- Email: [email protected]
Red blood cell antioxidant levels in Wuchereria bancrofti infection
Experimental Parasitology 102 (2002) 81–88 www.elsevier.com/locate/yexpr
Red blood cell antioxidant levels in Wuchereria bancrofti infectionq R. Premaratna,a,* T.G.A.N. Chandrasena,b W. Abeyewickreme,b L.G. Chandrasena,c S. Senarath,c N.R. de Silva,b and H.J. de Silvaa a
Department of Medicine, Faculty of Medicine, University of Kelaniya, P O Box 6, Thalagolla Rd., Ragama, Sri Lanka b Department of Parasitology, Faculty of Medicine, University of Kelaniya, Sri Lanka c Department of Biochemistry, Faculty of Medicine, University of Kelaniya, Sri Lanka Received 9 December 2001; received in revised form 12 August 2002; accepted 14 February 2003
Abstract The elimination of microfilariae of Wuchereria bancrofti is probably mediated by free radicals. Red cell catalase (C), glutathione peroxidase (GPX), and superoxide dismutase (SOD) activity levels were measured as an indirect method of assessing blood oxidant status in 29 asymptomatic microfilaraemics, 29 ‘‘endemic normals’’, and 29 controls living in a non-endemic area. Changes in the activity of these enzymes were also compared over a one month period in 22 asymptomatic microfilaraemics randomised to receive either single dose or 14 day treatment with diethyl carbamazine citrate (DEC). Red cell GPX activity levels were significantly higher in ‘‘endemic normals’’ when compared to mf positive cases and non-endemic controls. An early and significant increase in GPX activity (on days 3, 7 and 14 compared to pretreatment levels, p < 0:01) was observed after DEC in both treatment groups. Increases in the activity of catalase and SOD became significant only on days 14 and 30 respectively. The percentage reduction in microfilaraemia correlated significantly with the percentage increase in GPX activity levels (R2 ¼ 0:58; p ¼ 0:6 105 ). Our results may suggest a role for GPX related oxidant species in the elimination of microfilariae. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Filariasis; Endemic normals; Antioxidants; Glutathione peroxidase; DEC
1. Introduction Wuchereria bancrofti is a filarial worm known to cause lymphatic pathology in large areas of the tropics; more than 1.1 billion people are estimated to be at risk of infection worldwide (WHO, 1999). In any endemic region, a proportion of the population at risk remains asymptomatic despite harbouring adult worms and circulating microfilariae (asymptomatic microfilaraemics); another proportion remains persistently asymptomatic and negative for microfilaria (mf) and adult worms despite almost certain exposure to infection (endemic normals) (Day, 1991). This may be because these individuals are able to q Parts of this work were presented at the Oxford 2000 Conference on New Challenges in Tropical Medicine and Parasitology, 18th–22nd September, 2000 and the 11th European Congress of Clinical Microbiology and Infectious Diseases, 1–4th April, 2001. * Corresponding author. Fax: +94-1-958337. E-mail address: [email protected] (R. Premaratna).
destroy the infective larvae or developing stages of the worm before they reach sexual maturity. There is no single test that can be used to confirm the Ôendemic normalÕ status. The ICT filariasis test and OG4C3 ELISA test, both of which detect adult filarial antigens, are among the most sensitive tests available (Dreyer et al., 1996). Because W. bancrofti is a multicellular parasite host effector mechanisms for its destruction by the immune system are likely to involve oxidants, in a manner similar to schistosomulae (the infective stages of schistosomes) being very sensitive to oxidant mediated killing (LoVerde, 1998). The microfilaricidal action of diethyl carbamazine citrate (DEC) is poorly understood (de Silva et al., 1997). In vitro studies have shown that treatment with DEC results in the stimulation of platelets to release undefined (Ox)-type free radical species, the probable mechanism by which microfilaria are killed (Cesbron et al., 1987). This process seems to be antibody independent (Cesbron et al., 1987). Once platelets are stimulated by a single small dose of DEC, the
0014-4894/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4894(03)00028-6
82
R. Premaratna et al. / Experimental Parasitology 102 (2002) 81–88
microfilaricidal effect of platelets outlast the drug levels by more than a week. (Cesbron et al., 1987). This suggests that treatment with DEC results in an oxidant stress in vivo. In an event of high oxidant stress, antioxidant systems are up regulated to prevent adverse effects of excess free radicals (Halliwell, 2000; Harris, 1992; Kojima and Yamaoka, 1999). Thus, if DEC kills mf by stimulating the release of free radicals from platelets, red cell antioxidant enzyme activity levels should increase to face such an oxidant stress in vivo. Direct estimation of blood oxidant levels is difficult because of the very short half-life of free radicals, but oxidant stress can be estimated indirectly by measuring levels of antioxidants in blood (Knight, 1999; Sahnoun et al., 1997) or red blood cell antioxidant enzyme activity levels (Laaksonen et al., 1999). The most important anti-oxidant enzymes in red blood cells are catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPX) (Fridovich, 1983; Fridovich, 1986). Interestingly, the enzyme GP29, which is homologous to human GPX, is located in the subcuticular region of filarial nematodes. This enzyme is known to protect filarial nematodes against host oxidants (Wakelin, 1996). It is also known that GP29 is expressed on the parasite only in vivo and that the level of expression gradually increases from infective stage three larvae to the adult worm (Wakelin, 1996). The possibility of induction of antibodies against this protein to disrupt its action has been proposed as a protective mechanism against these infections (Bradley and Medeiros, 1994). The currently recommended treatment for bancroftian filariasis is DEC for 12–14 days (Ottesen, 1985; WHO, 1992), but it is apparent that a single 6 mg/kg dose of drug has similar macrofilaricidal efficacy (FigueredoSilva et al., 1996; Noroes et al., 1997) and produces comparable long term suppression of microfilaraemia (Andrade et al., 1995; Simonsen et al., 1995). Furthermore, spacing individual doses of DEC has been found to be more effective than administration of the same total dosage in consecutive daily doses (Meyrowitsch and Simonsen, 1998). Although most asymptomatic microfilaraemics show a rapid clearance of microfilaraemia after treatment with DEC, some show a slower clearance, and some individuals have a rapid re-emergence of microfilaraemia after treatment (Ottesen, 1985). These observations are yet to be explained. Studies on the role of oxidants and anti-oxidants in the interaction between lymphatic filarial worms and the human immune response have not been extensively reported. We first performed a case controlled study to assess red blood cell antioxidant enzyme status in asymptomatic microfilaraemics, Ôendemic normalsÕ and non-endemic controls, and in a second study, compared the changes in red blood cell antioxidant activity levels and microfilaraemia between single dose treatment and 14 day treatment with DEC.
