High Level of Plasma Tumour Necrosis Factor-α ( TNF-α ) in Pregnant Mice Infected with Plasmodium berghei is Strongly Related to Low Level of Hemoglobin but not Related to Fetal Low Weight

Malaria infection in pregnancy may increase the morbidity and mortality for both mother and fetus. In pregnant women, it can lead to severe anemia, cerebral malaria, pulmonary edema, renal failure and even death, while in the fetus it can cause abortion, premature birth, low birth weight, and fetal death. Elevated levels of tumor necrosis factor-α (TNF-α ) are associated with low birth weight and anemia in pregnant women. This study was conducted to measure the levels of TNF-α in plasma and hemoglobin levels as well as fetal weight to determine the relationship among them in P. berghei infected pregnant mice and normal pregnant mice. Seventeen BALB/c mice used in this study were divided into two groups, those were the study group (9 pregnant mice infected with P. berghei) and control group (8 pregnant mice not infected with P. berghei). Levels of TNF-α were measured using Enzyme Linked Immunosorbent assay (R&D Systems, catalog A00B MT). Hemoglobin levels were determined using flowcytometri, whereas fetal weights were measured with Mettler analytical balance AE 50. T-test statistical analysis showed that the levels of plasma TNF-α in study group were higher than control group (p=0,000). Hemoglobin levels in the study group were lower than control group (p=0,025). Fetal weights were also lower in fetuses of infected mice than fetuses of uninfected mice (p=0,002). Pearson correlation test showed that increasing plasma levels of TNF-α in infected P. berghei pregnant mice were related with the decreasing levels of Hb, (p=0,020; r=-0,748). However plasma levels of TNF-α were not associated with the incidence of fetal low weight (p=0,380, and p=0,365). It can be concluded that the increasing levels of TNF-α is associated with decreasing levels of hemoglobin (Hb), but not associated with fetal low weight.


INTRODUCTION
University of Brawijaya Malang.The hemoglobin levels were measured at the private laboratory.The level of TNF-Pregnant women are more susceptible than non pregnant α in plasma were measured using Quantikine ELISA (R & D women to malaria, and this susceptibility is highest in the Systems, catalog A00B MT).Hemoglobin levels were first pregnancy.The incidence of malaria infection and determined by hematoanalyzer and fetal birth weight anemia in primigravidae is higher compared with were measured using analytical scale (Mettler AE 50).His multigravidae subjects (1,2).Susceptibility to pregnancystudy had received ethical clearance from the Ethics associated malaria represents a combination of Committee of Health Researches of the Faculty of immunological and hormonal changes associated with Medicine, University of Brawijaya No. 104/EC/KEPKpregnancy (3)(4)(5).Malaria infection in pregnant women are S2/03/2013.more easily occurs because of changes in immunity status during pregnancy, from Th1 to Th2 dominance (6).
Preparations and Inoculation of P.berghei The manifestation of Plasmodium falciparum infection in Plasmodium berghei ANKA strain were obtained from pregnant women can also be more severe and Central Biomedical Laboratory, Faculty of Medicine complicated compared to the non pregnant population, University of Brawijaya.Pellet of erythrocytes infected includes severe anemia, fever illness, hypoglycemia, with P. berghei from the storage of liquid nitrogen tank cerebral malaria and pulmonary edema (4,7,8).Malaria with -135 C temperature was thawed and centrifuged at infection during pregnancy also increases the risk of 2000 rpm (0,26832 G) with Sentrifuse Hettich Mikro 2R for puerperal pyrexia, abortion, prematurity (9), anemia, 5 minutes, and washed twice in RPMI medium and diluted intrauterine death in pregnancy woman (10), and low as needed for inoculation intra-peritoneally (ip) to donor birth weight (11) as well as intrauterine growth mice.Mice in the study group were inoculated as much as 6 retardation babies (12,13).10 parasites in 0,2ml of blood per mice.
The strong Th1 responses during pregnancy especially The degrees of parasitemia were measured by examining increased levels of TNF have been associated with low slides of thin blood smear taken from the tip tail of mice birth weight (LBW), maternal anemia, spontaneous and stained with Giemsa under light microscope with abortions, and premature deliveries (14).The clearest 1000x magnification and counted the number of infected finding has been an association between raising levels of erythrocytes per 1000 erythrocytes by two different TNF-α and lowering of babies's birth weights (12,15) observers.including low birth weight due to fetal growth restriction Examination Levels of Plasma TNF-α (16) and preterm delivery (17).
The levels of TNF-α in plasma were determined by ELISA The mechanism of TNF-α in causing anemia and other (Enzyme Linked Immuno Assay).Fifty µL of assay diluent TNF-α related complications in pregnant women remains RD1-38 were added to each well that had been coated with unclear and the downstream consequence of high TNF-α the primary antibody.Standard, control, and samples was production has been linked to fetal growth restriction is added at 50µL per well, mixed by shaking the plate for 1 intriguing, since fetal growth restriction seems to result minute then covered with adhesive strip and incubated for from chronic infection (14).This study was performed to 2 hours at room temperature.Each samples were determine the effect of P.berghei infection in pregnant aspirated and then washed 4-5 times, by filling each well mice on the levels of plasma TNF-α, hemoglobin level, the with wash buffer (400µL) and spraying with dispenser.fetal birth weights and the relationship among them.
