High dissemination and hepatotoxicity in rats infected with Candida albicans after stress exposure: potential sensitization to liver damage

Silvia Graciela Correa1, María Cecilia Rodríguez-Galán1, Beatriz Salido-Rentería1, Roxana Cano1, Hugo Cejas2 and Claudia Elena Sotomayor1

1 Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas and 2 Cátedra de Patología, Servicio de Anatomía Patológica, Hospital Misericordia, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Córdoba, Argentina

Correspondence to: C. E. Sotomayor; E-mail: csotomay{at}bioclin.fcq.unc.edu.ar


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The liver constitutes the first barrier in the control of hematogenous dissemination for Candida albicans of intestinal origin. The ability of this organ to limit the growth of the yeast and to mount an efficient inflammatory reaction is crucial in determining the outcome of the fungal infection. When rats infected with C. albicans are exposed to chronic varied stress, the cell recruitment is impaired at the site of the infection, the tissue reaction is highly disorganized in target organs and the infection evolution is more severe. At hepatic level, higher fungal burden is associated with hyphal form and the consistent presence of steatosis (fatty liver). Herein we aimed at characterizing the steatosis associated with C. albicans infection and to provide molecular evidence of the correlation among liver injury markers, stress products and the initiation of the inflammatory tissue reaction. After 3 days of stress and infection, we observed micro and macro steatosis in acinar zone 1 (specific lipid stain), higher lipid peroxidation and increased levels of serum alanine aminotransferase and gamma glutamil transferase. While infection triggered hepatic NO production and arginase activity, stress down-modulated both. Remarkably, defects in levels of TNF-{alpha} and NO were observed during the first step of the inflammatory response. Our results demonstrate that stress mediators down-regulate the acute inflammatory reaction in the hepatic scenario, promoting a major liver injury with particular immunopathological traits.

Keywords: arginase, fungal infection, iNOS, lipid peroxidation, rats, steatosis, TNF-{alpha}


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Candida albicans is the leading cause of opportunistic fungal infections in immunocompromised individuals (13). A proper integration between innate and adaptive immune response is necessary to control this fungus (4,5), and disseminated infections by C. albicans constitute a clear marker of immune alterations. Regulation of the early fungal burden, cytokine production and expression of co-stimulatory molecules are possible pathways through which the innate immune system conditions the development of the adaptive response (1,5).

The gastrointestinal mucosa is probably the most common portal of entry of C. albicans for hematogenous dissemination. At early stages of the infection, the liver constitutes the first barrier for the control of fungal spreading (3). The ability of this organ to limit the growth of the yeast and to mount an efficient inflammatory reaction is crucial in determining the outcome of the fungal infection (3,6). Inflammatory mediators such as proteolytic enzymes, proinflammatory cytokines, nitric oxide (NO) and oxidative products are released with local and systemic effects (68). In several models of liver injury (9,10), the active and passive participation of hepatocytes, the functional status and the modulation of hepatic activity are considered markers of the different susceptibility to the damage. Liver function depends on the integrated activity of different hepatic cells and the injury of the organ could result in disintegrated intracellular functions (8).

Recently, we have reported severe alterations in the immune response against C. albicans in rats infected and exposed to stress during 10 days (1113). After 3 days of treatment, livers of infected and stressed rats showed increased fungal colonization and lesions were associated with acute steatosis consistent with lipid accumulation in the cytoplasm of hepatocytes (11). Herein, we used specific lipid staining (Sudan Black) to characterize the steatosis associated to C. albicans infection. We assessed alanine aminotransferase (ALT), gamma glutamil transferase ({gamma}-GT) and lipid peroxidation levels to determine the magnitude of liver injury. Finally, in order to evaluate the key immune mediators that participate in the efficient organization of the tissular reaction we assessed TNF-{alpha} levels, hepatic NO production and arginase activity. Our results demonstrate that stress mediators down-regulate the acute inflammatory reaction in the hepatic scenario promoting a major liver injury with particular immunopathological traits.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
Outbreed female Wistar rats (body weight, 100–150 g) were collectively housed in the experimental room for at least 3 days before experiments started. Rats were maintained at 22°C under a 12 h light–dark cycle with continuous access to food and water except when food was removed from the stressed groups as part of the stress procedure.