2. Methods During the period June–September 1999, 352 people in 72 households in the Colombo and Gampaha districts, in the Western Province of Sri Lanka, were screened for microfilaraemic status (both districts are highly endemic for W. bancrofti; there is no Brugian filariasis in Sri Lanka). From among those screened, 29 asymptomatic microfilaraemics (index cases) and 29 persons who were considered Ôendemic normalsÕ were recruited. Index cases were identified using a standard night thick blood film. We considered persons to be Ôendemic normalsÕ for the purpose of recruitment for this study if they fulfilled the following criteria: negative for filarial antigenaemia by the immuno-chromatographic card test (which detects an antigen secreted by the adult worm) [ICT-AMRAD, Australia (Weil et al., 1997)] using a 100 ll capillary blood sample, same ethnic group as the index case, had been living for more than 10 years in the same household as the index case, had neither experienced filariasis specific symptoms or signs (using an expert validated questionnaire) nor received antifilarial treatment at any time in their life, presently in good health, closest possible match for age and sex with the index case. Nine (28.1%) of Ôendemic normalsÕ was blood relatives of the index cases. A control group matched with Ôendemic normalsÕ for age, sex, and socioeconomic status (Central Bank of Ceylon 1981/82) was recruited from a non-endemic area (Hantana near Kandy, in the Central Province). Red blood cell antioxidant levels and micofilaraemia was assessed using venous blood as described below. Blood samples for red cell antioxidant levels were obtained from index cases and Ôendemic normalsÕ at the same time. To investigate changes in red blood cell antioxidant levels and microfilaraemia after DEC treatment, a randomized controlled study was performed during the period from February to August 2000, in 22 asymptomatic microfilaraemics, who had not been treated with DEC within the past two years, and who were not on any long term treatment for chronic illness. Using block randomization these 22 individuals were assigned to receive either a single dose (300 mg) or 14 day treatment course (total 4.2 g) of DEC. In all of them, microfilaraemia levels were assessed pretreatment and one month post treatment. Enzyme assays were carried out pretreatment, and on days 3, 7, 14, and 30 after commencing treatment. The ICT filariasis test was carried out on each blood sample to assess filarial antigenaemia. We used Banocide, the diethyl carbamazine citrate manufactured by Glaxo Wellcome Pharmaceuticals for the trial. Patients were requested to indicate the development of any symptoms after treatment was commenced, on a form supplied to them. Compliance with treatment was ensured by frequent home visits and ap-
R. Premaratna et al. / Experimental Parasitology 102 (2002) 81–88
pointing an ÔobserverÕ from among their family members. They were requested to abstain from alcohol but there was no dietary restrictions, as was not practical to assign them the same diet. All blood samples were obtained using disposable syringes and needles, under aseptic conditions. For the case controlled study to assess red blood cell antioxidant enzyme status in asymptomatic microfilaraemics, Ôendemic normalsÕ and non-endemic controls, a single sample of 3 ml venous blood was obtained at night (9 pm–12 midnight) from each participant, 1 ml of which was collected into an EDTA bottle for parasitological investigations. The other 2 ml was collected into a separate EDTA bottle and was immediately stored at 8 °C. This blood sample was centrifuged within half an hour at 3000 rpm to separate the upper layer of red cells. The separated red cells were stored at 8 °C until antioxidant assays were performed. For the study comparing changes in red blood cell antioxidant activity levels and microfilaraemia between single dose treatment and 14 day treatment with DEC, while a night blood sample was collected for parasitological investigations in a similar manner to that described above, 2 ml of blood was collected into an EDTA bottle at 8.00—9.00 in the morning (before breakfast) for red cell antioxidant assays. Antioxidant assays were carried out within 6 h of collection of the blood sample. All participants of the study were from the Sinhalese ethnic group. Informed consent was obtained from participants at the point of recruitment to the study. A Nuclepore membrane (3 lm) filtration was performed to assess the degree of parasitaemia. Red blood cell antioxidant enzyme activity levels were assayed by spectrophotometry using the following commercially available kits, according to manufacturerÕs instructions; Ransel-Randox (UK) for GPX, Ransod-Randox (UK) for SOD, and Catalase was measured by the method of GÕoth (1992). For the SOD activity assays, 0.5 ml of separated RBCs was washed four times with 3 ml of 0.9% saline, centrifuging for 10 min at 3000 rpm after each wash. Washed RBCs were mixed with 2 ml of cold distilled water and left to stand at 4 °C for 15 min. The lysate was used to determine SOD activity using Ransel kits and the absorbance of the final sample was read at 505 nm against the reagent blank. For the GPX activity assay 500 ll of separated red blood cells was diluted with 1 ml of diluting agent. The diluent, which was incubated for 5 min and then mixed well with double strength Drabkins reagent, was used for GPX assays and the absorbance of the final sample was read at 340 nm against the reagent blank. For estimation of Catalase activity, 20 ll of separated blood cells was diluted with 4 ml of 60 mmol/L sodium potassium phosphate buffer (pH 7.4). Then 100 ll saponine solution (20 g/L) was added to the solution. Thirty ll of lysate was mixed with 1 ml subs-
83
tarte (65 lmol/L H2 O2 ) and incubated for 37 °C for 60 s. One ml of 32.4 mmol/L ammonium molybdate was added and the absorbance of the final sample was read at 405 nm against the blank reagent. 2.1. Statistical methods Data were entered on Microsoft Excel. Differences in anti-oxidant levels in the three groups were tested using the t test assuming equal variances in the two samples under comparison. Antioxidant responses on selected days after treatment were compared with pretreatment levels using the t test for paired samples for means and correlation between percentage reduction in microfilaraemia and percentage increase in GPX levels was assessed by regression analysis using the following formulae: ½% increase GPX ¼
ðGPX level on day 3 Pre treatment GPXÞ 100; Pre treatment GPX
½% reduction in mf ðPre treatment mf post treatment mf Þ 100: ¼ Pre treatment mf 2.2. Ethics Ethical approval for the study was obtained from the Ethics Committee of the Faculty of Medicine, University of Kelaniya, Sri Lanka.
3. Results In the first study, there were no significant differences in the age or sex distribution between the 29 asymptomatic microfilaraemic index cases, 29 Ôendemic normalsÕ who fulfilled the inclusion criteria, and 29 nonendemic controls (Table 1). The mean mf count, as assessed by membrane filtration, in the asymptomatic microfilaraemic index cases was 417/ml (range 12—2337/ ml). All Ôendemic normalsÕ and non-endemic controls were negative for filarial antigenaemia and for mf by ICT and membrane filtration, respectively. Live mf were still present in all samples of EDTA blood drawn from mf positive individuals until red cells were washed and separated for enzyme analysis. The red blood cell anti-oxidant activities are shown in Table 2. Levels of GPX activity were significantly higher in Ôendemic normalsÕ when compared to asymptomatic microfilaraemics (index cases) and non-endemic controls. As there were more males among index cases than among Ôendemic normalsÕ, a subanalysis was performed to investigate whether an individualÕs sex influenced the level of antioxidants. We found no significant
84
R. Premaratna et al. / Experimental Parasitology 102 (2002) 81–88
Table 1 Demographic data of the population; Study 1
Sample size Sex distribution Mean age [SD (range)] Socioeconomic status Food habits Past malaria Present intestinal worm infections Acute/chronic illness
Index cases
Endemic normals
Controls
29 0.7 38.2 years [16 (16–75)] Lower Rice/vegetables/fish/meat 1 (10 years back) 2 (1; round worms, 1; hook worms) None
29 1.2 34.6 years [14 (18–75)] Lower Rice/vegetables/fish/meat None 1 (hook worms) None
29 1.2 32.8 years [14 (15–68)] Lower Rice/vegetables/fish/meat None None None
mf Positive index cases and the endemic normals were from the same households. The selected areas for the study are non-endemic for malaria.
Table 2 Blood antioxidant levels and cell counts of the three groups mf Positive index cases Mean
SD
Endemic normals Range
Mean
Non-endemic controls
SD
Range
Mean
SD
Range
Catalase (KAU/L) 0:82 105 0.17 0.52–1.15 0:89 105 0.24 0.51–1.27 0:61 105 0.19 mf Positive vs controls, p < 0:001 ; endemic normals vs controls, p < 0:001 ; mf positive vs endemic normals, p ¼ 0:2
0.31–1.1
Superoxide dismutase (IU/ml) 2:2 102 0.52 1.6–3.4 2:02 102 0.58 1.3—2.9 2:2 102 mf Positive vs controls, p ¼ 0:7 ; endemic normals vs controls, p ¼ 0:2 ; mf positive vs endemic normals, p ¼ 0:2
0.2
1.7—2.5
Glutathione peroxidase (IU/L) 5:6 103 1.9 3.1–10.8 7:8 103 2.1 4.6–12.4 5:7 103 1.8 mf Positive vs controls, p ¼ 0:89 ; endemic normals vs controls, p < 0:001 ; mf positive vs endemic normals, p < 0:001
2.9–10.2
Mean glutathione peroxidase levels in males and females Female Male p Value Female 5:8 103 5:4 103 p ¼ 0:56 8:4 103
Male 7:6 103
Glutathione peroxidase levels within mf positive index cases mf Count < 100/ml (n ¼ 10) mf Count > 500 (n ¼ 11) Mean SD Range Mean SD 5:01 103 1.9 3.08–8.28 6:11 103 1.5 *
p Value p ¼ 0:3
Female 5:4 103
Range 5.0–8.4
p Value p ¼ 0:16
Male 5:8 103
p Value p ¼ 0:6
p Values calculated by StudentÕs t test assuming equal variance in the two population.
