Erythrocyte antioxidative enzyme activities and ... - Semantic Scholar

1 downloads 0 Views 4MB Size Report
Summary: Although cutaneous leishmaniasis (CL) is a local infection, the cellular immune response against the disease is systemic, and reactive oxygen ...
Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/2003103277

ERYTHROCYTE A N T I O X I D A T I V E IN

E N Z Y M E ACTIVITIES A N D LIPID P E R O X I D A T I O N

PATIENTS W I T H

CUTANEOUS

LEVELS

LEISHMANIASIS

K O C Y I G I T A.*, GUREL M.** & ULUKANLIGIL M.***

Summary: Although cutaneous leishmaniasis (CL) is a local infection, the cellular immune response against the disease is systemic, and reactive oxygen intermediates (ROis) are an important part of cellular immune response involved in killing the parasite. However, whether these intermediates cause oxidative damage in host is unknown. In this study, the metabolism of ROIs were investigated in patients with CL, and compared with healthy subjects. Erythrocyte lipid peroxidation was determined, as an index of oxidative damage, by measurement of malondialdehyde (MDA) levels. Erythrocyte MDA levels and SOD activities were found to be significantly higher in CL patients than in control subjects (p < 0.01 and p < 0 . 0 1 , respectively). However, CAT and GSHPx activities were significantly lower in theCLgroup (p < 0 . 0 5 and p < 0 . 0 1 , respectively). There was a tendency to decreased hemoglobin (Hb) levels, but values did not reach statistical significance. These findings suggest that changes in antioxidant enzyme activities may amplify the leishmanicidal effect in patients with CL However, these changes may not only cause the killing of parasite but also may cause oxidative damage in the other cells or tissues. KEY WORDS : cutaneous leishmaniasis, lipid peroxidation, antioxidative enzymes.

Résumé : ACTIVITÉ ENZYMATIQUE ANTIOXYDANTE DES ÉRYTHROCYTES ET DEGRÉ DE PEROXYDATION DES LIPIDES DE PATIENTS ATTEINTS DE LEISHMANIOSE CUTANÉE

Bien que la leishmaniose cutanée (LC) soit une infection locale, il existe une réponse immunitaire systémique cellulaire, et des intermédiaires réagissant avec l'oxygène représentent une part importante de cette réponse visant à détruire le parasite. Cependant, la possibilité de lésions oxydatives liées à ces intermédiaires chez l'hôte n'est pas élucidée. Leur métabolisme a été étudié chez des patients atteints de LC et comparé à celui de sujets sains. La peroxydation des lipides des érythrocytes a été déterminée, comme index de lésion oxydative, par la mesure du niveau de malondialdéhyde (MDA). Le niveau de MDA et l'activité superoxyde dismutase erythrocytaire ont été trouvés significativement plus élevés chez les porteurs de LC que chez les sujets témoins (p < 0,01 et p < 0,01, respectivement). Cependant, les activités catalase et glutathion peroxydase sont significativement plus basses dans le groupe LC (p < 0,05 et p < 0,01, respectivement). Il y a une tendance (non significative) à la baisse du taux d'hémoglobine. Ces résultats suggèrent que des modifications de l'activité enzymatique antioxydante pourraient amplifier la réponse immunitaire des porteurs de LC contre le parasite. Cependant, ces modifications pourraient également causer des lésions oxydatives chez d'autres cellules et tissus de l'hôte. MOTS CLÉS : leishmaniose cutanée, peroxydation de lipides, enzyme antioxidant.

C

u t a n e o u s leishmaniasis (CL) is c a u s e d b y the

T h e m e c h a n i s m ( s ) by w h i c h d e f e n s e cells kill microor­

protozoan

ganisms

parasite o f the Leishmania

genus.

has been

t h e subject

o f intense

research.

