IJMM July 07.Indd
IndianJuly-September Journal of 2007 Medical Microbiology, (2007) 25 (3):267-71 267 Brief Communication
THE USE OF MTT [3-(4, 5-DIMETHYL-2-THIAZOLYL) -2, 5-DIPHENYL - 2H- TETRAZOLIUM BROMIDE]-REDUCTION AS AN INDICATOR OF THE EFFECTS OF STRAIN-SPECIFIC, POLYCLONAL RABBIT ANTISERA ON CANDIDA ALBICANS AND C. KRUSEI
*SN Arseculeratne, DN Atapattu, R Kumarasiri, D Perera, D Ekanayake, J Rajapakse Abstract
There is only scanty data on the effects of speciÞ c antibody, with or without complement, on Candida albicans or Candida krusei in cell-free systems in vitro, although previously published work has shown that speciÞ c antibody mediates anti- Candida immunity in vivo by inhibition of adherence to host cells or surfaces and by the promotion of phagocytosis and intra-phagocytic killing.
The MTT (3-[4, 5-dimethyl-2-thiazolyl] -2, 5-diphenyl -2H- tetrazolium bromide)-reduction method as a test of the viability of fungi was used to investigate the effect of complement, normal serum and immune serum on these two species of Candida that are of increasing importance as opportunistic pathogens. We report that normal rabbit serum or strain-speciÞ c, polyclonal anti-Candida rabbit antibody, with or without guinea pig complement, did not cause the reduction of total cell- mass or of the viability of either C. albicans or C. krusei, in vitro as determined by the MTT-reduction test. Complement alone without speciÞ c antibody, also, had no such effect on these two Candida species.
Key words: Strain-speciÞ c antibody, complement, Candida albicans, Candida krusei, viability, in vitro
Candida albicans and C. krusei are of increasing Candida monoclonal antibodies in vitro; that appears to be the importance as opportunistic pathogens on account of the only report based on the use of MTT for this purpose. increasing prevalence of states of immune compromisation. This investigation was done to provide a greater understanding The investigations described here with C. albicans and of humoral defences against infections by Candida species. C. krusei and their strain-specific antibodies, were done to investigate the effect of sera containing strain-speciÞ c As far as we are aware, there are only two reports on anti-Candida antibodies on the viability of C. albicans and the effects of speciÞ c antibody on Candida cells in vitro.1,2 C. krusei, in vitro. The tests were based on the capacity of It is generally assumed that antibody and complement viable cells to reduce MTT to formazan which was assayed alone (i.e., without cells) cannot kill Candida3 and that, in by spectrophotometric quantitation of optical density (OD) general, the role of antibody immunity in fungal(www.medknow.com). infections after its extraction with acid-propanol, with the OD taken as a is a controversial subject.4 Although the role of B-cells and measure of the metabolic status and the total, viable mass of antibody has been studied, understanding of its relative the Candida cells. importance has not ensued.5 This PDFa site is hosted available by forMedknow freeMaterials download and Publications Methods from The MTT [3-(4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl -2H- tetrazolium bromide]-reduction test has been used C. albicans and C. krusei were isolates from clinical earlier on Candida albicans after treatment with heat, material and identiÞ ed by the API system. Stock cultures on formaldehyde, hypochlorous acid or amphotericin B6 and to Sabouraud agar were sub-cultured on brain heart infusion o determine the sensitivity of Candida albicans and Aspergillus (BHI) broth for –three to six days at 37 C. 7 spp. to therapeutic anti-fungal agents. MTT-reduction was Preparation of anti-Candida sera demonstrated to correlate with the viability of the antifungal agent-treated Candida cells on culture.6 Moragues et al2 also Strain-specific antibodies were used in view of the used the MTT-reduction to investigate the effects of anti- heterogeneity of strains of C. albicans and because the protective efÞ cacy of antibodies is dependent on epitope- *Corresponding author (email:
www.ijmm.org 268 Indian Journal of Medical Microbiology vol. 25, No. 3 with antigen (100 µg protein per dose) in Freund’s complete complement with and without complement preservative. adjuvant, followed after one week by antigen in Freund’s 7. Cells with 10% formalin (40% formaldehyde) in PBS incomplete adjuvant, weekly for four weeks followed by two incubated at room temperature for 18h were used as a booster doses with an interval of one week. positive-control for inhibition of MTT-reduction.9
