INCREASED TUMOR NECROSIS FACTOR ALPHA AND INTERLEUKIN-6 SERUM LEVELS AND THEIR CORRELATION WITH LABORATORY PARAMETERS IN PATIENTS WITH IMPORTED MALARIA

In malaria, blood concentrations of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6, are increased. In a study which included 34 patients, TNF-α and IL-6 were examined in two phases, immediately after the admission of patients, and at the end of antimalarial therapy, when the parasitemia was negative. The results show a significant increase of TNF-α and IL-6 in the first phase, before the effects of antimalarial therapy. A very strong correlation between TNF-α and IL-6 is also confirmed, which suggests their coordinated production. Increased TNF-α values were correlated with an older age, the level of parasitemia, the number of platelets and leukocytes, elevated values of procalcitonin, D-dimer and lactate dehydrogenasе, and lower values of serum iron and antithrombin. Increased values of IL-6 were correlated with the level of parasitemia, the number of platelets and leukocytes, and elevated values of D-dimer and lactates.


INTRODUCTION
Patients with malaria, especially in severe forms, have an increased concentration of proinflammatory cytokines, of which TNF-α has been the most often examined (Chen et al., 2000).The increasing concentration of TNF-α in malaria was first recorded in 1986 (Scuderi et al., 1986).Kwiatkowski et al. (1989) demonstrated a significant increase in TNF-α levels in children with malaria in Gambia.In malaria, as well as in other systemic infections and diseases, the release of proinflammatory cytokines produces a systemic inflammatory re-sponse.Previous research related to sepsis and malaria has shown that TNF-α, the prototype of proinflammatory cytokines, increased in both diseases, causing hyperlactatemia.It is well known that increased regulation of proinflammatory cytokines in the process of adhesion to endothelial cells attracts circulating blood elements on the inner wall of the vessel (Sarangi et al., 2011).In many diseases, including malaria, this includes activated leukocytes and platelets, which play an important role in procoagulant activity.It is thought that proinflammatory cytokines increase the expression of molecules on the endothelial cells adhered to by parasitizing erythrocytes.Proinflammatory cytokines enhance the expression of tissue factor on the endothelial cells and monocytes, and by generating the occurrence of thrombin, a molecule with significant roles in cross-reactions of inflammation and coagulation (Clark et al., 2006).The speed, time and intensity of release of cytokines varies considerably in different stages of the disease and between different individuals, thus forming a characteristic clinical feature.It is believed that proinflammatory cytokines are responsible for the occurrence of fever, nausea, vomiting, diarrhea, anorexia, myalgia, thrombocytopenia, immunosuppression, coagulopathy and neurological manifestations (Jennings et al., 2006).In severe forms of malaria, the concentrations of proinflammatory cytokines, such as TNF-α, IL-1, IL-6, and IL-8, are elevated in the serum.The high level of TNF-α in patients with falciparum malaria correlates with the severity of the disease, with the appearance of hypoglycemia, hyperparasitemia, jaundice, kidney damage, cardiovascular complications and death (Mordmüller et al., 2007).
The aim of this study was to determine the serum levels of TNF-α and interleukin-6, and to establish their correlation with other clinical and laboratory parameters.