difference in antioxidant activity levels between males and females in any of the three groups (only data for GPX are shown in Table 2). A further subanalysis of GPX activity levels was performed within mf positive index cases to investigate whether persistence of live mf in blood samples until red cells were separated had an effect on GPX activity. We observed no significant differences in GPX activity between those with an mf count of less than 100/ml and those with an mf count of more than 500/ml. Blood catalase activity levels were significantly higher in both mf positive index cases and Ôendemic normalsÕ compared to non-endemic controls, but there was no significant difference in catalase activity between index cases and Ôendemic normalsÕ. SOD activity levels were similar in all three groups. In the second study, after randomization of 22 individuals with microfilaraemia (all from lower socioeconomic background), 11 individuals (5 males) where median age was 32.8 years (range 15–59) received either a single dose DEC treatment and other 11 (6 males)
where median age was 34 years (range 16–57) received 14 day treatment with DEC. One person in the 14 day treatment arm was lost to follow up after the 3rd day of treatment. None of the subjects had any vomiting within four hours of taking DEC. Two individuals had used paracetamol (500 and 1500 mg) for Ôaches and painsÕ after commencement of DEC. Both developed these symptoms within 24 h of DEC treatment. A significant increase in red blood cell GPX activity was observed by the 3rd day in both treatment groups (p < 0:01) (Fig. 1A). The increasing trend in GPX activity continued up to the 14th day after commencement of treatment and showed a gradual decline thereafter. Although the mean GPX activity was higher in the 14 day treatment arm when compared to the single dose arm at 14 and 30 days after commencing treatment, these differences were not statistically significant. There was also a gradual increase in catalase and SOD activity after treatment (Figs. 1B and C). However, when compared to the pre-treatment levels, a significant difference
R. Premaratna et al. / Experimental Parasitology 102 (2002) 81–88
85
Fig. 1. Changes in red cell antioxidant activities and microfilaraemia after treatment.
was observed only after 14 and 30 days of treatment. At the end of one month, mean microfilaraemia was reduced by 46 and 42% with single dose and 14 day treatments, respectively (Fig. 1D). The comparison between GPX responses and the reduction in mf counts showed no significant differences between the two treatment arms (Figs. 1A and D). There was a significant correlation between the percentage reduction in microfilaraemia and the percentage rise in GPX levels [by the 3rd day of treatment R2 ¼ 0:58 ðp ¼ 0:6 105 Þ] (Fig. 2). The ICT filariasis test was positive in all individuals at recruitment. The test was negative in three persons after one month of DEC treatment.
4. Discussion Fig. 2. Percentage reduction in microfilaraemia vs percentage increase in GPX.
Our results showed significantly higher levels of red blood cell GPX activity in Ôendemic normalsÕ compared
86
R. Premaratna et al. / Experimental Parasitology 102 (2002) 81–88
to asymptomatic microfilaraemics (index cases) and non-endemic controls. Catalase activity was significantly higher in individuals living in the endemic area (asymptomatic microfilaraemics and Ôendemic normalsÕ) when compared to non-endemic controls. There was no significant difference in SOD activity between the three groups. We considered persons to be Ôendemic normalsÕ if they had a negative ICT filariasis test and were negative for circulating microfilaria. However, as these tests do not have a sensitivity of 100%, we admit that there could have been a small percentage of persons who could still have harboured adult filarial worms. Although this could have had an influence on the results, our observations seem to suggest that GPX activity is induced in Ôendemic normalsÕ (due in turn to generation of GPX related oxidant species; further supported by our second study). Participants in this study were all from a low socioeconomic background and Ôendemic normalsÕ were selected from the same household as a mf positive index case. It is therefore unlikely that differences in diet would have interfered with these results. There was also no significant difference in the presence of other infections or ill health within these groups. However, blood samples of mf positive index cases contained live mf until red cells were separated for antioxidant activity assays. If microfilarial antioxidant enzymes were present in lysates they could neutralize blood oxidants, and this may down regulate red cell GPX activity levels in mf positive index cases. However, if this were the case, blood samples with high mf counts should have lower levels of red cell GPX activity than samples with low mf counts. We did not observe this trend in the subanalysis of samples with mf counts less than 100/ml and more than 500/ml (Table 2). Therefore, the observation of higher GPX activity in ‘‘endemic normals’’ compared to mf positive cases and nonendemic controls may suggest that GPX is induced in ‘‘endemic normals.’’ Although the exact mechanism for this is unclear, it is possible that higher GPX activity is due to generation of GPX related oxidant species in ‘‘endemic normals’’ due to persistent direct stimulation of platelets by filarial antigens. Low red blood cell GPX in asymptomatic microfilaraemics may be due to a defect in generation of GPX related oxidant species from platelets or a defect in platelet activation by filarial antigens. Catalase has been demonstrated to participate in the IgE dependant cytotoxic pathway of platelets (Joseph et al., 1986). The significantly higher levels of catalase in the blood of individuals living in an endemic area when compared to non-endemic controls could be due to continued priming of the host IgE mediated immune pathway by parasite antigens. However, addition of catalase to macrophage cultures or to media testing DEC action on parasites has failed to prevent parasite killing (Cesbron et al., 1987; Taylor et al., 1996) sug-
gesting a lack of participation of catalase related oxidants in parasite elimination. This is consistent with our finding of similar catalase activity levels in both mf positive index cases and Ôendemic normals.Õ There was no significant difference in SOD activity levels among these three groups, suggesting against an active role for SOD related oxidant species in the elimination of the filarial parasite. This is also supported by the previously mentioned study on DEC action (Cesbron et al., 1987), where addition of SOD to the test media failed to prevent parasite killing. We observed an increase in all three red cell antioxidant enzyme activities (catalase, GPX, and SOD) after treatment with DEC. This may reflect the oxidative burst initiated by DEC treatment (Laaksonen et al., 1999). Of the antioxidant enzymes, GPX activity showed the earliest significant increase, within 3 days of commencing treatment. The activity of catalase and SOD rose to significant levels only by the 14th and 30th days after commencing treatment, respectively. The difference in behaviour of these antioxidants could be because, although the three enzymes assayed carry out a similar biological function (neutralization of oxidants), there are specific substrate free radicals for each of them (Harris, 1992). Catalase and GPX are probably under the regulation of the same regulon and also have similar functional properties, but GPX neutralizes organic as well as non-organic peroxides, unlike catalase, which acts exclusively on hydrogen peroxide (Harris, 1992). Therefore, different levels of GPX activity may reflect availability of such oxidant precursors. Furthermore, these antioxidant enzymes may be located at different sites within a biological system. The rapid increase in levels of GPX related oxidant species may suggest a special role for them in the microfilaricidal action of DEC. The persistence of high GPX activity for about two weeks after a single dose of DEC may be due to continued release of related oxidant species by platelets for an extended period, as observed in in vitro studies (Cesbron et al., 1987). The late rise in catalase and SOD activity after treatment with DEC may suggest either activation of specific humoral immunological pathways against the parasite or an immunological response to dead parasite antigens, as these two enzymes have been shown to participate in the IgE mediated immunological pathway of the host–parasite relationship (Cesbron et al., 1987). However, further studies are necessary to investigate these differences in individual antioxidant levels before firm conclusions can be arrived at. The efficacy of DEC in reducing microfilaraemia varied greatly within each of the treatment arms, but with no significant difference between them. In some patients there was a 100% elimination of microfilaraemia, whereas in a few the mf counts remained unchanged at follow up 30 days after treatment. However, there was a statistically significant reduction in the mean
R. Premaratna et al. / Experimental Parasitology 102 (2002) 81–88
mf counts compared to pretreatment counts (46% for single dose treatment and 42% for 14 day treatment). This type of result is known to occur in the treatment of W. bancrofti infection with DEC (Weil et al., 1988; Cartel et al., 1990). As was the case with reduction in mf counts, there was no significant difference in the increased activity of antioxidants between the two treatment arms. There was a moderately significant correlation between the increase in GPX activity and the reduction in microfilaraemia. This could be due to differences in the ability of parasite antioxidants to counter oxidative stress, which would mean that the generation of similar levels of free radicals could still have different efficacies in killing microfilariae. This would explain why a few individuals with relatively strong GPX responses had a low percentage reduction in microfilaraemia (Fig. 2). In conclusion, the ability to generate GPX related oxidant species seems to play a role in the determination of microfilaraemia in filarial infection. Low levels of red blood cell GPX activity in asymptomatic microfilaraemics may be due to a defect in generation of such GPX related oxidant species from platelets or a defect in platelet activation by filarial antigens. The efficacy of single dose treatment with DEC appears to be similar to that of continuous 14 day treatment, as measured both by reduction in microfilaraemia and by anti-oxidant responses.