T h e s p e c t r u m o f disease varies from a s p o n t a ­

Although, it is n o w apparent that nitric o x i d e ( N O ) has

n e o u s l y healing lesion to c h r o n i c a n d multilating cuta­

an important role, reactive oxygen intermediates (ROIs)

n e o u s and m u c o c u t a n e o u s ulcer a n d , rarely a c h r o n i c

have also an important role in parasite killing (Assruey

diffuse c u t a n e o u s disease in w h i c h i m m u n e system is

et al.,

implicated in the p a t h o g e n e s i s o f CL (Raziuddin et al.,

that organisms p r o d u c e ROIs such as hydrogen per­

1 9 9 4 ) . According

oxide ( H 0 ) ,

to the World Health

Organisation

1994). Numerous

2

2

superoxide

studies (O ) 2

have

demonstrated

and hydroxyl ( O H )

( W H O ) , various forms o f the disease currently affect

radicals as a host d e f e n s e strategies to kill intracellular

12 million p e o p l e in 8 8 countries, with an i n c i d e n c e

and extracellular parasites (Li et al.,

o f 1-1.5 million n e w c a s e s p e r year.

In healthy organisms, there is a delicate balance a m o n g

1992).

oxidants a n d antioxidants such as s u p e r o x i d e dismu* Department of Biochemistry. ** Department of Dermatology. *** Department of Microbiology. Medical Faculty, Harran University, Sanliurfa, Turkey. Correspondence: Abdurrahim Kocyigit, MD. Harran University, Medical Faculty. Department of Clinical Biochemistry. 63200 San­ liurfa, Turkey. Fax: +90-414-3151181 - E-mail: [email protected]

tase

(SOD),

catalase

(CAT), glutathion

( G S H - P x ) . vitamins E, C a n d A. U n d e r

peroxidase pathological

conditions t h e b a l a n c e m a y b e tilted toward oxidative side, with the e n d result b e i n g uncontrolled oxidative d a m a g e (Mc Cord, 1 9 9 3 ) . Recently, enzyme

w e have

demonstrated

activities c h a n g e

that

antioxidative

in patients with

CL. p r o -

277

bably as host d e f e n s e (Erel et al., 1 9 9 9 ) . However, w e have not determined whether these changes cause oxi­ dative damage in these patients. In this study, the c h a n g e s of erythrocyte antioxidant e n z y m e activities and the levels o f lipid peroxidation, as an index o f oxi­ dative damage, w e r e evaluated in patients with CL.

MATERIALS A N D METHODS

T

he study was conducted at Harrankapi Leish­ mania Treatment Center in Sanliurfa, which is a h y p e r e n d e m i c area for leishmaniasis in sou­ theastern Anatolia in T u r k e y (Gurel et al., 2 0 0 2 ) . 32 patients with localized CL, 19 man and 13 w o m e n , aged 10 to 34 years ( 2 8 . 2 +- 4 . 3 ) w e r e enrolled in the study. Admission criteria for the patient group w e r e as follows: no pregnancy and no prior intralesional or sys­ temic antimonial therapy or n o bacterial infection. All patients had nodular or noduloulcerative leishmanial lesions. Patients with lesions o f six months or greater duration w e r e e x c l u d e d from the study b e c a u s e of the possibility o f s p o n t a n e o u s healing and immunity. T h e clinical diagnosis was confirmed by laboratory demonstration o f the parasite in the lesions by direct smears. Lesions were cleaned with ethanol, and punc­ tured at the margins o f the lesions with a sterile lancet. Biopsy material was smeared, dried in air and fixed by methanol. Parasitologic diagnosis was based on visua­ lisation of parasites in Giemsa-stained smears, prepared with material aspirated from borders of skin lesions and from tissue imprints from biopsy. In order confirm the diagnosis, the materials were also cultured Novy-MacNeal-Nicolle (NNN) medium f o r up t o three w e e k s t o detect leishmanial promastigotes. The parasites were classified by izoenzyme analyses by using starch gel elec­ trophoresis techniques (Gramicia et al. 1984). In all cases the strains corresponded to the Leishmania tropica. A t o t a l o f 3 0 h e a l t h y v o l u n t e e r s , 17 m a n a n d 13 w o m e n , aged 11 to 33 years (27.3 ± 4.5 years), served as controls from in the s a m e area. None had received any systemic disease and n o n e had received either mineral or vitamin drugs.