MATERIALS AND METHODS
The study included 34 patients with imported malaria who were treated from 2007 to 2010 at the Clinic of Infectious and Tropical Diseases, Clinical Center of Serbia.From the total number of patients, 27 (79.41%)had falciparum malaria, while only 7 (20.59%)had malaria caused by the other type of plasmodium (vivax ovale).
The following data sources were used in this study: medical records, hematological, biochemical, parasitological and immunological analysis.
The most important parasitological analysis used in the study was the examination of thick and thin peripheral blood smears, stained with Giemsa.The level of parasitemia may be expressed either as a per-centage of parasitized erythrocytes or as the number of parasites per microliter of blood.
The immunological analyses of the cytokines (TNF-α and IL-6) were performed at the Center for Medical Biochemistry, Clinical Center of Serbia, using the method of Quantikine ELISA Kit (R&D Systems).
The serum samples of patients for cytokine analysis were taken in two stages: immediately after the admission and at the end of antimalarial therapy, when the parasitemia was negative.After centrifugation, they were separated and stored at -70˚C until use.
The method by which the cytokines were determined was the quantitative sandwich enzyme immunoassay technique.A monoclonal antibody specific for TNF-α/IL-6 was pre-coated onto a microplate.Standards and samples are pipetted into the wells and any TNF-α/IL-6 present was bound by the immobilized antibody.After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for TNF-α/IL-6 was added to the wells.Following a wash to remove any unbound antibodyenzyme reagent, a substrate solution was added to the wells and color developed in proportion to the amount of TNF-α/IL-6 bound in the initial step.The color development was stopped and the intensity of the color was measured.
The following methods were used in statistical analysis: methods of descriptive statistics: mean value, standard deviation; Spearman's rank correlation to determine the correlation between the levels of cytokines and the other clinical and laboratory parameters; analysis of variance (ANOVA) to compare the levels of TNF-α in patients with malaria in relation to the control values; Kolmogorov-Smirnov test and Mann-Whitney test were used to compare the levels of IL-6 in patients with malaria in relation to the control values.

RESULTS
The study included 34 patients.The cytokines TNF-α and IL-6 were examined at two phases: immediately after the admission of patients (1) and at the end of antimalarial therapy, when the parasitemia was negative (2).
Using the methods of statistical analysis (ANO-VA, Kolmogorov-Smirnov and Mann-Whitney test) we have proved the increased values of TNF-α (p=0.027) and IL-6 (p=0.006) in the first phase.
The ANOVA test confirmed that there was no statistically significant difference in the values of TNF-α (p=0.974) and ) between the two phases, i.e. before and after treatment.
Additional laboratory parameters were also taken in each phase to determine the correlation and for monitoring dynamics in relation to the values of cytokines.
The values of D-dimer and C-reactive protein (CRP) were increased in the first phase, while the values of antithrombin III (AT3) and lactates were in the reference range.
In the second phase, there was a decrease in the value of laboratory parameters of the first phase, Ddimer and CRP.The values of AT3 and lactates were increased, but within the reference range.
The results show that there is a very strong correlation between TNF and IL-6 in the first phase.Increased TNF-α levels correlated with an older age, the level of parasitemia, as did the elevated values of procalcitonin, D-dimer and LDH, but also with the lower values of platelets, leukocytes, serum iron, AT3 and Hb.
The increased values of IL-6 correlated with the level of parasitemia, elevated values of D-dimer and lactates.A negative correlation was observed for thrombocytopenia and leucopenia.Unlike the TNF-α, elevated values of IL-6 poorly correlated with age.
The values of TNF-α in the second phase strongly correlated with the number of platelets.The values of IL-6 in the second phase strongly correlated with the value of D-dimer.It was concluded that in the first phase, before starting treatment, TNF-α and IL-6 correlated more with the laboratory parameters of the same phase.