Acknowledgments We thank Drs. T. Liyanage, S. Sivakumara, M.S. Subasinghe, N.S.H. Liyanage, and K.L. Gamagedara, the Public Health Inspectors, and Field Assistants of the Anti-Filariasis Campaign Offices in Colombo and Gampaha, Mr. Hemantha Sudasinghe, Mrs. G Wickramarachchi, Mrs. K Rodrigo, and Mrs. S. Jayaweera of the Faculty of Medicine, University of Kelaniya for help with the study, and Prof. A.S. Dissanayale, Emeritus Professor of Parasitology, University of Colombo, for advice.
References Andrade, L.D., Medeiros, Z., Pires, M.L., Pimentel, A., Rocha, A., Figueredo-Silva, J., Coutinho, A., Dreyer, G., 1995. Comparative efficacy of three different diethyl carbamazine regimens in lymphatic filariasis. Transactions of the Royal Society of Tropical Medicine and Hygiene 89, 319–321. Bradley, J.E., Medeiros, 1994. Filariasis from the lab to the field. Parasitology Today 10, 87–89. Cartel, J.L., Celerier, P., Spiegel, A., Burucoa, C., Roux, J.F., 1990. A single diethylcarbamazine dose for treatment of Wuchereria bancrofti carriers in French Polynesia: efficacy and side effects. South East Asian Journal of Tropical Medicine and Public Health 21, 465–470.
87
Cesbron, J.Y., Capron, A., Vargaftig, B.B., Lagarde, M., Pincemail, J., Braquet, P., Taelman, H., Joseph, M., 1987. Platelets mediate the action of diethylcarbamazine on microfilariae. Nature 325, 533–536. Day, K.P., 1991. The endemic normal in lymphatic filariasis: a static concept. Parasitology Today 7, 341–343. de Silva, N., Guyatt, H., Bundy, D., 1997. Antihelminthics; a comparative review of their clinical pharmacology. Drugs 53, 769–788. Dreyer, G., Santos, A., Noroes, J., Rocha, A., Addiss, D., 1996. Amicrofilaraemic carriers of adult Wuchereria bancrofti. Transactions of the Royal Society of the Tropical Medicine and Hygiene 90, 288–289. Figueredo-Silva, J., Jungmann, P., Noroes, J., Piessens, W.F., Coutinho, A., Brito, C., Rocha, A., Dreyer, G., 1996. Histological evidence for adulticidal effect of low dose diethyl carbamazine citrate in bancroftian filariasis. Transactions of the Royal Society of Tropical Medicine and Hygiene 90, 192–194. Fridovich, I., 1983. Superoxide radical: an endogenous toxicant. Annual review of Pharmacology and Toxicology 26, 239–257. Fridovich, I., 1986. Biological effects of the superoxide radical. Archives of Biochemistry and Biophysics 247, 1–11. GÕoth, L., 1992. Two cases of acatalasemia in Hungary. Clinica Chimica Acta 207, 155–158. Halliwell, B., 2000. The antioxidant paradox. Lancet 355, 1179–1180. Harris, E.D., 1992. Regulation of antioxidant enzymes. FASEB Journal 6, 2675–2683. Joseph, M., Capron, A., Ameisen, J.C., Capron, M., Vorng, H., Pancre, V., Kusnierz, J.P., Auriault, C., 1986. The receptor for IgE on blood platelets. European Journal of Immunology 16, 306–312. Knight, J.A., 1999. In: Free Radicals Antioxidants Aging and Disease. Free Radicals: Their Presence in Biological Systems. AACC press, Washington, pp. 21–42. Kojima, S., Yamaoka, K., 1999. Adaptive responses of living cells towards low doses of radiation; induction of endogenous antioxidant systems and its applicable possibility for treatment of diseases. Journal of Health Sciences 45, 133–143. Laaksonen, D.E., Atalay, M., Niskanen, L., Uusitupa, M., Hanninen, O., Sen, C.K., 1999. Blood glutathione homeostasis as a determinant of resting and exercise induced oxidative stress in young men. Redox Report 4, 53–59. LoVerde, P.T., 1998. Do antioxidants play a role in schistosome host– parasite interactions? Parasitology Today 14, 284–289. Meyrowitsch, D.W., Simonsen, P.E., 1998. Long term effect of mass diethylcarbamazine chemotherapy on bancroftian filariasis:results at four years after start of treatment. Transactions of the Royal Society of Tropical Medicine and Hygiene 92, 98–103. Noroes, J., Dreyer, G., Santos, A., Mendes, V.G., Medeiros, Z., Addis, D., 1997. Assessment of the efficacy of diethyl carbamazine on adult Wuchereria bancrofti in vivo. Transactions of the Royal Society of Tropical Medicine and Hygiene 91, 78–81. Ottesen, E.A., 1985. Efficacy of diethylcarbamazine in eradicating infection with lymphatic-dwelling filariae in humans. Reviews of Infectious Diseases 7, 341–356. Sahnoun, Z., Jamoussi, K., Zeghal, K.M., 1997. Free radicals and anti-oxidants: human physiology, pathology and therapeutic aspects. Therapie 52, 251–270. Simonsen, P.E., Meyrowitsch, D.W., Makunde, W.H., Magnussen, P., 1995. Selective diethylcarbamazine chemotherapy for control of bancroftian filariasis in two communities of Tanzania: compared efficacy of a standard dose treatment and two semi-annual single dose treatments. American Journal of Tropical Medicine and Hygiene 53, 267–272. Taylor, M.J., Cross, H.F., Mohammed, A.A., Trees, A.J., Bianco, A.E., 1996. Susceptibility of Brugia malayi and Onchocerca lienalis microfilariae to nitric oxide and hydrogen peroxide in cell free
88
R. Premaratna et al. / Experimental Parasitology 102 (2002) 81–88
culture and from INFc activated macrophages. Parasitology 112, 315–322. Wakelin, D., 1996. In: Immunity to Parasites—How Parasitic Infections are Controlled. Nematodes that Invade Tissues. Cambridge University press, Cambridge, pp. 146–162. Weil, G.J., Lammie, P.J., Weiss, N., 1997. The ICT filariasis test: a rapid-format antigen test for diagnosis of bancroftian filariasis. Parasitology Today 13, 401–404. Weil, G.J., Sethumadhavan, K.V.P., Santhanam, S., Jain, D.C., Ghosh, T.K., 1988. Persistance of parasite antigenemia following diethylcarbamazine therapy of bancroftian filariosis. American Journal of Tropical Medicine and Hygiene 38, 589–595.
WHO, 1992. Lymphatic filariasis: the disease and its control: Fifth Report of the WHO Expert Committee on Filariasis. World Health Organization, Geneva, Technical Report Series, No. 821. WHO, 1999. Lymphatic filariasis: reasons for hope. Division of Control of Tropical Diseases, WHO, Geneva.
