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ARTIGO/ARTICLE
Revista da So ciedade Br asileir a de Medicina Tr o pical 3 7 ( 2 ) :1 6 5 - 1 6 8 , mar- abr, 2 0 0 4
Evaluation of the phagocytic activity of per ipher al blood monocytes
of patients with Jor ge Lobo’s disease
Avaliação da atividade fagocítica dos monócitos do sangue periférico
de pacientes com doença de Jorge Lobo
Fátima Regina Vilani- Mo r eno1
, Luciana Mo r eir a Silva1
and Dilto r Vladimir Ar aújo Opr o mo lla2
ABSTRACT
Stu di e s o n ho st- pa ra si te i n te ra c ti o n i n Jo rge Lo b o ’s di se a se a re sc a rc e , wi th n o re po rt i n the li te ra tu re o n the pha go c yto si s o f Lac azia loboi b y pha go c ytic m o n o n u c le a r c e lls. Thu s, the o b je c tive o f the pre se n t stu dy wa s to a sse ss the pha go c ytic a c tivity o f b lo o d m o n o c yte s i n the pre se n c e o f L. lo b o i i n pa ti e n ts wi th the di se a se a n d i n he a lthy su b je c ts ( c o n tro ls) o ve r 3 a n d 24 h o u rs o f i n c u b a ti o n . Sta ti sti c a l a n a lyse s o f th e re su lts sh o we d n o si gn i f i c a n t d i f f e re n c e i n p e rc e n t p h a go c yto si s o f th e fu n gu s b e twe e n pa tie n t a n d c o n tro l m o n o c yte s. With re spe c t to in c u b a tio n tim e , ho we ve r, the re wa s a sign ific a n t diffe re n c e , i n th a t p e rc e n t p h a go c yto si s wa s h i gh e r a t 3 h o u rs th a n a t 2 4 h o u rs ( p < 0 .0 1 ) . Th e se re su lts su gge st th a t m o n o c yte s f ro m p a ti e n ts wi th th e m yc o si s a re a b le to p h a go c yte th e f u n gu s, a s a lso o b se rve d i n c o n tro l i n d i vi d u a ls.
Ke y-words: Jo rge Lo b o ’s di se a se . Pha go c yto si s. Mo n o c yte . Lac azia lo b o i.
RESUMO
Os e stu d o s e n vo lve n d o a i n te ra ç ã o h o sp e d e i ro - p a ra si ta n a d o e n ç a d e Jo rge Lo b o sã o e sc a sso s; a té o m o m e n to , n ã o e x i ste n e n hu m tra b a lho a b o rda n do a fa go c ito se do fu n go La c a zia lo b o i pe la s c é lu la s m o n o n u c le a re s fa go c itá ria s. Assim , o pre se n te e stu d o te ve c o m o o b je ti vo a va li a r a a ti vi d a d e f a go c í ti c a d o s m o n ó c i to s sa n gü í n e o s f re n te a o L. lo b o i ta n to e m p a c i e n te s p o rta d o re s d a d o e n ç a c o m o e m i n d i ví d u o s sa d i o s ( gru p o c o n tro le ) , u ti li za n d o 3 e 2 4 h o ra s d e i n c u b a ç ã o . A a n á li se e sta tí sti c a d o s re su lta d o s re ve lo u q u e n ã o h o u ve d i f e re n ç a si gn i f i c a n te e n tre o p e rc e n tu a l d e f a go c i to se d o f u n go p e lo s m o n ó c i to s de pa c i e n te s e do gru po c o n tro le , po ré m e m re la ç ã o a o s te m po s de i n c u b a ç ã o , ho u ve di f e re n ç a si gn i f i c a n te , i sto é , à s 3 h o ra s o p e rc e n tu a l d e f a go c i to se f o i m a i o r q u e o o b ti d o à s 2 4 h o ra s ( p < 0 ,0 1 ) . Este s re su lta d o s su ge re m q u e m o n ó c ito s de pa c ie n te s po rta do re s da m ic o se sã o há b e is e m fa go c ita r o fu n go , à se m e lha n ç a do q u e fo i visto e m in divídu o s do gru po c o n tro le .
Palavr as-chave s: Do e n ç a de Jo rge Lo b o . Fa go c i to se . Mo n ó c i to . Lac azia lo bo i.