were destroyed by the addition of four volumes o f cold redistilled water in order to measure antioxidative enzyme, SOD, GSH-Px and CAT activities. Suspension was centrifuged twice to obtain erythrocyte membranes and hemolysate: first for 10 min in a tube centrifuge at 1,500 x g at 4 ° C , and then in an eppendorf centrifuge at 5,000 x g for five min at 4° C. Clear supernatant was obtained as hemolysate. Remaining erythrocyte mem­ brane was used to measure MDA contend. Blood h e m o ­ globin (Hb) levels and. erythrocyte counts were mea­ sured by automatic blood analyzer (Coulter, Stakes, USA) and hemolysate hemoglobin ( H b ) levels w e r e measured by the cyanmethemoglobin method (Von Kompen & Ziljlstra, 1961). All enzyme activities were measured from the hemolysate. Hemolysate antioxidant SOD and GSH-Px enzyme activities were measured with commercially available kits (Randox, Lab. Ltd. Ireland Cat. No. SDI 25 and RS505 respectively). DETERMINATION OF ERYTHROCYTE SUPEROXYDE DISMUTASE ACTIVITY Erythrocyte SOD activity was determined with a Randox test combination. Xanthine and xanthine oxidase w e r e used to generate superoxide radicals reacting with 2-(4iodophenyl) 3-(4-nitrophenol) - 5-phenyl tetrazolium chloride (INT) to form a red formazan dye (McCord & Fridovich, 1 9 6 9 ) . Superoxide dismutase inhibits this reaction by converting the superoxide radical to oxygen. Superoxide dismutase activity was measured at 505 nm on a cessil 3000 spectrometer in hemolysates o f washed erythrocytes. Results were expressed in S O D U/g Hb. DETERMINATION OF ERYTHROCYTE GLUTATHION PEROXIDASE ACTIVITY Measurement of GSH-Px activity was based on the fol­ lowing principle: GSH-Px catalyses the oxidation o f glu­ tathione by c u m e n e hydroperoxide (Paglia & Valentine, 1967). In the presence of glutathione reductase and nico­ tinamide adenine dinucleotide phosphate (NADPH) the oxidized glutathione is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP . The decrease in absorbance at 340 nm is mea­ sured. Erythrocyte GSH-Px activity was expressed as U/g Hb. The enzyme unit of GSH-Px ( U ) is defined as the number of mmoles o f reduced NADPH oxidized per minute at 37° C by 1 g of Hb under standard assay condi­ tions. Measurements were performed by automatic ana­ lyzer (Hitachi 9 1 1 , Boehringer Mannheim, Germany) according to the Randox application procedure. +

SAMPLE PREPARATION In total, 10 ml of heparinized venous blood was drawn after overnight fasting and mixed with 10 ml of Dextran T 500 6 % w/v in saline 0.9 % w/v in a 25 ml tube and kept at room temperature for 30 min before the leuko­ cyte-rich plasma layer was removed. After the leukocyterich plasma was discharged, the remaining erythrocytes were washed repeatedly with an isotonic solution of NaCl (0.9 % ) until a colorless supernatant was observed. Ery­ throcyte membrane malondialdehyde (MDA) levels were measured, as a marker of lipid peroxidation, in 100 µl of packed erythrocytes. 500 µl of packed erythrocytes

278

DETERMINATION OF ERYTHROCYTE CATALASE ACTIVITY CAT activity in erythrocyte w a s assayed by a method described by Goth

(Goth, 1 9 9 1 ) . O f the hemolysate

0.2 ml erythrocyte hemolysate was incubated in 1.0 ml substrate (65 mmol per H 0 in 60 mmol/L sodiumpotassium phosphate buffer, pH 7.4) at 37° C for 6 0 s. T h e enzymatic reaction was stopped with 1.0 ml o f 32.4 mmol/l a m m o n i u m molybdate ((NH ) M o 0 . 4 H 0 ) and the yellow c o m p l e x o f molybdate and H 0 , was measured at 405 nm against blank 3. Erythrocyte CAT activity is linear up to 100 kU/L. If the CAT activity e x c e e d e d 100 kU/L the serum w a s diluted with the phosphate buffer (two to 10 fold) and the assay was repeated. O n e unit CAT d e c o m p o s e s 1 mmol o f H 0 / L min under these conditions. 2