Microorganism and infection
The pathogenic C. albicans strain N° 387 was grown on Sabouraud glucose agar slant at 28°C, maintained by weekly subculture and periodically checked for assimilation pattern and virulence (1113). For infection, after 48 h of culture, yeasts were harvested in sterile 0.15 M NaCl–0.1% gentamicin, counted and diluted to the desired concentration.

Stress procedure
In our model, rats are infected and exposed to a stress paradigm that involves a different stressor during 10 days (1114). Considering that in this work we evaluated the inflammatory response and hepatic injury observed after 3 days of treatment, the stress procedure described below corresponds to the first 3 days of the stress paradigm (13) and includes: Day 0, swim (4°C for 5 min); day 1, restraint (for 2 h); and day 2, food deprivation (for 24 h). The Animal Experimentation Ethics Committee, Faculty of Chemical Science, National University of Cordoba approved the protocols.

Experimental design
Rats were assigned to four groups: Normal uninfected and unstressed (N), stressed (S), C. albicans-infected (Ca) and infected and stressed (CaS). Rats were infected i.p. with a 1 ml inoculum (3 x 108 yeast/ml) on day 0 and stressed immediately after the infection and during the following 2 days. On day 3, animals were weighed and killed by decapitation (1113). Blood was obtained and livers were removed, placed on individual Petri dishes, weighed and processed for histological examination and biochemical assays.

Histological studies
Livers were fixed with 10% formalin in PBS for at least 24 h, dehydrated in alcohol, cleared in xylol and embedded in paraffin. Six-micrometer specimens were sectioned and stained with hematoxylin–eosin (HE). For specific lipid detection, Sudan Black stain was performed and livers were prepared as described (15). Briefly, the fresh tissue was frozen using dry ice, cut with microtome and stained with Sudan Black. This technique was used to evaluate the distribution, extension and morphology of lipid droplets and to classify the pathological injury using an arbitrary scale as follows: 0 (no damage), 1 (light), 2 (moderate) and 3 (intense). Pathological parameters were measured in at least three separate experiments (with 4–6 rats per group).

Assay of hepatic enzymes activity, proteins and TNF-{alpha} levels
Serum activities of ALT and {gamma}-GT were measured with commercial kits (Wiener Lab. Rosario, Argentina) according to the manufacturer's recommended protocols. Separation of serum proteins (16) was performed by zone electrophoresis using the Microtech Series Electrophoresis System 4.13 (Enfolab Interlab, Rome, Italy) that provides the electrophoretical separation of serum proteins and values of total and individual protein fractions. TNF-{alpha} quantities were determined by solid-phase sandwich ELISA protocol (12). The amount of TNF-{alpha} was extrapolated from the standard curve which was generated in 1:2 dilutions. Results are expressed in ng/ml.

Assay for hepatic lipid peroxidation
Liver homogenates (1 ml) were mixed with 2 ml of a working solution containing 15% (w/v) trichloroacetic acid–0.375% (w/v) thiobarbituric acid–0.25 N HCl and heated for 15 min in boiling water. After cooling, the precipate was removed by centrifugation at 1000 g for 10 min. Absorbance was determined at 535 nm. Levels of malonaldehyde (MDA) were measured as an indicator of lipid peroxidation using 1,1,3,3-tetrametoxypropane as standard. Results expressed as mmol of MDA/mg of protein are mean values ± SEM of five rats/group (17).

Immunoblot analysis
iNOS and arginase I expression were assessed in liver homogenates prepared with PBS containing phenylmethane sulfonyl fluoride (100 µg/ml), aprotinin (1 µg/ml) and leupeptin (1 µg/ml). Equal amounts of protein (30 µg/ lane) were fractionated by 10 or 15% SDS–polyacrylamide gel electrophoresis, respectively, and proteins were electrotransferred onto nitrocellulose membranes. Membranes were incubated with a 1:1000 dilution of a rabbit antibody against iNOS (Santa Cruz Biotechnology Inc., Santa Cruz, California, CA) or a 1:500 dilution of a mouse antibody against human arginase I (BD Pharmingen, Chicago, IL) that detects arginase I in rat macrophages (18). Membranes were washed with TBS–0.05% Tween 20 and incubated with goat anti-rabbit or anti-mouse conjugated horseradish peroxidase (1:1000) (Pharmingen, San Diego, CA). Immunodetection was performed using Western Blot Chemiluminescence reagent kit (NEN Life Science, Boston, MA).

