Elevated tumor necrosis factor alpha and interleukin-6 serum levels as markers for complicated Plasmodium falciparum malaria

PURPOSE: Tumor necrosis factor alpha (TNF-alpha) has been implicated in the pathology of experimental malaria. To establish its relevance to human malaria, we studied serum levels of two monocyte-derived cytokines, TNF-alpha and interleukin-6 (IL-6), as well as of the lymphocyte-derived mediator interferon gamma (IFN-gamma) in patients with malaria before and during antiparasitic treatment. PATIENTS AND METHODS: One hundred twenty serum samples of 40 patients with malaria (Plasmodium falciparum [n = 32], Plasmodium vivax [n = 8]) were analyzed. IL-6 was measured by a highly sensitive and specific bioassay, TNF-alpha by immunoradiometric assay, and IFN-gamma by radioimmunoassay. RESULTS: Elevated cytokine levels could be detected in the majority of patients with P. falciparum malaria before treatment (31 of 32, 21 of 32, and 21 of 32 for TNF-alpha, IL-6, and IFN-gamma, respectively), but only in some patients with P. vivax malaria (four of eight, one of eight, and zero of eight for TNF-alpha, IL-6, and IFN-gamma, respectively). Serum concentrations of the monokines TNF-alpha and IL-6 correlated significantly with parasitic density (p less than 0.001). No such correlation was obtained with the circulating IFN-gamma concentration. The levels of monokines TNF-alpha and IL-6 were markedly elevated in 18 P. falciparum-infected patients with complicated clinical courses (median values for TNF-alpha 172 pg/mL, for IL-6 16 U/mL, peak values: 896 pg/mL and 1,000 U/mL, respectively). The correlation between TNF-alpha and IL-6 concentrations in serum (n = 40, r = 0.56, p = 0.0002) suggests co-ordinate production of those mediators. CONCLUSION: Organ impairment in human malaria was found to be correlated with the amount of circulating cytokine levels of TNF-alpha and IL-6. Thus, imbalances of the cytokine network in untreated P. falciparum infection serve as markers of severity of disease. Modulation of cytokine response could represent a novel approach to the treatment of severe organ dysfunctions in human malaria.     

Soluble tumor necrosis factor receptors correlate with parasitemia and disease severity in human malaria.

Elevated serum or plasma concentration of immunoreactive tumor necrosis factor (TNF) is consistently detected in patients with malaria. TNF levels correlate with high parasitemia and clinical severity but not always with outcome. Since the effects of TNF may be neutralized by soluble TNF receptors, sera of 30 nonimmune patients with malaria were analyzed before and during antimalarial therapy. High concentrations of receptors R1 (55 kDa) and R2 (75 kDa) were detected immunologically in all sera of untreated patients. Levels of immunoreactive TNF correlated closely with levels of soluble TNF R1 and R2 (r = .75 and .59, respectively). In contrast, sera lacked cytotoxic activity against target cells in the TNF bioassays. Soluble TNF receptor levels remained elevated for days after treatment. These results suggest that excessive release of TNF induced by the asexual stage of malaria parasites is controlled by a subsequent shedding of soluble TNF receptors that may bind and deactivate biologically functional TNF.     

Elevated levels of circulating cachectin/tumor necrosis factor in patients with acquired immunodeficiency syndrome

PURPOSE: In order to evaluate the relationship between cachectin/tumor necrosis factor (TNF) and cachexia in the acquired immunodeficiency syndrome (AIDS), we studied the serum levels of endogenous cachectin/TNF in subjects with human immunodeficiency virus (HIV) infection. PATIENTS AND METHODS: Fifty-three serum samples were obtained from 39 HIV-seropositive patients. The condition of each patient was clinically classified as either asymptomatic, lymphadenopathy syndrome (LAS), AIDS-related complex (ARC), or AIDS. Control sera were obtained from 29 healthy male blood donors. A double antibody radioimmunoassay was used to measure the serum levels of cachectin/TNF. RESULTS: Cachectin/TNF levels were within the reference range of the control values in all (eight of eight) asymptomatic HIV-infected subjects and in 11 of 13 of the patients with LAS. In contrast, all patients with AIDS (nine of nine) and five of nine of the patients with ARC had raised levels of cachectin/TNF. Fluctuation of the levels of cachectin/TNF occurred during follow-up, but initially raised levels remained elevated. CONCLUSION: Since cachectin/TNF suppresses lipoprotein lipase in adipocytes in vitro, and causes weight loss under experimental conditions, the findings of raised levels of cachectin/TNF in patients with AIDS may have relevance to the pathogenesis of cachexia.     