2

4

7

2 4

6

2

2

2

CAT

2

(kU/gHb) =

A(sample) - A(blank 1) A(blank 2) - A(blank 3)

x 271

lesions was 3,4 ± 1 . 2 months. As s e e n in the table II, erythrocyte MDA levels and erythrocyte S O D activities w e r e significantly higher in the patient group than in healthy subjects (p < 0.01 and p < 0.01, respectively). However, GSH-Px and CAT activities were found to b e significantly lower in the patient group than in the controls (p < 0.01 and p < 0.05, respectively). There was a tendency to decreased Hb levels (Fig. 1) and erythrocyte counts in patients group, h o w e v e r values did not reach statistical significance (p > 0 . 0 5 ) . There was a w e a k inverse relationship b e t w e e n erythrocyte MDA levels and Hb content in the erythrocytes of CL patients (r = - 0.389. p < 0 . 0 5 ) . However, erythrocyte MDA levels did not correlate with any o f the antioxi­ dant e n z y m e levels.

Blank 1 contained 1.0 ml substrate, 1.0 ml molybdate and 0.2 ml hemolysate: blank 2 contained 1.0 ml substrate, 1.0 ml molybdate and 0.2 ml buffer: blank 3 contained 1.0 ml buffer, 1.0 ml molybdate and 0.2 ml buffer.

DETERMINATION OF ERYTHROCYTE MALONDIALDEHYDE LEVELS Erythrocyte m e m b r a n e MDA levels w e r e measured in terms o f thiobarbituric acid ( T B A ) reactive substances. MDA. an e n d product of fatty acid peroxidation, can react with T B A to form a colored c o m p l e x that has a m a x i m u m a b s o r b a n c e at 532 nm (Jain et al, 1990).

STATISTIC T h e m e a n values obtained in the different groups w e r e c o m p a r e d by Student's t test. Pearson's correla­ tion test was used to evaluate the relationship b e t w e e n the two variables. All statistical analyses w e r e per­ formed with the program Statistical Package for the Social Sciences (SPSS) for Windows, version 7.5. All results w e r e e x p r e s s e d as mean values ± SE; signifi­ c a n c e w a s defined as p < 0.05.

RESULTS A N D DISCUSSION

T

he CL patients and matched controls had similar ranges in terms of age, height, body weight, and b o d y mass index ( B M I ) ( T a b l e I ) . Onset o f

Patients n = 32 Mean ± SD Sex ( F/M) Age (year) Height (cm) Weight (kg) BMI (kg/m-)

19/13 28.2 ± 4.3 169 ± 6 64.1 ± 8.9 23.4 ± 3.6

Healthy Subjects n - 30 Mean ± SD 17/13 27.3 ± 4.5 167 ± 5 65 ± 12.4 24.7 ± 4.4

p > > > >

0.05 0.05 0.05 0.05

Table I - Physical characteristics of CL patients and healthy subjects.

Fig. 1. - Blood hemoglobin (Hb) levels of patient and control groups (mean ± SD).

Although CL is a local infection, the host generates s o m e systemic immune defense m e c h a n i s m ( s ) against CL infection. It has b e e n demonstrated that immune mediators such as interferon-gamma (IFN-y) and tumor necrosis factor-alpha ( T N F - α ) produced by the host against Leishmania infection, regulate the metabolisms o f nitric oxide ( N O ) and ROIs (Liew et a l . , 1990; Liew & O ' D o n n e l , 1993). Owing to the fact that the effects o f cytokines in the circulation are systemic, other cells are likely to b e affected. Erythrocytes abundantly contain S O D , CAT and GSH-Px enzymes and they reflect alterations in the activities o f these enzymes well. T h e erythrocyte m e m b r a n e is also sensitive to lipid peroxidation b e c a u s e it contains unsaturated fatty acids(Van Asbeck et al., 1985). Therefore, w e measured the c h a n g e s of these antioxidant enzyme activities, Hb levels and erythroyte counts, and the levels of lipid per­ oxidation in erythrocytes to evaluate ROIs and oxida­ tive damage in patients with CL. Previous studies have demonstrated that ROI can kill intracellular and extracellular Leishmania parasite (Ham-

