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Keywords:

  • adjuvants;
  • complete Freund's adjuvant;
  • experimental autoimmune encephalomyelitis;
  • Dark Agouti rats;
  • T lymphocytes

SUMMARY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Experimental autoimmune encephalomyelitis (EAE) is a well-recognized model for multiple sclerosis (MS) in humans. However, adjuvants used with encephalitogens to induce EAE produce non-specific effects interfering with the mechanisms involved in the autoimmune response to the central nervous system (CNS) tissue. It is therefore important to establish a more suitable model of EAE for analysis of autoimmune phenomena resembling those operative in MS. Here we report that EAE can be induced regularly in Dark Agouti (DA) strain of rats with spinal cord tissue without any adjuvant, as judged by both clinical and histological parameters. The incidence and severity of EAE depended on the origin of the encephalitogen, the rat versus guinea pig spinal cord homogenate being more efficient. Furthermore, EAE could be reinduced in animals which had recovered from disease that had been induced actively with encephalitogen alone, suggesting the role of adjuvant-generated non-specific mechanisms in resistance to reinduction of EAE. Thus, EAE induced in DA rats with encephalitogen alone provides a reproducible model for defining pathogenically relevant events in CNS autoimmunity devoid of the potentially misleading effects of adjuvants.


INTRODUCTION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Experimental  autoimmune  encephalomyelitis  (EAE)  is  an organ-specific cell-mediated autoimmune demyelinating disease of the central nervous system (CNS). It is characterized clinically by neurological deficits and histologically by lymphocytic and mononuclear cell infiltration of the CNS, an increase in blood–brain barrier (BBB) permeability and often demyelination (for review, see [1]). Based on the shared similarities in the disease course and histology, EAE has been used extensively as an animal model for the human disease multiple sclerosis (MS). There is considerable evidence that the initiating effector lymphocyte is a CD4+ T cell and that activation of a Th1-type cytokine cascade with resulting recruitment to the CNS and activation in situ of large numbers of lymphocytes and macrophages plays a key role in disease pathogenesis (for review, see [2]). EAE can be induced in susceptible strains of different species by sensitization with CNS myelin antigens or by the adoptive transfer of CNS myelin antigen-specific CD4+ T cells into naive syngeneic recipients (for review, see [3]). Active induction of EAE was accomplished with a number of myelin proteins, including myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), myelin-associated glycoprotein (MAG) and myelin oligodendrocyte basic protein [3]. However, rapid and regular induction of EAE with a single injection of an encephalitogen requires the co-administration of an adjuvant. The most frequently used induction protocol is based on complete Freund's adjuvant (CFA), which has greatly facilitated the study of this and many other organ-specific autoimmune diseases. However, CFA induces a strong inflammatory response and exerts, besides adjuvant activity, numerous immunomodulatory properties (reviewed in [4]). CFA also possesses immunogenicity by itself generating a strong anti-PPD response and may induce another autoimmune disease, adjuvant arthritis, in susceptible strains of animals [5]. Pretreatment with CFA can prevent several spontaneous and induced autoimmune diseases such as type I diabetes in non-obese diabetic (NOD) mice [6], experimental allergic encephalomyelitis [7,8], experimental autoimmune uveitis [9] and pristane-induced arthritis [10]. CFA injections generally result in severe skin pathology, including granuloma formation and necrosis. Following injection of CFA into the hind paw, responsiveness to noxious thermal and mechanical stimuli were greatly enhanced and CFA induced inflammation is a model used frequently to study chronic pain and the mechanisms of nociception [11]. These effects might blur clinical signs of EAE, such as ascending impairment of tail nociception [12]. CFA injection also increases BBB permeability with consequent perivascular extravasation of serum proteins in the CNS [13], thus interfering with the process of EAE induction. The observed alterations in both functional and molecular properties of the BBB were ascribed to the high production of anti-mannan antibodies in CFA-treated animals [14] or to the peripheral inflammation and consecutive inflammatory pain induced by injection of CFA [15].

