A comparative histopathological study of systemic candidiasis in association with experimentally induced breast cancer

Transcription

1 oncology letters 1: , A comprtive histopthologicl study of systemic cndidisis in ssocition with experimentlly induced brest cncer Z.W. Choo 1, S. Chkrvrthi 2, S.F. Wong 2, H.S. Ngrj 3, P.M. Thnikchlm 2, J.W. Mk 2, A. Rdhkrishnn 2 nd A. Ty 2 1 Deprtment of Post Grdute Studies, Reserch Lb; Deprtments of 2 Pthology nd 3 Humn Biology, Fculty of Medicine, Interntionl Medicl University, Kul Lumpur, Mlysi Received September 2, 2009; Accepted November 2, 2009 DOI: /ol_ Abstrct. Systemic cndidisis is fungl infection which coupled with solid mlignncies plces ptients t high risk of succumbing to the disese. Few studies hve shown evidence of the reltionship between systemic cndidisis nd mlignncy-induced immunosuppression disese especilly in brest cncer. At present, niml studies tht exclusively demonstrte this reltionship hve yet to be conducted. The exct custive mechnism of systemic cndidisis is currently under much specultion. This study therefore imed to demonstrte this reltionship by observing the histopthologicl chnges of orgns hrvested from femle Blb/c mice which were experimentlly induced with brest cncer nd inoculted with systemic cndidisis. The mice were rndomly ssigned to five different groups (n=12). The first group (group 1) ws injected with phosphte buffer solution, the second (group 2) with systemic cndidisis, the third (group 3) with brest cncer nd the finl two groups (groups 4 nd 5) hd both cndidisis nd brest cncer t two different doses of cndidisis, respectively. Inocultion of mice with systemic cndidisis ws performed by n intrvenous injection of Cndid lbicns vi the til vein following successful culture methods. Induction of mice with brest cncer occurred vi injection of 4T1 cncer cells t the right xillry mmmry ftpd fter effective culture methods. The prepred slides with orgn tissues were stined with hemtoxylin nd eosin, periodic cidic schiff nd gomori methenmine silver stins for histopthologicl nlysis. Grding of primry tumour nd identifiction of metsttic deposits, s well s scoring of inflmmtion nd congestion in ll the respective orgns ws conducted. Sttisticl tests performed to compre groups 2 nd 4 showed tht group 4 exhibited highly sttisticlly significnt increse in orgn inflmmtion nd congestion Correspondence to: Dr Srikumr Chkrvrthi, Deprtment of Pthology, Fculty of Medicine, Interntionl Medicl University, Kul Lumpur, Mlysi E-mil: srikumr_chkrvrthi@imu.edu.my Key words: systemic cndidisis, brest cncer, Cndid lbicns, 4T1 mouse cncer model, metstsis, immunosuppression (p<0.01). The medin severity of cndidisis in the kidneys nd liver lso incresed in group 4 s compred to group 2. In conclusion, bsed on the bove evidence, systemic cndidisis significntly incresed in mice with brest cncer. Introduction Cndidisis is disese cused by Cndid sp which re prt of the norml flor found in the upper respirtory, gstrointestinl nd femle genitl trct of the humn body. Most cses of Cndid infection result from Cndid lbicns, which is n opportunistic infection s it does not induce disese in immunocompetent individuls but cn only do so in those with n impired host immune defence system. The Cndid infection is generlly clssified into superficil nd deep. It commonly infects the nils, skin nd mucous membrnes, especilly the orophrynx, vgin, oesophgus nd gstrointestinl (GI) trct. Occsionlly, Cndid sp invde the bloodstrem nd spred to other deep structure orgns in the body such s kidneys, lungs nd brin, cusing systemic cndidisis (1). Although decrese in bloodstrem infection hs been noted, the number of risk fctors which my eventully led to systemic cndidisis hve been on the increse (2). Risk fctors for systemic cndidisis include immunosuppression due to chemotherpy or corticosteroid therpy, dibetes mellitus, low birth weight in neontes, brod spectrum ntibiotics, longterm ctheteriztion, hemodilysis nd prenterl nutrition. However, significnt observtion ws tht the three min groups of ptients ssocited with systemic cndidisis re those with neutropenic cncer, orgn or stem cell trnsplnt ptients nd those undergoing intensive cre procedures. Brest cncer is the most common cncer mong Mlysin women, with pproximtely 1 in 20 developing brest cncer in their lifetime (3). There is mrked geogrphicl difference in the worldwide incidence of brest cncer, with higher incidence in developed countries compred to developing countries. In survey conducted in two prominent hospitls in Mlysi, the ge incidence ws similr. Moreover, it ws discovered tht on verge, hlf of the cses were delyed in presenttion. This dely ws possibly ttributed to strong belief in trditionl medicine, the negtive perception of the disese, poverty nd poor eduction, coupled with fer nd denil (3,4).