1 . Eq uipe Té c nic a de Imuno lo gia do Instituto Laur o de So uza Lima, B aur u, SP, B r asil. 2 . Divisão de Pe sq uisa e Ensino do Instituto Laur o de So uza Lima, B aur u, SP, B r asil.
This r e se ar c h has b e e n appr o ve d b y the Ethic al Co mmitte e o f the Instituto Laur o de So uza Lima, pr o c e ss 2 1 /0 0 .
Addr e ss to: Dr ª Fátima Re gina Vilani Mo r e no . Eq uipe Té c nic a de Imuno lo gia do Instituto Laur o de So uza Lima. Ro d. Co mte . J o ão Rib e ir o de B ar r o s, k m 2 2 5 /2 2 6 , 1 7 0 3 4 - 9 7 1 B aur u, SP, B r asil.
Fax: 1 4 3 1 0 3 - 5 9 1 4 , Te l: 1 4 3 1 0 3 - 5 8 7 2 e -mail: imuno lo gia@ ilsl.b r
Re c e b ido par a pub lic aç ão e m 3 /4 /2 0 0 3 Ac e ito e m 1 6 /2 /2 0 0 4
Jo rge Lo bo ’s disease is a c utaneo us-subc utaneo us myc o sis with a c hr o nic e vo lutio n c ause d b y the fungus La c a zi a lo b o i
(L. lo b o i)8 1 8. The myc o sis pr edo minantly o c c ur s in B r azil b ut c ase s have b e e n r e po r te d, in de c r e asing o r de r, in Co lo mb ia, Surinam, Frenc h Guyana, Venezuela, Panama, Costa Ric a, Peru, Ec uador, Bolivia, Mexic o, and Europe1 2. Ac c ording to Opromolla
e t a l1 1, the estimated number of c ases of the disease is 4 5 8 , 2 9 5
and those that occurred in Brazil ( 2 9 5 cases) were predominantly in the Amazon region.
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Vila ni- Mo r e no FR e t a l
individuals, with no evidenc e of greater susc eptibility to infec tion in a given ethnic gr o up7.
Clinic ally, the myc osis is c harac terized by c utaneous lesions of with a c heloid-like, wart-like infiltrative, ulc erous or gummy aspec t, with the po ssib le o c c ur r enc e o f mo r e than o ne type o f le sio n in the same patie nt1 6. Ulc e r ate d le sio ns se e m to b e a result o f traumatic inj uries mo re than to primary spo ntaneo us ulc eratio n and therefo re are c linic ally sec o ndary1 1.
Histo patho lo gic ally, Jo r ge Lo b o ’s dise ase is c har ac te r ize d b y an inte nse diffuse histio c ytic r e ac tio n c o ntaining lar ge numb e r s o f fo r e ign b o dy giant c e lls and Langhans’ c e lls, and nume r o us intr a o r e xtr ac e llular par asite s. Small numb e r s o f lympho c ytes, plasmo c ytes and neutro phils are also present7 1 0. Ultrastruc tural studies of the c utaneous lesions have revealed that, after the fungus is endoc ytosed by the phagoc ytic c ell and suffers the ac tion of lysosomal enzymes, remnants of the c ell wall o f L. lo b o i c an be detec ted in the c ytoplasm. These c ell wall remnants progressively ac c umulate inside the mac rophages, c onferring a foamy aspec t, as observed by light mic rosc opy1 4 1 5. Mac rophages are c ells c ommonly assoc iated with the defense mec hanisms of the host against invading mic roorganisms. They belo ng to the mo no nuc lear phago c ytic system and, in additio n to defending the ho st thr o ugh phago c yto sis and c ell digestio n, they also interac t with and stimulate lymphoc ytes to partic ipate in the de fe nse me c hanisms, thus playing a fundame ntal r o le bo th in no nspec ific mec hanisms o f defense and in the spec ific immune respo nse1.