Therefore the application of CFA, although irreplaceable component of induction protocols of many experimental animal models of autoimmune diseases, imposes obvious limitations for understanding the basic mechanisms involved in generation of autoimmune response to the CNS tissue. The aim of this study was to establish reproducible model of EAE that can be induced without the aid of CFA or any other adjuvant and to analyse the requirements for such induction as well as the characteristics of the induced disease.

MATERIALS AND METHODS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Experimental animals

Inbred Dark Agouti (DA) rats were maintained in the animal facility of the Institute for Biological Research ‘Sinisa Stankovic’ (Belgrade) in accordance with institutional guidelines. Animals were used when they were 10–16 weeks old, matched by age and sex and divided randomly into groups of three to five per cage. Rats were housed under conventional conditions with laboratory chow and water ad libitum and were given water by hand during the period of paralysis.

EAE induction

For induction of EAE, the encephalitogenic emulsion was prepared by mixing rat or guinea pig spinal cord homogenate (RSCH or GPSCH, respectively; 50% w/v in saline), and equal amount of saline (PBS) or complete Freund's adjuvant containing 1 mg/ml Mycobacterium tuberculosis (CFA, Difco Laboratories, Detroit, MI, USA) supplemented additionally with 4 mg/ml heat-killed M. phlei. Rats were immunized with 0·1 ml of the emulsion, given as a single intradermal (i.d.) injection in the right hind footpad. In some experiments, in order to reinduce the disease, the animals were injected i.d. in the left hind footpad with 0·1 ml of the emulsion containing RSCH + CFA, as indicated in the Results.

Clinical evaluation

Rats were monitored daily for clinical signs and clinical scoring from 0 to 4 was used as follows: 0 = no clinical signs; 1 = flaccid tail; 2 = hindlimb paresis; 3 = complete bilateral hindlimb paralysis often associated with incontinence, and 4 = moribund state or death of an animal. Intermediate scores were assigned if neurological signs were of lower severity than observed typically. Several parameters of disease were examined to evaluate the severity of EAE: incidence, the number of rats within a group that developed a clinical score of 1 or greater in comparison to the starting number of rats in that group; mean day of onset, the mean day that affected rats within a group first developed clinical signs of disease; clinical score, clinical signs graded from 0 to 4 for individual rat within a group on a given day; mean maximum severity, the mean of the maximum daily score that each rat in a group developed over the course of the experiment; and mortality, the number of rats within a group that died or were sacrificed as a result of severe EAE.

Histological analysis

For histological evidence of EAE, three to five rats from each group were sacrificed on day 18 post-immunization. Spinal cords were removed, fixed in 10% neutral buffered formalin and embedded for sectioning. Serial transverse sections (5 µm in thickness) were stained with haematoxylin and eosin. The presence of infiltrating cells was determined by a trained investigator, in the blinded fashion. All sections were examined using standard bright-field optics.

T cell proliferation assay

T cell proliferative responses were assessed in draining popliteal lymph nodes (DPLN) isolated from rats 2 weeks after immunization. Cells were cultured in flat-bottomed 96-well plates at a density of 3 × 105 per well. Culture medium consisted of RPMI-1640 supplemented with 5% fetal calf serum (FCS), l-glutamine and antibiotics (all from Sigma Aldrich Chemie GmbH, Steinheim, Germany). Cultures were incubated at 37°C for 72 h in the presence or in the absence of the antigens in concentrations as indicated in the Results. Antigens used for stimulation were rat MBP (kindly provided by Professor H. Wekerle, Max-Planck Institute for Neurobiology, Martinsried, Germany), PLP peptide 139–154 and MOG peptide 35–55 (generous gift from Dr D. Sun, Kentucky Lions Eye Center, University Louisville, Louisville, KY, USA). The cultures were pulsed with 1 µCi of [3H]methyl-thymidine (specific activity 6·7 Ci/mmol; ICN Pharmaceuticals, Irvine, CA, USA) for the final 18 h. Cells were then harvested on glass fibre filters and incorporation of [3H]methyl-thymidine was measured by liquid scintillation counting. The results were expressed as the average counts/min (cpm) of cultures set in triplicates with or without in vitro stimulation ± standard deviation of the mean (s.d.).