2 216 choo et l: systemic cndidisis with brest cncer Tble I. Chrcteristics of the five groups. Group no. group description concentrtion per dose durtion before of 0.1 ml (cells/ml) dissection (weeks) 1 Control group (injected with PBS only) Mice inoculted with Cndid lbicns 5x Mice induced with brest cncer 1x Mice induced with brest cncer 1x10 5 of 4T1 brest cncer cells nd subsequently inoculted nd 5x10 6 for Cndid lbicns with Cndid lbicns 5 Mice induced with brest cncer 1x10 5 of 4T1 brest cncer cells nd subsequently inoculted nd 5x10 8 for Cndid lbicns with Cndid lbicns While the exct mechnism leding to systemic cndidisis is not known, the initition nd progression of systemic cndidisis cn be viewed s n imblnce in the host-pthogen reltionship in fvour of Cndid lbicns. Previous studies showed tht invsive cndidisis is common nd serious compliction of cncer nd its therpy (5). In cncer ptients, it hs been hypothesized tht it develops from initil GI coloniztion with subsequent trnsloction into the bloodstrem. It is uncler wht components of the innte immune system re necessry for the prevention of Cndid lbicns dissemintion from the GI trct, but it is hypothesized tht neutropeni nd GI mucosl dmge re criticl in llowing widespred invsive Cndid lbicns disese (6). Few studies hve documented the co-existence nd plusible reltionship between brest cncer nd systemic cndidisis (7-10). However, there hve been no uthentic studies on systemic cndidisis nd its reltionship with brest cncer in experimentlly induced mice. This study imed to estblish hypotheticl reltionship between the most common type of cncer in women in Mlysi nd systemic cndidisis by using mouse brest cncer model with Cndid inocultion. Results from this study will provide the groundwork from which further studies, such s immunology, cn be crried out to better understnd the pthogenesis of Cndid in cncer ptients. It my lso help bring better insight into the current tretment nd pthophysiology of cncer which hs itself been shown to be risk fctor to the predisposition of cndidisis. Mterils nd methods Experimentl nimls. Femle Blb/c mice were used for the investigtion, fter prior pprovl from the Ethics committee. The mice were divided into five groups (Tble I). Dosing begn when the mice were 10 weeks old nd weighed g. They were housed in groups of 6 mice for ech metl cge locted within the Animl Housing Fcility in the Interntionl Medicl University. The mice were fed with stndrd mice chow nd were given free ccess to wter. The weight of the mice ws recorded t the strt, once every week therefter nd finlly t the end of the experiment. Culture of Cndid yest cells. The Cndid yest cells were obtined from ptient clinicl isoltes (Interntionl Medicl University Reserch Lb, Wong et l). Smples were used fter permission ws obtined from the investigtor. The cells were then subcultured onto solid medi of Sbourud gr by streking methods nd stored in n incubtor t 37 C. Before hrvesting the colonies for inocultion, one of the Cndid colonies ws subcultured in the YPD broth nd left for 72 h in shking incubtor (Certomt S11) fixed t 100 rpm t controlled temperture of 37 C. After 3 dys or on the stipulted dy of inocultion, serum ws dded into the broth to llow for germ tube formtion to occur nd left in the shking incubtor for n dditionl 3 h with similr settings. The colonies were then hrvested by mens of centrifugtion. The volume nd concentrtion needed for inocultion were prepred by dilutions nd clculted using hemocytometer. Inocultion of mice with systemic cndidisis. The mice were first plced inside retiner. A 27G needle syringe ws then used to inject 0.1 ml of Cndid blstospores suspended in phosphte buffer solution (PBS) t concentrtion of 5x10 6 cells/ml vi the til vein using ethnol swp. This step ws repeted with nother group of mice t concentrtion of 5x10 8 cells/ml. Culture of 4T1 brest cncer cells. The brest cncer cells (4T1 cell line; Interntionl Medicl University Reserch Lb, Rdhkrishnn