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SYNERGISTIC ANTIFUNGAL EFFECT OF CASPOFUNGIN AND HUMAN POLYMORPHONUCLEAR LEUKOCYTES (PMNs) FROM RENAL TRANSPLANT RECIPIENTS AND CHRONIC HAEMODIALYSED PATIENTS AGAINST CANDIDA ALBICANS

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INTRODUCTION

Fungal opportunistic infections characterized by high morbidity and mortality, related to an impairment of the phagocytic response, are a clinical significant problem especially in immunocompromised subjects, such as renal transplant recipients. Hence, the optimal antifungal drug should combine good in vitro activity with the capacity to act in concert with the immune system in a way that potentiates the host’s defences [1, 2, 3]. Since polyenes and azoles, the mainstay of fungal infection treatment, show actually clinical drawbacks due to the emergence of drug resistant strains, there is the need to study new antifungal drugs [4]. Among new drugs for fungal infection treatment, recently echinocandins, such as caspofungin, stand out exhibiting an inhibitory activity of fungal cell wall glucan. In particular, caspofungin shows a marked antifungal activity against Candida spp., some filamentous and dimorphic fungi related to its favorable pharmacodynamic and pharmacokinetic characteristics [5, 6].

This study was designed to evaluate the potential effect of caspofungin on the functions of polymorphonuclear cells (PMNs) harvested from both healthy subjects (HSs) and renal transplant recipients (RTRs) or haemodialysed patients (HDs) against Candida albicans often associated with serious fungal infections in immunocompromised patients.

PATIENTS AND METHODS

Patients: All patients participating in this study gave their informed consent. Blood samples were obtained from 30 healthy subjects (HSs), as controls, and from 54 patients that had undergone renal transplantation (RTRs)- 31 males and 23 females (mean age 53.9 years), and 66 patients on chronic dialysis (HDs)- 42 males, 24 females (mean age 69.3 years), followed at Nephrology and Dialysis Unit of the Ivrea Hospital (Turin, Italy). The mean time since transplantation was 83.94 months (range 7 - 248 months); the mean serum creatinine at the time of the study was 1.7 ± 0.8 mg/dl. Post-transplant immunosuppressive treatment was in the majority of patients  FK, MMF and prednisone (P), in 2 patients cyclosporine A (Cy A) and MMF; in 7 patients Cy A, P, sirolimus; in 3 P and FK, in 4 patients FK and MMF, in 5 patients FK alone and in 1 patient Cy A alone.

The mean time on dialysis was 52.8 months (range 1–359 months) and the causes of renal failure were as follows: chronic glomerulonephritis (8 cases); nephroangiosclerosis (18 cases); polycystic kidney disease (2 cases); diabetic nephropathy (12 cases); chronic renal failure (18 cases); kidney myeloma (2 cases); interstitial nephritis (1 cases); and other (5 cases). The mean normalised dose of dialysis/treatment (Kt/V single-pool = 1.44) and the protein catabolic rate (PCR = 1.15) indicated an adequate dialysis prescription and nutrition. The dialyser membrane was modified cellulose without reuse.

Yeasts: Clinical C. albicans strain, identified by biochemical methods were subcultured on Sabouraud dextrose (SAB) agar (Oxoid S.p.A., Milan, Italy) to ensure viability and purity. Yeast cultures consisted entirely of blastoconidia and had a slight tendency to differentiate into pseudohyphae during the course of the experiments.

Drug: Caspofungin acetate (Merck Sharp&Dohme Ltd., Hoddesdon,UK) was dissolved in pyrogen-free water and stored at -20° C. Antifungal susceptibility testing was performed with both inoculum of 10CFU/ml, in accordance with the CLSI M27-A3 [7], and inoculum of 106CFU/ml to perform tests with phagocytes.

PMNs: PMNs were separated from lithium heparinized venous blood using Ficoll–Paque (Pharmacia S.p.A., Milan, Italy) and adjusted to 106 cells/ml in RPMI 1640 (Gibco Laboratories, Grand Island, NY, USA) [8]. Viability, determined by trypan blue exclusion, was greater than 95 %.

Influence of caspofungin on PMN phagocytosis and intracellular killing: The effect of caspofungin on the phagocytosis of radiolabelled yeasts [3H-uracil (specific activity: 1270 GBq/mmol; NEN Life, Milan, Italy)] by PMNs was investigated by incubating the yeasts (106 CFU/ml) and PMNs (106cells/ml) at 37°C in a shaking water bath in the presence of the MIC of caspofungin. Caspofungin-free controls were included. After 30, 60 or 90 min, phagocytosis was assessed [3, 9]. PMNs were centrifuged twice at 1200 rpm for 5 min to remove free blastoconidia and suspended in sterile water for 5 min; 100 µl samples were placed in scintillation fluid (Atomlight, NEN) and counted by spectophotometry. Radioactivity was expressed as counts/minute/sample. The percentage of phagocytosis at a given sampling time was calculated as follows: % phagocytosis = [(cpm in PMNs pellet)/(cpm in total fungal pellet)]×100 [3,9]. Intracellular killing was investigated by incubating yeasts and PMNs (1:1 ratio) for 30 min to allow phagocytosis to proceed. The PMN-yeast mixtures were centrifuged at 1200 rpm for 5 min and washed to remove extracellular blastoconidia. An aliquot of PMNs was lised by adding sterile water, and an intracellular yeast viable count was performed (time0). The killing values were expressed as the survival index (SI), which was calculated by adding the number of surviving yeasts at time0 to the number of survivors at timex, and dividing by the number of survivors at time0. According to this formula, if fungal killing was 100% effective, the SI would be 1 [3,9].