279

C o n t r o l Group n - 30

Patient Group n - 32

Parameters

4.16 ± 425.4 ± 49.2 ± 143.22 ± 12.98 ± 3.97 ±

8.68 ± 1.44

Erythrocyte pellet MDA, nmol MDA/Hb Erythrocyte SOD, U/g Hb Erythrocyte GSH-Px, U/g Hb Erythrocyte CAT, kU/gHb Hemoglobin g/dL Erythrocyte (x 10 )

570.9 31.5 8 115.70 11.32 3.48

6

± ± ± ± ±

23.76 3.60 10.22 2.70 0.35

p < < < < > >

1.161 24.26 4.74 9.97 2.57 0.34

a

0.01 0.01 0.01 0.05 0.05 0.05

Values are given Mean ± SD Table II. - Comparative analysis of lipid peroxidation of - as assessed by MDA content - and the monitoring of three anti-oxidant enzymes in erythrocytes recovered from either healthy subjects or patients with active cutaneous leishmaniasis.

moda et al, 1996; Clark et al, 1986). Specially H 0 , which is a product of the macrophage respiratory burst, is the major killing mechanism of microorganisms (Li et al., L992). An antioxidant enzyme, S O D , converts superoxide radicals into H 0 . O n the other hand, CAT and GSH-Px convert H 0 into H 0 and 0 GSH-Px also plays a role in the detoxification of intracellular h y d r o p e r o x i d e s . T h e r e f o r e , i n c r e a s e d S O D activity results in increased conversion of the superoxide anion into H 0 . At the same time, decrease in GSH-Px and CAT activities decrease the degradation o f H 0 , so that it remains in the medium for a long time at a high concentration in their in vitro study (Rockett et al., 1991). Studies have also demonstrated that S O D can effectively e n h a n c e vascular relaxation and the half-life o f NO (Gryglewski et al., 1986; Assreuy et al., 1 9 9 4 ) . On the contrary, it was reported that CAT inhibits m a c r o p h a g e leishmanicidal activity and this correlates with a reduction in NO synthesis (Li et al, 1992). T h e ­ refore, increased S O D activity, and decreased CAT and GSH-Px activities may cause to the elevated production o f NO and H , 0 via two independent pathways to kill parasite. Anyway, it was demonstrated that the addi­ tion o f S O D to m a c r o p h a g e s e n h a n c e d leishmanicidal activity (Assreuy et al, 1 9 9 4 ) . 2

2

2

2

2

2

2

2

2

2

2

2

2

T h e differences o f antioxidant e n z y m e activities in the CL patient may b e a d e f e n s e strategy o f the organism induced b y cytokines, such as IL-1, IL-2 and T N F - a . It was demonstrated that S O D activity was significantly induced by IL-1 and T N F - a (Rubany & Vanhoutte, 1986), and GSH-Px activity induced b y IL-2r (Reimund et al, 2 0 0 0 ) . Although, w e could not determine these immunocytokins, s o m e in vivo and in vitro observa­ tions s h o w n that the production o f IL-1 and T N F - a w e r e also induced b y CL infection (Pirmez et al, 1993; Cillarie et al, 1 9 8 9 ) . Contrary to our findings, S O D activity has b e e n found to b e decreased along with GSH-Px and CAT activities in Leishmania donovani infection (Biswas et al, 1997). This difference in findings between visceral and CL may b e due to a distinct pathology and immune response

280

in these two species o f Leishmania, as IL-1 has also b e e n found to b e lower in Leishmania donovani infec­ tion in contrast to CL infection (Reiner, 1 9 8 7 ) . Stimulation o f lipid peroxidation of red cell m e m b r a n e was evident from the increased levels of MDA in the erythrocytes of CL patients. B e c a u s e , the production o f ROIs to kill microorganism may also c a u s e oxida­ tive d a m a g e in the other cells or tissues such as ery­ throcytes at the s a m e time. Likewise w e found to b e a higher erythrocyte m e m b r a n e MDA levels and a w e a k inverse correlation b e t w e e n and Hb and ery­ throcyte MDA content. Biswas et al ( 1 9 9 7 ) also indi­ cated that there was a severe peroxidative injury in the erythrocytes and an anemia o f indian kala-azar patients, but Leishmania donovani infection is a g e n e ­ ralized systemic infection than CL infection. T o our k n o w l e d g e , this is the first report indicating peroxi­ dative injury in the erythrocytes o f CL patients.