Statistical analysis

The results were expressed as the mean ± s.d. Significant differences in disease parameters were evaluated by anova (daily mean clinical score and disease index), Student's t-test (mean day of onset) and χ2 test (incidence of the disease, mortality and body weight loss). Significant differences were accepted for *P < 0·05 and **P < 0·005.

RESULTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Clinical signs of EAE in rats immunized with and without CFA

Initial studies were conducted in DA rats by comparing the ability of encephalitogenic emulsion containing CFA versus equal amount of encephalitogen without adjuvant to induce EAE. All control rats immunized with homologous SCH with adjuvant containing Mycobacteria developed disease, thus confirming a high susceptibility to EAE of this strain. The intensity of clinical signs of EAE varied from extremely severe in some experiments, as shown in Fig. 1 depicting disease course in individual animals (Fig. 1b), with high mortality rate (5/6) to milder forms characterized with paresis and paralysis only (not shown). Interestingly, when rats were injected with RSCH without any adjuvant, the clinical signs of EAE also appeared in all tested animals (Fig. 1a). There was no significant difference in the day of onset between groups of rats, but in comparison to animals immunized with SCH and CFA the disease induced in the absence of adjuvant was usually milder, with lower maximal disease score (Fig. 1). Development of EAE signs was accompanied by body weight loss in all experimental groups and maximum weight loss coincided with paralysis (not shown).

image

Figure 1. Disease course in individual animals immunized with RSCH in the presence or absence of CFA. Groups of six rats were immunized with RSCH in saline alone (a), or with RSCH-CFA (b). Clinical score for each individual rat was recorded daily throughout 28 days. Indicates death of the animal.

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Eight separate experiments were conducted involving immunization without CFA of a total of 67 DA rats (Table 1). The overall incidence of disease was 87·5%. The mean day of onset ranged between 9·2 and 15·3 days over all eight experiments. In only one of eight experiments mean maximal score of diseased animals was below 2, while all others ranged between 2·3 and 3 (Table 1). Thus, induction of EAE in DA rats with rat SCH in PBS was highly efficient and reproducible.

Table 1.  Clinical EAE in DA rats immunized with RSCH-PBS
Exp. no.IncidenceaOnsetbMaximal scorec
  • a

    Number of rats with clinical signs of EAE/total number of rats;

  • b

    mean day ± s.d. when first signs of EAE appeared;

  • c

    mean maximal clinical score ± s.d. in diseased rats.

1 6/610·5 ± 2·22·7 ± 0·5
212/1910·7 ± 2·62·3 ± 1·0
3 5/510·8 ± 1·12·6 ± 0·5
4 4/4 9·2 ± 0·53·0 ± 0·0
5 6/611·0 ± 1·32·7 ± 1·0
610/1010·1 ± 1·82·6 ± 0·6
7 7/1015·3 ± 2·61·2 ± 0·5
8 6/7 9·2 ± 0·72·7 ± 0·2

Susceptibility to EAE induction without adjuvant depends on the origin of encephalitogen

In order to characterize further a model of EAE that can be induced without the aid of CFA or any other adjuvant and determine whether the susceptibility to EAE is influenced by species origin of encephalitogen, we studied comparatively susceptibility to EAE induction in DA rats immunized with either RSCH or GPSCH. Table 2 illustrates the higher incidence, severity and mortality that resulted from immunization with homologous SCH compared to immunization with GPSCH. Clinical signs of EAE (mean score 2·5) appeared in the majority of rats injected with RSCH (77%), while GPSCH induced a mild disease (mean score 1·3) in only 10% of rats.

Table 2.  Susceptibility to EAE induction without CFA in DA rats depends on the origin of encephalitogen a
Spinal cordIncidenceb (%)OnsetcMaximal scored
  1. a Cumulative data obtained from two separate experiments; b number of rats with clinical signs of EAE/total number of rats (percentage); c mean day ± s.d. when first signs of EAE appeared; d mean maximal clinical score ± s.d. in diseased rats; *statistically significant by χ2 test (P = 0·013).

