Antiarthritic effect of VIP in relation to the host resistance against Candida albicans infection
Yana Zafirova1,
Martin Yordanov2 and
Reni Kalfin1
1 Laboratory of Neuropeptides, Institute of Physiology, Bulgarian Academy of Sciences, 23 G. Bonchev Str., 1113 Sofia, Bulgaria
2 Department of Immunology, Institute of Microbiology, 26 G. Bonchev Str., 1113 Sofia, Bulgaria
Correspondence to: R. Kalfin; E-mail: rkalfin{at}hotmail.com
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Abstract
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Vasoactive intestinal peptide (VIP) is one of the prospective candidates for clinical application in rheumatoid arthritis (RA). Its antiarthritic effect is associated with the suppression of inflammatory and autoimmune responses. The ability of VIP to trigger a shift towards Th2 immunity suggests that anti-infectious host resistance might be affected. In the present study VIP was applied at the initiation and at the established phase of collagen-induced arthritis (CIA). Mice developed Th2 dominant anti-collagen response. The susceptibility to primary and secondary Candida albicans infection was determined after VIP administration at the established CIA. The percentage of survivors, kidney colonization, cytokine secretion by splenocytes and specific antibody synthesis were assessed. Reduced TNF-
production but not IFN-
and IL-10 was observed after the first challenge with the pathogen in CIA mice treated with VIP while the percentage of survivors was not significantly changed. The adaptive immune response was impaired in VIP-treated mice as they were more susceptible to reinfection, showed increased kidney colonization and suppressed anti-Candida IgG antibody production.
Keywords: anti-infectious resistance, cytokines, collagen-induced arthritis
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Introduction
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The antirheumatic drugs currently in use have various undesirable effects and even after long term treatment do not stop joint destruction. Arthritic disease includes two components, inflammatory and autoimmune, so agents capable of inhibiting both processes would be of best value. One approach to gain a remission of the arthritic process is the suppression of proinflammatory cytokine secretion or blocking of their cell surface receptors (13). An alternative strategy explores the changes in Th1/Th2 profile. In general, it is considered that Th1 cytokines have a pathogenic role in rheumatoid arthritis (RA), while Th2-derived cytokines possess suppressive properties (47). The alteration of the balance to Th2 response can ameliorate the arthritic process.
Vasoactive intestinal peptide (VIP) seemed to be a very promising candidate for the treatment of RA due to its ability to induce a shift towards Th2 anti-inflammatory response (8). One of the first immunomodulatory effects ascribed to VIP concerned the production of different cytokines (9). Delgado et al. (10,11) by using a murine model of collagen-induced arthritis (CIA) demonstrated that VIP reduces the frequency of arthritis, the severity of symptoms and prevented joint damage. VIP downregulates the proinflammatory cytokines and upregulates anti-inflammatory acting cytokines. The physiological significance of VIP is confirmed by the observation that its level is elevated in serum and joints in correlation with the progression of CIA (11).
The main goal of new antiarthritic therapies is the influence on specific immune responses without causing a generalized immunosuppression. The efforts to gain a shift to Th2 response in order to reduce autoimmunity, may provoke the manifestation of opportunistic pathogens. The significant reduction of key cytokines which play an important role in host defense mechanisms, like TNF-
and IFN-
, leads to an increased susceptibility to infections. Candida albicans is one of the most common opportunistic pathogens in humans. Under normal conditions C. albicans is considered to be a part of the normal gastrointestinal flora (12). The incidence of mucosal and systemic infections has raised dramatically due to the widespread use of immunosuppressive agents (13). In murine models of experimental C. albicans infection the development of protective immune response is mediated by Th1 cells, while Th2 dominance leads to a nonprotective response (1417). The aim of the present study was to examine the effect of VIP administration in mice with CIA on the host resistance against C. albicans infection. We found that mice treated with VIP at the established phase of CIA expressed partially impaired protective response to the yeast, observed as higher kidney colonization, increased susceptibility to reinfection and downregulation of anti-Candida IgG antibody production. These data indicate that the antiarthritic effect of VIP might be attended with a predisposition to lethal Candida infection at least at the early period after its administration.