Fe w studie s o n the ho st-par asite inte r ac tio n ar e availab le fo r J o r ge Lo b o ’s dis e a s e a n d n o s tudy e x is ts a b o ut th e phago c yto sis o f L. lo b o i b y mo no nuc le ar phago c ytic c e lls. Ho wever, this to pic has been studied in o ther myc o ses suc h as parac oc c idioidomyc osis3 4 and c andidiasis1 3, demonstrating the impo r tant r o le o f mac r o phages in ho st defense. Thus, the aim of the present study was to assess the phagoc ytosis of the fungus
L. lo b o i b y pe r iphe r al b lo o d mo no c yte o f patie nts with Jo r ge Lo bo ’s disease.
PATIENTS AND METHODS
Patients. The study was conducted on 1 0 patients with Jorge Lobo’s disease from the State of Acre and examined by the clinical staff of the Lauro de Souza Lima Institute, in Bauru-SP, with a diagnosis of the mycosis confirmed by anatomicopathological examination. Fifteen healthy adults, employees of the Lauro de Souza Lima Institute, with similar age to that of the patients, were used as control group.
Blood collection. Twenty ml of venous blood was c ollec ted fr o m e ac h patie nt and c o ntr o l sub j e c t with 2 Vac utaine r ( B D, Mi n a s Ge r a i s , B r a zi l ) tu b e s c o n ta i n i n g h e p a r i n a s a n antic oagulant.
Mo no nuc le a r c e ll pr e pa r a tio n. Mo no nuc le ar c e lls we r e se par ate d o n a Fic o ll-Hypaque de nsity gr adie nt2 ( Sigma, Misso ur i, USA) . The c e lls we r e r e suspe nde d in 1 m l RPMI m e dium c o ntaining L- glutam ine and 1 5 m M HEPES b uffe r ( Gib c o B RL, São Paulo , B r azil) supple me nte d with pe nic illin
( 1 0 0 UI/ml) -streptomyc in ( 1 0 0
µ
g/ml) ( Gibc o BRL) . Monoc ytes were c ounted in a Neubauer c hamber by 1 :8 dilution of the c ell suspensio n with 0 .0 2 % neutr al r ed dye in saline so lutio n. The pr e par atio n was le ft to stand at 3 7oC fo r 2 0 min and the final c o nc e ntr atio n o f the suspe nsio n was adj uste d to 5 x 1 05 monoc ytes/ml.Monocyte cultur e. One ml of the monoc yte suspension was distributed in duplic ate among the wells of flat-bottomed 2 4 -well tissue c ulture plates ( Corning, New York, USA) c ontaining a previously sterilized c overslip measuring 1 3 mm in diameter ( Glasstéc nic a, São Paulo, Brazil) . After 2 hours, the supernatants were removed and eac h well was washed with RPMI medium to r e m o ve n o n - a dh e r e n t c e lls . Ne xt, 1 m l o f RPMI m e dium supplemented with 1 5 % fresh AB+ pooled serum ( obtained by combining sera from 5 healthy donors under conditions preserving complement activity) and with antibiotics was added. A 100ml aliquot of the L. lo bo i suspension containing 5 x 1 05 fungi at the proportion of 1 c ell for 1 fungus was added to eac h well.
The La c a zi a lo b o i suspe nsio n was o b taine d fr o m sk in le sio ns pr e vio usly c o lle c te d b y sur gic al r e mo val and sto r e d fr o ze n. The b io psie s we r e pr o c e sse d in 0 .8 5 % saline so lutio n and the fungal suspensio n o btained was filtered thro ugh gauze fo r the r e mo val o f tissue r e mnants and the n auto c lave d. The fungi pr e se nt in the suspe nsio n we r e c o unte d in a Ne ub aue r c hamber.
The plate was inc ubated at 3 7oC in a 5 % CO
2 atmosphere for 3 and 2 4 hours. After these inc ubation periods, the c overslips were removed, washed with 0 .8 5 % saline and stained with Giemsa.
E va l u a t i o n o f p h a go c yt i c a c t i vi t y. Two h un dr e d monoc ytes were examined in c overslips at 1 0 0 0 x magnific ation fo r enumer atio n o f the c ells c o ntaining fungus o r no t.
Statistical analyses. Two-way analyses of varianc e was used to determine the signific anc e of the differenc e in perc ent fungus phagoc ytosis, c omparing patient and c ontrol monoc ytes1 9. The same analyses was also used to determine the effec t of time on the perc entage of phagoc ytosis regardless of the group studied. In all c irc umstanc es, the level of signific anc e was set at p < 0 .0 1 .