Rat19/24 (79)11·4 ± 3·52·3 ± 0·9
Guinea pig 3/30* (10)13·0 ± 10·41·3 ± 0·5

Histological evidence of mononuclear cell infiltration

The CNS histopathology of spinal cords from rats immunized in the absence of adjuvant is shown in Fig. 2. At day 18 after immunization, a large number of infiltrating cells were detected in RSCH-saline immunized rats, confirming that characteristic pathohistology developed even in the absence of any adjuvant.

imageimage

Figure 2. Representative microscopic photographs of the spinal cord stained with haematoxylin and eosin showing mononuclear cell infiltration (arrowheads).

T cell response in rats immunized with RSCH without the aid of adjuvant

While the strong inflammatory reaction, as judged by redness and swelling of the injected hind paw, was observed in rats immunized with SCH-CFA, no macroscopic evidence of local inflammation was present in rats injected with encephalitogen alone. Similarly, cellularity of popliteal lymph node draining the site of injection was consistently different between the two examined groups in several repeated experiments. Thus, the mean numbers ± s.d. of cells in the lymph node draining the site of inoculation in control animals immunized with emulsion containing CFA on days 8, 15 and 22 were 21·1 ± 4·9, 38·9 ± 2·6 and 26·5 ± 11·7 million cells per rat, while in rats immunized without adjuvant, the numbers were 6·1 ± 5·4, 7·2 ± 4·2 and 3·0 ± 1·8 millions, respectively (P = 0·002). However, the functional studies of lymphocytes, as evaluated by [3H]thymidine incorporation of in vivo primed T cells stimulated in vitro with myelin antigens did not show correlation of activation of autoreactive T cells with the inflammatory reaction. Proliferation of DPLN cells derived from individual rats SCH-immunized with or without CFA 15 days before was assessed after the 72 h of culture in the presence of rat MBP, PLP- and MOG-peptides, or without antigen. As shown in Fig. 3, in vitro T cell-proliferative responses to three different myelin antigens were obtained, regardless of the absence or presence of adjuvant during in vivo priming. These data indicate that in spite of lack of inflammation, antigen-specific lymphocytes were primed efficiently.

image

Figure 3. Proliferative response of DPLN lymphocytes to antigens. Rats were immunized with RSCH with (full bars) or without CFA (open bars). DPLN lymphocytes were collected after 15 days of immunization and 3 × 105 cells were incubated at 37°C in medium alone (0), or in the presence of 10 µg/ml of rat MBP, PLP-peptide 139–154 and MOG-peptide 35–55, as indicated. Proliferation was assessed with [3H]thymidine incorporation after 72 h of culture. Results from a representative experiment are presented as the mean cpm (s.d. for three animals per group.

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Reinduction of EAE in rats immunized with RSCH-PBS

It has been shown previously [16,17] that rats recovered from EAE induced by conventional immunization with adjuvant containing Mycobacteria are resistant to the reinduction of the disease. In contrast to this, in three separate experiments animals that had been immunized with SCH in the absence of adjuvant developed signs of disease after a second challenge with encephalitogenic emulsion containing CFA (Table 3). However, reimmunized rats showed significantly milder clinical signs in comparison to control immunized rats, but interestingly with earlier onset, although this difference did not reach the level of statistical significance (Table 3). Thus, it appears that encephalitogen alone does not possess the capacity to induce suppressive mechanisms capable to completely prevent reinduction of EAE in DA rats.

Table 3.  Reinduction of EAE in DA rats previously immunized with RSCH-PBS
Exp. no.I immunizationaII immunizationa
Maximal IncidencebOnsetcscoredMaximal IncidencebOnsetcscored
  • a

    Rats were immunized with RSCH-PBS (I immunization) and 30–35 days later with RSCH-CFA (II immunization);

  • b

    number of rats with clinical signs of EAE/total number of rats;

  • c

    mean day ± s.d. when first signs of EAE appeared;

  • d

    mean maximal clinical score ± s.d. in diseased rats;

  • *

    statistically significant by Student's t-test (P < 0·001) versus corresponding control group of rats.

