This study aimed to compare the caspofungin immunomodulating activities against Candida albicans on polymorphonuclear cells (PMNs) from renal transplant recipients (RTRs) and healthy subjects (HSs). RTR PMNs showed a significantly reduced fungicidal activity compared with that of HS PMNs. Addition of caspofungin to RTR PMNs significantly potentiated the yeast intracellular killing rate, achieving values similar to those observed for HS PMNs. These data show that caspofungin is suitable for invasive candidiasis treatment in patients with immune system-impaired components

Allizond, Valeria

Banche, Giuliana

Cuffini, Annamaria

Garneri, G

Giacchino, G

Mandras, Narcisa

Merlino, Chiara

Roana, Janira

Scalas, Daniela

Tullio, Viviana Cristina

English

PubMed

Institutional Research Information System University of Turin

  Published Ahead of Print 3 October 2011. 10.1128/AAC.00521-11. 2011, 55(12):5936. DOI:Antimicrob. Agents Chemother. Narcisa Mandras, Janira Roana and Anna Maria CuffiniMerlino, Daniela Scalas, Vivian Tullio, Giuseppe Garneri, Valeria Allizond, Giuliana Banche, Franca Giacchino, Chiara Caspofungin on Polymorphonuclear Cells Transplant Recipients: Effect of Candida albicans Infections in Renalhttp://aac.asm.org/content/55/12/5936Updated information and services can be found at: These include:REFERENCEShttp://aac.asm.org/content/55/12/5936#ref-list-1This article cites 23 articles, 7 of which can be accessed free at:CONTENT ALERTS more»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when newhttp://aac.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders: http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to:  on November 18, 2011 by DIP DI SANITA PUBBLICA E MICROBIOLOGIAhttp://aac.asm.org/Downloaded from ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 2011, p. 5936–5938 Vol. 55, No. 120066-4804/11/$12.00 doi:10.1128/AAC.00521-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.Candida albicans Infections in Renal Transplant Recipients: Effect ofCaspofungin on Polymorphonuclear CellsValeria Allizond,1 Giuliana Banche,1 Franca Giacchino,2 Chiara Merlino,1 Daniela Scalas,1Vivian Tullio,1 Giuseppe Garneri,2 Narcisa Mandras,1Janira Roana,1 and Anna Maria Cuffini1*Department of Public Health and Microbiology, University of Turin, Via Santena 9, 10126, Turin, Italy,1 and Nephrology andDialysis Unit, Civil Hospital, Piazza della Credenza 2, 10015, Ivrea, Turin, Italy2Received 18 April 2011/Returned for modification 12 July 2011/Accepted 23 September 2011This study aimed to compare the caspofungin immunomodulating activities against Candida albicans onpolymorphonuclear cells (PMNs) from renal transplant recipients (RTRs) and healthy subjects (HSs). RTRPMNs showed a significantly reduced fungicidal activity compared with that of HS PMNs. Addition ofcaspofungin to RTR PMNs significantly potentiated the yeast intracellular killing rate, achieving valuessimilar to those observed for HS PMNs. These data show that caspofungin is suitable for invasive candidiasistreatment in patients with immune system-impaired components.Renal transplant recipients (RTRs) are highly susceptible toinvasive fungal infections (IFIs) mainly due to Candida spp.,which continue to be the most frequent cause of death (60%)in the posttransplant period (1, 2, 13–15, 21). In fact, Candidaspp. may easily find ideal conditions for growing and invadingtissues in relation with both long-term immunosuppressivetreatment and decreased defense mechanisms (1, 7).Among newer antifungals, echinocandins such as caspofun-gin can be used for IFI treatment, thanks to their favorablepharmacodynamic/pharmacokinetic characteristics, lower tox-icity, and immunopharmacologic mode of action (4, 6, 9–12,20, 23). This study aimed to compare the immunomodulatingactivities of caspofungin against Candida albicans on PMNsfrom RTRs and healthy subjects (HSs).