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Methods
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Mice
Male mice strain ICR (CD-2) were used for the study. Animals were housed under standard conditions of temperature (21 ± 2°C), relative humidity (55 ± 10%) and kept on a standard commercial pellet diet with water ad libitum. The animal protocols were approved by the Animal Care Commission at the Institute of Microbiology in accordance with the Helsinki declaration.
Induction of CIA and VIP treatment
CIA is usually induced by an intradermal immunization with CII in CFA followed by i.p. boost after 21 days. We have used a previously described model of arthritis induced in newborn mice (18). Briefly, bovine type II collagen (bCII, Sigma Chemicals Co., St Louis, MO) was solubilized to 1 mg/ml by stirring overnight in 0.1 M acetic acid at 4°C. Animals were injected subcutaneously at the back with 50 µg bCII two days after birth. Arthritis development was observed by inspection three times a week until 7 weeks of age. Clinical severity of arthritis was qualified according to a grade scale from 0 to 3: 0, no swelling; 1, slight swelling; 2, well expressed swelling; 3, strong swelling and erythema. A mean arthritic score value among arthritic mice was calculated at the end of observation. Arthritic mice were sacrificed, and the hind paws were fixed in 10% buffered formalin and decalcified in HNO3 for 12 days. The tissue was then processed and embedded in paraffin. Five-micrometer sections were stained with hematoxylin and eosin (H&E) using standard methodology. Histopathological changes were graded on a scale from 03, based on the cell infiltration in the synovial cavity and synovial tissue, cartilage destruction and bone erosion.
VIP (Sigma Chemicals Co., St Louis, MO) was injected i.p. in a dose of 2 nmol in 50 µl PBS at days 3, 5, 7, 9 and 12 after birth or i.p. in a dose of 10 nmol (in 0.1 ml)/daily from day 42 to day 48.
Experimental infection
A virulent clinical isolate 562 of Candida albicans (Institute of Infectious and Parasitic Diseases, Sofia, Bulgaria) was used in the study. Prior to inoculation, the yeast was grown for 48 h on Saubouraud's broth at 37°C. For infection, cells were washed twice in saline, and diluted to the desired density to be injected via the tail vain in a volume of 0.2 ml/mouse. The concentration of yeast suspension was determined by measuring the extinction at OD620. A standard curve was drawn by plotting the extinction against the number of colony forming units (CFU) and was used to calculate inoculum concentration. Mice with CIA were treated i.p. with 10 nmol/daily of VIP from day 42 to day 48. Three days later animals were inoculated i.v. with different doses of C. albicans (primary infection). At day 14 the survivors (1 x 105 CFU in primary infection) were reinfected i.v. with 1 x 106 or 2 x 105 CFU (secondary infection). The course of infections was followed over 14 days, when the percentage of survivors was calculated. Subgroups of five animals were killed on day 8 of each infection and both kidneys were removed aseptically and homogenized in sterile saline in a tissue grinder. The number of viable C. albicans cells was determined by plating serial dilutions on Sabouraud agar plates. The CFU were counted after 48 h of incubation at 37°C, and expressed as log CFU/kidney. Individual sera were collected on day 14 of primary and secondary infections for determination of anti-Candida IgG antibody levels.
Cytokine assays
Cell suspensions were prepared from freshly removed spleens and erythrocytes were depleted by lysing buffer (0.15 M NH4Cl/10 mM KHCO3/0.1 mM EDTA, pH 7.2). Splenocytes (5 x 106 cells/ml) were incubated in 24-well Falcon plates (Becton Dickinson, Switzerland) in RPMI-1640 media (Gibco BRL, Grand Island, NY), containing 10% fetal bovine serum (Sigma Chemical Co., St. Louis, MO), 100 U/ml penicillin, 100 µg/ml streptomycin (Sigma), 2 mM L-glutamine, 25 mM ß-mercaptoethanol, and 10 mM HEPES. Cytokine concentration was determined in the supernatants after 4 days of culture in the presence or absence of 50 µg/ml bCII. As a negative control splenocytes were cultured with 50 µg/ml of OVA. Quantitative ELISA for IL-4 was performed using paired mAbs according to the manufacturer's recommendation (Pharmingen, San Diego, CA). ELISA kits were used for TNF-
, IFN-
, IL-2 and IL-10 (Euroclone, Paignton, Devon, UK). Minimum detectable concentration was less than 25 pg/ml for TNF-
, 15 pg/ml for IFN-
, 5 pg/ml for IL-2, 10 pg/ml for IL-4 and 20 pg/ml for IL-10.