Further reading Central Bank of Ceylon, 1984. Report on consumer finances and socioeconomic survey 1981/82, Sri Lanka, Part 1, Appendix 1.
Red blood cell antioxidant levels in Wuchereria bancrofti infectionq R. Premaratna,a,* T.G.A.N. Chandrasena,b W. Abeyewickreme,b L.G. Chandrasena,c S. Senarath,c N.R. de Silva,b and H.J. de Silvaa a
Department of Medicine, Faculty of Medicine, University of Kelaniya, P O Box 6, Thalagolla Rd., Ragama, Sri Lanka b Department of Parasitology, Faculty of Medicine, University of Kelaniya, Sri Lanka c Department of Biochemistry, Faculty of Medicine, University of Kelaniya, Sri Lanka Received 9 December 2001; received in revised form 12 August 2002; accepted 14 February 2003
Abstract The elimination of microfilariae of Wuchereria bancrofti is probably mediated by free radicals. Red cell catalase (C), glutathione peroxidase (GPX), and superoxide dismutase (SOD) activity levels were measured as an indirect method of assessing blood oxidant status in 29 asymptomatic microfilaraemics, 29 ‘‘endemic normals’’, and 29 controls living in a non-endemic area. Changes in the activity of these enzymes were also compared over a one month period in 22 asymptomatic microfilaraemics randomised to receive either single dose or 14 day treatment with diethyl carbamazine citrate (DEC). Red cell GPX activity levels were significantly higher in ‘‘endemic normals’’ when compared to mf positive cases and non-endemic controls. An early and significant increase in GPX activity (on days 3, 7 and 14 compared to pretreatment levels, p < 0:01) was observed after DEC in both treatment groups. Increases in the activity of catalase and SOD became significant only on days 14 and 30 respectively. The percentage reduction in microfilaraemia correlated significantly with the percentage increase in GPX activity levels (R2 ¼ 0:58; p ¼ 0:6 105 ). Our results may suggest a role for GPX related oxidant species in the elimination of microfilariae. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Filariasis; Endemic normals; Antioxidants; Glutathione peroxidase; DEC
1. Introduction Wuchereria bancrofti is a filarial worm known to cause lymphatic pathology in large areas of the tropics; more than 1.1 billion people are estimated to be at risk of infection worldwide (WHO, 1999). In any endemic region, a proportion of the population at risk remains asymptomatic despite harbouring adult worms and circulating microfilariae (asymptomatic microfilaraemics); another proportion remains persistently asymptomatic and negative for microfilaria (mf) and adult worms despite almost certain exposure to infection (endemic normals) (Day, 1991). This may be because these individuals are able to q Parts of this work were presented at the Oxford 2000 Conference on New Challenges in Tropical Medicine and Parasitology, 18th–22nd September, 2000 and the 11th European Congress of Clinical Microbiology and Infectious Diseases, 1–4th April, 2001. * Corresponding author. Fax: +94-1-958337. E-mail address: [email protected] (R. Premaratna).
destroy the infective larvae or developing stages of the worm before they reach sexual maturity. There is no single test that can be used to confirm the Ôendemic normalÕ status. The ICT filariasis test and OG4C3 ELISA test, both of which detect adult filarial antigens, are among the most sensitive tests available (Dreyer et al., 1996). Because W. bancrofti is a multicellular parasite host effector mechanisms for its destruction by the immune system are likely to involve oxidants, in a manner similar to schistosomulae (the infective stages of schistosomes) being very sensitive to oxidant mediated killing (LoVerde, 1998). The microfilaricidal action of diethyl carbamazine citrate (DEC) is poorly understood (de Silva et al., 1997). In vitro studies have shown that treatment with DEC results in the stimulation of platelets to release undefined (Ox)-type free radical species, the probable mechanism by which microfilaria are killed (Cesbron et al., 1987). This process seems to be antibody independent (Cesbron et al., 1987). Once platelets are stimulated by a single small dose of DEC, the
0014-4894/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4894(03)00028-6
82
R. Premaratna et al. / Experimental Parasitology 102 (2002) 81–88
microfilaricidal effect of platelets outlast the drug levels by more than a week. (Cesbron et al., 1987). This suggests that treatment with DEC results in an oxidant stress in vivo. In an event of high oxidant stress, antioxidant systems are up regulated to prevent adverse effects of excess free radicals (Halliwell, 2000; Harris, 1992; Kojima and Yamaoka, 1999). Thus, if DEC kills mf by stimulating the release of free radicals from platelets, red cell antioxidant enzyme activity levels should increase to face such an oxidant stress in vivo. Direct estimation of blood oxidant levels is difficult because of the very short half-life of free radicals, but oxidant stress can be estimated indirectly by measuring levels of antioxidants in blood (Knight, 1999; Sahnoun et al., 1997) or red blood cell antioxidant enzyme activity levels (Laaksonen et al., 1999). The most important anti-oxidant enzymes in red blood cells are catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPX) (Fridovich, 1983; Fridovich, 1986). Interestingly, the enzyme GP29, which is homologous to human GPX, is located in the subcuticular region of filarial nematodes. This enzyme is known to protect filarial nematodes against host oxidants (Wakelin, 1996). It is also known that GP29 is expressed on the parasite only in vivo and that the level of expression gradually increases from infective stage three larvae to the adult worm (Wakelin, 1996). The possibility of induction of antibodies against this protein to disrupt its action has been proposed as a protective mechanism against these infections (Bradley and Medeiros, 1994). The currently recommended treatment for bancroftian filariasis is DEC for 12–14 days (Ottesen, 1985; WHO, 1992), but it is apparent that a single 6 mg/kg dose of drug has similar macrofilaricidal efficacy (FigueredoSilva et al., 1996; Noroes et al., 1997) and produces comparable long term suppression of microfilaraemia (Andrade et al., 1995; Simonsen et al., 1995). Furthermore, spacing individual doses of DEC has been found to be more effective than administration of the same total dosage in consecutive daily doses (Meyrowitsch and Simonsen, 1998). Although most asymptomatic microfilaraemics show a rapid clearance of microfilaraemia after treatment with DEC, some show a slower clearance, and some individuals have a rapid re-emergence of microfilaraemia after treatment (Ottesen, 1985). These observations are yet to be explained. Studies on the role of oxidants and anti-oxidants in the interaction between lymphatic filarial worms and the human immune response have not been extensively reported. We first performed a case controlled study to assess red blood cell antioxidant enzyme status in asymptomatic microfilaraemics, Ôendemic normalsÕ and non-endemic controls, and in a second study, compared the changes in red blood cell antioxidant activity levels and microfilaraemia between single dose treatment and 14 day treatment with DEC.