Assessment of arginine metabolism
To determine NO levels, liver homogenates were treated with 1 N HCLO4 and centrifuged at 100 000 g for 60 min; supernatants were stored at –20°C until use. NO was determined as nitrite via a microplate-assay method using Griess reagent (19). Absorbance was measured at 540 nm in a microplate reader. Nitrite was measured using NaNO2 as a standard. Each sample was tested in triplicate and results are expressed in nmol/mg of protein.

Arginase activity was assessed in liver homogenates as described (20). Each sample was tested in triplicate. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 µmol of urea per minute. Results are expressed as mU/mg of tissue.

Statistical analysis
Differences between group means were assessed using ANOVA followed by Student–Newman–Keuls test for multiple comparisons. A P-value < 0.05 was considered statistically significant.


    Results
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 Abstract
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 Methods
 Results
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 References
 
Histopathological findings
The occurrence of steatosis in infected and stressed animals was reported recently by our group (11). This relevant and new finding prompted us to characterize the liver steatosis associated with C. albicans infection by evaluating the histopathological damage. Livers of CaS group showed a remarkable number of fat vacuoles in the cytoplasm of hepatocytes (Fig. 1B and D). In infected animals (Ca) instead, the scarce lipid droplets were clearly visualized after high magnification (Fig. 1A and C). Infiltrating polymorphonuclear neutrophils (PMNs) recruited in response to fungal infection are also shown (C and D).



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Fig. 1. Histopathological findings. Representative microphotographs of H&E-stained liver sections removed 3 days after infection. (A and C) Infected (Ca) group, magnification x10 and x40, respectively. (B and D) Infected and stressed (CaS) group, magnification x10 and x40, respectively. Ca group shows clearly visualized fat vacuoles in the cytoplasm of hepatocytes (steatosis) at x40 magnification (C). Liver of CaS shows remarkable number of lipid droplets at both magnifications (B and D). Arrows: PMNs infiltrating the parenchyma. Arrowhead: lipid droplets in the cytoplasm of hepatocyte.

 
Sudan Black, one of the most specific and sensitive dyes for lipids (15), was used to evaluate the distribution, extension and morphology of lipid droplets and to classify the pathological injury. No alterations were observed in liver sections of N and S rats (Fig. 2A and B). Microscopic observation disclosed a striking difference between Ca and CaS groups: animals only infected but not stressed developed diffuse microvesicular steatosis that extended all over hepatic acini (Fig. 2C and E; Table 1). In CaS group instead, most of the lipid accumulation was found in acinar zone 1 and the pattern of lipid deposition showed a mixed reaction with micro and macro steatosis (Fig. 2D and F; Table 1). A strong association between histopathological characteristics and treatment was evident and it was possible to reliably differentiate animals from infected and infected-stressed groups on the basis of histology.



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Fig. 2. Histopathological findings. Representative microphotographs of Sudan black-stained liver sections removed 3 days after treatment. (A) Normal group, magnification x10. (B) Stressed group, magnification x10. (C and E) Infected (Ca) group, magnification x10 and x40, respectively. (D and F) Infected and stressed (CaS) group, magnification x10 and x40, respectively. Note the different pattern of steatosis in Ca and CaS groups: in Ca animals microvesicular steatosis distributed in all hepatic acinus was observed (C and D). CaS exhibited micro and macrovesicular steatosis with acinar distribution in zone 1 (D and F).

 

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Table 1. Histopathological findings in livers of animals infected with Candida albicans

 
To quantify the hepatic damage we considered several parameters: the absolute and relative liver weights, the hepatotoxicity in arbitrary units, and the type and localization of steatosis after Sudan Black stain (Table 1). Animals infected and exposed to stress showed a clear profile: lower weight of the organ (P < 0.01 vs Ca), higher hepatotoxicity with a histological grade 3 and different type and localization of steatosis.