et l) were mintined nd subcultured into 25-cm 3 culture flsk until the cells were helthy nd hd chieved stedy replictive rte. They were then hrvested by mens of centrifugtion nd kept suspended in the culture medium. The volume nd concentrtion needed for inocultion ws prepred by dilutions nd clculted using hemocytometer. Inducing mice with 4T1 cncer cells. The mice were first nesthesized with diethyl ether before n injection of 0.1 ml of 1x10 5 cells/ml ws dministered subcutneously into the mmmry ftpd t the xill of the right rm. Smple collection. The mice were weighed t the end of the experiment before being scrificed with diethyl ether in

3 oncology letters 1: , Tble II. Results of the pired t-test for gross weight of mice t the beginning nd end of the experiment. Group Men initil weight (g) Men finl weight (g) Asymptote significnce (p<0.05) b b b The men gross weight in grms, noted t the beginning nd end of the experiment, ws included. Significnt difference t p<0.05 nd b p<0.01. Tble III. Results obtined from the Kruskl-Wllis test for globl comprison of orgn metstses mong the groups nd the Mnn-Whitney U test for comprison between groups 3 nd 4 for the extent of orgn metstses. Orgns Asymptote significnce (p<0.05) kruskl-wllis test of globl comprison Mnn-Whitney U test for comprison between groups 3 nd 4 Brin Kidneys b b Lungs b Liver b Spleen b Significnt difference t p<0.05 nd b p<0.01. desicctor. The orgns hrvested were spleen, kidneys, lungs, hert nd brin. The tumours from groups 3, 4 nd 5 were lso hrvested. They were subsequently fixed in 10% formlin for t lest 2 dys. Tissue processing. The fixed orgns were then sectioned nd processed to prffin blocks. Sections of 4 µm were tken on glss slides nd were stined with hemtoxylin nd eosin, periodic cid schiff (PAS) nd gomori methenmine silver (GMS). The sections were then dehydrted, clered nd mounted with cover slips using DPX mountnt medi. Results The slides were observed under light microscope in order to grde the primry tumour, presence of metsttic deposits, nd extent of cndidisis in ll the orgns by comprtively exmining both the hemtoxylin nd eosin-stined slides, PAS nd GMS, nd the extent of orgn inflmmtion nd congestion. A correltion ws then mde between the pthologicl lesions observed in the groups nd the men gross weight chnges. The histopthologicl scoring of inflmmtion nd congestion chnges in these orgns ws bsed on the technique employed by Kh Heng et l nd Blck et l (11,12) (Figs. 3 nd 4). Scoring of cndidisis ws with reference to Blck et l nd Blish et l (12,13) (Figs. 2 nd 3). Grding of the primry tumour ws crried out using the conventionl method of nlyzing the similrity of the cells to its tissue of origin s poorly, modertely nd severely differentited (14) (Figs. 1 nd 4). Sttisticl nlysis. In this study, 60 smples were studied nd nlyzed. Anlyticl dt were expressed s men with stndrd devition nd 95% confidence intervl. The level of significnce ws set t Sttisticl tests used were: The i) pired t-test for comprison of initil nd finl men weight of mice in ech group, ii) Kruskl-Wllis test for globl comprison of groups for ll the prmeters, iii) nonprmetric Mnn-Whitney U test for comprison between two groups for ech prmeter nd iv) Spermn's rho test for the correltion of cndidisis, cncer metstses, inflmmtion nd congestion. The sttisticl tests were conducted with the id of SPSS Sttisticl softwre version 16. For the individul tests, p<0.05 ws considered to be significnt. The pired t-test is prmetric method employed to test for ny significnt difference between the mens on the sme or relted subject over time or in differing circumstnces. From the test conducted, it ws found tht p<0.05 in ll the groups, with groups 1, 2 nd 3 showing p<0.01. Thus, significnt difference in the weight of the mice in ll the groups t the beginning nd the end of the experiment ws noted (Tble II). Bsed on globl comprison for metstsis in ech of the orgns for ll groups, the Mnn-Whitney U test for comprison between groups 3 nd 4 showed significnt difference in ll the orgns except the brin (p<0.01; Tble III). The kidneys showed greter level of significnce (p<0.01) s compred to