2. Methods During the period June–September 1999, 352 people in 72 households in the Colombo and Gampaha districts, in the Western Province of Sri Lanka, were screened for microfilaraemic status (both districts are highly endemic for W. bancrofti; there is no Brugian filariasis in Sri Lanka). From among those screened, 29 asymptomatic microfilaraemics (index cases) and 29 persons who were considered Ôendemic normalsÕ were recruited. Index cases were identified using a standard night thick blood film. We considered persons to be Ôendemic normalsÕ for the purpose of recruitment for this study if they fulfilled the following criteria: negative for filarial antigenaemia by the immuno-chromatographic card test (which detects an antigen secreted by the adult worm) [ICT-AMRAD, Australia (Weil et al., 1997)] using a 100 ll capillary blood sample, same ethnic group as the index case, had been living for more than 10 years in the same household as the index case, had neither experienced filariasis specific symptoms or signs (using an expert validated questionnaire) nor received antifilarial treatment at any time in their life, presently in good health, closest possible match for age and sex with the index case. Nine (28.1%) of Ôendemic normalsÕ was blood relatives of the index cases. A control group matched with Ôendemic normalsÕ for age, sex, and socioeconomic status (Central Bank of Ceylon 1981/82) was recruited from a non-endemic area (Hantana near Kandy, in the Central Province). Red blood cell antioxidant levels and micofilaraemia was assessed using venous blood as described below. Blood samples for red cell antioxidant levels were obtained from index cases and Ôendemic normalsÕ at the same time. To investigate changes in red blood cell antioxidant levels and microfilaraemia after DEC treatment, a randomized controlled study was performed during the period from February to August 2000, in 22 asymptomatic microfilaraemics, who had not been treated with DEC within the past two years, and who were not on any long term treatment for chronic illness. Using block randomization these 22 individuals were assigned to receive either a single dose (300 mg) or 14 day treatment course (total 4.2 g) of DEC. In all of them, microfilaraemia levels were assessed pretreatment and one month post treatment. Enzyme assays were carried out pretreatment, and on days 3, 7, 14, and 30 after commencing treatment. The ICT filariasis test was carried out on each blood sample to assess filarial antigenaemia. We used Banocide, the diethyl carbamazine citrate manufactured by Glaxo Wellcome Pharmaceuticals for the trial. Patients were requested to indicate the development of any symptoms after treatment was commenced, on a form supplied to them. Compliance with treatment was ensured by frequent home visits and ap-
R. Premaratna et al. / Experimental Parasitology 102 (2002) 81–88
pointing an ÔobserverÕ from among their family members. They were requested to abstain from alcohol but there was no dietary restrictions, as was not practical to assign them the same diet. All blood samples were obtained using disposable syringes and needles, under aseptic conditions. For the case controlled study to assess red blood cell antioxidant enzyme status in asymptomatic microfilaraemics, Ôendemic normalsÕ and non-endemic controls, a single sample of 3 ml venous blood was obtained at night (9 pm–12 midnight) from each participant, 1 ml of which was collected into an EDTA bottle for parasitological investigations. The other 2 ml was collected into a separate EDTA bottle and was immediately stored at 8 °C. This blood sample was centrifuged within half an hour at 3000 rpm to separate the upper layer of red cells. The separated red cells were stored at 8 °C until antioxidant assays were performed. For the study comparing changes in red blood cell antioxidant activity levels and microfilaraemia between single dose treatment and 14 day treatment with DEC, while a night blood sample was collected for parasitological investigations in a similar manner to that described above, 2 ml of blood was collected into an EDTA bottle at 8.00—9.00 in the morning (before breakfast) for red cell antioxidant assays. Antioxidant assays were carried out within 6 h of collection of the blood sample. All participants of the study were from the Sinhalese ethnic group. Informed consent was obtained from participants at the point of recruitment to the study. A Nuclepore membrane (3 lm) filtration was performed to assess the degree of parasitaemia. Red blood cell antioxidant enzyme activity levels were assayed by spectrophotometry using the following commercially available kits, according to manufacturerÕs instructions; Ransel-Randox (UK) for GPX, Ransod-Randox (UK) for SOD, and Catalase was measured by the method of GÕoth (1992). For the SOD activity assays, 0.5 ml of separated RBCs was washed four times with 3 ml of 0.9% saline, centrifuging for 10 min at 3000 rpm after each wash. Washed RBCs were mixed with 2 ml of cold distilled water and left to stand at 4 °C for 15 min. The lysate was used to determine SOD activity using Ransel kits and the absorbance of the final sample was read at 505 nm against the reagent blank. For the GPX activity assay 500 ll of separated red blood cells was diluted with 1 ml of diluting agent. The diluent, which was incubated for 5 min and then mixed well with double strength Drabkins reagent, was used for GPX assays and the absorbance of the final sample was read at 340 nm against the reagent blank. For estimation of Catalase activity, 20 ll of separated blood cells was diluted with 4 ml of 60 mmol/L sodium potassium phosphate buffer (pH 7.4). Then 100 ll saponine solution (20 g/L) was added to the solution. Thirty ll of lysate was mixed with 1 ml subs-
83
tarte (65 lmol/L H2 O2 ) and incubated for 37 °C for 60 s. One ml of 32.4 mmol/L ammonium molybdate was added and the absorbance of the final sample was read at 405 nm against the blank reagent. 2.1. Statistical methods Data were entered on Microsoft Excel. Differences in anti-oxidant levels in the three groups were tested using the t test assuming equal variances in the two samples under comparison. Antioxidant responses on selected days after treatment were compared with pretreatment levels using the t test for paired samples for means and correlation between percentage reduction in microfilaraemia and percentage increase in GPX levels was assessed by regression analysis using the following formulae: ½% increase GPX ¼
ðGPX level on day 3 Pre treatment GPXÞ 100; Pre treatment GPX
½% reduction in mf ðPre treatment mf post treatment mf Þ 100: ¼ Pre treatment mf 2.2. Ethics Ethical approval for the study was obtained from the Ethics Committee of the Faculty of Medicine, University of Kelaniya, Sri Lanka.
3. Results In the first study, there were no significant differences in the age or sex distribution between the 29 asymptomatic microfilaraemic index cases, 29 Ôendemic normalsÕ who fulfilled the inclusion criteria, and 29 nonendemic controls (Table 1). The mean mf count, as assessed by membrane filtration, in the asymptomatic microfilaraemic index cases was 417/ml (range 12—2337/ ml). All Ôendemic normalsÕ and non-endemic controls were negative for filarial antigenaemia and for mf by ICT and membrane filtration, respectively. Live mf were still present in all samples of EDTA blood drawn from mf positive individuals until red cells were washed and separated for enzyme analysis. The red blood cell anti-oxidant activities are shown in Table 2. Levels of GPX activity were significantly higher in Ôendemic normalsÕ when compared to asymptomatic microfilaraemics (index cases) and non-endemic controls. As there were more males among index cases than among Ôendemic normalsÕ, a subanalysis was performed to investigate whether an individualÕs sex influenced the level of antioxidants. We found no significant
84
R. Premaratna et al. / Experimental Parasitology 102 (2002) 81–88
Table 1 Demographic data of the population; Study 1
Sample size Sex distribution Mean age [SD (range)] Socioeconomic status Food habits Past malaria Present intestinal worm infections Acute/chronic illness
Index cases
Endemic normals
Controls
29 0.7 38.2 years [16 (16–75)] Lower Rice/vegetables/fish/meat 1 (10 years back) 2 (1; round worms, 1; hook worms) None
29 1.2 34.6 years [14 (18–75)] Lower Rice/vegetables/fish/meat None 1 (hook worms) None
29 1.2 32.8 years [14 (15–68)] Lower Rice/vegetables/fish/meat None None None
mf Positive index cases and the endemic normals were from the same households. The selected areas for the study are non-endemic for malaria.
Table 2 Blood antioxidant levels and cell counts of the three groups mf Positive index cases Mean
SD
Endemic normals Range
Mean
Non-endemic controls
SD
Range
Mean
SD
Range
Catalase (KAU/L) 0:82 105 0.17 0.52–1.15 0:89 105 0.24 0.51–1.27 0:61 105 0.19 mf Positive vs controls, p < 0:001 ; endemic normals vs controls, p < 0:001 ; mf positive vs endemic normals, p ¼ 0:2
0.31–1.1
Superoxide dismutase (IU/ml) 2:2 102 0.52 1.6–3.4 2:02 102 0.58 1.3—2.9 2:2 102 mf Positive vs controls, p ¼ 0:7 ; endemic normals vs controls, p ¼ 0:2 ; mf positive vs endemic normals, p ¼ 0:2
0.2
1.7—2.5
Glutathione peroxidase (IU/L) 5:6 103 1.9 3.1–10.8 7:8 103 2.1 4.6–12.4 5:7 103 1.8 mf Positive vs controls, p ¼ 0:89 ; endemic normals vs controls, p < 0:001 ; mf positive vs endemic normals, p < 0:001
2.9–10.2
Mean glutathione peroxidase levels in males and females Female Male p Value Female 5:8 103 5:4 103 p ¼ 0:56 8:4 103
Male 7:6 103
Glutathione peroxidase levels within mf positive index cases mf Count < 100/ml (n ¼ 10) mf Count > 500 (n ¼ 11) Mean SD Range Mean SD 5:01 103 1.9 3.08–8.28 6:11 103 1.5 *
p Value p ¼ 0:3
Female 5:4 103
Range 5.0–8.4
p Value p ¼ 0:16
Male 5:8 103
p Value p ¼ 0:6
p Values calculated by StudentÕs t test assuming equal variance in the two population.
