Acta Tropica, 47(1990)47-51
Elsevier 47 ACTROP 00047
Plasma levels of tumor necrosis factor during a longitudinal survey in an endemic area of malaria F. Peyron 1, J.P. Vuillez 2, G. Barbe 2, C. Boudin 1, S. Picot 1 and P.
Ambroise-Thomas' 1Department of Parasitology, A. Michallon Hospital, Grenoble, France and 2Biophysics and Nuclear Medicine Department, A. Michallon Hospital, Grenoble, France
(Received 27 February 1989; revised version received 22 May 1989; accepted 16 June 1989)
The plasma levels of tumor necrosis factor were measured during a longitudinal survey of 84 subjects living in an endemic area of malaria. In most cases, the plasma tumor necrosis factor was found at its highest level during the malaria transmission peak and became normal again during the dry season. Children having suffered from malarial attack keep low tumor necrosis factor levels compared to adults and asymptomatic children. These results suggest that tumor necrosis factor could be associated with the development of resistance against malaria. Key words: Plasmodium falciparum; Tumor necrosis factor; Radioimmunoassay; Longitudinal survey
Introduction T u m o r Necrosis F a c t o r ( T N F ) , secreted by activated m a c r o p h a g e s a n d lymphocytes, plays a role in m a l a r i a l a t t a c k (Clark et al., 1987). This cytokine seems to have b o t h a protective action, by inhibiting the d e v e l o p m e n t o f parasites in vivo (Playfair, 1987), a n d a p a t h o g e n i c action in murine m a l a r i a ( G r a u et al., 1987). In h u m a n s T N F a p p e a r s to be associated with the severity o f cerebral m a l a r i a ( G r a u a n d L a m b e r t , 1988). P l a s m a levels o f T N F were r e c o r d e d in a seasonal e n d e m i c a r e a o f m a l a r i a d u r i n g a l o n g i t u d i n a l survey. O n l y children were subject to m a l a r i a l a t t a c k s in this area. A d u l t s , a l t h o u g h c a r r y i n g a low p a r a s i t a e m i a , were clinically healthy. T h e a i m o f this s t u d y was to check if T N F level was linked to this a s y m p t o m a t i c state.
Materials and Methods This investigation t o o k place in the s a v a n a a r e a a r o u n d B o b o - D i o u l a s s o , B u r k i n a F a s o ( W e s t A f r i c a ) where m a l a r i a l t r a n s m i s s i o n occurs f r o m M a y to O c t o b e r ( R o b e r t et al., 1985). 87 subjects were followed f r o m June 1987 to J a n u a r y 1988. The p r o t o c o l included a weekly clinical c h e c k - u p as well as a p e r i o d i c a l search for p a r a s i t e s which was Correspondence address: F. Peyron, Department of Parasitology, A. Michallon Hospital, 38700 Grenoble,
France. 0001-706X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
48 systematic after pyrexia. For each subject a clinical record was kept by a doctor. Chloroquine was handed out by a medical doctor during malarial attacks, thus its consumption by the inhabitants was totally controlled. Blood samples for T N F level were collected on three occasions: period A: June 87 (beginning of transmission); period B: late August, early September 87 (peak of transmission); period C: January 88 (after transmission). Plasmas were frozen on the spot and stored in liquid nitrogen. Patients were divided according to: (1) age: adults (age > 14 years) and children (age _< 14 years); (2) presence or absence of malarial attacks: patients M+ are defined as subjects who presented 1 malarial attack at least during the whole survey, and patients M - as subjects who never presented malarial attack during the survey. Malarial attack was defined as: fever > 37.7°C, parasite count > 10 000 parasites/~l, and cured with chloroquine.