Statistical analysis: Statistical analysis was performed using GraphPad Prism v.3.00 for Windows (GraphPad Software, San Diego, CA). Results are expressed as mean ± standard error of the mean for 10 separate experiments each performed in quadruplicate. Evaluation of differences between test and control results was performed using Student’s unpaired t-test. P-values of <0.05 were considered statistically significant.

RESULTS

In all experiments the viability of PMNs remained unchanged throughout. MICs of CAS for the C. albicans clinical strain were 0.5 mg/mL with an inoculum of 103 CFU/mL and 2 mg/mL with an inoculum of 106 CFU/mL.

Based on our in vitro results, a diminished phagocytic efficiency was detected in PMNs from renal transplant recipients (RTRs), with reduced both phagocytosis (data not shown) and fungicidal activity towards intracellular C. albicans  in comparison with those of PMNs from healthy subjects (HSs; TABLE 1). In fact, PMNs harvested from HSs were able to kill ingested C. albicans  in ranges between 46 - 48 % (SIs = 1.52-1.54) during the 90 minutes of incubation, whereas PMNs from RTRs were able to kill C. albicans between 26 - 46 % (SIs = 1.54-1.74). These results confirm literature data that report an impairment of the phagocytic response in RTRs responsible for a highly susceptibility to fungal opportunistic infections [1,3,10]. Analogous result was done in haemodialysed patients (TABLE 2).

As the majority of systemically acting antifungal drugs, caspofungin did not significantly improve the fungal uptake by PMNs from HSs, RTRs and HDs, since yeast cells were phagocytozed at rates that were similar to those of the drug-free controls (data not shown).

Conversely, in the same experimental conditions, caspofungin had a marked effect on intracellular killing by PMNs of HSs (TABLES 3,4), resulting in increased numbers of killed blastoconidia for all three incubation times compared with those of the drug-free controls: during the 90 minute period the intracellular blastoconidial load was reduced by 71-75% (SIs = 1.25-1.29; p<0.01). When caspofungin was added to PMNs harvested from RTRs and HDs, the depressed fungicidal activity of PMNs was totally restored resulting in killing values of 56-67% (SIs = 1.33-1.44; p<0.01) for RTRs (TABLE 3)  and of 37 - 70 % (SIs = 1.30-1.63; <0.01) for HDs (TABLE 4)

 

Table 1. Intracellular killing of PMNs from healthy subjects (HSs) and renal transplant recipients (RTRs) against C. albicans.

  Survival Index ± SEM
Time HSs RTRs

30’

1.54 ± 0.03 (46%) 1.54 ± 0.1 (46%)

60’

1.53 ± 0.02 (47%) 1.65 ± 0.14 (35%)

90’

1.52 ± 0.03 (48%) 1.74a ± 0.08 (26%)

 

(%)  Percentages of yeasts killed by PMNs
a Significantly different from HS controls (p < 0.01)

 

Table 2. Intracellular killing of PMNs from healthy subjects (HSs) and  haemodialysed patients  (HDs) against  C. albicans.

  Survival Index ± SEM
Time HSs HDs

30’

1.54 ± 0.03 (46%) 1.70a ± 0.03 (30%)

60’

1.53 ± 0.02 (47%) 1.75a ± 0.02 (25%)

90’

1.52 ± 0.03 (48%) 1.73a ± 0.05 (27%)

 

(%)  Percentages of yeasts killed by PMNs
a Significantly different from HS controls (p ≤ 0.01)

Table 3. Effect of caspofungin on intracellular killing of PMNs from healthy subjects (HSs) and renal transplant recipients (RTRs) against C. albicans.