After the last wash, the remaining wash buffer were removed by inverting the plate on a clean paper towel.METHODS Furthermore 100µL of secondary antibody which had been conjugated with biotin was added to each well.The plate Research Design and Samples was covered with a new adhesive strip and incubated for 2 This experimental study was conducted using 50 adult hours at room temperature.The washing process was female BALB/c mice 13-15 weeks old, 20-30 grams in repeated 3 times.After that, 100µL substrate solution was weight, obtained from the Integrated Research and added to each well and incubated for 30 min at room Testing Laboratory University of Gadjah Mada.In order to temperature and protected from light.Finally, 100µL stop get a group of pregnant mice with the same gestation, solution was added to each well.Within 30 minutes the their estrus were synchronized cycles using Leebot, plate was read under ELISA reader at a wavelength of Pheromone and Whitten effects and then mated 450nm.simultaneously in pairs within one night (18).They were then divided into two groups and observed daily.On the RESULTS ninth day post mating (estimated to be the second trimester of pregnancy), the study group was infected by Among those of 50 female mice used in this research, th Plasmodium berghei.when the operation was performed on the 18 days post th mating, there were only 17 successfully pregnant mice, On the 18 day post mating (estimated to be the third those were nine mice from the study group and eight mice trimester of pregnancy), the mice were sacrificed from the control group.humanely by placing them into plastic cages containing th cotton wool soaked with chloroform.Intra-cardiac The degree of parasitemia on 18 days post mating in puncture for blood collections were performed pregnant mice that inoculated successfully with P.berghei immediately, the gravid uterus were weighted individually were examined by two observers.Data show that the and the placentas were isolated and stored in -80 C. Group macrophages, T, B and mast cells.This molecule is not only involved in immuno-protection against infection, but also The comparison between measured variables is shown on plays a role in inflammation, autoimmune and patho-Table 2. There are significant differences between the physiology of many diseases (19).levels of TNF-α plasma in the study group and control group (p=0,000).The level of plasma TNF-α in infected During malaria infection, IFN-γ produced by activated pregnant mice is significantly higher than non infected lymphocytes activates macrophages to produce TNF-α and pregnant mice (p=0,000).
induce phagocytosis (4).TNF-α is pro-inflammatory cytokine that has been most closely investigated in malaria and usually act as homeostatic agents, but can cause The level of TNF -α plasma as well as fetal body pathology if produced excessively (20).weight was higher compare to control group.In contacy Decrease Hemoglobin (Hb) Level in the Study Group the control group has higher hemoglobin level compare to the study group A decrease in Hb level in pregnant mice infected with Plasmodium berghei evidenced by low level of Hb in treatment group (Figure 2A).Statistical analysis using independent T test, showed that Plasmodium berghei Table 2.The differences between level of plasma TNFinfection can cause a decrease in Hb levels of study group α, hemoglobin and fetal body weights in study group compared to the control group (p=0,025).Previous studies and control group in humans showed that decreasing hemoglobin levels could lead to anemia, Malaria was associated with lower mean hemoglobin on antenatal visit and delivery (21,22).Other studies in human showed that the mean of haematological values were significantly lower among parasitized pregnant women compared to non-parasitized pregnant and non pregnant subjects.A positive correlation was observed between the level of parasitaemia and anemia (21,23).Microcytic and hypochromic anemia were significantly higher in pregnant and parasitized subjects The relationship between the degree of parasitemia with (2).levels of plasma TNF-α is not significant (p=0,257; r=-In human malaria infection, many uninfected red cells 0,423) as well as the relationship between the degree of were destroyed, in the spleen and quite possibly in the parasitemia with hemoglobin levels (Hb) (p=0,086; liver.This destruction had been identified as the major r=0,602).Relationship analysis between levels of TNF-α in contributor to malarial anemia (24).During malaria plasma and hemoglobin with fetal weights using 5% infection the obvious loss of infected erythrocytes is confidence, interval (α) shows that there are no through recognition by macrophages (25).The activity and relationships between levels of plasma TNF-α with fetal the number of macrophages were also increased during body weight (p=0,380; r=-0,334), but there is a significant human malarial infection, and may therefore contribute to relationship between levels of plasma TNF-α with the increased removal of uninfected cells (26).hemoglobin levels (Hb) (p=0,020; r=-0,748).There is also In murine malaria infections, high parasitemias with no significant relationship between the fetal weights and intravascular hemolysis of infected erythrocytes were the level of hemoglobin (p=0,774; r=0,112).frequently observed.Due to the high level of parasitemia, it may appear that the clearance of uninfected RBCs for the development of anemia is less significant in mice than in the majority of human malaria infections.In the mouse α model, both lethal and non-lethal malaria infections induce ineffective erythropoiesis, with alterations in erythropoietic progenitor and precursor populations, as well as in the sites of erythropoiesis, erythropoietic suppression and dyserythropoiesis.Each of these mechanisms had been implicated in both human and mouse malarial anemia (25).