CONCLUSION

A

lthough CL is a local parasitic infection, s o m e systemic antioxidative e n z y m e activities c h a n g e p r o b a b l y as a p a r t o f t h e h o s t d e f e n s e . However, oxidant/antioxidant b a l a n c e may b e tilted towards the oxidative side, with the e n d result being uncontrolled peroxidative d a m a g e .

REFERENCES ASSREUY J . , CUNHA F . Q . , O DONNELL C A . ,

EPPERLEIN M . , NORONHA-DUTRA A . ,

LIEW F . Y .

& MONCADA S . P r o d u c t i o n

of

nitric o x i d e a n d s u p e r o x i d e b y activated m a c r o p h a g e s a n d

killing o f Leishmania major. European nology, 1994, 24, 6 7 2 - 6 7 6 .

Journal

of

Immu-

BISWAS T . , GHOSH D . K . , MUKHERJEE N . & GHOSAL J . Lipid per­ o x i d a t i o n o f erythrocytes in visceral leishmaniasis,

of Parasitology,

fournal

1997, 83, 1 5 1 - 1 5 2 .

CLARK I . A . , HUNT H . N . & COWDEN W . B . O x y g e n - d e r i v e d free

radicals in t h e p a t h o g e n e s i s o f parasitic d i s e a s e .

Advances

RAZIUDDIN S., ABDALLA R.E., EL-AWAD E . H . & AL-JANADI M .

CILLARIE E., DIELI M , MALTASE E., MILANO S., SALERNO A. &; LIEW

duction in visceral a n d c u t a n e o u s leishmaniasis, j o u r n a l

1 9 8 6 , 25, 1 - 4 4 .

in Parasitology, F.Y.

Enhancement

Leishmania

of macrophage

IL-1 p r o d u c t i o n b y

infection in vitro a n d its inhibition b y

major

IFN-g. Journal

Immunoregulatory

1 9 8 9 , 143,

of Immunology,

2001-2005.

EREL O , KOCYIGIT A, BULUT V . ¿4 GUREL M.S. Reactive nitrogen and o x y g e n intermediates in patients with c u t a n e o u s leish­ maniasis. Memorias

do Instuto

Oswaldo

Cruz,

1 9 9 9 , 94,

Diseases,

GOTH L. A s i m p l e m e t h o d for d e t e r m i n a t i o n o f s e r u m catal a s e activity a n d revision 1 9 9 1 , 196,

Chim Acta,

o f reference range.

Clinical

43-52.

GRAMICCIA M, BETTINI S. & YASAROL S. I z o e n z y m e characteri­ zation o f leishmania isolates from h u m a n c a s e s o f cuta­ n e o u s lesihmaniasis in Urfa, south-east T u r k e y .

Transac-

tions of the Royal Society of Tropica! Medicine

Hygiene.

and

1 9 8 4 , 78, 5 6 8 - 5 6 9 . GRYGLEWSKI R.J., PALMER R.M. & MONCADA S. S u p e r o x i d e anion is involved in the b r e a k d o w n o f e n d o t h e l i u m - d e r i v e d vas­ cular relaxing factor. Nature,

1 9 8 6 , 320,

454-456.

GUREL M.S., ULUKAXLIGIL M. & OZBILGE H. C u t a n e o u s leish­ maniasis in Sanliurfa: Epidemiological a n d clinical features o f t h e four years ( 1 9 9 7 - 2 0 0 0 ) . International 2 0 0 2 , 41,

matology,

journal

of Der-

32-37.

HAMMODA N.A.. NEGM A.Y., HUSSEIN E . D . , EL-TEMSAMY M.M. & RASHWAN E.A. Leishmania

major,

leishmanicidal activity o f

activated m a c r o p h a g e s b y interferon g a m m a a n d t u m o r n e c r o s i s factor alpha, fournal 1 9 9 6 , 26,

of Egyptian

Society of

Para-

1 9 9 4 , 170,

cytokine

pro­

1037-1040.