19/914·7 ± 2·11·6 ± 0·9 9/910·8 ± 0·92·8 ± 0·6*
13/1312·2 ± 3·23·8 ± 0·4
23/3 8·3 ± 0·92·7 ± 0·9 3/312·3 ± 0·92·5 ± 0·9
 4/411·0 ± 4·23·5 ± 2·2
37/710·4 ± 5·32·5 ± 2·1 7/7 9·0 ± 7·71·2 ± 1·6*
 4/412·0 ± 2·83·0 ± 0·0

DISCUSSION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

In the present study we have demonstrated for the first time that EAE can be induced efficiently in DA rats with spinal cord homogenate without the aid of CFA or any other adjuvant. The susceptibility of DA rats to EAE induction with SCH-PBS depended on the origin of CNS tissue, the homologous tissue being more efficient encephalitogen. The clinical manifestations of disease were accompanied by histological lesions. Moreover, EAE could be reinduced in animals recovered from disease which appeared after immunization with SCH-PBS.

The use of encephalitogen emulsified in CFA is the most conventional way of EAE induction and the severity of induced disease depends on the amount of Mycobacteria in CFA [18]. However, DA rats have been shown to develop EAE when spinal cord homogenate [19] or MBP peptide 63–81 [20] were administered in IFA, while this procedure in other susceptible strains and species generally led to tolerance induction [21]. To this we add evidence that EAE can be induced in DA rats without any adjuvant if a single injection of homologous spinal cord tissue was used as encephalitogen. Although the first model of acute disseminated encephalomyelitis was induced experimentally in monkeys without adjuvant, repeated injections of brain extracts were required to produce neurological signs [22]. Later on only a few reports showed a respectable incidence of EAE in rats following sensitization to spinal cord alone [23,24] although the development of disease in these models required several injections of spinal cord homogenate and was enhanced by i.p. pretreatment with Bordetella pertussis[23]. Even more, omission of CFA from the challenge inoculum precluded the development of clinical encephalomyelitis and also conferred solid resistance against induction of disease by subsequent challenge with nervous system tissue plus CFA in otherwise susceptible strains of laboratory animals [25].

The other bacteria or bacterial components were used as efficient adjuvants for the induction of EAE such as B. pertussis[26], pertussis toxin (PT) [27], muramil dipeptide (MDP) [28] and mycobacterial glucolipid 6, 6′-trechalose-dimycolate [17]. Similarly to CFA, beside adjuvant activity they all possess multiple properties which may interfere with and preclude the pathogenesis of EAE. Thus, pretreatments with both MDP [29] and PT [30] were shown to protect otherwise susceptible animals against EAE.

Bacterial adjuvants also interfere with the actual large-scale analyses of messenger RNA levels at the site of disease [31]. The comparison of data obtained from studies in MS [31,32] and EAE [33] have been precluded by the fact that CFA and PT used as adjuvants in the latter model might have contributed to the alterations in gene transcription that were observed in CNS tissue, as suggested by Steinman and Zamvil [34]. Therefore, the induction of EAE described here provides the model which avoids all inconveniences imposed by the use of bacterial adjuvants mentioned above.

Recent studies have identified the molecular nature of many bacterial adjuvants, including CFA, and ascribed most of their effects to the triggering the nonclonal receptors of the innate immune system such as toll-like receptors [35], thus activating dendritic cells to provide signals necessary for lymphocyte activation and differentiation [36]. We cannot rule out the contamination of encephalitogenic spinal cord homogenate used in our experiments with bacteria and/or their components that might signal ‘infectious non-self’ and induce maturation of dendritic cells, thus activating autoimmune cells. However, products released by necrotic cells and stressed or damaged tissues can act as powerful adjuvants as well [37]. It might be assumed that necrotic cells within spinal cord homogenate provided the requisite maturation signal to dendritic cells. Our results suggest that the threshold in the magnitude of either ‘infectious non-self’ or ‘danger’ signals necessary for dendritic cell maturation might be extremely low in DA rats, contrary to other susceptible strains and species which require much stronger signal to achieve the same effect.