Lamikanra et al mentioned that the most decreasing Hb levels in relation to blood parasitemia occurred in malaria-DISCUSSION infected primigravid and reduced in accordance with the increase in parity.Mean parasite densities were Increasing Levels of PlasmaTumor Necrosis Factor Alpha significantly higher in pregnant women compared with (TNF-α) in the Study Group non pregnant women for both P. falciparum and P. vivax The levels of plasma TNF-α of pregnant mice infected with infection.Pregnant women with falciparum or vivax Plasmodium berghei demonstrated a higher value in the malaria were significantly more anemic than non infected treatment group than control group (Table 2; Fig. 1).
pregnant women or infected non pregnant women (27).Statistical analysis using T test, showed that Plasmodium Despite the effect of malaria on maternal Hb, which was berghei infection could cause elevation of TNF-α levels in expectedly lower, neonatal Hb levels were not decreasing plasma (p=0,000).In normal condition, increased level of in the cord blood of malaria infected compared to TNF-α occurs in any human infections.The TNF-α is a uninfected women.In addition, cord Hb values were not pyrogenic and multifunctional cytokine, produced by correlated to maternal Hb concentration in either group or on the level of hemoglobin (p=0,025) and fetal body weights between the study group and the control group (p=0,001). .

Table 3. The correlation among parasitemia degree, TNF-haemoglobin, and fetal body weight
There is a significant difference TNF-α is associated with impaired fetal growth and preterm birth, due to chronic infection, levels of TNF-α The Relationship between Increased Levels of TNF-α with have been associated with LBW and anemia (14,33).

Decreasing Levels of Hemoglobin
The Relationship between Hemoglobin Levels with Fetal Increased levels of TNF-α of plasma and placental tissue in Weight pregnant mice infected with Plasmodium berghei relate with the decrease in hemoglobin levels.Analysis using the Malaria increases the risk of preterm delivery and stillbirth Pearson correlation test (Figure 3) shows a significant through fever and contribution to severe anemia rather correlation between elevated levels of plasma TNF-α with than through parasitemia (10,34).Abrams showed a the decrease of hemoglobin (p=0,020; r=-0,748).Malaria similar result with this study.Maternal and cord infection may increase the destruction of red blood cells hemoglobin levels and malaria status had no effect on and suppression of erythropoiesis.These effects may be birth outcome although increasing cord ferritin was mediated in part by an increase in pro-inflammatory associated with significantly decreasing birth weight and cytokines such as TNF-α, which is related to the anemia of gestational length (28).malaria-infected pregnant women (12,14).
In Plasmodium vivax, infection can increase the weight of During the acute phase of both human and mouse hematological abnormalities such as anemia and infections there is a strong inflammatory response, which thrombocytopenia, as well as miscarriage and premature results in increases in TNF-α and IFN-γ (29,30).Interferonbirth.There was a report of the clinical features and γ were over-expressed in the bone marrow of Fanconi pregnancy outcomes in 12 cases of P. vivax infection in anemia patients and TNF-α suppresses erythropoiesis in pregnant women complicated in some by either vitro (31,32).
miscarriages or premature deliveries, some of them The Relationship between TNF-α Level and Fetal Weight developed significant degrees of anemia and thrombocytopenia during the malaria episode (35).It can There is a significant difference of fetal weight between be concluded that the increased levels of TNF-α is treatment group and control group, the data range and associated with decreased levels of hemoglobin (Hb), but median of treatment group were less than the control not associated with fetal low weight.group.In previous study, the average weight of neonates from infected mothers was less than that of neonates ACKNOWLEDGEMENTS from non-infected mothers.This difference in birth weight Aimed to University of Brawijaya which had provided was statistically significant for both P. falciparum and P.
funding through the Competitive Grant Program (PHB).vivax infection (27).However the decrease of fetal weight Also to Mrs. Heni Triwahyuni, A,Md and Mr. Wahyuda in treatment group of this study was not significantly Ngatiril Lady, S.si from Central Biomedical Laboratory, associated with increase level of TNF-α in plasma.
Faculty of Medicine, University of Brawijaya Malang, who Previous research suggested that increased levels of TNFα associated with spontaneous abortion, high levels of provide a good technical assistance in ELISA method.
average parasitemia degree is 41,93±21,55 with nonhomogeneous distribution.The measurement of levels of TNF-α, and fetal weight were conducted at the Laboratory of Parasitology and The Comparison between Level of Plasma TNF-Α, Central Biomedical Laboratory Faculty of Medicine, Hemoglobin and Fetal Weights in Study Group and Control °°High Level of Plasma Tumour Necrosis... Jurnal Kedokteran Brawijaya, Vol. 28, No. 4