ELISA assays for detection of serum IgG anti-collagen and anti-Candida antibodies
For determination of anti-collagen antibodies in sera, plates were coated with 0.1 ml of bovine cartilage derived type II collagen (Sigma) solution in PBS (10 µg/well) at 4°C overnight. Then the plates were washed and blocked with 2% BSA at 37°C for 2 h. After washing, serum samples were diluted 1:100 in PBSTween 0.5% BSA and added to the wells (0.1 ml). After incubation overnight at 4°C the plates were washed and 0.1 ml of peroxidase-labelled goat anti-mouse IgG (1:10 000 diluted) or anti-mouse IgG1 (1:1000), or IgG2a (1:2000) (Cappel, Durham, NC) were added for 2 h. The reaction was developed by o-phenylenediamine and after 30 min stopped by adding of 4N H2SO4. The absorbance was measured at 492 nmol in ELISA reader.
For determination of anti-Candida IgG level in serum the ELISA test plates were coated with 1 x 105/ml glutaraldehyde-inactivated yeast cells (treated overnight at 4°C with 2% glutaraldehyde). After that they were washed with PBS/0.05% Tween 20 and blocked by adding 0.1 ml of PBS/2% BSA for 1 h at room temperature. Test sera appropriately diluted in PBS/1% BSA (0.1 ml) were added for 2 h at 37°C. The assay was proceeded further as described above for anti-bCII IgG antibodies.
DTH reaction
Mice with CIA untreated or treated with 2 nmol VIP (days 3, 5, 7, 9, 12 after birth) were injected at day 42 with 100 µg bCII into the hind paw. After 24 h the footpad thickness of the injected paw was measured and the difference with the control paw, injected with 0.01 M acetic acid, was calculated in mm.
Statistical analysis
Results are expressed as mean ± SD and were evaluated by Student's t-test. Differences in number of survivors were estimated by
2 test.
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Results
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VIP altered the anti-bCII response during the induction and established phase of CIA
Arthritis was induced by immunization of newborn mice with a high dose of heterologous collagen. In order to determine whether VIP could prevent the development of anti-collagen response, the peptide was administered on days 3, 5, 7, 9 and 12 after birth in a dose of 2 nmol. Alternatively, mice were treated on five consecutive days with 10 nmol VIP in the established phase of CIA (days 4248). Clinical signs of arthritis and histopathological joint changes were determined at the age of 67 weeks. Mice treated with VIP showed lower incidence of CIA compared to the untreated group (Fig. 1). Only 25% of the animals injected as newborns with VIP and 20% of mice treated as adult expressed swelling without erythema. Histopathologically in untreated mice there was an occurrence of infiltrating cells in the synovial cavity and tissue, cartilage inflammation, and in some cases bone erosion was observed (Fig. 1B). In VIP-treated mice less cell infiltration and synovial inflammation was registered.

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Fig. 1. Mean arthritic and histological score in control CIA mice and after VIP application at the onset of arthritis and at the established phase (A). In parenthesis the percentage of mice with CIA in each group is given. Typical joint changes in control mice (B, left) and in VIP-treated mice (B, right) 78 weeks after the induction of CIA. Data are representative of three experiments, 79 animals per group.