RESULTS
The pe r c e ntage s o f pe r iphe r al b lo o d mo no nuc le ar c e lls phago c yting L. lo b o i fro m patients and c o ntro ls are presented in Table 1 . Statistic al analyses of the results showed no signific ant differenc e in perc ent phagoc ytosis by the monoc ytes of patients and c o ntr o ls. Ho we ve r, the r e was a signific ant diffe r e nc e between the time po ints evaluated, with a higher perc entage o f phago c yto sis at 3 ho ur s than at 2 4 ho ur s ( p < 0 .0 1 ) . Figur e 1 illustrates L. lo b o i phagoc ytosis by peripheral blood monoc ytes fr o m a patient with Jo r ge Lo b o ’s myc o sis.
1 6 7 Revista da So ciedade Br asileir a de Medicina Tr o pical 3 7 :1 6 5 - 1 6 8 , mar- abr, 2 0 0 4
DISCUSSION
The phago c yto sis o f mic ro o rganisms represents o ne o f the no nspe c ific de fe nse me c hanisms o f vital impo r tanc e fo r the host. The monoc yte/mac rophage c ell lines, c ommonly referred to as professional phagoc ytes, c an neutralize, engulf and destroy par tic le s, inc luding infe c tio us age nts, thus pr e se nting a high phago c ytic po te ntial1. In this r e spe c t, the se c e lls have b e e n fr e que ntly e valuate d fo r phago c ytic and lytic c apac ity against pathogenic mic roorganisms.
In the present study, the evaluation of the phagoc ytic c apac ity of blood monoc ytes from patients with Jorge Lobo’s disease revealed that the monocytes of both patients and healthy individuals ( c ontrols) presented a similar perc entage of phagoc ytosis, with no statistic ally signific ant differenc e between them.
A survey of the literature did not loc ate any report c onc erning th e ph a go c yto s is o f L. lo b o i b y m o n o c yte s o f pa tie n ts with the m yc o sis, altho ugh this to pic has b e e n inve stigate d fo r o the r myc o se s. In par ac o c c idio ido myc o sis, a study o n the phagoc ytosis of P. b ra silie n sis by mouse alveolar mac rophages revealed that 6 4 .2 % of the mac rophages phagoc ytized the fungus. When the c ultures were treated with interferon gamma ( INF-
γ
) there was no signific ant differenc e in perc ent phagoc ytosis over a perio d o f 4 ho urs, but the perc entage inc reased signific antly afte r 2 4 ho ur s. Similar ly, the fungic idal c apac ity o f ac tivate d mac r o phages was also higher after 2 4 ho ur s3.In a later study, Brummer e t a l4 evaluated the phagoc ytic and fungic idal po te ntial o f mur ine pe r ito ne al mac r o phage s
ac tivated or not with INF-
γ
and observed that after 4 hours 9 2 % of the fungi had been phagoc ytized by mac rophages regardless of whether or not they had been ac tivated. However, only ac tivated mac rophages were able to fully eliminate P. b ra silie nsis within a period of 4 8 hours. Ac c ording to the c ited investigators, ac tivated m a c r o pha ge s pla y a de c isive r o le in the r e sista nc e to P. b ra silie n sis. More rec ently, Mosc ardi-B ac c hi e t a l9 evaluated the pha go c yto s is o f b lo o d m o no c yte s fr o m pa tie nts with parac oc c idioidomyc osis and detec ted a phagoc ytic rate of 8 0 % . They also observed that monoc ytes ac tivated with INF-γ
markedly inhibited the multiplic ation of P. b ra silie nsis.In the present study, the phagoc ytosis of L. lo b o i was assessed at 3 and 2 4 hours and statistic al analyses of the results showed that, regardless of the group studied, phagoc ytosis of the fungus was higher at 3 hours than at 2 4 hours. These results are similar to tho se o b taine d b y B r umme r e t a l4 who de te c te d 9 2 .7 % phagoc ytosis of P.b ra silie nsis by mouse peritoneal mac rophages at 4 hours, 6 1 % at 2 4 hours, and 1 0 .2 % at 4 8 hours.
