Indian Journal of Medical Microbiology, (2005) 23 (1):14-19
Special Article
HUMAN ANTI-RHINOSPORIDIAL ANTIBODY DOES NOT CAUSE METABOLIC INACTIVATION OR MORPHOLOGICAL DAMAGE IN ENDOSPORES OF RHINOSPORIDIUM SEEBERI, IN VITRO
*SN Arseculeratne, DN Atapattu, NB Eriyagama Abstract
This report describes the use of the MTT-reduction and Evan’s blue-staining tests for the assessment of the viability and morphological integrity, respectively, of rhinosporidial endospores after exposure to sera from rhinosporidial patients with high titres of anti-rhinosporidial antibody. Sera from three patients, with nasal, ocular and disseminated rhinosporidiosis respectively were used, with human serum without anti-rhinosporidial antibody for comparison, with or without added fresh guinea pig serum as a source of complement. All four sera tested, with or without guinea-pig serum, had no effect on the morphological integrity or the viability of the endospores and it is suggested that anti-rhinosporidial antibody has no direct protective role against the endospores, the infective stage, in rhinosporidiosis. This finding is compatible with the occurrence of chronicity, recurrence and dissemination that are characteristic of rhinosporidiosis despite the presence of high titres of anti-rhinosporidial antibody in patients with these clinical characteristics. The possible occurrence of humoral mechanisms of immunity that involve anti-rhinosporidial antibody with cells such as leucocytes and NK cells, in vivo, cannot yet be discounted, although the presence of high titres of anti-rhinosporidial antibody in patients with chronic, recurrent and disseminated lesions might indicate that such antibody is non-protective in vivo.
Key words – Rhinosporidium seeberi, anti-rhinosporidial antibody
Rhinosporidiosis in humans presents several Rhinosporidium seeberi and for the establishment of a enigmas that have yet to be explained: (a) the chronicity model of rhinosporidiosis in experimental animals, has of the disease that could last for several years, (b) contributed to the paucity of studies on the role of recurrence after surgical excision of the initial polyps that immune mechanisms in recovery from rhinosporidiosis. most commonly occur in the anterior respiratory There are also, hitherto, no standardised methods passages, and (c) occasional dissemination from these described for the assessment of the viability of sites to the limbs and viscera. Cell-mediated immune rhinosporidial ontogenic elements on which studies on (CMI) responses were recently demonstrated by the the protective effect of CMI and anti-rhinosporidial assay of lymphoproliferative responses to rhinosporidial antibody could be made in vitro. antigens and the T-cell mitogen Concanavalin A of peripheral blood lymphocytes of patients with There has been only one report on the viability rhinosporidiosis1 and by the foot-pad delayed-type assessment of rhinosporidial endospores that stated that hypersensitivity response to rhinosporidial antigens in the Nigrosin-Eosin stain was capable of differentiating 4 experimental mice.2 The occurrence of CMI responses between living and dead endospores. The was also suggested by the marked presence in monotetrazolium salt MTT (3-[4,5-dimethylthaizol-2-yl]- rhinosporidial polyps of CD8+ lymphocytes and TIA1- 2,5-diphenyltetrazolium bromide) has been used to containing CD8+ and CD57+ NK cells, with numerous assess the viability of mammalian cells, especially 5-7 8, 9 macrophages; these host-cell infiltrates were often lymphocytes and of mycelial and yeast fungi. This associated with degenerate rhinosporidial sporangia. 1 method depends on the production of a deep purple- The role of antibody mediated anti-rhinosporidial coloured formazan by the reduction of the MTT by immunity has however not been investigated although mitochondrial dehydrogenases of viable cells. This 8 high titres of anti-rhinosporidial antibody have been method was used by Levitz and Diamond to assess the detected in patients with localized or disseminated sensitivity of mycelial fungi to hydrogen peroxide, and 9 disease.3 The lack of a method for in vitro culture of by Jahn et al to assess the susceptibility of Candida albicans and Aspergillus spp. to anti-fungal agents.