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The secretion of Th1 and Th2 cytokines by splenocytes was determined
1 month after the last VIP application. Data in Fig. 2 show that the level of TNF-
was not affected in both schedules. IFN-
secretion was strongly diminished as a result of VIP administration. When mice were treated in the established phase of CIA, IFN-
level was sometimes lower than in untreated animals. In contrast, the levels of Th2 cytokines were significantly elevated. The amount of IL-4 was very low on day 42 of CIA, but it was twice as high in the VIP-treated group, simultaneously with increased IL-10 level. IL-4 was nearly undetectable in CIA mice at day 80 and it was slightly enhanced in mice treated with VIP as adult. On day 80 of CIA, the VIP-untreated group showed an elevated level of IL-10 and a twice higher level was observed in the VIP-treated group. Cytokine response was bCII-specific, since no reaction to OVA was detected.

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Fig. 2. Mice were immunized 2 days after birth with 50 µg bCII and divided into four groups (911 mice/group). Spleen cells were harvested from 42 day old mice, (newborn CIA); treated with 2 nmol VIP on days 3, 5, 7, 9 and 12 after birth (newborn CIA + VIP); from 80 day old mice (adult CIA) and 80 day old mice, treated with 10 nmol VIP daily from day 42 to 48 (adult CIA + VIP). Splenocytecs (5 x 106/ml) were cultured in the presence of bCII (50 µg/ml) or OVA (50 µg/ml) for 4 days. As the cytokine secretion by OVA stimulated splenocytes was under 20 pg/ml these data are omitted. Spontaneous cytokine release in the absence of stimuli was below the detection limit of the assay. Each bar represents the mean ± SD from three determinations, *P < 0.001.
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The development of CIA is attended with the production of anti-collagen antibodies, characterized by a high IgG2a to IgG1 ratio. The administration of VIP in the initial phase of arthritis did not affect autoreactive IgG2a synthesis, but elevated IgG1 level (Fig. 3A). The ability of VIP to influence the cellular immunity was confirmed by the weak DTH reaction to bCII observed in VIP-treated mice (Fig. 3B).

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Fig. 3. Anti-bCII specific IgG, IgG1 and IgG2a levels (A) in sera collected on day 42 after immunization with bCII (2 days postpartum, CIA) or immunized with bCII and treated with 2 nmol VIP on days 3, 5, 7, 9 and 12 (CIA + VIP). DTH-reaction to bCII (B) on day 42. Data represent the means ± SD from groups of seven mice. *P < 0.01.
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Course of C. albicans infection in CIA mice treated with VIP
Mice with CIA were treated with VIP (10 nmol/daily) from day 42 to day 48 of age and 3 days after the last injection, the animals were i.v. inoculated with C. albicans. The course of infection was similar in control mice and CIA mice treated or untreated with VIP (Fig. 4A). Further, the survivors at day 14 were challenged i.v. with 1 x 106 CFU or 1 x 105 CFU of C. albicans. CIA mice treated with VIP showed higher susceptibility to secondary infection as compared to control and untreated with VIP groups (Fig. 4B and C).

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Fig. 4. Mice immunized with bCII were treated from day 42 to 48 with 10 nmol VIP daily and 3 days after the last treatment were i.v. inoculated with 1 x 105 C. albicans cells (primary infection, n = 15). The course of infection was followed 14 days when the survivors were reinfected i.v. with 1 x 106 or 1 x 105 CFU (secondary infection, n = 10). *P < 0.01, 2 test vs control with infection.
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Next, we assessed the level of Th1 and Th2 cytokine production in cultures of splenocytes obtained from mice 14 days after the first (1 x 105 cells) or second (1 x 106 cells) inoculation, and stimulated in vitro with inactivated yeast (Fig. 5). The primary infection was characterized by high levels of TNF-
and IFN-
, simultaneously with high secretion of IL-10 and minimal amount of IL-4. In CIA mice treated with VIP the secretion of TNF-
was powerfully suppressed, while the release of IFN-
and IL-4 was increased. IL-10 was detected in significant amount in both, control and VIP-treated groups. In respect to reinfection, TNF-
levels were similar in control and VIP-treated mice, but IFN-
production was significantly reduced in the latter one. The secretion of IL-4 and IL-10 was augmented in arthritic mice receiving VIP.