DISCUSSION
The role of cytokines in the pathogenesis of malaria has been clearly confirmed, but not completely understood.It is thought that many factors impact on their level, which in the course of malaria depends on the circadian rhythm, and in accordance with this, there are large variations in the serum cytokine levels.TNF-α appears to be the most important in the early immune response and subsequent pathological manifestations.It has been proved that its concentration regulates the levels of the other cytokines as well, particularly in relation to IL-6 (Angulо and Fresno, 2002).Thus we could explain the results of this study, where the levels of TNF-α correlated to a high degree with other laboratory parameters in relation to IL-6, especially when taking into account the parameters that indicate the more difficult forms of disease (anemia, thrombocytopenia, high value of LDH).The results show a significant increase in TNF-α and IL-6 in the first phase, in the presence of parasitemia, and before the effects of antimalarial therapy.In addition, there is a very strong correlation between the levels of TNF-α and IL-6 in the first phase and their significant association with the level of parasitemia.Many other authors have also established their mutual coordination and association with the degree of parasitemia in patients with malaria (Shaffer et al., 1991;McGuire et al., 1998;Zeyrek et al., 2006).
According to a study from Hamburg (Kern et al., 1989), an increase in TNF-α and IL-6 was observed mainly in patients with falciparum malaria, while the concentrations of TNF-α and IL-6 correlated with the degree of parasitemia and the occurrence of complications.Thereby, they found a correlation between increased levels of these cytokines and anemia, hy-poglycemia, hyperbilirubinemia and acute renal failure, which is consistent with the results of this study.A study from Pakistan showed almost identical results in falciparum malaria, but there was no correlation with the degree of parasitemia (Gandapuр and Malik, 1996).Research conducted in Mali, in patients with falciparum malaria, showed a significant increase in cytokines in patients, including TNF-α and IL-6, but the correlation between hyperparasitemia and anemia with increased concentrations of IL-6 has not been proven (Lyke et al., 2004).
In the enlisted patients, increased levels of cytokines were in a stronger (TNF-α) or weaker (IL-6) correlation with the age.Authors from Pakistan (Gandapuр and Malik, 1996) indicated that    et al., 2001;Krabbe et al., 2004).It is thought that in a relatively healthy elderly population, increasing levels of circulating IL-6 present a systemic response to local proinflammatory activity; however, when inflammatory diseases associated with old age progress, the level of circulating TNF-α increases, which gradually becomes a stronger marker of morbidity than IL-6 (Brüünsgaard and Pedersen, 2003).
The increased levels of TNF-α and IL-6 correlate with thrombocytopenia and a high concentration of D-dimer in both phases, i.e. before and after treatment, which is consistent with the pathophysiological mechanisms that occur in the course of this disease.In fact, the activation of the coagulation system leads to the appearance of fibrin degradation products (FDP), i.e.D-dimer in the circulation, which we found in malaria as well, mainly in those caused by Plasmodium falciparum.Initiation of the coagulation cascade and fibrinolytic system is caused by parasitized erythrocytes and/or activated platelets (Francischetti, 2008;Ghosh and Shetty, 2008).Parasitized erythrocytes adhere to the capillary endothelium, causing further activation which results in platelet consumption and the activation of coagulation, which may culminate in the appearance of DIC (disseminated intravascular coagulation).IL-6 stimulates platelets, which are manifested by an increased activation of thrombin and increased procoagulant activity, and coagulation markers detect changes as a response to the effects of TNF-α (Esmon et al., 1999).In the first phase, we found strong correlation between TNF-α and AT3, whose values are often reduced in falciparum malaria, especially its severe forms.
These results are expected when we have in mind the pathophysiological mechanism in which cytokine production and parasitized erythrocytes cause the consumption of AT3 (Mohanty et al., 1997).In contrast to the correlation of TNF-α and AT3, in the first phase we registered a correlation between increased levels of IL-6 and the increase of lactates.Some authors also find an association between increased concentrations of IL-6 and IL-10 and lactates, before starting antimalarial therapy (Day et al., 1999).Sarthou et al. (1997) have shown a decline in the concentrations of TNF-α and IL-6 after antimalarial treatment.According to the results of this study, the average values of TNF-α and IL-6 in the second phase were much lower than values before therapy, but the difference in values was not statistically significant.The time interval between the determination of cytokine concentrations before and after treatment, i.e. during the presence or absence of parasitemia, was an average of 7 days, but for some patients this period was shorter.Specifically, these were patients who were taking antimalarial therapy on their own initiative, and the time of treatment was shorter.Most probably, this is the reason for the observed disparities between the levels of cytokines before and after treatment.Namely, after the therapy, they were generally reduced in all patients, albeit not equally.

Fig. 1 .
Fig. 1.Correlation on TNF-a with IL-6, the age and laboratory parameters in the first phase (1)

Table 1 .
The values of cytokines in the first (1) and second (2) phase

Table 2 .
The values of cytokines of the controls

Table 3 .
The values of additional laboratory parameters (phase 1)

Table 4 .
The values of additional laboratory parameters (phase 2)

Table 5 .
Correlation of TNF-α and IL-6 in the second phase (2)