(This study was presented in part at the XLVII ERA-EDTACongress, abstr. Sa283, Munich, Germany, 25 to 28 June 2010).All patients participating in this study gave their writteninformed consent. Blood samples were obtained from 30 HSs(controls) and from 54 RTRs, 31 males and 23 females (meanage, 53.9 years), without any evidence of infection, being fol-lowed at Ivrea Hospital (Turin, Italy). The mean time sincetransplantation was 83.94 months (range, 7 to 248 months; for2/54 patients, less than 12 months since transplant), and theprimary renal diseases which infected these patients were asfollows: nephroangiosclerosis (n  16), chronic glomerulone-phritis (n  6), interstitial nephritis (n  5), polycystic kidneydisease (n  4), diabetic nephropathy (n  4), chronic renalfailure (n 10), and other (n 9). The mean serum creatininelevel at the time of the study was 1.7  0.8 mg/dl. Posttrans-plant immunosuppressive treatment used included the follow-ing: for the majority of patients, tacrolimus (FK), mycopheno-late mofetil (MMF), and prednisone (P); for 2 patients,cyclosporine (CyA) and MMF; for 7 patients, CyA, P, andsirolimus; for 3 patients, P and FK; for 4 patients, FK andMMF; for 5 patients, FK alone; and for 1 patient, CyA alone.A clinical C. albicans strain, isolated from blood and iden-tified by biochemical methods, was subcultured on Sabourauddextrose agar (Oxoid S.p.A., Milan, Italy) to ensure viabilityand purity (20). Caspofungin acetate (Merck Sharp & DohmeLtd., Hoddesdon, United Kingdom) was dissolved in pyrogen-free water and stored at 20°C.Antifungal susceptibility testing was performed with an in-oculum of 103 CFU/ml, in accordance with CLSI M27-A3 (5),and an inoculum of 106 CFU/ml, used to perform tests withphagocytes.PMNs separated from lithium-heparinized venous blood us-ing Ficoll-Paque (Pharmacia S.p.A., Milan) were suspended inRPMI 1640 medium (106 PMNs/ml) (Gibco Laboratories, NY)as described previously (3). PMN viability was greater than95%.Both phagocytosis of radiolabeled C. albicans ([3H]uracil[specific activity, 1,270 GBq/mmol; NEN Life Science Prod-ucts, Milan]) and intracellular blastoconidia killing by PMNswere investigated by incubation of yeast cells (106 yeast cells/ml) and PMNs (1:1 ratio) at 37°C in a shaking water bath for30, 60, and 90 min in the presence of one times the MIC (2g/ml), one-half times the MIC, and one-fourth times the MICof caspofungin. Drug-free controls were included. Phagocyto-sis and intracellular killing were assessed by the methods de-scribed previously (3, 20). Radioactivity was expressed ascounts per minute (cpm) per sample. The percentage ofphagocytosis at a given sampling time was calculated as fol-lows: percentage of phagocytosis  [(number of cpm in PMNpellet)/(number of cpm in total fungal pellet)]  100 (3, 20).The PMN killing values were expressed as the survival index(SI), which was calculated by adding the number of survivingyeast cells at time zero to the number of survivors at time x anddividing by the number of survivors at time zero (3, 20). Ac-cording to this formula, if fungal killing was 100% effective, theSI would be 1.Results were expressed as the means  the standard errors* Corresponding author. Mailing address: Department of PublicHealth and Microbiology, Microbiology Section, University of Turin,Via Santena 9, 10126 Turin, Italy. Phone: 39/0116705638. Fax: 39/0112365638. E-mail: annamaria.cuffini@unito.it. Published ahead of print on 3 October 2011.5936 on November 18, 2011 by DIP DI SANITA PUBBLICA E MICROBIOLOGIAhttp://aac.asm.org/Downloaded from of the means (SEM) from 10 separate experiments, each per-formed in quadruplicate. Statistical evaluation of the differ-ences between test and control results was performed byTukey’s test. A P value of 0.05 was considered significant.RTR PMNs showed phagocytosis toward C. albicans similarto that registered for HS PMNs (Table 1), whereas they dis-played significant reduced fungicidal activity within 90 min(P  0.01), confirming our previous findings, which show thatin RTRs, the number of PMNs was normal, but several met-abolic and functional alterations were observed, resulting in ahigh incidence of infections in these patients (19).As observed for other systemic antifungal drugs (8, 16, 19,20), no synergistic effect between PMNs and caspofungin onphagocytosis toward C. albicans in RTR or HS PMNs (Table 1)throughout the observation period occurred, indicating thatcaspofungin did not adversely interfere with PMN phagocyticcapacity, probably due to a dramatic effect on the integrity