*Corresponding author (email:
www.ijmm.org January, 2005 Arseculeratne et al – R.seeberi and anti-rhinosporidial antibody 15 finding that only the latter produced the formazan homogenate was centrifuged to deposit the endospores, derivative in both the endospore’s cytoplasm and in a the deposit was washed once in distilled water and the proportion of the spherical bodies within the endospore final pellet re-suspended in PBS to a density of that were provisionally identified as the electron dense approximately 2.5 x 106 endospores/mL as counted in an bodies – EDBs.11 We have also used the MTT method improved Neubauer blood cell counting chamber; a few to assess the sensitivity of rhinosporidial endospores developing endospores that were in the stage of early to biocides 12 and to anti-microbial drugs.13 juvenile sporangia were present.
This report describes the application of the MTT Immature and mature sporangia were absent; they and Evan’s blue-staining methods to the investigation were probably ruptured by the homogenisation. The of the effects of human sera from rhinosporidial patients, mature sporangia would have released their content of with or without added guinea pig serum as a source of endospores on rupture. complement, on the metabolic activity and morphological integrity of rhinosporidial endospores in vitro; untreated Test sera endospores and those treated with anti-rhinosporidial Sera from three human patients with serum, with or without added complement, showed rhinosporidiosis, stored at – 20°C, were used in normal staining with MTT and with Evan’s blue. We comparison with serum from a 1 year-old infant (obtained conclude that neither sera from normal (non- from a serum bank) that showed no anti-rhinosporidial rhinosporidial) persons nor anti-rhinosporidial sera from antibody. The rhinosporidial sera showed the following patients with rhinosporidiosis that contained anti- titres on dot-blot ELISA assays with anti-human rhinosporidial antibody inhibited MTT-reduction by the immunoglobulin-phosphatase conjugates3 – endospores and hence anti-rhinosporidial antibody was incapable of inactivating the endospores; this result is Disseminated rhinosporidiosis- IgG 1/640, IgM 1/60 in accordance with the occurrence of the predominant Recurrent nasal rhinosporidiosis – IgG 1/320, IgM 1/130 clinical characteristics of rhinosporidiosis – chronicity, Recurrent palpebral rhinosporidiosis – IgG 1/640, IgM 1/30, recurrence and dissemination despite the presence of IgA 1/60 high titres of anti-rhinosporidial antibody. 3 Incubation with sera Materials and Methods The normal and rhinosporidial sera were diluted 1/ Buffers 10 in PBS and used without heat-inactivation or added fresh, guinea pig serum as a source of complement. In 0.2M phosphate buffered-saline (PBS) at pH 7.2 was other experiments, the unheated sera were used at the used to suspend endospores, to dilute the normal and same dilutions in PBS containing fresh 1/10 normal rhinosporidial sera, and to dissolve MTT and Evan’s guinea pig serum. blue. To the centrifuged deposit from approximately 50 µL Endospores of the endospore suspension in 1.5 mL, capped, plastic microfuge tubes, was added approximately 150 µL of the Only endospores were examined as the possible diluted serum, followed by side-tapping of the tube for target of antibody-mediated destruction because (1) dispersion of the endospores. The tubes were incubated these are considered to be the infective stage of R. at room temperature (28°C ambient) for 36 hours. seeberi that gains access to susceptible sites from the pathogen’s natural habitat – ground water, and because Endospores, deposited from the original suspension, “auto-inoculation” 14 occurs on mucosal surfaces by re-suspended in PBS and incubated in a similar manner endospores when they are released from traumatised or were used as controls. excised polyps leading to the formation of satellitic rhinosporidial lesions, (2) sporangia have a pore for the The MTT test exit of mature endospores, through which passive In the assessment of lymphocyte proliferation, MTT ingress of the dyes might have occurred; (3) MTT- is used at a concentration of 5 mg/mL, which is diluted staining of sporangia was observed only in the to 0.5 mg/mL when the dye-solution is added to the wells endospores they contain. containing growth medium and lymphocytes. Since, in our tests, the dye-solution was added to the supernatant- Fresh endospores were obtained by homogenising free deposit of endospores, the concentration of MTT nasal rhinosporidial tissues, soon after surgery, in was reduced to 0.5 mg/mL. The MTT solutions were distilled water to lyse osmotically labile host cells. The freshly prepared.