4 218 choo et l: systemic cndidisis with brest cncer Tble IV. Histopthologicl scoring of cndidisis in hemtoxylin nd eosin. Experimentl group Medin of severity of cndidisis Brin kidneys lungs liver Group 2- Mice with Cndid (5x10 6 cells/ml) Group 4- Mice with brest cncer nd Cndid (5x10 6 cells/ml) Absent (-), mild (+), moderte (++) nd severe (+++). Tble V. Results of Kruskl-Wllis for globl comprison mong groups nd Mnn-Whitney U test for comprison between groups 2 nd 4 for orgn inflmmtion response. Orgns Asymptote significnce (p<0.05) kruskl-wllis test Mnn-Whitney U test for of globl comprison comprison between groups 2 nd 4 Brin b b Kidneys b b Lungs b b Liver b b Significnt difference t p<0.05 nd b p<0.01. Tble VI. Results of Kruskl-Wllis for globl comprison mong groups nd Mnn-Whitney U test for comprison between groups 4 nd 5 for orgn congestion. Orgns Asymptote significnce (p<0.05) kruskl-wllis test Mnn-Whitney U test for of globl comprison comprison between groups 4 nd 5 Brin b b Kidneys b b Lungs b b Liver b b Significnt difference t p<0.05 nd b p<0.01. the other orgns. This shows tht the presence of cndidisis, s in the cse of group 4, hs n effect on the extent of the metsttic growth in these orgns. By compring the medin severity of cndidisis between groups 2 nd 4, significnt difference in severity ws observed in the kidneys nd liver. In the kidneys of group 2, the severity of cndidisis ws mild, while tht in group 4 ws moderte (Tble IV). These observtions were lso observed in slides stined in PAS nd GMS. The Kruskl-Wllis test used for globl comprison between groups 2 nd 4, nd 4 nd 5 for inflmmtion nd congestion showed significnt difference of p<0.01 in ll the orgns (Tbles V nd VI). The Mnn-Whitney U test used for comprison between groups 2 nd 4 for inflmmtion response showed significnt difference in ll the orgns. This shows tht the co-existence of both cndidisis nd cncer in the mice hd heightened effect on the severity of inflmmtion s compred to mice with cndidisis lone. The Mnn-Whitney test ws used for comprison between groups 4 nd 5 to exmine the extent of cndidisis. The findings showed tht the increse in the Cndid dosge injected into group 5 t concentrtion of 5x10 8 cells/ml compred to

5 oncology letters 1: , Tble VII. Results of hte Mnn-Whitney U test for comprison between groups 4 nd 5 for the extent of cndidisis, orgn inflmmtion nd orgn congestion. Orgns Asymptote significnce (p<0.05) Extent of cndidisis Orgn inflmmtion Orgn congestion Brin b b Kidneys Lungs b Liver b Significnt difference t p<0.05 nd b p<0.01. Tble VIII. Correltion coefficient between cndidisis with cncer metstses, orgn inflmmtion nd orgn congestion. Comprison Spermn rho correltion Spermn rho correltion Spermn rho correltion orgns coefficient between cndidisis coefficient between cndidisis coefficient between cndidisis nd cncer metstses nd orgn inflmmtion nd orgn congestion Brin 0 (no correltion) b Kidneys b b b Lungs b b Liver b b b Correltion is significnt t the 0.05 (2-tiled) nd b 0.01 levels (2-tiled). 5x10 6 cells/ml in group 4, showed sttisticlly significnt difference in the brin nd lungs (p<0.01; Tble VII). The Mnn-Whitney U test for comprison between groups 4 nd 5 for inflmmtion nd congestion showed significnt difference in the inflmmtory response t the liver with p<0.01. This mens tht the incresed dose in group 5 showed sttisticlly significnt effect on the inflmmtory response noted in the liver nd congestion observed in the brin nd the lungs. The correltion between cndidisis nd cncer metstses ws positive in the kidneys nd liver for significnce level of 0.01 (Tble VIII). However, no correltion ws found in the brin, while in the lungs the correltion ws not significnt. The correltion between cndidisis nd inflmmtion ws