ofthe yeast cell wall. The expression of surface molecules thatlink to cleavage products of complement could be altered withdirect consequences for phagocytosis (8, 16, 20).On the contrary, the addition of caspofungin significantly(P  0.01) elicited fungicidal activity against intracellular C.albicans by HS PMNs compared to that observed in drug-freecontrols (Table 1). Analogously, when caspofungin was addedto RTR PMNs, their depressed fungicidal activity was com-pletely restored, with rates quite similar to those of HS PMNs,indicating that caspofungin synergized for fungal killing withPMNs being able to kill C. albicans at values significantlyhigher than those observed in drug-free RTR PMNs (P 0.01)(Table 1). A similar pattern was also detected when RTRPMNs and yeast cells were exposed to sub-MICs of caspofun-gin (data not shown), indicating the absence of a drug dose-dependent effect. The increased PMN killing activity observedat various caspofungin concentrations is related to a directdrug action on both C. albicans and phagocytes, as previouslydemonstrated (20) by the following: caspofungin is able both tounmask a fungal virulence factor in the yeast cell wall, makingintracellular blastoconidia more susceptible to PMN lyticmechanisms, and to enter phagocytes remaining available in abiologically active form against proliferating yeast cells.Taken together, the results of this study provide evidencethat caspofungin is able to elicit the depressed phagocytic re-sponse of PMNs from RTRs, with the potential to totallyrestore their primary functions in vitro. These findings supportand could explain the improvement of survival in neutropenichigh-risk patients with IFIs undergoing an empirical treatmentwith caspofungin versus liposomal amphotericin B, as de-scribed in the literature (17, 18, 24). In spite of the high cost ofcaspofungin, which nowadays limits its use to azole-resistantcases of candidal infections (22, 23), all these data permit theconclusion that caspofungin, in addition to its antifungal activ-ity, possesses immunomodulating properties that make ithighly suitable for fungal infection treatment in patients withimpaired components of the immune system, which representa high-risk population.To better assess these encouraging findings, further studiesby testing both other Candida clinical isolates and other im-munocompromised patients are needed.This work was supported by a grant from the Regione Piemonte,Ricerca Sanitaria Finalizzata (2008 bis), Italy.We are grateful to the staff of both the Nephrology and Dialysis Unit(Ivrea Hospital, Ivrea, Turin, Italy) and Banca del Sangue e del Plasma(A.S.O. San Giovanni Battista, Turin, Italy) for their help in conduct-ing this study.We have no conflicts of interest to declare.REFERENCES1. Anees, M. M., et al. 2010. Enhanced enzymatic activity of Candida speciesresponsible for oral candidiasis in renal transplant recipients. Mycoses doi:10.1111/j.1439-0507.2010.01874.x.2. Badiee, P., P. Kordbacheh, A. Alborzi, and S. A. Malekhoseini. 2007. Inva-sive fungal infection in renal transplant recipients demonstrated by panfun-gal polymerase chain reaction. Exp. Clin. Transplant. 5:624–629.3. Banche, G., et al. 2006. Effect of dialysis membrane biocompatibility onpolymorphonuclear granulocyte activity in dialysis patients. Nephrol. Dial.Transplant. 21:3532–3538.4. Chiller, T., K. Farrokhshad, E. Brummer, and D. A. Stevens. 2001. Theinteraction of human monocytes, monocyte-derived macrophages, and poly-morphonuclear neutrophils with caspofungin (MK-0991), an echinocandin,for antifungal activity against Aspergillus fumigatus. Diagn. Microbiol. Infect.Dis. 39:99–103.5. Clinical and Laboratory Standards Institute. 2008. Reference method forbroth dilution antifungal susceptibility testing of yeasts, 3rd ed. Approvedstandard M27-A3. Clinical and Laboratory Standards Institute, Wayne, PA.6. Deresinski, S. C., and D. A. Stevens. 2003. Caspofungin. Clin. Infect. Dis.36:1445–1457.7. Dongari-Bagtzoglou, A., et al. 2009. Oral Candida infection and colonizationin solid organ transplant recipients. Oral Microbiol. Immunol. 24:249–254.8. Frank, U., M. Greiner, I. Engels, and F. D. Daschner. 