In the literature, mo st studies o n fungus phago c yto sis used pe r io ds o f time r anging fr o m half an ho ur to 4 ho ur s3 5 1 3 1 7. Thus, in the pr e se nt study we o pte d fo r the e valuatio n o f phago c yto sis afte r 3 ho ur s and the n at 2 4 ho ur s.
The de sign o f the pr e se nt study to o k into c o nside r atio n what the ideal pro po rtio n o f phago c ytic c ell:fungi wo uld be fo r the c ultures. Literature data have revealed wide variatio ns5 1 3 1 7 and the r e fo r e we o pte d fo r the use o f 1 c e ll:1 fungus. In this r e spe c t, Sawye r1 3 asse sse d the phago c ytic ac tivity o f mo use alve o lar mac r o phage s against C. a lb i c a n s using 1 :1 and 5 :1 phago c ytic c e ll:fungus pr o po r tio ns and the ir r e sults sho we d that the r ate s o f phago c yto sis we r e highe r whe n the 1 c e ll:1 fungus pr o po r tio n was use d.
In another study, Gaziri e t a l5 used the proportions of 1 :3 , 1 : 6 and 1 : 9 c e ll: fungus fo r the e valuatio n o f C. a lb i c a n s
phagoc ytosis by mouse peritoneal mac rophages and obtained b e tte r r e s ults wh e n th e 1 : 9 pr o po r tio n wa s us e d. Th e s e investigators demonstrated the important role of the c omplement system in the opsonization of C. a lb ic a ns and later phagoc ytosis by mac rophages. They showed that phagoc ytic ac tivity was muc h higher when fresh serum was added to the c ultures as a sourc e of c omplement. When fresh serum was added, phagoc ytosis was 5 0 .6 % and when heat-inac tivated serum was used, phagoc ytosis was 5 .4 % using the proportion of 1 c ell:3 fungi.
I n the pr e se nt study, m o no c yte c ultur e s c o ntaine d fr e sh AB+ se r um as a so ur c e o f c o mple me nt. As also do ne b y Gazir i
e t a l5, we had the opportunity to assess the phagoc ytic index of two patie nts with Jo r ge Lo b o ’s dise ase b y adding fr e sh and inac tivate d se r um o f the patie nt himse lf to the c ultur e s. The results obtained suggest that the antibody and c omplement favor the o pso nizatio n and phago c yto sis o f the fungus. Ho we ve r, a lar ger numb er o f patients is nec essar y fo r a b etter study o f the par tic ipatio n o f the se o pso nins in L. lo b o i phago c yto sis.
Whate ve r, the r e sults r e po r te d he r e r e pr e se nt a fir st assay for the evaluation of one of the nonspec ific defense mec hanisms o f the ho st and reveal that the mo no c ytes o f patients with Jo rge Lo b o ’s dise ase ar e ab le to phago c yte L. lo b o i, the r e was no Ta b le 1 - Pe rc e nt pha go c yto sis o f L. lo b o i b y b lo o d m o no c yte s fro m pa tie nts
with Jo rge Lo b o ’s dise a se a nd c o ntro ls.
Perc ent phagoc ytosis ( mean ± SD)
3 ho ur s 2 4 ho ur s
Patients 3 9 .3 ± 6 .5 3 4 .0 ± 7 .0
Co ntr o l 3 7 .5 ± 4 .9 2 9 .3 ± 8 .1 Statistic al analyses: patient = c ontrol ( F = 1 .8 3 6 6 7 ; p = 0 .1 8 9 7 4 1 )
3 hours> 2 4 hours ( F = 2 0 .1 1 3 7 1 ; p = 0 .0 0 0 2 0 4 ) *
* statistic ally signific ant differenc e ( p < 0 .0 1 )
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signific ant differenc e between patients and c o ntro ls. They also show that the rate of fungal phagoc ytosis was higher during the period of 3 hours c ompared to 2 4 hours, suggesting that during the later per io d the phago c ytes may be invo lved in the pr o c ess o f lysis o f the phago c ytize d fungus.
ACKNOWLEDGMENTS
We wish to thank Pro f. Jo sé Ro berto Pereira Lauris, Dental Sc ho o l, Univer sity o f São Paulo , B aur u, fo r statistic al analyses o f the data.
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