shown to be positively correlted with significnce t level of 0.01 in the kidneys, lungs nd liver, while the brin only showed significnce t level of The correltion between cndidisis nd congestion ws positive t significnce level of 0.01 in ll of the orgns. Discussion Few studies hve been conducted on experimentl systemic cndidisis in mice. Informtion obtined from these studies on the necessry dosges, s well s previous observtions were used to mke comprisons with this study (15-17). Some of these studies hve been dedicted to the observtions of the correltion between systemic cndidisis nd other forms of immunosuppression such s chemotherpy, steroid therpy, ntibiotic therpy, s well s other types of mlignncies such s leukemi nd esophgel cncer. However, n exclusive study on systemic cndidisis nd its reltionship with brest cncer hs yet to be conducted, even though there re few epidemiologicl studies tht hve shown co-existence of brest cncer nd systemic cndidisis in humns (7,10,18). This study imed to focus on the reltionship between systemic cndidisis nd brest cncer by exmining the behviour of cndidisis when the body is subjected to chronic disese stte. Therefore, brest cncer ws not only selected s n idel representtion of chronic illness, but lso one tht is cpble of suppressing the host immune system (19-22). To study how the presence of chronic illness such s brest cncer cn, by itself, be ttributed to the incresed severity of cndidisis, this novel study focused on the growth of systemic cndidisis following the induction of mice with brest cncer. Scores were clculted bsed on the severity of cndidisis nd grding of the primry tumour, nd identifiction of their metsttic deposits ws conducted. Other prmeters tken into considertion included gross weight of the mice t the beginning nd end of the study, s well s inflmmtion nd congestion in the respective orgns which were studied by scoring on semi-quntittive scle using n estblished technique s mentioned erlier. Systemic cndidisis. In group 2, the mice were solely inoculted with systemic cndidisis by intrvenous injection vi the til vein for durtion of two weeks. During the course of the experiment, signs of the disese were noted in these mice. Their eyeblls protruded, their fur roughened nd they were generlly less ctive s compred to the norml group.

6 220 choo et l: systemic cndidisis with brest cncer Figure 1. Poorly differentited brest crcinom cells with multiple mitotic figures in the primry tumour, extending into chest wll muscle (x100, H&E). Figure 3. A colony of Cndid in the centrl region of huge bscess in the liver, surrounded by inflmmtion (x100, PAS). Figure 2. Growth of lrge colony of Cndid yest cells nd hyphe in the renl prenchym (x1000, PAS). Figure 4. Extensive metstsis of cncer in the liver mong micro-bscesses filled with chronic inflmmtory cells, Cndid nd debris (x200, PAS). Moreover, incresed group huddle nd sleep were noted. The mice lso ppered very wek nd thin with the curvtures of the bony structures beneth the mice visible to the nked eye. In ddition, the weight tken t the beginning nd end of the experiment showed tht there ws sttisticlly significnt reduction in their men weight. This ws ttributed to the possible loss of ppetite nd generl cchexic stte of the mice. Histopthologiclly, fvourble growth of the Cndid colonies in the form of hyphe, yest cells nd pseudohyphe were discovered in the kidneys, pelvis nd tubule region, but not in mny of the other orgns. This ws ttributed to the mild dose of 5x10 6 cells/ml Cndid cells injected nd the short durtion of the experiment s lso shown by Wong et l (16). Brest cncer study. In group 3, the mice were injected t the mmmry ftpd with 4T1 cncer cells in the right xill region t concentrtion of 1x10 6 cells/ml (23). After four weeks of growth nd metstses, the mice were scrificed for nlysis. During the course of the investigtion, the weights of the mice were reduced for the first week before grdully incresing in