Dynamics of fever and serum levels of tumor necrosis factor are closely associated during clinical paroxysms in Plasmodium vivax malaria.

Paroxysms are sharp episodes of high fever accompanied by chills and rigors that occur periodically, once in every 48 hr in Plasmodium vivax infections. We have measured the changing levels of serum tumor necrosis factor (TNF) during paroxysms in non-immune patients infected with P. vivax malaria. The changes in TNF levels closely paralleled the rise and fall in temperature during the paroxysms but tended to precede them by 30-60 min. These observations suggest that the rise and fall in temperature during P. vivax paroxysm may be directly related to the periodic changes in TNF levels induced during these infections. The peak TNF levels reached during P. vivax infections were much higher than even those which have been recorded during severe and fatal P. falciparum infections in which TNF has been postulated to contribute to the severe manifestations of this disease.     

Predicted conformations for the immunodominant region of the circumsporozoite protein of the human malaria parasite Plasmodium falciparum.

The circumsporozoite protein of the human malaria parasite Plasmodium falciparum contains multiple tandem repeats of the amino acid sequence Asn-Ala-Asn-Pro. The repeated sequence encompasses the immunodominant region of the protein, and antibodies raised against it are potent inhibitors of invasion and development of sporozoites in cultured hepatocytes. Using a modified build-up procedure, we have explored a large number of possible helical and near-helical conformations of a terminally blocked tetraicosapeptide, consisting of six repeats of the sequence Asn-Ala-Asn-Pro, and conclude that two helical conformations are energetically favored to the exclusion of all others. One of these conformations is longer, thinner, and left-handed and is likely to be adopted in nonpolar environments, while the other is shorter, broader, and right-handed and should be favored in aqueous solutions. We propose that the immunodominant region of the circumsporozoite protein of P. falciparum adopts one of these conformations in vivo.     

Effect of interleukin-1 and tumor necrosis factor/cachectin on glucose turnover in the rat

We studied the effect of recombinant human interleukin-1 beta (IL-1) and recombinant human tumor necrosis factor alpha/cachectin (TNF) on glucose kinetics in healthy rats by means of a primed constant infusion of D-(6-3H)glucose and D-[U-14C]glucose. During the isotope (6-hour) and monokine (4-hour) infusion, plasma levels of glucagon and insulin were determined and correlated with changes in glucose metabolism. The rates of glucose appearance (Ra) and disappearance (Rd) were elevated only with IL-1 and were associated with an increase in glucagon and a concomitant decrease in the ratio of insulin to glucagon. Plasma glucose concentration was increased early after IL-1 administration and coincided with the peak in the Ra. The augmentation of the metabolic clearance rate (MCR) and percent of flux oxidized by IL-1 suggest that this monokine induces the utilization of glucose as a substrate. TNF administration failed to modify the Ra or Rd, percent of flux oxidized, or MCR. TNF-treated rats increased the percent of glucose recycling, but not the total rate of glucose production. The results of this experiment suggest that endogenous macrophage products participate in the diverse alterations of carbohydrate metabolism seen during injury and/or infection.     

X-linked inhibitor of apoptosis protein levels and protein kinase C activity regulate the sensitivity of human endometrial carcinoma cells to tumor necrosis factor alpha-induced apoptosis

Endometrial carcinomas are often chemoresistant. TNFalpha shows potent antitumor activity against various cancers, and if it demonstrates good antitumor activity against endometrial cancer, the cytokine could represent a valuable alternative therapeutic approach. We have tested the ability of TNFalpha to induce apoptosis in endometrial carcinoma cells, and examined a putative role for X-linked inhibitor of apoptosis protein (XIAP) in regulating cellular sensitivity to the cytokine. Exposure to TNFalpha triggered TNF-R1-dependent activation of caspases-8, -9, and -3, down-regulated Akt and XIAP proteins and induced dose-dependent and time-dependent apoptosis in Ishikawa cells. On the opposite, TNFalpha up-regulated XIAP in Hec-1A cells; in these cells, the cytokine induced delayed TNF-R1-dependent activation of caspase-8, and failed to activate caspases -9 and -3 and to induce apoptosis. However, XIAP small interfering RNA restored TNFalpha-induced caspase signaling and apoptosis in Hec-1A cells; XIAP small interfering RNA also increased TNFalpha-induced apoptosis in Ishikawa cells. In addition, inhibition of protein kinase C activity enhanced TNFalpha-induced down-regulation of XIAP and potentiated apoptosis induction, in both Ishikawa and Hec-1A cells. Finally, we found XIAP immunoreactivity in epithelial cells from a large number of human endometrial tumor tissue samples, indicating that XIAP is produced by endometrial tumor cells in vivo. This could allow XIAP to play a putative in vivo role in counteracting TNFalpha-induced apoptosis in endometrial tumor cells; in this case, direct or indirect targeting of XIAP should potentiate the antitumor effect of TNFalpha.     