difference in antioxidant activity levels between males and females in any of the three groups (only data for GPX are shown in Table 2). A further subanalysis of GPX activity levels was performed within mf positive index cases to investigate whether persistence of live mf in blood samples until red cells were separated had an effect on GPX activity. We observed no significant differences in GPX activity between those with an mf count of less than 100/ml and those with an mf count of more than 500/ml. Blood catalase activity levels were significantly higher in both mf positive index cases and Ôendemic normalsÕ compared to non-endemic controls, but there was no significant difference in catalase activity between index cases and Ôendemic normalsÕ. SOD activity levels were similar in all three groups. In the second study, after randomization of 22 individuals with microfilaraemia (all from lower socioeconomic background), 11 individuals (5 males) where median age was 32.8 years (range 15–59) received either a single dose DEC treatment and other 11 (6 males)
where median age was 34 years (range 16–57) received 14 day treatment with DEC. One person in the 14 day treatment arm was lost to follow up after the 3rd day of treatment. None of the subjects had any vomiting within four hours of taking DEC. Two individuals had used paracetamol (500 and 1500 mg) for Ôaches and painsÕ after commencement of DEC. Both developed these symptoms within 24 h of DEC treatment. A significant increase in red blood cell GPX activity was observed by the 3rd day in both treatment groups (p < 0:01) (Fig. 1A). The increasing trend in GPX activity continued up to the 14th day after commencement of treatment and showed a gradual decline thereafter. Although the mean GPX activity was higher in the 14 day treatment arm when compared to the single dose arm at 14 and 30 days after commencing treatment, these differences were not statistically significant. There was also a gradual increase in catalase and SOD activity after treatment (Figs. 1B and C). However, when compared to the pre-treatment levels, a significant difference
R. Premaratna et al. / Experimental Parasitology 102 (2002) 81–88
85
Fig. 1. Changes in red cell antioxidant activities and microfilaraemia after treatment.
was observed only after 14 and 30 days of treatment. At the end of one month, mean microfilaraemia was reduced by 46 and 42% with single dose and 14 day treatments, respectively (Fig. 1D). The comparison between GPX responses and the reduction in mf counts showed no significant differences between the two treatment arms (Figs. 1A and D). There was a significant correlation between the percentage reduction in microfilaraemia and the percentage rise in GPX levels [by the 3rd day of treatment R2 ¼ 0:58 ðp ¼ 0:6 105 Þ] (Fig. 2). The ICT filariasis test was positive in all individuals at recruitment. The test was negative in three persons after one month of DEC treatment.
4. Discussion Fig. 2. Percentage reduction in microfilaraemia vs percentage increase in GPX.
Our results showed significantly higher levels of red blood cell GPX activity in Ôendemic normalsÕ compared
86
R. Premaratna et al. / Experimental Parasitology 102 (2002) 81–88
to asymptomatic microfilaraemics (index cases) and non-endemic controls. Catalase activity was significantly higher in individuals living in the endemic area (asymptomatic microfilaraemics and Ôendemic normalsÕ) when compared to non-endemic controls. There was no significant difference in SOD activity between the three groups. We considered persons to be Ôendemic normalsÕ if they had a negative ICT filariasis test and were negative for circulating microfilaria. However, as these tests do not have a sensitivity of 100%, we admit that there could have been a small percentage of persons who could still have harboured adult filarial worms. Although this could have had an influence on the results, our observations seem to suggest that GPX activity is induced in Ôendemic normalsÕ (due in turn to generation of GPX related oxidant species; further supported by our second study). Participants in this study were all from a low socioeconomic background and Ôendemic normalsÕ were selected from the same household as a mf positive index case. It is therefore unlikely that differences in diet would have interfered with these results. There was also no significant difference in the presence of other infections or ill health within these groups. However, blood samples of mf positive index cases contained live mf until red cells were separated for antioxidant activity assays. If microfilarial antioxidant enzymes were present in lysates they could neutralize blood oxidants, and this may down regulate red cell GPX activity levels in mf positive index cases. However, if this were the case, blood samples with high mf counts should have lower levels of red cell GPX activity than samples with low mf counts. We did not observe this trend in the subanalysis of samples with mf counts less than 100/ml and more than 500/ml (Table 2). Therefore, the observation of higher GPX activity in ‘‘endemic normals’’ compared to mf positive cases and nonendemic controls may suggest that GPX is induced in ‘‘endemic normals.’’ Although the exact mechanism for this is unclear, it is possible that higher GPX activity is due to generation of GPX related oxidant species in ‘‘endemic normals’’ due to persistent direct stimulation of platelets by filarial antigens. Low red blood cell GPX in asymptomatic microfilaraemics may be due to a defect in generation of GPX related oxidant species from platelets or a defect in platelet activation by filarial antigens. Catalase has been demonstrated to participate in the IgE dependant cytotoxic pathway of platelets (Joseph et al., 1986). The significantly higher levels of catalase in the blood of individuals living in an endemic area when compared to non-endemic controls could be due to continued priming of the host IgE mediated immune pathway by parasite antigens. However, addition of catalase to macrophage cultures or to media testing DEC action on parasites has failed to prevent parasite killing (Cesbron et al., 1987; Taylor et al., 1996) sug-
gesting a lack of participation of catalase related oxidants in parasite elimination. This is consistent with our finding of similar catalase activity levels in both mf positive index cases and Ôendemic normals.Õ There was no significant difference in SOD activity levels among these three groups, suggesting against an active role for SOD related oxidant species in the elimination of the filarial parasite. This is also supported by the previously mentioned study on DEC action (Cesbron et al., 1987), where addition of SOD to the test media failed to prevent parasite killing. We observed an increase in all three red cell antioxidant enzyme activities (catalase, GPX, and SOD) after treatment with DEC. This may reflect the oxidative burst initiated by DEC treatment (Laaksonen et al., 1999). Of the antioxidant enzymes, GPX activity showed the earliest significant increase, within 3 days of commencing treatment. The activity of catalase and SOD rose to significant levels only by the 14th and 30th days after commencing treatment, respectively. The difference in behaviour of these antioxidants could be because, although the three enzymes assayed carry out a similar biological function (neutralization of oxidants), there are specific substrate free radicals for each of them (Harris, 1992). Catalase and GPX are probably under the regulation of the same regulon and also have similar functional properties, but GPX neutralizes organic as well as non-organic peroxides, unlike catalase, which acts exclusively on hydrogen peroxide (Harris, 1992). Therefore, different levels of GPX activity may reflect availability of such oxidant precursors. Furthermore, these antioxidant enzymes may be located at different sites within a biological system. The rapid increase in levels of GPX related oxidant species may suggest a special role for them in the microfilaricidal action of DEC. The persistence of high GPX activity for about two weeks after a single dose of DEC may be due to continued release of related oxidant species by platelets for an extended period, as observed in in vitro studies (Cesbron et al., 1987). The late rise in catalase and SOD activity after treatment with DEC may suggest either activation of specific humoral immunological pathways against the parasite or an immunological response to dead parasite antigens, as these two enzymes have been shown to participate in the IgE mediated immunological pathway of the host–parasite relationship (Cesbron et al., 1987). However, further studies are necessary to investigate these differences in individual antioxidant levels before firm conclusions can be arrived at. The efficacy of DEC in reducing microfilaraemia varied greatly within each of the treatment arms, but with no significant difference between them. In some patients there was a 100% elimination of microfilaraemia, whereas in a few the mf counts remained unchanged at follow up 30 days after treatment. However, there was a statistically significant reduction in the mean
R. Premaratna et al. / Experimental Parasitology 102 (2002) 81–88
mf counts compared to pretreatment counts (46% for single dose treatment and 42% for 14 day treatment). This type of result is known to occur in the treatment of W. bancrofti infection with DEC (Weil et al., 1988; Cartel et al., 1990). As was the case with reduction in mf counts, there was no significant difference in the increased activity of antioxidants between the two treatment arms. There was a moderately significant correlation between the increase in GPX activity and the reduction in microfilaraemia. This could be due to differences in the ability of parasite antioxidants to counter oxidative stress, which would mean that the generation of similar levels of free radicals could still have different efficacies in killing microfilariae. This would explain why a few individuals with relatively strong GPX responses had a low percentage reduction in microfilaraemia (Fig. 2). In conclusion, the ability to generate GPX related oxidant species seems to play a role in the determination of microfilaraemia in filarial infection. Low levels of red blood cell GPX activity in asymptomatic microfilaraemics may be due to a defect in generation of such GPX related oxidant species from platelets or a defect in platelet activation by filarial antigens. The efficacy of single dose treatment with DEC appears to be similar to that of continuous 14 day treatment, as measured both by reduction in microfilaraemia and by anti-oxidant responses.