TNF assay IRE M E D G E N I X (ref: 30 175000) radioimmunoassay was used. This assay uses human recombinant TNF-~ as standards and 125I-TNF-~ as tracer. Anti-TNF-~ antiserum was raised in rabbits. All reagents are lyophilized and reconstitued at the time of use in a 0.05 M phosphate buffer, pH 7.5, containing 0.05% N a N 3 and 0.5% BSA. Vial of tracer contains approximately 158 kBq of I/SI-TNF-~ and is reconstituted with 10.5 ml of buffer. After reconstitution of each vial of standard with 1.0 ml of buffer the concentrations in TNF-~ are respectively 0, 0.1, 0.2, 0.5, 1, 2 and 5 ng/ml. Recommended assay procedure is as following: 100 lal of patient plasma sample are mixed with 100 ~tl of anti-TNF-~ antiserum and the tubes incubated for 18 24 h at room temperature. Then 100 lal of IzsI-TNF-~ are dispensed into each tube which is incubated for 4 h at room temperature. Separation is performed by a 20 min incubation with D A - P E G (anti-rabbit 7-globulin antibodies, polyethylene glycol, cellulose and Tween 20), at room temperature, centrifugation (15 rain, 1500 x g), and aspiration of supernatant. This original protocol was modified as follows: antiserum was diluted 1/2; the lyophilized tracer was reconstituted with 12 ml of buffer instead of 10.5 ml; and the incubations lasted 24 h. These modifications gave a better sensitivity: detection was possible above 40 pg/ml. Normal values determined in 53 healthy subjects were: 61.1 _+31.0 pg/ml (m_+2 SD) with extreme values ranging from < 40 to 97 pg/ml. The variation coefficient was 5.9% intra-assay and 6.1% inter-assay.
Stat&tical analysis The variance analysis and the Student t means comparison test for standard and paired series were used.
Results The protocol was completed for 84 subjects, i.e. 37 children from 4 to 14 years of age: 22 boys and 15 girls (17 under age 8), and 47 adults above 14 years of age: 21 men and 26 women (mean age + DS = 30.1 +_ 13.1).
49
Malarial attack
Only 21 children (57% of children population) suffered from at least one malarial attack as defined above; these subjects are referred to as M +. The whole of the adult population and 16 children remained asymptomatic; these are referred to as M - . The two groups did not differ according to sex, or other pathologies, or origin. Parasitaemia
Parasitaemia was extremely variable with time. The mean level of parasites was calculated for each group during the plasma collecting Periods (an average of 20 days) so as to have a good estimation of the parasite density. Only asexual forms of P.falciparum, the prevalent species in the study area, were counted. Results are in Table 1. T N F levels
The variance analysis, for the studied population, revealed two significant variability factors: (Fig. 1) Period of sample collection. The highest mean levels, for population on the whole, coincided with the malaria transmission peak (period B) and came back to normal levels in January. Levels were already significantly higher than usual in June. (Fig. 2) With or without malarial attack. Asymptomatic subjects were the only ones to have an increase of the T N F levels during the seasonal transmission period. No significant variations were recorded for symptomatic patients. This came out as a significant difference of the mean levels between the two groups for period B. No differences linked to age or sex appeared (data not shown).
Discussion
Our results show that mean T N F levels vary according to seasonal malarial transmission. Other diseases may provoke the secretion of this cytokine. But it should be noted that T N F levels became normal again after the transmission period and that the clinical approach made it possible (especially in the adult group) to set aside other
TABLE 1 Percentage of subjects with positive parasite count for the 3 periods. Average parasite count (number of parasites/txl of blood, mean values __+SEM).
Adults (all M - ) Children M Children M +
PERIOD A
PERIOD B
PERIOD C
0% (0) 25% (12.5 +8) 38% (12 963_+3 955)
10% (176+78) 50% (425+326) 95% (27 354_+ 14 038)
14% (67+27) 68% (628 +349) 19% (384_+ 165)
M + : symptomatic. M - : asymptomatic. Period A = June (before transmission), period B = August and September (during transmission), period C = January (after transmission).