Activation of p38 mitogen-activated protein kinase contributes to the early cardiodepressant action of tumor necrosis factor.

OBJECTIVES: The purpose of this study was to determine whether p38 mitogen-activated protein kinase (p38-MAPK) contributes to tumor necrosis factor-alpha (TNFalpha)-induced contractile depression. BACKGROUND: Tumor necrosis factor has both beneficial and detrimental consequences that may result from the activation of different downstream pathways. Tumor necrosis factor activates p38-MAPK, a stress-responsive kinase implicated in contractile depression and cardiac injury. METHODS: In isolated hearts from mice lacking the p38-MAPK activator, MAPK kinase 3 (MKK3), perfused at constant coronary pressure or flow, we measured the left ventricular developed pressure (LVDP) and the relationship between end-diastolic volume and LVDP in the presence and absence of 10 ng/ml TNFalpha. RESULTS: Within 15 min at constant pressure, TNFalpha significantly reduced LVDP and coronary flow in outbred and mkk3(+/+) mice. This early negative inotropic effect was associated with a marked phosphorylation of both p38-MAPK and its indirect substrate, HSP27. In hearts lacking MKK3, TNFalpha failed to activate p38-MAPK or to cause significant contractile dysfunction. The actions of TNFalpha were similarly attenuated in MAPK-activated protein kinase 2 (MK2)-deficient hearts, which have a marked reduction in myocardial p38-MAPK protein content, and by the p38-MAPK catalytic site inhibitor SB203580 (1 micromol/l). Under conditions of constant coronary flow, the p38-MAPK activation and contractile depression induced by TNFalpha, though attenuated, remained sensitive to the absence of MKK3 or the presence of SB203580. The role of p38-MAPK in TNFalpha-induced contractile depression was confirmed in isolated murine cardiac myocytes exposed to SB203580 or lacking MKK3. CONCLUSIONS: Tumor necrosis factor activates p38-MAPK in the intact heart and in isolated cardiac myocytes through MKK3. This activation likely contributes to the early cardiodepressant action of TNFalpha.     

A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray

Abs are central to malaria immunity, which is only acquired after years of exposure to Plasmodium falciparum (Pf). Despite the enormous worldwide burden of malaria, the targets of protective Abs and the basis of their inefficient acquisition are unknown. Addressing these knowledge gaps could accelerate malaria vaccine development. To this end, we developed a protein microarray containing ∼23% of the Pf 5,400-protein proteome and used this array to probe plasma from 220 individuals between the ages of 2–10 years and 18–25 years in Mali before and after the 6-month malaria season. Episodes of malaria were detected by passive surveillance over the 8-month study period. Ab reactivity to Pf proteins rose dramatically in children during the malaria season; however, most of this response appeared to be short-lived based on cross-sectional analysis before the malaria season, which revealed only modest incremental increases in Ab reactivity with age. Ab reactivities to 49 Pf proteins measured before the malaria season were significantly higher in 8–10-year-old children who were infected with Pf during the malaria season but did not experience malaria (n = 12) vs. those who experienced malaria (n = 29). This analysis also provided insight into patterns of Ab reactivity against Pf proteins based on the life cycle stage at which proteins are expressed, subcellular location, and other proteomic features. This approach, if validated in larger studies and in other epidemiological settings, could prove to be a useful strategy for better understanding fundamental properties of the human immune response to Pf and for identifying previously undescribed vaccine targets.     

Identification and characterization of a membrane-bound cytotoxin of murine cytolytic lymphocytes that is related to tumor necrosis factor/cachectin.