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Fig. 5. Mice with CIA were treated with 10 nmol VIP from day 42 to day 48 of age. Three days later they were inoculated i.v. with 1 x 105 C. albicans (primary infection) and the survivors were reinfected at day 14 with 1 x 106 cells (secondary infection). Splenocytes were obtained on day 14 in both infections and cytokine secretion was determined after in vitro stimulation with heat-inactivated C. albicans. Data are the mean ± SD from five mice in a group and are representative for one of two experiments. *P < 0.05.
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The contamination of kidneys with the yeast was determined at the peak of infection (day 8). Higher yeast burden showed in VIP-treated mice in both, primary and secondary infections as compared to control and CIA-mice (Fig. 6A).

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Fig. 6. Kidney colonization and serum anti-Candida IgG level. On day 8 of primary (1 x 105 CFU) and secondary (1 x 106) i. v. infections, kidneys were excised and the contamination was determined according to CFU (A). Data represent the mean ± SD from seven mice per group (two experiments). On day 14 sera from primary or secondary infected mice were collected and anti-Candida antibody titers were determined (B). Data represent the mean ± SD from nine individual sera in each group. *P < 0.05.
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The humoral immune response was also affected after the first and second challenge with the pathogen as a result of VIP application. While in primary infection the serum IgG anti-Candida antibody level was slightly reduced in VIP-treated mice, the suppressive effect was clearly expressed after the reinfection (Fig. 6B).
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Discussion
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Collagen-induced arthritis in mice is an experimental model that in many but not all aspects resembles rheumatoid arthritis in humans (19). Different schedules used for its induction in susceptible strains of mice include the application of CIA. Previously, we have established that the single immunization of newborn mice with a high dose of bCII provokes an arthritic inflammation leading to joint destruction (18). In this model the additive effect of adjuvant is avoided. The arthritis proceeded in a mild form in dependence to a greater extent on T cell immunity than on humoral response. The present results revealed that 6 weeks after bCII injection Th1 cytokines, TNF-
and IFN-
prevailed upon Th2 cytokines, IL-4 and IL-10. In the late established phase of CIA, Th1 response to bCII was dominant, although IL-10 level was also increased. In the present experiments, VIP was applied under the schemes and doses similar to those used by Delgado et al. (10) in CIA model. In order to determine the influence of VIP on the onset of arthritis, mice were treated with VIP as newborns, just after the collagen injection. In another group of mice, VIP was administered at the age of 6 weeks, when the arthritis was already established. Under both schedules VIP strongly decreased the incidence of arthritis simultaneously with a lesser extent synovial and cartilage inflammation.
In both schedules of treatment VIP did not affect TNF-
secretion while IFN-
level was greatly suppressed. Simultaneously, VIP successfully stimulated IL-4 and IL-10 when applied at the earliest phase while the late treatment potentiated only IL-10 secretion. The antiarthritic effect of VIP was confirmed by the changed IgG1 to IgG2a ratio of anti-bCII antibodies in favor of IgG1 isotype. The inhibited DTH reaction to bCII reflects VIP action on bCII sensitive T cells. The ability of VIP to favor Th2 immunity raised the question of how the anti-infectious host resistance was affected. Candida albicans is an appropriate pathogen to study the relationships between antiarthritic effect of VIP and the susceptibility to infection in the light of Th1/Th2 balance. Studies in murine candidiasis suppose that the resistance to infection is associated with Th1 response and the production of IFN-
. A failure to develop protective immunity is a consequence of predominant Th2 response and the production of IL-4 and IL-10 (2022). As it is not proven that the same Th1 or Th2 populations participate in autoimmune and protective responses, direct elevation of host resistance as a result of arthritic state, should not be postulated. Our results showed that the percentage of survivors in the primary and secondary infections was not statistically different in mice with CIA compared to nonarthritc. The high amounts of TNF-
and IFN-
secreted by the splenocytes from CIA mice in vitro, reflect the existence of collagen-specific cells capable to generate Th1 response, but it did not result in increased protection. CIA mice treated with VIP and infected 3 days later showed similar susceptibility to primary infection, but were less resistant to reinfection compared to nonarthritic and to the CIA group that did not received VIP. The simple classification of cytokines as protective vs suppressive (nonprotective) does not describe the pleiotropic role that several cytokines express during the course of infection. TNF-
