nervous system to convulsive activity after S. dysenteriae administration. Similar mechanisms may trigger neurologic disturbances in other infectious diseases.     

Downregulation by cryptococcal polysaccharide of tumor necrosis factor alpha and interleukin-1 beta secretion from human monocytes.

The regulation by Cryptococcus neoformans encapsulation of interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) production by human monocytes was investigated. By using encapsulated and acapsular C. neoformans, we demonstrated that both strains induce cytokine production, although the acapsular strain was a better stimulator than the thinly encapsulated strain. The cytokine levels produced by cells stimulated by the two strains were lower and followed a different kinetic than those stimulated by lipopolysaccharide (LPS). Purified capsular polysaccharide inhibits TNF-alpha secretion induced by LPS or acapsular C. neoformans. In contrast, no regulator effect on IL-1 beta was observed when LPS was used. The secretory response of these cytokines follows different pathways of macrophage activation; in fact, complete inhibition of TNF-alpha does not affect IL-1 beta production and vice versa. These data indicate that purified capsular polysaccharide of C. neoformans could contribute to the in vivo progress of cryptococcosis by suppressing cytokine production of macrophages and suggest that a therapeutic approach to address the suppressive effect of cryptococal polysaccharide could be devised.     

Production of gamma interferon by natural killer cells from Toxoplasma gondii-infected SCID mice: regulation by interleukin-10, interleukin-12, and tumor necrosis factor alpha.

Previous studies of mice have implicated natural killer (NK) cells as mediators of protective activity against Toxoplasma gondii through their production of gamma interferon (IFN-gamma). In the present study, we have compared NK-cell activity in infected and uninfected SCID mice. Our data reveal that infection results in increased levels of IFN-gamma in serum and elevated NK-cell activity but that these NK cells were not cytotoxic for T. gondii-infected P815 cells. Treatment with anti-IFN-gamma antibody abrogated the increase in NK-cell activity and resulted in earlier mortality of infected mice. In vivo treatment with anti-asialo GM1 antiserum reduced NK cell activity and levels of IFN-gamma in serum but did not alter time to death. Spleen cells from infected mice produced higher levels of IFN-gamma than those from uninfected mice when stimulated in vitro with live T. gondii or parasite antigen preparations. Further analysis revealed that interleukin 10 (IL-10) inhibited, whereas tumor necrosis factor alpha (TNF-alpha) and IL-12 enhanced, IFN-gamma production by spleen cells from infected or uninfected mice. The combination of IL-12 and TNF-alpha induced higher levels of IFN-gamma from whole spleen cells of infected mice than from those of uninfected mice. Depletion of the adherent cell population from the spleen cells of infected mice led to a significant reduction in the levels of IFN-gamma produced after stimulation with IL-12 plus TNF-alpha. Similar results did not occur with cells from uninfected mice. These data indicate that other cytokines produced by the adherent cell population from infected mice may be involved in maximal production of IFN-gamma by NK cells stimulated with IL-12 and TNF-alpha. To assess the importance of endogenous IL-12, a polyclonal anti-IL-12 was administered to infected SCID mice. This treatment led to earlier mortality, indicating that endogenous IL-12 mediates resistance to T. gondii.     

The A/T-specific DNA alkylating agent adozelesin inhibits Plasmodium falciparum growth in vitro and protects mice against Plasmodium chabaudi adami infection

There is an urgent need for new anti-malarial drugs to combat the resurgence of resistance to current therapies. To exploit the A/T richness of malaria DNA as a potential target for anti-malarial drugs we tested an A/T-specific DNA synthesis inhibitor, adozelesin, for activity against Plasmodium falciparum in vitro and Plasmodium chabaudi adami in mice. Adozelesin is a DNA alkylating agent that exhibits specificity for the motif A/T, A/T and A. In P. falciparum 3D7 cultures, adozelesin acts as a powerful inhibitor of parasite growth (IC(50) of 70 pM) and is equally potent at killing the drug-resistant strains FCR3 and 7G8. Using a real-time PCR assay, we show that treatment with adozelesin in vitro results in damage of P. falciparum genomic DNA. In synchronized cultures, adozelesin exhibits a concentration-dependent effect on parasitemia and on the development of parasites through the asexual cycle. In asynchronous cultures, parasites arrest at all stages of the asexual cycle suggesting that adozelesin exerts other anti-parasitic effects in addition to inhibiting DNA replication. These anti-parasite effects are irreversible since cultures exposed to adozelesin for more than 6h fail to recover upon removal of the drug. Furthermore, adozelesin is very effective at suppressing malaria infection in vivo; growth of P. c. adami DK in mice was highly impaired by a single injection of adozelesin (25 microg/kg) at 4 days post-infection. These results demonstrate that adozelesin irreversibly blocks parasite growth in vitro and suppresses parasite infection in vivo, suggesting that A/T-specific DNA damaging agents represent a new class of compounds with potential as anti-malarials.