Cytotoxic T lymphocytes (CTLs) kill their targets by a contact-dependent mechanism. We investigated the possibility that the CTL membranes themselves could exert direct cytotoxic activity. Murine CTLs that had been fixed with paraformaldehyde retained a slow cytotoxic activity toward various target cells that are also sensitive to another cytokine, tumor necrosis factor (TNF)/cachectin. This cytotoxic activity was neutralized by antibodies specific for TNF. Membrane fractions obtained from CTLs were cytotoxic to TNF-sensitive targets but not to several TNF-resistant cell lines. Immunoblot analysis revealed a membrane protein band of 50-60 kDa from CTLs that reacts with anti-TNF antibodies. The surface localization of this cytokine was further ascertained by flow cytometry, indirect immunofluorescence, and immunoelectron microscopy studies using TNF-specific antibodies. Radioiodination of CTL surface proteins followed by immunoprecipitation with anti-TNF antibodies confirmed the presence of a TNF-related cytokine in the plasma membranes of CTLs that migrated with an apparent molecular mass of 50-60 kDa under disulfide-reducing conditions. This cytokine can be removed from membranes by treatment with detergents but not with high-salt buffers, suggesting that it may be an integral membrane protein.     

Dynamics of asymptomatic Plasmodium vivax infections and Duffy binding protein polymorphisms in relation to parasitemia levels in Papua New Guinean children

The interaction between Plasmodium vivax Duffy binding protein II (PvDBPII) and human erythrocyte Duffy antigen is necessary for blood stage infections. However, PvDBPII is highly polymorphic. We recently observed that certain recombinant DBPII variants bind better to erythrocytes in vitro. To examine the hypothesis that haplotypes with enhanced binding have increased parasitemia levels, we followed 206 Papua New Guinean children biweekly for six months with a total of 713 P. vivax samples genotyped. Twenty-seven PvDBPII haplotypes were identified, and 3 haplotypes accounted for 57% of the infections. The relative frequencies of dominant haplotypes remained stable throughout the study. There was no significant association with PvDBPII alleles or haplotypes with P. vivax parasitemia. The dominant haplotype (26% of samples), however, corresponded to a high-binding haplotype. Thus, common haplotypes are not likely to have arisen from increased fitness as measured by greater parasitemia levels. The restricted number of common haplotypes increases the feasibility of a PvDBPII-based vaccine.     

Synthetic peptides based on motifs present in human band 3 protein inhibit cytoadherence/sequestration of the malaria parasite Plasmodium falciparum.

Synthetic peptides patterned on the amino acid sequences found in two exofacial regions of band 3 protein (residues 824-829 of loop 7 and residues 547-553 of loop 3) blocked, in a dose-dependent fashion, the in vitro adherence of Plasmodium falciparum-infected erythrocytes to C32 amelanotic melanoma cells. Intravenous infusion of these synthetic peptides into Aotus and Saimiri monkeys infected with sequestering isolates of P. falciparum resulted in the appearance of mature forms of the parasite in the peripheral circulation. The finding that the peptides were effective as adhesion blockers in the micromolar range suggests that cerebral malaria could be managed through antiadhesion therapy.     

Polymorphism of the tumor necrosis factor region in relation to disease: an overview.

HLA antigens have been shown to be associated with several immunoinflammatory diseases. The mechanisms by which these antigens confer susceptibility to disease continue to be of major interest. Rapid progress has been made in the elucidation of the structure and function of class I and II MHC molecules, and several genes located within the HLA complex have been identified which are potentially involved in immunologic processes. Because of the HLA localization of the TNF-alpha and -beta genes and the biologic activities of the gene products, recent investigation has focused on a possible role of polymorphic TNF genes in the pathogenesis of HLA-associated diseases. Allelic variations have only been detected in the TNF-beta gene. No evidence has been found so far that a particular TNF-beta allele contributes significantly in the susceptibility to the diseases studied. Although it has been postulated that the TNF beta*2 allele contributes to susceptibility to IDDM in HLA-DR3, 4 heterozygous individuals, a larger group of HLA-typed patients and controls is needed to provide more conclusive evidence for this hypothesis. The increasing number of genes of unknown function encoded by the class III region leaves the possibility that the observed HLA associations in some diseases may be related to the presence of these genes. In AS, the lack of association with the TNF-beta alleles furthermore supports the function of the HLA-B27 molecule in the disease and underlines the improbability that HLA-B27 is merely a marker for a closely linked susceptibility gene.     