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After incubation, the tubes were centifuged at 400 x g at room temperature (RT), and the endospore-pellets were washed thrice with PBS. To the pellets were added approximately 50 µL of MTT (0.5 mg/mL), the mixtures were agitated by side-tapping of the tubes and then incubated at 37oC in air for 3 hr. After incubation, a drop of the suspension was placed on a microscope slide under a cover-slip for microscopy at x 1000, under oil. The intensity of the deep purple formazan-staining in the Fig.1 Fig.2 cytoplasm and the spherical bodies (EDBs) within the endospore and the distribution of positively-stained endospores were assessed qualitatively and taken as indices of metabolic activity of the endospores. Quantitative counts of staining were not done as the host tissue cells in the endospore-suspension obscured some endospores. In addition to the intensity of staining, the morphology of the endospores (walls and spherical bodies) were also observed.
The Evan’s blue test. Fig.3 Fig.4 Two drops of 0.2 % Evan’s blue in PBS were added to the washed pellets of control (untreated) or serum- treated endospores, and the mixtures after agitation, were incubated at RT for 1 hr. A drop of the mixture was then placed on a microscope slide under a cover-slip for examination at x 1000 under oil. Results Control, serum-free endospores showed intense purple-staining of their EDBs with MTT, indicating their Fig.5 Fig.6 metabolic activity and hence viability after 36 hour of incubation in PBS (Fig.1) in over 95% of the endospores. Figure 1: Fresh endospores of R. seeberi after 36 hr. in PBS, With Evan’s blue the cytoplasm showed moderate stained with MTT, showing deep-purple staining of EDB (arrows) and cytoplasm. Initial magnification x 1000. staining while the EDBs stained deeply in endospores incubated with normal serum (Fig. 2). Figure 2: Fresh endospores of R. seeberi after 36 hr. of incubation with normal human serum without anti-rhinosporidial Endospores incubated in normal human serum antibody, stained with Evan’s blue showing staining of cytoplasm and EDBs. Unstained spherical bodies (arrows) are probably Lipid without added guinea pig serum (Fig. 3) and endospores Bodies. Initial magnification x 1000. incubated in normal human serum with added guinea pig Figure 3: Fresh endospores of R. seeberi after 36 hr. of serum (Fig. 4) showed deep staining of the EDBs and incubation with normal human serum without anti-rhinosporidial the cytoplasm with MTT in a similar proportion as in antibody, without guinea pig serum, stained with MTT showing untreated endospores. staining of EDBs and cytoplasm. Initial magnification x 1000. Figure 4: Fresh endospore of R. seeberi after 36 hr. of incubation Endospores that were exposed to a human serum with normal human serum without anti-rhinosporidial antibody, that contained high titres of anti-rhinosporidial antibody with fresh guinea pig serum, stained with MTT showing staining without added guinea pig serum (Fig. 5) or with added of EDBs and cytoplasm. Initial magnification x 1000. guinea pig serum (Fig. 6) showed deep staining of the Figure 5: Rhinosporidial endospores 36 hr. after incubation in EDBs and cytoplasm. The intensities of the staining of PBS with serum from a case (No.1) of disseminated the EDBs in the control endospores and its distribution, rhinosporidiosis with anti-rhinosporidial antibody, without guinea as well as of the serum-treated endospores were similar pig serum, showing deep-purple staining with MTT and hence indicating that neither the anti-rhinosporidial antibody- absence of inactivation. Initial magnification x 1000. free human serum nor the high-titred anti-rhinosporidial Figure 6: Rhinosporidial endospores 36 hr. after incubation in PBS with serum from a case (No.1) of disseminated serum inactivated the endospores. Negative results were rhinosporidiosis with anti-rhinosporidial antibody, with guinea pig obtained with all 3 sera from patients with serum, showing deep-purple staining with MTT and hence absence rhinosporidiosis. of inactivation. Initial magnification x 1000.