the 3rd week. The growth of the primry tumour ws detected s plpble mss s erly s the 10th dy nd s lte s the 14th dy. The mice were generlly ctive for the first two weeks with no pprent devitions from those usully observed in the norml control group. However, by the 3rd week, the mice begn to exhibit signs of lethrgy nd did not move s often. Furthermore, the mss of tumour begn to pper significntly enlrged to the nked eye by the middle of 3rd week. Generl ppetite ws good. No distinct chnges to the fur, eyes or prominent curvtures of the bony structures were noted. Grding for the primry tumour showed it to be moderte to poorly differentited with the mjority of the tumours being poorly differentited. Metsttic deposits were discovered in the lungs, liver nd spleen with vrying frequencies mong the mice. The gross morphology of the other orgns did not exhibit ny crude chnges, with the exception of the spleen which ws mrkedly enlrged s compred to tht found in the norml group. The tumour ppered hrd nd smooth in texture with glistening surfce. Scoring for inflmmtion showed tht the medin of severity of the entire group ws mild in both the lungs nd liver. The medin severity of congestion found in the kidneys, lungs nd liver were mild, while congestion found in the brin ws mild to moderte. In the liver, however, the micro-bscesses tht were observble in group 2, were not noted in the group s whole. Therefore, in the group with brest cncer, the severity of inflmmtion nd congestion observed in the orgns were mostly mild in severity with metsttic deposits found in the lungs, liver nd spleen. Correltion between systemic cndidisis nd brest cncer. In group 4, the mice were first induced with brest cncer

7 oncology letters 1: , for three weeks nd subsequently inoculted with Cndid t concentrtion of 5x10 6 cells/ml for one week. The time of induction with brest cncer ws set t three weeks, bsed on studies showing tht by this period, dequte metstses hve occurred in ll these orgns (23,24). The initil stges of tumour growth nd chnges in the mice were similr to those found in group 3. However, when Cndid ws injected, chnges in group 2 were noted within dys insted of the 2nd week s in the cse of group 2. These chnges included protruding eyes, roughened fur nd generl inerti, with incresed huddle nd sleep. In the finl stges of the investigtion, surge in the growth of tumour size ws observed. Grding crried out for the primry tumour showed poorly differentited tissue with typicl cells nd high number of mitotic figures. Metsttic deposits were lso found in the lungs, liver, spleen nd even in the kidneys t higher frequency s compred to tht of group 3. These differences were sttisticlly significnt (p<0.05). Thus, n incresed frequency of metsttic deposits occurred in these orgns in group 4 s compred to tht in group 3. This suggests possible role of Cndid cusing immunosuppression which, by itself, ttributed to the incresed metsttic deposits of the cncer found in these orgns. It lso explins the lte surge in tumour growth noted lte in the investigtion. Notble chnges in the kidneys include cndidisis involvement in the renl prenchym, renl tubules nd pelvis. Within the liver prenchym nd vsculture, distinct chnges such s micro-bcesses, chronic inflmmtion nd congestion were observed t greter level in this group s compred to tht noted in group 2. This group lso showed n incresed group medin of severity in Cndid infection in the kidneys nd liver. The kidneys showed moderte severity s compred to mild one in group 2, while the liver showed moderte severity of cndidisis s compred to the bsence of cndidisis noted in group 2. Thus, this group showed extr involvement of the liver compred to only the kidneys s ws the cse in group 2. This observtion holds true in scoring performed for both PAS nd GMS. Scoring of inflmmtion showed moderte severity s noted in the brin, kidneys nd lungs, while the liver showed severe chnges s compred to only mild ones found