REIMUND J . M . , HIRTH C , KOEHL C , BAUMMAN R. & DUCLOS B . Antioxidant a n d i m m u n e status in active Crohn's d i s e a s e . A p o s s i b l e relationship. Clinical

Nutritien,

2 0 0 0 , 19, 4 3 -

48. REINER N.E. Parasite a c c e s s o r y cell interactions in murine leishmaniasis.

179-183.

sitology,

of Infectious

and proinflammatory

I. E v a s i o n a n d s t i m u l u s - d e p e n d e n t

p r e s s i o n o f the m a c r o p h a g e

interleukin

Leishmania

of Immunology,

donovani.

Journal

sup­

1 response by 1 9 8 7 , 138,

1919-1925. ROCKETT K.A., AWBURN M . M . , COWDEN W . B . & CLARK I.A. Killing o f Plasmodium derivatives. Infect

falciparum

Immun,

in vitro b y nitric o x i d e

1991. 59. 3280-3283.

RUBANYI G . M . & VANHOUTTE P . M . O x y g e n - d e r i v e d f r e e radi­ cals, e n d o t h e l i u m , a n d r e s p o n s i v e n e s s o f vascular s m o o t h m u s c l e . American

journal

of Physiology,

1 9 8 6 , 250, 8 1 5 -

821. VAN ASBECK B . S . , HOIDAL J . , VERCELLOTTI G . M . , SCHWARTZ B . A . , MOLDOW C F . & ACOB H . S . P r o t e c t i o n against lethal hypero x i a b y tracheal insufflation o f erythrocytes: role o f r e d cell glutathione. Science,

1 9 8 5 , 227,

756-759.

VON KOMPEN E.J. & ZILJLSTRA W . G . Standardization o f h e m o globinometry. II. T h e h e m o g l o b i n c y a n i d e method. Chim Acta.

Clinical

1961. 6, 538-544. Reçu le 2 2 o c t o b r e 2 0 0 2 A c c e p t é le 9 mai 2 0 0 3

555-566.

JAIN S . K . , LEYINE S.N., DUETT J . & HOLLIER B . Elevated lipid p e r o x i d a t i o n levels in red b l o o d cells o f s t r e p t o z o t o c i n treated d i a b e t i c rats. Metabolism.

1 9 9 0 . 39,

971-975.

LI Y . , SEXTRN A., ROGERS M.V., PALMER R.M., MONCADA S. & LIEW F . Y . Catalase inhibits nitric o x i d e synthesis a n d the killing o f intracellular Leishmania European

fournal

major

in murine m a c r o p h a g e s .

of Immunology,

1 9 9 2 , 22, 4 4 1 - 4 4 6 .

LIEW F . Y . , PARKINSON C , MILLOR S., SENTRN A. ik CARRIER M. T u m o r n e c r o s i s factor ( T N F - a ) in leishmaniasis

1.TNF-a

m e d i a t e s host protection against c u t a n e o u s leishmaniasis. Immunology,

1 9 9 0 , 69, 5 7 0 - 5 7 3 .

LIEW F . Y . & O'DONNEL C.A. I m m u n o l o g y o f leishmaniasis. Advances

in Parasitology,

1 9 9 3 , 32, 162-222.

MCCORD J . M . & FRIDOVICH I. S u p e r o x i d e dismutase. An enzy­ matic function for e r y t h r o c u p r e i n ( h e m o c u p r e i n ) . of Biological

Chemistry,

MCCORD J . M . H u m a n

1 9 6 9 , 244,

fournal

6049-6055.

d i s e a s e , free radicals, a n d t h e oxi­

dant/antioxidant b a l a n c e . Clinical

Biochemistry,

1 9 9 3 , 26,

351-357. PAGLIA D . E . & VALENTINE W.N. Studies o n the quantitative a n d qualitative characterization o f erythrocyte glutathione p e r ­ o x i d a s e . Journal

of Laboratory

Clinical Medicine,

1 9 6 7 , 70,

158-169. PIRMEZ C , YAMAMURA M., UYEMURA K . , PAES-OLIVEIRA M., CONCEICAO-SILVA F. & MODLIN R.L. Cytokine patterns in t h e p a t h o g e n e s i s o f h u m a n leishmaniasis, j o u r n a l of Investigations.

1 9 9 3 , 91.

Clinical

1390-1395.

281