50 [TNFJ pg/ml 300 -
200'
100'
A (June)
i
i
B
C
PERIOD
(August-September) (Janua~/)
Fig. 1. Mean TNF levels (+ SEM) in plasma for population studied during 3 differents periods of malarial transmission. *, significant variance (P
Elsevier 47 ACTROP 00047
Plasma levels of tumor necrosis factor during a longitudinal survey in an endemic area of malaria F. Peyron 1, J.P. Vuillez 2, G. Barbe 2, C. Boudin 1, S. Picot 1 and P.
Ambroise-Thomas' 1Department of Parasitology, A. Michallon Hospital, Grenoble, France and 2Biophysics and Nuclear Medicine Department, A. Michallon Hospital, Grenoble, France
(Received 27 February 1989; revised version received 22 May 1989; accepted 16 June 1989)
The plasma levels of tumor necrosis factor were measured during a longitudinal survey of 84 subjects living in an endemic area of malaria. In most cases, the plasma tumor necrosis factor was found at its highest level during the malaria transmission peak and became normal again during the dry season. Children having suffered from malarial attack keep low tumor necrosis factor levels compared to adults and asymptomatic children. These results suggest that tumor necrosis factor could be associated with the development of resistance against malaria. Key words: Plasmodium falciparum; Tumor necrosis factor; Radioimmunoassay; Longitudinal survey
Introduction T u m o r Necrosis F a c t o r ( T N F ) , secreted by activated m a c r o p h a g e s a n d lymphocytes, plays a role in m a l a r i a l a t t a c k (Clark et al., 1987). This cytokine seems to have b o t h a protective action, by inhibiting the d e v e l o p m e n t o f parasites in vivo (Playfair, 1987), a n d a p a t h o g e n i c action in murine m a l a r i a ( G r a u et al., 1987). In h u m a n s T N F a p p e a r s to be associated with the severity o f cerebral m a l a r i a ( G r a u a n d L a m b e r t , 1988). P l a s m a levels o f T N F were r e c o r d e d in a seasonal e n d e m i c a r e a o f m a l a r i a d u r i n g a l o n g i t u d i n a l survey. O n l y children were subject to m a l a r i a l a t t a c k s in this area. A d u l t s , a l t h o u g h c a r r y i n g a low p a r a s i t a e m i a , were clinically healthy. T h e a i m o f this s t u d y was to check if T N F level was linked to this a s y m p t o m a t i c state.
Materials and Methods This investigation t o o k place in the s a v a n a a r e a a r o u n d B o b o - D i o u l a s s o , B u r k i n a F a s o ( W e s t A f r i c a ) where m a l a r i a l t r a n s m i s s i o n occurs f r o m M a y to O c t o b e r ( R o b e r t et al., 1985). 87 subjects were followed f r o m June 1987 to J a n u a r y 1988. The p r o t o c o l included a weekly clinical c h e c k - u p as well as a p e r i o d i c a l search for p a r a s i t e s which was Correspondence address: F. Peyron, Department of Parasitology, A. Michallon Hospital, 38700 Grenoble,
France. 0001-706X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
48 systematic after pyrexia. For each subject a clinical record was kept by a doctor. Chloroquine was handed out by a medical doctor during malarial attacks, thus its consumption by the inhabitants was totally controlled. Blood samples for T N F level were collected on three occasions: period A: June 87 (beginning of transmission); period B: late August, early September 87 (peak of transmission); period C: January 88 (after transmission). Plasmas were frozen on the spot and stored in liquid nitrogen. Patients were divided according to: (1) age: adults (age > 14 years) and children (age _< 14 years); (2) presence or absence of malarial attacks: patients M+ are defined as subjects who presented 1 malarial attack at least during the whole survey, and patients M - as subjects who never presented malarial attack during the survey. Malarial attack was defined as: fever > 37.7°C, parasite count > 10 000 parasites/~l, and cured with chloroquine.