Acknowledgments We thank Drs. T. Liyanage, S. Sivakumara, M.S. Subasinghe, N.S.H. Liyanage, and K.L. Gamagedara, the Public Health Inspectors, and Field Assistants of the Anti-Filariasis Campaign Offices in Colombo and Gampaha, Mr. Hemantha Sudasinghe, Mrs. G Wickramarachchi, Mrs. K Rodrigo, and Mrs. S. Jayaweera of the Faculty of Medicine, University of Kelaniya for help with the study, and Prof. A.S. Dissanayale, Emeritus Professor of Parasitology, University of Colombo, for advice.
References Andrade, L.D., Medeiros, Z., Pires, M.L., Pimentel, A., Rocha, A., Figueredo-Silva, J., Coutinho, A., Dreyer, G., 1995. Comparative efficacy of three different diethyl carbamazine regimens in lymphatic filariasis. Transactions of the Royal Society of Tropical Medicine and Hygiene 89, 319–321. Bradley, J.E., Medeiros, 1994. Filariasis from the lab to the field. Parasitology Today 10, 87–89. Cartel, J.L., Celerier, P., Spiegel, A., Burucoa, C., Roux, J.F., 1990. A single diethylcarbamazine dose for treatment of Wuchereria bancrofti carriers in French Polynesia: efficacy and side effects. South East Asian Journal of Tropical Medicine and Public Health 21, 465–470.
87
Cesbron, J.Y., Capron, A., Vargaftig, B.B., Lagarde, M., Pincemail, J., Braquet, P., Taelman, H., Joseph, M., 1987. Platelets mediate the action of diethylcarbamazine on microfilariae. Nature 325, 533–536. Day, K.P., 1991. The endemic normal in lymphatic filariasis: a static concept. Parasitology Today 7, 341–343. de Silva, N., Guyatt, H., Bundy, D., 1997. Antihelminthics; a comparative review of their clinical pharmacology. Drugs 53, 769–788. Dreyer, G., Santos, A., Noroes, J., Rocha, A., Addiss, D., 1996. Amicrofilaraemic carriers of adult Wuchereria bancrofti. Transactions of the Royal Society of the Tropical Medicine and Hygiene 90, 288–289. Figueredo-Silva, J., Jungmann, P., Noroes, J., Piessens, W.F., Coutinho, A., Brito, C., Rocha, A., Dreyer, G., 1996. Histological evidence for adulticidal effect of low dose diethyl carbamazine citrate in bancroftian filariasis. Transactions of the Royal Society of Tropical Medicine and Hygiene 90, 192–194. Fridovich, I., 1983. Superoxide radical: an endogenous toxicant. Annual review of Pharmacology and Toxicology 26, 239–257. Fridovich, I., 1986. Biological effects of the superoxide radical. Archives of Biochemistry and Biophysics 247, 1–11. GÕoth, L., 1992. Two cases of acatalasemia in Hungary. Clinica Chimica Acta 207, 155–158. Halliwell, B., 2000. The antioxidant paradox. Lancet 355, 1179–1180. Harris, E.D., 1992. Regulation of antioxidant enzymes. FASEB Journal 6, 2675–2683. Joseph, M., Capron, A., Ameisen, J.C., Capron, M., Vorng, H., Pancre, V., Kusnierz, J.P., Auriault, C., 1986. The receptor for IgE on blood platelets. European Journal of Immunology 16, 306–312. Knight, J.A., 1999. In: Free Radicals Antioxidants Aging and Disease. Free Radicals: Their Presence in Biological Systems. AACC press, Washington, pp. 21–42. Kojima, S., Yamaoka, K., 1999. Adaptive responses of living cells towards low doses of radiation; induction of endogenous antioxidant systems and its applicable possibility for treatment of diseases. Journal of Health Sciences 45, 133–143. Laaksonen, D.E., Atalay, M., Niskanen, L., Uusitupa, M., Hanninen, O., Sen, C.K., 1999. Blood glutathione homeostasis as a determinant of resting and exercise induced oxidative stress in young men. Redox Report 4, 53–59. LoVerde, P.T., 1998. Do antioxidants play a role in schistosome host– parasite interactions? Parasitology Today 14, 284–289. Meyrowitsch, D.W., Simonsen, P.E., 1998. Long term effect of mass diethylcarbamazine chemotherapy on bancroftian filariasis:results at four years after start of treatment. Transactions of the Royal Society of Tropical Medicine and Hygiene 92, 98–103. Noroes, J., Dreyer, G., Santos, A., Mendes, V.G., Medeiros, Z., Addis, D., 1997. Assessment of the efficacy of diethyl carbamazine on adult Wuchereria bancrofti in vivo. Transactions of the Royal Society of Tropical Medicine and Hygiene 91, 78–81. Ottesen, E.A., 1985. Efficacy of diethylcarbamazine in eradicating infection with lymphatic-dwelling filariae in humans. Reviews of Infectious Diseases 7, 341–356. Sahnoun, Z., Jamoussi, K., Zeghal, K.M., 1997. Free radicals and anti-oxidants: human physiology, pathology and therapeutic aspects. Therapie 52, 251–270. Simonsen, P.E., Meyrowitsch, D.W., Makunde, W.H., Magnussen, P., 1995. Selective diethylcarbamazine chemotherapy for control of bancroftian filariasis in two communities of Tanzania: compared efficacy of a standard dose treatment and two semi-annual single dose treatments. American Journal of Tropical Medicine and Hygiene 53, 267–272. Taylor, M.J., Cross, H.F., Mohammed, A.A., Trees, A.J., Bianco, A.E., 1996. Susceptibility of Brugia malayi and Onchocerca lienalis microfilariae to nitric oxide and hydrogen peroxide in cell free
88
R. Premaratna et al. / Experimental Parasitology 102 (2002) 81–88
culture and from INFc activated macrophages. Parasitology 112, 315–322. Wakelin, D., 1996. In: Immunity to Parasites—How Parasitic Infections are Controlled. Nematodes that Invade Tissues. Cambridge University press, Cambridge, pp. 146–162. Weil, G.J., Lammie, P.J., Weiss, N., 1997. The ICT filariasis test: a rapid-format antigen test for diagnosis of bancroftian filariasis. Parasitology Today 13, 401–404. Weil, G.J., Sethumadhavan, K.V.P., Santhanam, S., Jain, D.C., Ghosh, T.K., 1988. Persistance of parasite antigenemia following diethylcarbamazine therapy of bancroftian filariosis. American Journal of Tropical Medicine and Hygiene 38, 589–595.
WHO, 1992. Lymphatic filariasis: the disease and its control: Fifth Report of the WHO Expert Committee on Filariasis. World Health Organization, Geneva, Technical Report Series, No. 821. WHO, 1999. Lymphatic filariasis: reasons for hope. Division of Control of Tropical Diseases, WHO, Geneva.
Further reading Central Bank of Ceylon, 1984. Report on consumer finances and socioeconomic survey 1981/82, Sri Lanka, Part 1, Appendix 1.