Pre-immunisation and post-immunization sera were Twenty-Þ ve µL of PBS were added when necessary to assayed for antibody with their corresponding Triton-extracted equalize total volumes. Cells with heated guinea pig serum antigen by the Immuno dot-blot method, with monoclonal rather than cells in PBS alone, were used as a basal control anti-rabbit total Ig-peroxidase conjugate (Sigma, MO, USA) for comparison with cells that were treated with pre- or post- as tracer and 3-amino-9-ethyl carbazole (Sigma, MO, USA) as immunisation sera and/or complement because the former substrate-chromogen. The antisera against C. albicans and C. suspensions showed signiÞ cantly higher cell-content probably krusei had titres of anti-Candida antibody of over 800. due to their protein content and were therefore comparable with the serum-containing samples. The initial dilutions of antisera (1/100) in PBS were heated at 56oC for 20 min to inactivate residual complement, The tubes, after side-tapping to mix the cell-deposit with before addition to the Candida cell deposits. Each batch of the reagents, were incubated statically at room temperature guinea pig serum was assayed for complement activity by the (RT, 28oC ambient) for 18h. conventional anti-sheep red cell haemolysin method. Estimation of total cell mass and cell-viability The pre-immunization sera had no detectable anti-Candida antibody at this initial dilution (1/100). Total cell-counts in blood-cell counting chambers, as indicators of lysis, were not done as the Candida cells were Reagents often clumped. The MTT-reduction test, on the other hand, 0.2M phosphate buffered saline (PBS) at pH 7.4 was used provided a measure of both the total viable cell mass and its to dilute sera and dissolve MTT. Anti-Candida antibodies were metabolic capacity, irrespective of clumping of the yeast cells titrated by Immuno-dot-blot assay.8 and hence only this test was used as a measure of total cells and their viability in these experiments. Aliquots of freshly obtained guinea pig (gp) sera without added preservative were stored at -20oC. Aliquots The MTT test of guinea pig sera preserved with Richardson’s complement The centrifuged deposits of control or treated cells o preservative, were also stored at -20 C. The guinea pig serum were washed twice with PBS and were then treated with that was used as a source of complement had no detectable approximately 50 µL of the MTT solution (0.5 mg/mL PBS); anti-Candida antibody. the tubes, after mixing of the contents by side-tapping, were o Solutions containing 0.5 mg/mL of MTT (Sigma, MO, incubated at 37 C for three hours. The tubes containing MTT- USA) in PBS were used for viability assays on the deposits of cell mixtures were centrifuged (400 x g for 10 minutes) to the Candida cells. deposit the cells, the supernatant MTT solution was pipetted (www.medknow.com).out and then acid-isopropanol (95 mL isopropanol with 5 Tests with antisera mL 3N HCl) was added to the coloured cell deposit. After stirring of the acid-alcohol-treated deposit with a glass rod, Centrifuged (400g x 10 minutes) deposits from 25 µL of the mixture was allowed to react for Þ ve minutes, followed the Candida cellsThis in triplicate PDFa site or quadruplicateis hosted available were by treated forMedknow freeby centrifugation download Publications at 400 g forfrom 10 min. One hundred microlitre with 25 µL volumes of the following reagents in a typical of the purple-blue coloured supernatant that contained the experiment: solubilized formazan in each sample was added to a well in a 96-well microtitre plate for spectrophotometry at 570 nm 1. heated guinea pig serum (1/20, basal control). in an ELISA reader. Acid-isopropanol was used as a blank in 2. unheated guinea pig serum (1/20, complement) the readings. 3. pre-immunization (rabbit) serum (1/100) 4. pre-immunization (rabbit) serum (1/100) with added (1/20) Tests were repeated with each Candida strain, with or guinea pig serum without complement, at least twice. 5. post-immunization (rabbit) serum (1/100) Statistical comparisons 6. post-immunization (rabbit) serum (1/100) with added (1/20) guinea pig serum Statistical analysis was done using Student’s “t” test for the The diluted rabbit sera in (c), (d), (e) and (f) were heated, comparison of mean values (SPSS, version 10.01) of the ODs after dilution, at 56oC for 20 min to inactivate residual of the supernatants from tube-1 as basal cell control versus complement. Tests were done on each Candida species with tubes 2-7, for each species of Candida.