Markers of liver injury, lipid peroxidation and acute phase response
Markers of liver injury such as serum activities of the enzymes ALT and {gamma}-GT were assessed in different experimental groups. While a 2-fold increase in the cytoplasmatic enzyme ALT was observed after the infection (Ca and CaS vs N and E, P < 0.05), levels of the membrane enzyme {gamma}-GT were significantly higher in all treated groups compared with N rats (P < 0.01) (Fig. 3A and B). Remarkably, stress per se triggered a significant increase in {gamma}-GT activity (i.e. S group) with an additive effect in rats exposed to both infection and stress (Ca vs CaS, P < 0.05).



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Fig. 3. Markers of liver injury, lipid peroxidation and acute phase-response. Serum samples and livers were obtained after 3 days of treatment, and several parameters were evaluated as described in Methods. (A) Serum levels of ALT (*P < 0.05 vs N and S). (B) Serum levels of {gamma}-GT (*P < 0.01 vs N; #P < 0.05 Ca vs CaS). (C) Assay for hepatic lipid peroxidation, using malonaldehyde as an indicator, was performed in liver homogenates from all experimental groups (*P < 0.01 vs N; #P < 0.05 Ca vs CaS). Results are the mean and SEM of at least five rats/group; data from two experiments were pooled. (D). Serum level of albumin (black bars) and {alpha}2-macroglobulin (white bars) after serum electrophoresis of at least four samples/experimental groups (*P < 0.05 vs N and S).

 
Hepatic steatosis is frequently associated with oxidative stress and increased lipid peroxidation (21,22). We assessed the levels of malonaldehyde in liver homogenates as a marker of lipid peroxidation (21). Compared with N rats, all treated groups showed increased levels of malonaldehyde (Fig. 3C). However, the highest levels of lipid peroxidation were observed in infected and stressed animals (Ca vs CaS, P < 0.05).

Electrophoresis of serum proteins (Fig. 3D) showed that albumin and the typical rat acute-phase response protein, {alpha}2-macroglobulin (23) had a similar profile in infected groups, with a reduction in albumin (P < 0.05 vs N and S) and an increment in levels of {alpha}2-macroglobulin (P < 0.05 vs N and S).

TNF-{alpha} production
The release of TNF-{alpha} is crucial for the normal immune response against any invading microorganism (7,4) and it is a component of the early ‘danger signals’ necessary to mount a protective inflammatory reaction (24). Mannoprotein and other constituents of the C. albicans wall induce in vivo and in vitro TNF-{alpha} production (25). TNF-{alpha} has also been identified as a proximal mediator of endotoxin-induced injury of many tissues, including the liver (26,27). In C. albicans infected animals, serum concentration of TNF-{alpha} reached the maximal level on the first day of the infection (Fig. 4), showing that the fungal infection triggers the production of this proinflammatory cytokine (Ca vs N, P < 0.01). In CaS groups instead, TNF-{alpha} levels were ~30% lower on days 1 and 2 with similar values on day 3 for both.



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Fig. 4. TNF-{alpha} serum levels in infected and infected-stressed groups. Serum samples were collected 1, 2 and 3 days after treatment, and TNF-{alpha} was determined by using ELISA sandwich. Results are the mean and SEM of at least five rats/group; data from two experiments were pooled (*P < 0.01 Ca vs N).

 
Arginine metabolism in liver after C. albicans infection and stress exposure
In response to infection by several pathogens and inflammatory cytokines, noticeable changes occur in Mø arginine metabolism (20,28) that include increases in NO synthesis via inducible NO synthase (iNOS) and catabolism of arginine to ornithine and urea via arginase. The expression of iNOS and arginase isoform I in liver homogenates of different experimental groups is shown in Fig. 5(A and B), respectively. While an increased expression of iNOS was found in Ca rats, a lowest expression of the protein was observed in the CaS group. Similarly, for the arginase isoform I, CaS rats exhibited a slightly reduced expression of the enzyme compared with animals only infected. The activity of both enzymatic pathways was evaluated as NO production (panel C) and arginase activity (panel D). Again, infection triggered the two metabolic pathways of arginine (Ca vs N, P < 0.05), whereas the double stress-infection challenge reduced the activity of both enzymes (P < 0.05 vs Ca). Recently, we have reported that C. albicans triggers at the site of the infection the two metabolic pathways in peritoneal Mø, while stress products down-regulate the activity of both enzymes (13).