TNF assay IRE M E D G E N I X (ref: 30 175000) radioimmunoassay was used. This assay uses human recombinant TNF-~ as standards and 125I-TNF-~ as tracer. Anti-TNF-~ antiserum was raised in rabbits. All reagents are lyophilized and reconstitued at the time of use in a 0.05 M phosphate buffer, pH 7.5, containing 0.05% N a N 3 and 0.5% BSA. Vial of tracer contains approximately 158 kBq of I/SI-TNF-~ and is reconstituted with 10.5 ml of buffer. After reconstitution of each vial of standard with 1.0 ml of buffer the concentrations in TNF-~ are respectively 0, 0.1, 0.2, 0.5, 1, 2 and 5 ng/ml. Recommended assay procedure is as following: 100 lal of patient plasma sample are mixed with 100 ~tl of anti-TNF-~ antiserum and the tubes incubated for 18 24 h at room temperature. Then 100 lal of IzsI-TNF-~ are dispensed into each tube which is incubated for 4 h at room temperature. Separation is performed by a 20 min incubation with D A - P E G (anti-rabbit 7-globulin antibodies, polyethylene glycol, cellulose and Tween 20), at room temperature, centrifugation (15 rain, 1500 x g), and aspiration of supernatant. This original protocol was modified as follows: antiserum was diluted 1/2; the lyophilized tracer was reconstituted with 12 ml of buffer instead of 10.5 ml; and the incubations lasted 24 h. These modifications gave a better sensitivity: detection was possible above 40 pg/ml. Normal values determined in 53 healthy subjects were: 61.1 _+31.0 pg/ml (m_+2 SD) with extreme values ranging from < 40 to 97 pg/ml. The variation coefficient was 5.9% intra-assay and 6.1% inter-assay.
Stat&tical analysis The variance analysis and the Student t means comparison test for standard and paired series were used.
Results The protocol was completed for 84 subjects, i.e. 37 children from 4 to 14 years of age: 22 boys and 15 girls (17 under age 8), and 47 adults above 14 years of age: 21 men and 26 women (mean age + DS = 30.1 +_ 13.1).
49
Malarial attack
Only 21 children (57% of children population) suffered from at least one malarial attack as defined above; these subjects are referred to as M +. The whole of the adult population and 16 children remained asymptomatic; these are referred to as M - . The two groups did not differ according to sex, or other pathologies, or origin. Parasitaemia
Parasitaemia was extremely variable with time. The mean level of parasites was calculated for each group during the plasma collecting Periods (an average of 20 days) so as to have a good estimation of the parasite density. Only asexual forms of P.falciparum, the prevalent species in the study area, were counted. Results are in Table 1. T N F levels
The variance analysis, for the studied population, revealed two significant variability factors: (Fig. 1) Period of sample collection. The highest mean levels, for population on the whole, coincided with the malaria transmission peak (period B) and came back to normal levels in January. Levels were already significantly higher than usual in June. (Fig. 2) With or without malarial attack. Asymptomatic subjects were the only ones to have an increase of the T N F levels during the seasonal transmission period. No significant variations were recorded for symptomatic patients. This came out as a significant difference of the mean levels between the two groups for period B. No differences linked to age or sex appeared (data not shown).
Discussion
Our results show that mean T N F levels vary according to seasonal malarial transmission. Other diseases may provoke the secretion of this cytokine. But it should be noted that T N F levels became normal again after the transmission period and that the clinical approach made it possible (especially in the adult group) to set aside other
TABLE 1 Percentage of subjects with positive parasite count for the 3 periods. Average parasite count (number of parasites/txl of blood, mean values __+SEM).
Adults (all M - ) Children M Children M +
PERIOD A
PERIOD B
PERIOD C
0% (0) 25% (12.5 +8) 38% (12 963_+3 955)
10% (176+78) 50% (425+326) 95% (27 354_+ 14 038)
14% (67+27) 68% (628 +349) 19% (384_+ 165)
M + : symptomatic. M - : asymptomatic. Period A = June (before transmission), period B = August and September (during transmission), period C = January (after transmission).
50 [TNFJ pg/ml 300 -
200'
100'
A (June)
i
i
B
C
PERIOD
(August-September) (Janua~/)
Fig. 1. Mean TNF levels (+ SEM) in plasma for population studied during 3 differents periods of malarial transmission. *, significant variance (P