2004. Effects ofcaspofungin (MK-0991) and anidulafungin (LY303366) on phagocytosis,oxidative burst and killing of Candida albicans by human phagocytes. Eur.J. Clin. Microbiol. Infect. Dis. 23:729–731.9. Grover, N. D. 2010. Echinocandins: a ray of hope in antifungal drug therapy.Indian J. Pharmacol. 42:9–11.TABLE 1. Effect of 1 MIC of caspofungin against C. albicans on intracellular killing of PMNs from HSs and RTRsTime (min)Mean % phagocytosis  SEM SI  SEM (%)bControls PMNs treated withcaspofungin (2 g/ml) ControlsPMNs treated withcaspofungin (2 g/ml)HSs30 77.1 0.67 77.2 0.76 1.54  0.03 (46) 1.29 0.07 (71)a60 75.8 0.11 75.1 0.11 1.53  0.02 (47) 1.28 0.11 (72)a90 75.1 0.32 75.9 0.09 1.52  0.03 (48) 1.25 0.07 (75)aRTRs30 74.9 0.74 74.3 0.02 1.54  0.1 (46) 1.44 0.09 (56)60 76.4 0.46 76.1 0.15 1.65  0.14 (35) 1.42 0.11 (58)a90 76.4 0.58 76.7 0.31 1.74  0.08 (26) 1.33 0.11 (67)aa Significantly different from controls (P  0.01).b Values shown in parentheses represent the percentages of yeast cells killed by PMNs in the absence and in the presence of the antifungal drug.VOL. 55, 2011 CASPOFUNGIN AND RENAL TRANSPLANT RECIPIENT PMNs 5937 on November 18, 2011 by DIP DI SANITA PUBBLICA E MICROBIOLOGIAhttp://aac.asm.org/Downloaded from 10. Gullo, A. 2009. Invasive fungal infections: the challenge continues. Drugs69:65–73.11. Hohl, T. M., M. Feldmesser, D. S. Perlin, and E. G. Pamer. 2008. Caspo-fungin modulates inflammatory responses to Aspergillus fumigatus throughstage-specific effects on fungal beta-glucan exposure. J. Infect. Dis. 198:176–185.12. Lamaris, G. A., et al. 2008. Caspofungin-mediated beta-glucan unmaskingand enhancement of human polymorphonuclear neutrophil activity againstAspergillus and non-Aspergillus hyphae. J. Infect. Dis. 198:186–192.13. Matignon, M., et al. 2008. Outcome of renal transplantation in eight patientswith Candida sp. contamination of preservation fluid. Am. J. Transplant.8:697–700.14. Pappas, P. G., et al. 2010. Invasive fungal infections among organ transplantrecipients: results of the Transplant-Associated Infection Surveillance Net-work (TRANSNET). Clin. Infect. Dis. 50:1101–1111.15. Pourmand, G., et al. 2007. Infectious complications after kidney transplan-tation: a single-center experience. Transpl. Infect. Dis. 9:302–309.16. Richardson, M. D., G. Scott, and G. S. Shankland. 1992. Effect of cilofunginon phagocytosis and intracellular killing of Candida albicans by human neu-trophils. Eur. J. Clin. Microbiol. Infect. Dis. 11:22–26.17. Ru¨ping, M. J., J. J. Vehreschild, and O. A. Cornely. 2008. Patients at highrisk of invasive fungal infections: when and how to treat. Drugs 68:1941–1962.18. Ru¨ping, M. J., J. J. Vehreschild, and O. A. Cornely. 2008. Antifungal treat-ment strategies in high risk patients. Mycoses 51:46–51.19. Tullio, V., et al. 2003. Combined action of fluconazole and PMNs fromuremic patients in clearing intracellular Candida albicans. J. Chemother.15:301–303.20. Tullio, V., et al. 2010. Synergy of caspofungin with human polymorphonu-clear granulocytes for killing Candida albicans. Antimicrob. Agents Che-mother. 54:3964–3966.21. van Hal, S. J., et al. 2009. Candidemia following solid organ transplantationin the era of antifungal prophylaxis: the Australian experience. Transpl.Infect. Dis. 11:122–127.22. Veroux, M., et al. 2007. Caspofungin in kidney transplant recipients withrefractory invasive candidiasis. Nephrol. Dial. Transplant. 22:1487–1489.23. Veroux, M., et al. 2006. Caspofungin in the treatment of azole-refractoryesophageal candidiasis in kidney transplant recipients. Transplant. Proc.38:1037–1039.24. Walsh, T. J., et al. 2004. Caspofungin versus liposomal amphotericin B forempirical antifungal therapy in patients with persistent fever and neutrope-nia. N. Engl. J. Med. 351:1391–1402.5938 ALLIZOND ET AL. ANTIMICROB. AGENTS CHEMOTHER. on November 18, 2011 by DIP DI SANITA PUBBLICA E MICROBIOLOGIAhttp://aac.asm.org/Downloaded from 

Candida albicans infections in renal transplant recipients: effect of caspofungin on polymorphonuclear cells.

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Candida albicans infections in renal transplant recipients: effect of caspofungin on polymorphonuclear cells.

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