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Fig. 5. Assessment of arginine metabolism in livers from different experimental groups. Livers of animals were obtained after 3 days of treatment and processed as described in Methods. Expression of the enzymes was assessed by western blot analysis using specific antibodies against iNOS (A) and arginase I isoform (B). The NO production was evaluated in supernatant aliquots by a microplate assay method using the Griess reagent (#P < 0.05 vs N; *P < 0.05 Ca vs CaS) (C). Arginase activity was evaluated in the liver homogenate by a colorimetric assay as described in Methods (*P < 0.05 CaS vs Ca) (D). For (C) and (D), results are the mean and SEM of at least five rats/group; data from three experiments were pooled.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Candida albicans infection induces steatosis and liver damage. Stress exposure promotes a major liver injury
The liver plays a critical role in the inflammatory response to injury; however, whether the liver is affected and how it influences the rest of the immune system is not well understood. One striking finding of our experimental condition (11) is the presence of steatosis associated with C. albicans infection and its exacerbation after stress exposure (PubMed keywords: C. albicans, liver, steatosis, stress: No items found). In the present work, we characterized the steatosis pattern in both groups of infected animals: the Ca group showed microvesicular steatosis all over hepatic acini, while CaS rats had a mixed form with micro and macrovesicular steatosis distributed mainly in the zone 1 of hepatic acini. Fatty liver is a common histological finding in human liver biopsies and several risk factors have been identified such as the ingestion of certain drugs, alcohol abuse, diabetes and obesity (22,29). Widespread microvesicular steatosis is a characteristic acute condition in which impairment of fatty ß-oxidation reflects a more general perturbation of mitochondrial function (30). Macrovesicular steatosis instead, is typically related to a more long-standing disturbance of hepatic lipid metabolism (8,22,29), and it is frequently associated with the development of more advanced disease such as necroinflammation (31). The severity of the steatosis is considered an indicator of the intensity of the stimulus (22,29) as well as a marker of hepatotoxicity related to the immune status of the host. During Schistosoma mansoni infection of immunocompromised mice there is extensive hepatocyte damage (32) and in nude, SCID and T cell-depleted mice hepatic steatosis appears as microvesicular steatosis on hepatocytes (32,33). In agreement, histopathological findings showing a mixed steatosis pattern (micro and macrovesicular) in rats exposed simultaneously to both stress and infection, support the concept of a more severe condition.

The severity of steatosis correlates with the degree of lipid peroxidation in both animal and human models (22,29,34), and elevation of transaminases and hepatic enzymes are frequently associated with fatty liver and lipid peroxidation (35). In infected groups, we observed the increase in ALT activity without any effect associated with the stress exposure. Conversely, levels of {gamma}-GT and MDA exhibited a more complex pattern increased after stress exposure, infection or both. Again, the evaluated markers of hepatic injury showed the additive effect of the fungus and stress mediators in the severity of liver damage. Diminution in albumin and increment in {alpha}2-macroglobulin levels, two serum proteins, were observed in infected groups (Ca and CaS) as expected for an infectious process and stress products did not change the profile (23).

Involvement of TNF-{alpha} and L-arginase metabolism during C. albicans infection and stress exposure
Accumulative evidence points to TNF-{alpha} and/or NO as the main molecules involved in the resistance to infection. Regarding fungal infections, TNF-{alpha} participates in granuloma formation that restricts fungal dissemination (36,37), while NO participates in the killing process or down-regulates the immune response (36,38,39). Defects in TNF-{alpha} or iNOS correlate most closely with disease progression characterized by exacerbated bacterial growth; highly disorganized granulomas with delayed kinetics, and premature death (40). Olszewski et al., working with Cryptococcus neoformans, demonstrated the role of the early ‘danger signal’ mediated by TNF-{alpha} in the progression of this fungal disease (24,41). The net result of a decreased or absent early danger signal is limited inflammation, altered cytokine production, altered immune response and evolution to a chronic sate of the mycosis. One clear difference between the Ca and CaS groups was related to the ability to produce adequate levels of TNF-{alpha} and NO: in infected animals the TNF-{alpha} and NO production correlated with an acute inflammatory response, while in infected stressed rats, levels of NO similar to uninfected animals and transient production of TNF-{alpha} were observed.