www.ijmm.org July-September 2007 Arseculeratne et al - Antibody Effects on Candida species in vitro 269
Results strain-speciÞ c antibody, without complement (sample 5) and with complement (sample 6), no reduction of OD was found. C. krusei The absence of a reduction of OD with samples containing Table 1 shows the results from a typical experiment in antiserum, with or without complement, indicated that speciÞ c which C. krusei and preserved guinea pig serum as a source antibody did not cause the inactivation or lysis of C. krusei. of complement were used. Ten percent formalin, as a positive C. albicans control biocide, gave a highly signiÞ cant decrease of MTT- reduction. Similar results (Table 2) were obtained with C. albicans. With unheated gp serum in sample 2, the OD was higher Ten percent formaldehyde as a potent control biocide gave a than (not signiÞ cant) with heated gp serum (control sample 1) highly signiÞ cant reduction of OD (sample 7). With unheated probably indicating a higher growth-promoting effect of the gp serum (sample 2) the OD value was (non-signiÞ cantly) unheated serum. The absence of a reduction of OD indicated higher than in heated serum (control sample 1) probably that complement alone had no inhibitory effect and that there because the unheated serum was more growth-promoting than were no thermo labile agents with anti-Candida effects in the heated serum. The absence of a reduction of OD indicated normal serum. that complement alone had no inhibitory effect and that there were no thermolabile agents with anti-Candida effects in Pre-immunization serum alone (sample 3) also did not normal serum. reduce the OD; on the contrary the OD was signiÞ cantly higher than that with the control sample 1 containing heated With pre-immunization (rabbit) serum (sample 3) the OD gp serum (and with sample 2 that contained unheated gp was signiÞ cantly higher suggesting, that rabbit serum was more serum), an effect that could conceivably be due to higher growth-promoting than gp serum. In sample 4 that contained growth promoting capacity of rabbit over guinea pig serum. both rabbit and gp sera the OD was signiÞ cantly higher probably due to increase of growth resulting from the increased volume Pre-immunization serum with complement (sample 4) of serum (50 µL) than in samples 1, 2 or 3 (25 µL). SpeciÞ c showed a minor, non-significant reduction of OD. With antibody without complement (sample 5) or with complement
Table 1: Formazan-formation from MTT by Candida krusei after exposure to strain-speciÞ c polyclonal rabbit antiserum with or without preserved guinea pig (p-gp) complement and to 10% formalin Sample treatment Mean OD ± SD* Comparison (P) Inference Heated p-gp serum 388.75 ± 57.99 p-gp serum 417.50 ± 38.23 1-2 (0.44) NS, no reduction of OD Pre-immunization serum 506.75 ± 77.13 1-3 (0.05) S, no reduction of OD Pre-immunization serum with p-gp serum 381.25 ± 13.77 1-4 (0.810) NS, reduction Immune serum 478.00 ±(www.medknow.com). 71.75 1-5 (0.101) NS, no reduction of OD Immune serum with p-gp serum 394.00 ± 42.28 1-6 (0.888) NS, no reduction of OD 10% formalin 83.00 ± 8.49 1-7 (<0.000) S, marked reduction of OD Mean values of opticalThis density PDFa (OD) site from is hosted quadruplicates,available by *OD forMedknowmeasured free at 570 download nm, PublicationsNS - Non-signi Þ fromcant, S - SigniÞ cant
Table 2: Formazan-formation from MTT by Candida albicans after exposure to strain-speciÞ c polyclonal rabbit antiserum with or without preserved guinea pig (p-gp) complement and to 10% formalin Sample treatment with Mean OD ± SD* Comparison (P) Conclusion Heated p-gp serum 513.50 ± 11.15 p-gp serum 521.00 ± 26.17 1-2 (0.617) NS, no reduction of OD Pre-immunization serum 593.25 ± 31.38 1-3 (0.003) S, no reduction of OD Pre-immunization serum 654.50 ± 70.26 1-4 (0.007) S, no reduction with p-gp serum of OD Immune serum 631.50 ± 34.31 1-5 (0.001) S, no reduction of OD Immune serum with p-gp serum 711.50 ± 89.91 1-6 (0.005) S, no reduction of OD 10% formalin 13.50 ± 6.35 1-7 (<0.000) S, marked reduction of OD Mean values of optical density (OD) from quadruplicates, *OD measured at 570 nm, NS - Non-signiÞ cant, S - SigniÞ cant
www.ijmm.org 270 Indian Journal of Medical Microbiology vol. 25, No. 3
(sample 6) did not cause a reduction of OD indicating that there cells to be protected by chitin with carbohydrate polymers; was no antibody-mediated lysis or inactivation of C. albicans. endospores of R. seeberi that possess chitin in their walls as The signiÞ cantly higher OD values in samples 4, 5 and 6 would, in Candida, is also resistant to damage by speciÞ c antibody, again, probably be due to a greater growth-promoting capacity in vitro.14 of rabbit over guinea pig serum, for C. albicans more than for C. krusei. The increase rather than a decrease of OD of the Kozel described the role of Mannan-binding protein formazan in some samples could indicate a higher cell mass (MBP), in normal sera that binds, lectin-wise, to carbohydrate due to transformation of the yeast phase to the mycelial phase in structures in yeasts leading to activation of the antibody- the presence of the respective sera; our experiments, however, C1q-independent classical pathway, but stated that “direct were concerned primarily with an inhibition of the yeast and killing via formation of a membrane-attack-complex has not 15 therefore a reduction of formazan-OD. The yeast-mycelial been convincingly demonstrated with pathogenic fungi”. transformation would be of relevance to the protective role of Fungi are resistant to lysis by the terminal components of