The other, non-mutually exclusive explanations for the observed results are that DA lymphocytes have much lower requirements for activation and/or differentiation of autoreactive lymphocytes, and that the balance between proinflammatory and down-regulatory cytokines produced in this strain favours the first. This is in accordance with results previously published by us and others showing exquisite susceptibility of DA rats to the induction of several organ-specific autoimmune diseases such as EAE [38], experimental autoimmune uveitis [39], adjuvant arthritis [40] and multiple low-dose streptozotocin-induced diabetes mellitus [41], related probably to unique immunological and/or genetic characteristics such as high production of IL-2 [42] IFN-γ[43] and TNF-α[44] by DA cells. Indeed, in spite of significant difference in local inflammatory reaction in rats challenged with encephalitogen containing CFA or without it, both groups of rats mounted significant T cell-proliferative response to all tested myelin antigens.

Although organ-specific antigens are generally highly conservative, some discrete amino acid variations between species might influence the pathogenic potential of these antigens in inbred strains of experimental animals. Numerous studies have convincingly documented the striking difference between different species and strains within species concerning their respective capacities to develop EAE when immunized with same encephalitogen (for review, see [45]). There is ample evidence that guinea pig myelin antigens are the most potent encephalitogens in Lewis rats [46]. However, several differences between DA and Lewis rats, both susceptible to EAE, have been reported [20,47,48]. In order to better characterize our model and find out whether the susceptibility of DA rats to EAE induced without adjuvant is influenced by species origin of encephalitogen we compared encephalitogenic properties of rat or guinea pig SCH. Our results, showing the stronger encephalitogenic potential of homologous versus heterologous SCH in DA rats are in accordance of the data obtained when classical procedure of immunization with encephalitogenic emulsion containing CFA was applied in this strain (our unpublished results and [49]]. These findings might be explained as a consequence of previously described strain differences between Lewis and DA rats in presentation of encephalitogenic epitopes and/or TCR recognition events leading to EAE [47,48].

It is well known that animals after recovery from actively induced EAE become resistant to the active reinduction of disease and various mechanisms have been postulated to account for this phenomenon (for review, see [3]). However, in this study we demonstrated that DA rats which recovered from EAE that had been induced with homologous SCH without adjuvant and then immunized with encephalitogenic emulsion containing CFA, developed clinical signs of disease. Neurological signs in rechallenged rats followed a typical ascending course of EAE. The disease was milder but first signs appeared earlier in rechallenged rats than in naive control animals, thus resembling disease actively induced in Lewis rats after passive transfer of MBP-sensitized cells [50]. The earlier onset of EAE observed in DA rats after rechallenge might be ascribed to the reactivation of memory cells. The overall reduction in disease severity in rechallenged rats may be explained by specific suppressive mechanisms acting either on the periphery [51] or at the level of target tissue [50]. However, all non-specific mechanisms otherwise induced with CFA in first immunization and operative in resistance to reinduction were excluded in the model described here. Recent evidence demonstrated convincingly that immunoprotective effect of CFA requires inducible nitric oxide (NO) synthase production of large quantities of NO [52]. In classical attempts to actively reinduce EAE, mycobacterial antigen-specific memory T cells were reactivated and stimulated NO production which consecutively down-regulated T cell proliferation. These inhibitory mechanisms might be lacking in the absence of CFA in the first immunization and therefore the reinduction of EAE is possible.

Taken together, the data presented here demonstrate that EAE can be efficiently and reproductively induced in DA rats without the use of CFA, an immunologist's ‘dirty little secret’, as Janeway [53] named it. The disease induced in DA rats without any adjuvant provides reproducible experimental model for understanding the basic mechanisms involved in generation of autoimmune response to the CNS tissue, without limitations imposed by application of adjuvants, thus representing one of the most reliable rodent models of MS.

ACKNOWLEDGEMENTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

This work was supported by grants no 1664 and 2020 from the Ministry for Science, Technology and Development of Serbia.

REFERENCES

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
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