www.ijmm.org January, 2005 Arseculeratne et al – R.seeberi and anti-rhinosporidial antibody 17
The intensity of staining with Evan’s Blue of the without added complement in our experiments, is cytoplasm and the EDBs of the endospores exposed to therefore interpreted by us to mean that such antibody normal (anti-rhinosporidial antibody-free) serum or to has no inactivating, devitalising effect on rhinosporidial sera that contained anti-rhinosporidial antibody showed endospores; the morphological integrity of the no differences from that of the control endospores, endospores and their EDBs as also shown by staining indicating, as with the MTT stain, that no morphological with Evan’s blue, further indicates that anti-rhinosporidial abnormalities were produced by any of the sera tested. antibody does not cause morphological damage to the endospores. Discussion Some parallels probably exist between R. seeberi and In the absence of a method for the in vitro culture Toxoplasma. The encysted bradyzoites of Toxoplasma of R. seeberi, its “life-cycle” has been deduced from the persist for the life-time of the host 17, presumably evading appearances of the different ontogenic stages of the destruction by anti-Toxoplasma antibody that can pathogen in the rhinosporidial granulomata of diseased however kill the extracellular, non-encysted form.18 The hosts. The endospore that contains a thick wall with cyst that contains the bradyzoites is “surrounded by a chitin and cellulose, develops into the thin-walled dense argyrophilic wall enclosed within the host-cell juvenile sporangium (trophocyte) from which arise the cytoplasm” 19 and is thereby probably inaccessible to immature (intermediate and mature) sporangia. antibody; the bradyzoite cyst wall probably acts in a Endospore walls are different in structure and function manner similar to that of the rhinosporidial endospore’s from the walls of the cysts of parasites and the wall in excluding the penetration of antibody. A further endospores of bacteria that are resting, non-vegetative parallel of toxoplasmosis with rhinosporidiosis is that stages. The rhinosporidial endospores thus retain semi- despite high circulating antibody titres, permeability to nutrients and small molecules of dyes immunosuppressed patients develop disseminated such as MTT (MW 414). Endosporulation in the mature toxoplasmosis 19 recalling the fact that despite high anti- rhinosporidial sporangia leads to the formation of rhinosporidial antibody titres, dissemination occurs in thousands of endospores which repeat the life cycle rhinosporidiosis; in toxoplasmosis and rhinosporidiosis, when released from the mature sporangium. None of the the corresponding antibodies probably do not penetrate conventional in vitro culture methods has succeeded in into the cyst and the endospore respectively. providing serial propagation of the ontogenic stages and hence no culture methods are available for A further explanation of the failure of anti- assessment of their viability; hence we used the indirect rhinosporidial antibody to inactivate the endospores of method of MTT-staining for this purpose. R. seeberi, exists in a previous report that described the presence of human immunoglobulin bound to soluble 10 Evan’s blue was earlier shown to stain the antigens released by sonication of rhinosporidial cytoplasm and, more intensely, a proportion of the endospores and sporangia, and detected by immuno- spherical bodies within the endospore, that were blots and SDS-PAGE .20 It was pointed out that such 15, 16 regarded as the electron dense bodies – EDB. binding of host-protein could be a mechanism of evasion Spherical bodies that did not stain were regarded as the of immune attack by anti-rhinosporidial antibody in lipid bodies (LB). MTT was shown to stain areas in the diseased hosts. 