L-arginine can be metabolized by alternative pathways involving the enzymes iNOS and arginase (20,28). While NO is related to the fungal control, arginase activity is linked to the cell growth and connective tissue reaction and repair. Challenging the paradigm of reciprocal regulation of iNOS vs arginase demonstrated in many infections, we have recently reported that C. albicans triggers the two metabolic pathways in macrophages (13). Herein we demonstrate that, early in the infection, the two metabolic pathways are also activated in the liver but stress products down-modulate both. Accordingly, the induction of iNOS and arginase by inflammatory stimuli is largely abolished by dexamethasone (42).

Can the levels of inflammatory mediators account for the clear differences in hepatotoxicity? Independent studies show that the presence of fat per se may be causal in the development of more advanced liver pathology (22). Fatty livers have inherent immunologic alterations that may predispose them to damage from endotoxin and other insults that induce proinflammatory cytokine responses (43,44). Two possible mechanisms could explain hepatotoxic effects of proinflammatory cytokines: priming and sensitization. Priming occurs when cells, like tissue Mø, are primed to release TNF-{alpha} in response to stimuli, with some degree of liver injury. Sensitization is more related to a defective adaptation to oxidant stress and proinflammatory cytokines (10,45). According to this theory, obesity strongly associated with steatohepatitis increases susceptibility to endotoxin-mediated liver injury (10). Diehl et al. have recently reported that female fa/fa rats, which exhibit steatosis and the greatest hepatotoxicity after LPS administration, show the lowest TNF-{alpha} serum levels, even when compared with the lean control (10). It is important to point out here that while C. albicans infection induced steatosis in both groups (Ca and CaS), in absence of infection stress products profoundly affected the biology of liver cells with lipid peroxidation and increment in {gamma}-GT levels (S group). Together, our findings suggest that severe liver injury and hepatotoxicity in infected and stressed animals could result in the stress-induced sensitization to mediators released in a poorly developed inflammatory response. In this particular scenario the antagonistic relationships between host and fungal promote the development of a more aggressive pattern of this opportunistic fungal infection.

Given the complexity of biological systems, additional factors not disclosed in the present study such as other inflammatory mediators, chemokines and regulatory innate cell populations could also be involved in the described phenomenon. Interestingly, in obese animals that completely lack leptin (ob/ob mice), LPS induces a greater release of liver enzymes compared to that in fa/fa rats which overexpress leptin but have deficient leptin receptors (10,46). These findings support the provocative notion that leptin signaling may be a requisite for normal tissue sensitivity to proinflammatory cytokines. Our study did not evaluate the effect of leptin on the susceptibility to the fungal infection. We cannot exclude the possibility that leptin is modulating the innate response in infected and stressed animals.

Herein, our data suggest that the ultimate fate of the liver depends on the complex interplay of fungal aggression, proinflammatory mediators like TNF-{alpha} and NO, and stress. Given the importance of the innate immune system as a regulator of the hepatic cytokine milieu, and considering that this microenvironment will condition the liver acute response when the fungus challenges it, further work is required to define the sequence of events that take place in the liver after C. albicans infection and stress exposure.


    Acknowledgements
 
S. C. Correa and C. E. Sotomayor are members of career from Consejo Nacional de Investigaciones Cientificas & Tecnologicas (CONICET). The authors want to thank Dr C. M. Riera for discussion and critical reading of the manuscript; Mrs P. Icely for excellent technical assistance and L. Navarro for his help. This work was supported by grants from the International Society for Infectious Diseases (ISID-2002), Ag. Nacional de Promoción Científica y Tecnológica (Foncyt 05-05153), Secretaría de Ciencia y Tecnología (Secyt-2001-2002) and Ag. Cba Ciencia, Argentina.


    Notes
 
Transmitting editor: A. Falus

Received 19 May 2004, accepted 22 September 2004.


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 Introduction
 Methods
 Results
 Discussion
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