Higher production of tumor necrosis factor alpha in hemozoin-fed—human adherent monocytes is dependent on lipidic component of malarial pigment: new evidences on cytokine regulation in Plasmodium falciparum malaria

ObjectiveTo investigate whether the increase of tumor necrosis factor alpha is dependent on lipidic component of malarial pigment.MethodsAdherent human monocytes were fed for 3 hours with different meals (native hemozoin; lipid free hemozoin; and control latex particles), then tumor necrosis factor alpha was monitored in cell supernatants up to 48 hours through western blotting or specific enzyme-linked immunoadsorbent assay. In selected experiments, unfed monocytes were treated with different doses of 15(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid or 4-hydroxynonenal instead of phagocytosis.ResultsHemozoin-fed monocytes produced higher levels of tumor necrosis factor alpha than unstimulated and latex-fed cells, while lipid-free hemozoin did not reproduce these results. Additionally, hemozoin effects were mimicked dose-dependently by 15(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid, but not by 4-hydroxynonenal.ConclusionsPresent data suggest an essential role for lipids in hemozoin-dependent enhanced release of tumor necrosis factor alpha from monocytes, and 15(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid could be one possible specific mediator.     

Serum levels of the apoptosis-associated molecules, tumor necrosis factor-alpha/tumor necrosis factor type-I receptor and Fas/FasL, in sepsis

STUDY OBJECTIVE:s: Numerous reports suggest that apoptosis may play an important role in the sepsis syndrome. The objective of the present study was to examine the levels of molecules associated with apoptosis belonging to the tumor necrosis factor (TNF)-alpha/TNF type-I receptor (TNFRI) and Fas ligand (FasL)/Fas receptor (Fas) pathways in patients with sepsis. PATIENTS AND METHODS: Twenty-two patients with sepsis (14 patients with severe sepsis and 8 patients with sepsis), and 6 healthy volunteers were evaluated. Sequential analysis of the serum levels of TNF-alpha, TNFRI, FasL, and Fas were performed in these patients using enzyme-linked immunosorbent assays. RESULTS: Detectable levels of TNF-alpha were found in only 8 of 14 patients with severe sepsis. Patients with severe sepsis and sepsis had similarly increased levels of FasL, compared with healthy individuals (p < 0.05). Higher levels of TNFRI and Fas were found in patients with severe sepsis than in patients with sepsis and healthy volunteers (p < 0.001 and p < 0.01, respectively). Fas levels were also higher in patients who died than in those who survived (p < 0.01). A direct relationship was found between serum levels of TNFRI and Fas, and multiorgan dysfunction (sequential organ failure assessment score) [p < 0.0001]. CONCLUSIONS: These results suggest that the TNF-alpha/TNFRI and FasL/Fas systems may be involved in the pathogenesis of sepsis. Serum levels of the death-receptors, TNFRI and Fas, could serve as potential markers of the severity of human sepsis.     

Single-day, three-dose treatment with fixed dose combination artesunate/sulfamethoxypyrazine/pyrimethamine to cure Plasmodium falciparum malaria.

OBJECTIVES: Malaria kills approximately 1.5 to 2.7 million people each year. Despite the introduction of artemisinin-based combination therapies (ACTs), the treatment of malaria is hampered by problems such as inadequate efficacy, recrudescence, early re-infection, low patient compliance, and high cost price of drugs. This study tested the hypothesis that the co-formulated fixed dose combination (FDC) artesunate/sulfamethoxypyrazine/pyrimethamine (As/SMP) administered as a 24-hour therapy with a dose interval of 12 hours is as efficacious and safe as the administration of the same drug over 3 days given with a dose interval of 24 hours, for the treatment of uncomplicated Plasmodium falciparum malaria in Ivory Coast. METHOD: Two hundred and twenty-one patients presenting with uncomplicated P. falciparum malaria were randomly assigned to either one of the two dosing schemes. Treatment efficacy was assessed using the current 28-day World Health Organization protocol, success being determined by absence of recrudescence and parasitemia on day 28. RESULTS: Both treatment regimens were highly efficacious, with a success rate of 100% (111/111) for the 3-day therapy and 99% (109/110) for the 24-hour therapy. Only one patient in the 24-hour therapy group showed late treatment failure. No serious adverse events or significant laboratory abnormalities were seen. CONCLUSION: The 24-hour therapy is as well tolerated and efficacious as the same medicament administered over 3 days. This low cost and simplified three-pill treatment is certain to improve compliance.