 

  Survival Index ± SEM
Time HSs RTRs

 

Controls Caspofungin Controls Caspofungin

 

  MIC=2mg/ml   MIC=2mg/ml

30’

1.54 ± 0.03 (46%) 1.29a ± 0.07 (71%) 1.54 ± 0.1 (46%) 1.44 ± 0.09 (56%)

60’

1.53 ± 0.02 (47% 1.28a ± 0.11 (72%) 1.65 ± 0.14 (35%) 1.42a ± 0.11 (58%)

90’

1.52 ± 0.03 (48%) 1.25a ± 0.07 (75%) 1.74 ± 0.08 (26%) 1.33a ± 0.11 (67%)

 

(%)  Percentages of yeasts killed by PMNs in absence and in presence of the antifungal drug
Significantly different from controls without drug (< 0.01)

 

Table 4. Effect of caspofungin on intracellular killing of PMNs from healthy subjects (HSs) and haemodialysed patients  (HDs) against C. albicans


  Survival Index ± SEM
Time HSs HDs

 

Controls Caspofungin Controls Caspofungin

 

  MIC=2mg/ml   MIC=2mg/ml

30’

1.54 ± 0.03 (46%) 1.29a ± 0.07 (71%) 1.70 ± 0.03 (30%) 1.63 ± 0.06 (37%)

60’

  1.53±0.02     (47%) 1.28a ± 0.11 (72%) 1.75 ± 0.02 (25%) 1.47a ± 0.03 (53%)

90’

1.52 ± 0.03 (48%) 1.25a ± 0.07 (75%) 1.73 ± 0.05 (27%) 1.30a ± 0.03 (70%)

 

(%) Percentages of yeasts killed by PMNs in absence and in presence of the antifungal drug
a  Significantly different from controls without drug (< 0.01).

CONCLUSIONS

The findings of this study show that caspofungin, a new echinocandin, is able to reset the depressed intracellular killing of polymorphonuclear cells from renal transplant recipients and haemodialysed patients.

These data suggest that caspofungin possesses interesting beneficial properties which make it suitable for the treatment of IFIs in patients with impaired components of the immune system characterized by high morbidity and mortality.

REFERENCES

  1. Tullio V., Cuffini A.M., Giacchino F., Mandras N., Roana J., Comune L., Merlino C., Carlone N.A. 2003. Combined action of fluconazole and PMNs from uremic patients in clearing intracellular Candida albicans. J. Chemother. 15:301-303.
  2. Tullio V., Cuffini A.M., Banche G., Mandras N., Allizond V., Roana J., Giacchino F., Bonello F., Ungheri D., Carlone N.A. 2008. Role of fosfomycin tromethamine in modulating non-specific defence mechanisms in chronic uremic patients towards ESBL-producing Escherichia coli. Int. J. Immunopathol. Pharmacol. 21:153-160.
  3. Tullio V., Mandras N.,  Scalas D., Allizond V., Banche G., Roana J., Greco D., Castagno F., Cuffini A.M., Carlone, N.A. 2010. Synergy of caspofungin with human polymorphonuclear granulocytes for killing Candida albicans. Antimicrob. Agents Chemother. 54:3964-3966.
  4. Ramage G., VandeWalle K., Bachmann S.P., Wickes B.L., Lopez- Ribot J.L. 2002. In vitro pharmacodynamic properties of three antifungal agents against preformed Candida albicans biofilms determined by time-kill studies. Antimicrob. Agents Chemother. 46:3634-3636.
  5. DeresinskiS.C.and Stevens D.A. 2003. Caspofungin. Clin. Infect. Dis. 36:1445-1457.
  6. Letscher-Bru V. and Herbrecht R. 2003. Caspofungin: the first representative of a new antifungal class. J. Antimicrob. Chemother. 51:513-521.
  7. Clinical and Laboratory Standards Institute. 2008. Reference method for broth dilution antifungal susceptibility testing of yeasts; third edition. Approved standard M27-A3. Clinical and Laboratory Standards Institute,Wayne,PA.
  8. Banche G., Allizond V., Giacchino F., Mandras N., Roana J., Bonello F., Belardi P., Tullio V., Merlino C., Carlone N., Cuffini A.M. 2006. Effect of dialysis membrane biocompatibility on polymorphonuclear granulocyte activity in dialysis patients. Nephrol. Dial. Transplant. 21:3532-3538.
  9. Cuffini A.M., Tullio V., Giacchino F., Mandras N., Scalas D., Belardi P., Merlino C., Carlone N.A. 2001. Impact of co-amoxiclav on polymorphonuclear granulocytes from chronic hemodialysis patients. Am. J. Kidney Dis. 37:1253-1259.
  10. Dongari-Bagtzoglou A., Dwivedi P., Ioannidou E., Shaqman M., Hull D., Burleson J. 2009. Oral Candida infection and colonization in solid organ transplant recipients. Oral Microbiol. Immunol. 24:249-254.
release  1
pubblicata il  18 settembre 2013 
da Franca Giacchino¹, Giuliana Banche², Chiara Merlino², Valeria Allizond², Daniela Scalas², Janira Roana², Giuseppe Garneri¹, Rosaria Patti¹, Vivian Tullio², Narcisa Mandras² and Anna Maria Cuffini²
(¹Nephrology and Dialysis Unit, Civil Hospital, Ivrea, Turin, Italy; ²Department of Public Health and Pediatrics, University of Torino, Turin, Italy)
Parole chiave: dialisi, infezione fungina, terapia antimicotica, trapianto renale
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