in ll the orgns in group 2. Comprison of inflmmtion severity between the two groups ws sttisticlly significnt (p<0.01). As for congestion, group 4 showed moderte congestion in the brin nd kidneys s compred to mild one in group 2 nd while congestion in the lungs ws not observed in group 2, group 4 showed mild congestion. Furthermore, the liver showed severe congestion s compred to just moderte congestion found in group 2. A comprison between groups 2 nd 4 for congestion ws sttisticlly significnt (p<0.05). In conclusion, severity of cndidisis, inflmmtion nd congestion were found t greter levels in brest cncer induced mice with cndidisis s compred to mice with only cndidisis. Dose-dependent study. In group 5, the mice were first induced with brest cncer nd, subsequently, with cndidisis t higher dose of 5x10 8 cells/ml. These mice were similr to group 3 t the initil stges of cncer growth. However, when cndidisis ws injected, the mice died within the first week of inocultion t vried periods s compred to group 4 where the time of inocultion with cndidisis ws one week nd mice survived till the end of investigtion. The sudden immedite deth ws ttributed to septicemi. Grding on the primry tumour showed the tumours to be poorly differentited. Metsttic deposits were found in the kidneys, lungs, liver nd spleen. Scoring of cndidisis showed mild severity in the brin while the kidneys showed moderte nd the lungs severe cndidisis. The liver, on the other hnd, showed mild to moderte severity. Sttisticl tests compring differences in cndidisis severity between groups 4 nd 5 found significnt difference in the brin nd lungs (p<0.01). This mens tht with n incresed dose, the brin nd lungs exhibit cndidisis with incresed levels of severity. It is possible tht with higher dose, the higher reches of the body re more esily ccessed s the mount of dose eliminted by the liver or spleen is less. In the scoring for inflmmtion, the brin showed mild severity while the kidneys, lungs nd liver showed moderte severity. However, only the liver showed sttisticlly significnt difference when compred to group 4. Inflmmtion ws much less in severity compred to tht in group 4, which my be ttributed to the short period of inocultion time before the demise of the mice resulting in indequte time for chronic inflmmtion to occur. In the scoring for congestion, group 5 showed severe congestion in ll the orgns. However, only the brin nd the lungs showed sttisticlly significnt difference with group 4, which ws shown to hve greter level of congestion. This my be ttributed to the cute chnges noted in the host response to foreign pthogen. Correltion of cndidisis, cncer metstses, orgn inflmmtion nd orgn congestion. The correltion between cndidisis nd cncer metstses ws found to be significnt in the kidneys nd liver (p<0.01). This shows tht in the kidneys nd liver, n increse in cncer metsttic deposits ws ccompnied by n increse in cndidisis severity. When correlting cndidisis with inflmmtion nd congestion, it ws found to be sttisticlly significnt (p<0.05) in ll the orgns. Thus, incresed levels of cndidisis re ccompnied by incresed levels of inflmmtion nd congestion in the respective orgns studied. In conclusion, the mouse model of inducing brest cncer ws successful, s well s the method nd technique of inducing cndidisis ws effective. The mouse model nd method nd technique were ttributed to the efficient culture methods. Moreover, growth of brest cncer nd cndidisis were observble in ll the relevnt groups. The weight of the mice ws lso correlted with the pthology suffered by the mice. All the objectives were crried out with precision nd successfully chieved. An nlysis ws performed bsed on the scoring of cndidisis, grding of metsttic deposits, inflmmtion nd congestion in the brin, kidneys, lungs nd liver of the mice in ll the groups. The inflmmtion nd congestion prmeters showed sttisticlly significnt increse in severity in ll the orgns s compred to the group of mice with systemic cndidisis nd brest cncer, nd tht of systemic cndidisis lone. The medin severity of the entire group for cndidisis