the 16 antibody in vivo. complement system even in the presence of speciÞ c antibody. C. albicans activates complement on incubation with normal Replicate tests with both Candida species gave similar serum but does not undergo complement-mediated lysis or results. killing by serum.17 Our results also indicate that complement alone had no inhibitory or lytic effect on either Candida Discussion species in vitro. However, it was demonstrated experimentally in vivo, by Han et al.,18 that complement is essential for In our experiments, counts of yeast cells in blood-cell protection against haematogenously disseminated candidiasis counting chambers and viable counts in culture as additional by speciÞ c antibody. tests of cell viability, after their exposure to the antisera and complement, were abandoned on account of gross clumping of Protective effects of antibody in vivo could include the cells that caused wide variations in the counts in replicate inhibition of yeast-mycelial transformation,4 prevention samples. The pitfall of viable counts of Candida cells was of adherence to host cell surfaces,3 toxin neutralization, referred to by Chilgren et al.,1 who quoted the Þ ndings and antibody-dependent cell-mediated cytotoxicity,11 neutralization conclusions of Louria and Brayton (1964) who used viable of immuno-modulating fungal products, neutralization of counts of Candida albicans on culture plates. They interpreted extracellular proteases and promotion of opsonic phagocytosis. the reduction of counts of the yeast after exposure to normal Anti-Candida antibody with complement has been claimed human sera found by the latter authors as having indicated a to be protective against disseminated candidiasis, although lethal action of the sera on the yeast, whereas Chilgren et al.,1 Murphy10 earlier stated that the protective nature of anti- explained the reduction of counts as rather having been due Candida antibodies in both mucosal and systemic forms of the to clumping and not a lethal action. Moragues et al.,2 used disease is another unsettled issue. monoclonal anti-Candida antibodies in vitro with the MTT- reduction test as an indicator of cell viability and demonstrated The controversies regarding the anti-fungal effects three anti-Candida activities including a fungicidal effect. of antibodies appear to be explainable on the basis of the However, whether the antibodies they used were strain existence of multiple antibodies of varying epitope-speciÞ city, speciÞ c, was not stated. (www.medknow.com).their concentrations and ability to fix complement. Our demonstration of the absence of an in vitro anti-Candida effect Several non-speciÞ c antifungal agents have been described, of polyclonal serum needs re-investigation with monoclonal, including collectins, defensins and microbicidal peptides and strain-speciÞ c antibodies, in view of the comment that “the metal chelators Thisor iron-binding PDFa site proteins.is hosted available10,11 An inhibitoryby forMedknow freeuncertainty download regardingPublications the importancefrom of antibody in immunity factor that was active on Cryptococcus neoformans only against fungi despite several decades of study suggests that in high concentrations of serum, that caused disruption experiments with polyclonal sera may not be conclusive”.4 and fragmentation of the yeast cells and which was not It should, however, be noted that even among monoclonal complement or immunoglobulin, has been described.12 antibodies, protective and non-protective antibodies exist.4 Moreover, investigations with monoclonal antibodies in 1 On the other hand, Chilgren et al., used high concentratons vitro are needed because the identiÞ cation of the protective, of sera, 0.1 mL yeast cell-suspension to 1 mL of serum, non-protective or deleterious effects of these antibodies but found that normal sera neither affected the growth has thus far been made through in vivo rather than in vitro 13 of, nor were lethal to C. albicans. Lehrer and Cline also experimentation. reported that normal human serum did not kill C. albicans. GreenÞ eld, reviewing the literature, stated that human serum Our results indicate that complement alone or strain- is not fungicidal against Candida.5 Using the MTT-reduction speciÞ c antibody with or without complement, caused neither method, we too found no evidence of inhibition of either cell lysis nor metabolic inhibition of either C. krusei or C. Candida species by antibody-free rabbit or guinea pig sera in albicans as indicated by the MTT-reduction assay. Similar the higher dilutions that we tested. Murphy10 considered fungal conclusions were derived from experiments with complement,
www.ijmm.org July-September 2007 Arseculeratne et al - Antibody Effects on Candida species in vitro 271 speciÞ c antibody and endospores of Rhinosporidium seeberi.14 of Candida albicans and Aspergillus species by a simple It is noteworthy that rhinosporidial endospores and Candida microtiter menadione-augmented 3-(4,5-dimethyl-2-Thaizolyl)- cell walls are thick and possess chitin. 2,5-diphenyl-2H-tetrazolium bromide assay. J Clin Microbiol 1995;33:661-7. Our results with Candida spp. are in contrast to the lysis of 8. Arseculeratne SN, Kumarasiri PV, Rajapakse RP, Perera NA, gram negative bacteria, This contrast is probably attributable Arseculeratne G, Atapattu DN. Anti-rhinosporidial antibody to differences in the structure of the cell walls of Candida and levels in patients with rhinosporidiosis and in asymptomatic R. seeberi on the one hand and bacteria on the other. persons in Sri Lanka. Mycopathologia 2004;158:157-64.