21 This binding-phenomenon might also cytoplasm as well as some of the spherical bodies, that explain the absence of an inactivating effect of human were regarded as the EDB while the MTT-negative anti-rhinosporidial sera on the endospores, as found in 11 bodies were probably the LB. The reduction of MTT the present study. A similar phenomenon has been to the purple-coloured formazan depends on described of schistosomes in which older parasites have mitochondrial dehydrogenases of living cells and hence bound host-proteins; “They are only susceptible to the formazan production within the endospores was attack by specific antibody in vitro if they are not interpreted as indicating their viability. Levitz and coated with host protective antigens”. 22 On the other 8 Diamond used MTT to assess the effect of hydrogen hand, while binding was not found with rabbit peroxide on mycelial fungi and demonstrated that MTT- immunoglobulin, endospores in smears from negativity correlated with failure of growth of the rhinosporidial polyps were found 23 to be labelled with 9 peroxide-treated fungi in culture. Jahn et al used the experimental anti-rhinosporidial rabbit serum traced with MTT method to demonstrate that the effects of FITC-labelled anti-rabbit goat serum (Fig. 7) indicating antifungal agents on Candida albicans correlated well that endospores are antigenic and that they are reactive with MIC determinations. The retention of deep MTT- on their surface with anti-rhinosporidial serum that staining by the contents of the endospores despite contained anti-endospore antibody. However it is treatment with anti-rhinosporidial antibody, with or noteworthy that Figure 7 also illustrates the staining of
www.ijmm.org 18 Indian Journal of Medical Microbiology Vol.23, No.1 the endospore wall without staining the interior of the finding might imply the failure of such antibody to endospore; this failure might be attributable to the non- directly inactivate the endospores in vivo in infected penetration of the antibodies and the anti-rhinosporidial tissues. On the other hand other anti-rhinosporidial serum and tracer antibody-conjugate into the antibody-dependent mechanisms24 such as immune endospore’s interior, as perhaps with the endospores that opsonization, intra-phagocytic and extra-phagocytic failed to be inactivated in the tests with anti- killing, and antibody dependent cell-mediated rhinosporidial antibody. cytotoxicity could conceivably operate in vivo; the neutralization by antibody of soluble rhinosporidial products might also aid the process of resolution. Yet the presence of high titres of anti-rhinosporidial antibody in patients with chronic, recurrent and disseminated rhinosporidiosis 3 indicates that anti-rhinosporidial antibody is not protective.
The apparent ineffectiveness of anti-rhinosporidial antibody-containing sera from patients in inactivating endospores in vitro, might need further examination in view of recent findings on anti-mycobacterial antibody. In a reconsideration of the role of anti-Mycobacterium tuberculosis antibody in protection, Glatman, Freedman and Casadevall 25 pointed out that polyclonal serum preparations have had no consistently-demonstrable protective activity against some fungal infections, caused by Cryptococcus neoformans, Candida albicans and Pneumocystis carinii, but that monoclonal antibodies do have protective activity, with the possibility that in natural infections, a mixture of polyclonal antibodies with protective, non-protective or disease enhancing activities exists. It is a conceivable parallel that in rhinosporidiosis a similar effect operates, accounting for both the failure of antibodies in diseased Figure 7: Rhinosporidial endospores on a smear of discharge from a human naso-pharyngeal rhinosporidial polyp, treated patients to provide protection and polyclonal anti- with experimental anti-rhinosporidial rabbit serum followed by rhinosporidial sera in these patients to produce inhibition goat anti-rabbit IgG-FITC, showing labelling of endospore walls of R. seeberi, in vitro. (arrows), without staining of the endospore’s interior. Initial magnification x 1000. Acknowledgement
Our in vitro results suggest that anti-rhinosporidial We thank Dr P Balasooriya and Dr S Senanayake for antibody had no effect (sporicidal or sporolytic) on the supplies of excised rhinosporidial polyps, Dr NR de Silva for a gift of MTT. viability and integrity of rhinosporidial endospores; this
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