8 222 choo et l: systemic cndidisis with brest cncer scoring in the kidneys nd liver lso incresed for the group of mice with systemic cndidisis nd brest cncer. Therefore, bsed on these evidences, systemic cndidisis ppers to be more severe in experimentlly induced mice with brest cncer thn in mice without. Acknowledgements This investigtion ws funded by reserch grnt no. BMS I-02/2008 (12) from the Interntionl Medicl University, Kul Lumpur, Mlysi. References 1. Levinson W: Review of Medicl Microbiology nd Immunology. 9th edition. The McGrw-Hill Compnies, pp , Richrdson MD: Chnging ptterns nd trends in systemic fungl infections. J Antimicrob Chemother 56 (Suppl 1): 5-11, Yip CH, Tib NA nd Mohmed I: Epidemiology of brest cncer in Mlysi. Asin Pc J Cncer Prev 7: , Hishm AN nd Yip CH: Overview of brest cncer in Mlysin women: problem with lte dignosis. Asin J Surg 27: , DiNubile MJ, Hille D, Sble CA nd Krtsonis NA: Invsive cndidisis in cncer ptients: observtions from rndomized clinicl tril. J Infect 50: , Koh AY, Kohler JR, Coggshll KT, vn Rooijen N nd Pier GB: Mucosl dmge nd neutropeni re required for cndid lbicns dissemintion. PLoS Pthogens 4: 35-38, Gottfredsson M, Vredenburgh JJ, Xu J, Schell WA nd Perfect JR: Cndidemi in women with brest crcinom treted with high-dose chemotherpy nd utologous bone mrrow trnsplnttion. Cncer 98: 24-30, Ghoneum M nd Gollpudi S: Phgocytosis of Cndid lbicns by metsttic nd non metsttic humn brest cncer cell lines in vitro. Cncer Detect Prev 28: 17-26, Anderson LM, Krotz S, Weitzmn SA nd Thimmpy B: Brest cncer-specific expression of the Cndid lbicns cytosine deminse gene using trnscriptionl trgeting pproch. Cncer Gene Ther 7: , Sfdr A, Chturvedi V, Cross EW, Prk S, Bernrd EM nd Armstrong D: Prospective study of Cndid species in ptients t comprehensive cncer center. Antimicrob Agents Chemother 45: , Lee KH, Chen YS, Judson JP, Chkrvrthi S, Sim YM nd Er HM: The effect of wter extrcts of Euphorbi hirt on crtilge degenertion in rthritic rts. Mlys J Pthol 30: , Blck CA, Eyers FM, Russell A, Dunkley ML, Clncy RL nd Begley KW: Incresed severity of Cndid vginitis in BALB/c nu/nu mice versus the prent strin is not brogted by doptive trnsfer of T cell enriched lymphocytes. J Reprod Immunol 45: 1-18, Blish E: A URA3 null mutnt of Cndid lbicns (CAI-4) cuses oro-oesophgel nd gstric cndidisis nd is lethl for gnotobiotic, trnsgenic mice (Tgepsilon26) tht re deficient in both nturl killer nd T cells. J Med Microbiol 58: , Kumr VRSC nd Robbins SL: Robbins Bsic Pthology. 7th edition. Sunders, pp , De Repentigny L: Animl models in the nlysis of Cndid host-pthogen interctions. Curr Opin Microbiol 7: , Wong SF, Mk JW nd Pook CK: Potentil use of monoclonl ntibody for the detection of Cndid ntigens in n experimentl systemic cndidisis model. Hybridom 27: , Ashmn RB nd Ppdimitriou JM: Murine cndidisis. Pthogenesis nd host responses in geneticlly distinct inbred mice. Immunol Cell Biol 65: , Tlrmin JP, Boutoille D, Tttevin P, et l: Epidemiology of cndidemi: one-yer prospective observtionl study in the west of Frnce. Med Ml Infect 4: , Mndeville R, Lmoureux G, Legult-Poisson S nd Poisson R: Biologicl mrkers nd brest cncer. A multiprmetric study. II. Depressed immune competence. Cncer 50: , Semiglzov VF, Kondrt'ev VB, Mr'enko AI, L'Vovich EG nd Sofronov BN: Immunologic rectivity of brest cncer ptients. Vopr Onkol 24: 74-79, Contrers Ortiz O nd Stolir A: Immunologicl chnges in humn brest cncer. Eur J Gynecol Oncol 9: , Ds SN, Khnn NN nd Khnn S: A multiprmetric observtion of immune competence in brest cncer nd its correltion with tumour lod nd prognosis. Ann Acd Med Singpore 14: , Pulski BA nd Ostrnd-Rosenberg S: Mouse 4T1 brest tumor model. Curr Protoc Immunol 20: 2-11, To K, Fng M, Alroy J nd Shgin GG: Imgble 4T1 model for the study of lte stge brest cncer. BMC Cncer 8: , 2008.