While the protective role of speciÞ c anti-Candida antibody 9. Arseculeratne SN, Atapattu DN. The assessment of the viability remains controversial, a consensus exists that speciÞ c cell- of the endospores of Rhinosporidium seeberi with MTT (3- [4,5- mediated immunity rather than humoral immunity is the major dimethyl-2-thiazolyl] 2,5-diphenyl-2H-tetrazolium bromide). mechanism of adaptive immunity against fungal infections and Mycological Res 2004;108:1423-30. in candidiasis. 10. Murphy JW. Mechanisms of Natural Resistance to human Acknowledgement pathogenic fungi. Annu Rev Microbiol 1991;45:509-38. 11. Romani L. Immunity to fungal infections. Nat Rev Immunol We thank The National Science Foundation, Sri Lanka 2004;4:1-23. for a research grant to SNA, Gehan J. Panagoda for the two Candida strains, Sumati Puvanendiran for the guinea pig sera 12. Baum GL, Artis D. Growth inhibition of Cryptococcus neoformans and Navaratne B. Eriyagama for technical assistance. by cell-free human serum. Am J Med Sci 1961;241:613-6.
References 13. Lehrer RI, Cline MJ. Interaction with Candida albicans with human leucocytes and serum. J Bact 1969;98:996-1004. 1. Chilgren RA, Hong R, Quie PG. Human serum interactions with Candida albicans. J Immunol 1968;101:128-32. 14. Arseculeratne SN, Atapattu DN, Eriyagama NB. Human anti- rhinosporidial antibody does not cause metabolic inactivation or 2. Moragues MD, Omaetxebarria MJ, Elguezabal N, Sevilla MJ, morphological damage in endospores of Rhinosporidium seeberi Conti S, Polonelli L, et al. A monoclonal antibody directed in vitro. Indian J Med Microbiol 2005;23:14-9. against a Candida albicans cell wall mannoprotein exerts three anti-Candida albicans activities. Infect Immun 2003;71:5273-9. 15. Kozel TR. Activation of the complement system by pathogenic fungi. Clin Microbiol Rev 1996;9:34-46. 3. Challacombe S. Immunity in Oral Candidosis. In: Samaranayake LP, MacFarlane TW, editors. Oral Candidosis, Wright: London; 16. Levitz SM. Overview of host defenses in fungal infections. Clin 1990. p. 104-23, 113, 110. Infect Dis 1992;14:S37-42.
4. Casadevall A. Antibody immunity and invasive fungal infections. 17. Shoham S, Levitz SM. Immune responses to fungi. In: Rich RR, Infect Immun 1995;63:4211-8. Fleicher TA, Shearer WB, Kotzin BL, Schroeder HW Jr, editors. Clinical Immunology. Principles and Practice. vol. 1. Mosby: 5. GreenÞ eld RA. Host defence system interactions with Candida. New York; 2001. p. 31.1-31.7. J Med Vet Mycol 1992;30:89-104. (www.medknow.com). 18. Han Y, Cutler JE. Antibody response that protects against 6. Levitz SM, Diamond RD. A rapid colorimetric assay of disseminated Candidiasis. Infect Immun 1995;63:2714-9. fungal viability with the Tetrazolium salt MTT. J Infect Dis 1985;152:938-45.This PDFa site is hosted available by forMedknow free download Publications from Source of Support: Nil, Conß ict of Interest: None declared. 7. Jahn B, Martin E, Stueben A, Bhakdi S. Susceptibility testing
www.ijmm.org