is essential for the early control of infection as one of its actions is to induce an expression of costimulatory molecules on phagocytes (23). Deficiency is not related to the induction of Th2 response but correlated with the failure to develop protective Th1 response, associated with defective IL-12 responsiveness (24,25). In murine candidiasis, IFN-
is necessary to overwhelm IL-4-induced inhibition of IL12ß2 receptor in Th1 cells (26). Data about the role of IFN-
in the protection against C. albicans remain very controversial. The exogenous treatment of mice with IFN-
and its overproduction increased morbidity and mortality as well as yeast colonization in the kidneys (22,27). The resistance to primary infection is not affected in IL-4 or IL-10 deficient mice, while the susceptibility to reinfection is increased (28). Mice resistant to primary and secondary infections expressed increased production of IL-2 and IFN-
, and reduced production of IL-4 and IL-10 by CD4+ T cells (14) but for the optimal development of innate and adaptive anti-Candida response endogenous IL-4 and IL-10 are required (28,29). When IL-4 deficient mice are treated with IL-4 in the late phase of C. albicans infection their resistance to reinfection is greatly increased and fungal burden in organs is decreased. IL-4 directly affects neutrophils and macrophages to release IL-12. Also, IL-10 upregulates the expression of CD40 costimulatory molecule on accessory cells and IL-4 elevates the expression of B7-2 on macrophages, both sensitive to IL-12 (27). Thus, IL-4 and IL-10 may regulate the IL-12-dependent Th1 response in murine candidiasis. Moreover, the impaired Th1 reactivity early in infection correlated with IL-10 deficiency rather than that of IFN-
. Probably, the balance between the directive Th1 and Th2 cytokines, such as TNF-
, IL-4 and IL-10, defines the development of optimal protective response, not the absence of Th2 cytokines. The present results show that in primary infection the significant reduction of TNF-
and the increased IL-4 levels did not change the mortality of VIP-treated mice. In secondary infection the increased mortality paralleled with greatly lowered level of IFN-
concomitantly with higher levels of Th2 cytokines, IL-4 and IL-10. VIP-treated CIA mice expressed an increased yeast colonization in the kidneys after first and second inoculation. The suppressive action of VIP was observed in concern to specific anti-Candida antibody synthesis. At the onset of IgG response (day 14 after primary inoculation) the difference between VIP-treated and control groups was not significant, but after the second inoculation the serum antibody level of VIP-treated mice was 2-fold lower. The resistance to candidiasis is a result of antibody-dependent and antibody-independent mechanisms. The antibodies partially play a role in the restriction of the pathogen's growth, although it is not clear whether similar mechanisms operate in primary and secondary infection. In summary, the results show that VIP can modulate the autoimmune response in CIA through IFN-
suppression and stimulation of Th2 response. The antiarthritic effect is in parallel with partially impaired protective response against C. albicans expressed during secondary challenge with the yeast in a dose-dependent manner. VIP action on host resistance would be defined by very complex mechanisms dependent on the stage of CIA, the type of target cells and their role in infection or on the relationships between different types of cells. The well expressed antiarthritic effect of VIP does not elevate the susceptibility to primary C. albicans infection and it might be expected that the host resistance to the pathogen would not be dramatically decreased in the case of nonlethal secondary infection.
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Acknowledgements
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This work was financially supported by a Grant L-1305/2003 from the National Fund for Scientific Research (Republic of Bulgaria).
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Abbreviations
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BCII | bovine collagen type II |
CFA | complete Freund's adjuvant |
CFU | colony forming units |
CIA | collagen-induced arthritis |
DTH | delayed type hypersensitivity |
i.p. | intraperitoneally |
i.v. | intravenously |
OVA | chicken egg albumin |
RA | rheumatoid arthritis |
VIP | vasoactive intestinal peptide |
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Notes
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Transmitting editor: G. Trinchieri
Received 7 October 2003,
accepted 18 May 2004.
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