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Research Article |
Center of Electron Microscopy, School of Medical Sciences, National University of Córdoba, X5000HRA Córdoba, Argentina
ABSTRACT
Theprostate gland is the most inflammation-prone organ in the male reproductive tract. However, little information is available regarding the immunobiology of this gland. Toll-like receptor 4 (TLR4) is considered to be a major sensor of danger signals and a key trigger of the innate immune responses. TLRs have also been implicated in the development of different inflammatory diseases in organs in which epithelial-stromal interactions are critical for homeostasis. The purpose of this work was to evaluate the presence and regulation of TLR4 in the rat prostate. Western blot and immunocytochemical studies revealed that constitutive expression of TLR4 in the rat ventral prostate was localized in the epithelial cells, mainly associated with the rough endoplasmic reticulum, as well as in smooth muscle cells in the stroma. In addition, increased concentrations of TLR4 were found in castrated rats, predominantly in hypertrophied smooth muscle cells. On the other hand, using a bacterial prostatitis model, we observed an increment in the TLR4 cytoplasmic content and migration of this receptor to the apical plasmatic membranes of epithelial cells at 24 h and 48 h post-infection. These findings suggest that the prostate gland is able to recognize pathogens and to initiate immune responses. In addition, TLR4 appears to be implicated in the vital stromal-epithelial interactions that maintain prostate homeostasis during prostatitis, as well as following androgen deprivation.
immunology, male reproductive tract, prostate, testosterone
INTRODUCTION
As in the case of many other epithelia that are exposed continually to external insults, the epithelium lining the reproductive tract is equipped with a variety of antimicrobial, pro-inflammatory, and immunomodulatory compounds, which are key mediators of the innate immune system [1].
The discovery and characterization of a new host defense protein, the Toll-like receptor (TLR) in invertebrates and mammals, has greatly contributed to understanding how the host organism detects the presence of infectious agents and disposes of invaders, without destroying its own tissues [2]. TLRs are transmembrane proteins that distinguish specific patterns of microbial components, especially those from pathogens, and regulate the activation of both innate and adaptive immunity [3]. Once activated, the TLR intracellular signals culminate in the activation of NF-
B as well as MAPK, leading to the subsequent induction of various genes that function in host defense responses [4].
Among the eleven members of the TLR family (TLR1-TLR11) [5], the lipopolysaccharide (LPS) receptor Toll-like receptor 4 (TLR4) is a central player in signaling pathways of the innate immune response to infection by several pathogens. TLR4 is activated by the LPS [6] assembled in the wall of Gram-negative bacteria, such as Escherichia coli and Klebsiella pneumoniae. These receptors are widely distributed, not only in immune cells, such as macrophages [7] and dendritic cells [8], but also in the epithelia of the respiratory [9], digestive [10], and urinary tracts [11].
Prostatic inflammation, or prostatitis, represents an important problem for human health worldwide. The International Prostatitis Collaborative Research Network (funded by the NIH) has proposed the use of animal models to achieve a better understanding of the immunobiology of the prostate, in order to improve our knowledge of this disease [12]. In addition, the prostate gland is the most inflammation-prone organ in the male genito-urinary tract, and is frequently the main target of venereal diseases. Many peptides with innate immune activity have been found in this gland [1], although neither the presence of TLR4 nor its regulation by inflammatory stimuli has been evaluated.
In the present study, we investigated the expression of TLR4 in the rat prostate gland and its response to inflammation and androgens. Prostate morphology was assessed by electron microscopy and immunocytochemistry was performed with anti-vimentin and anti-smooth muscle alpha actin (ACTA2) antibodies.
MATERIALS AND METHODS
Animals
Adult 12-week-old male Wistar rats, weighing 250–350 g, were bred and housed at the Animal Research Facility of the National University of Córdoba, in air-conditioned quarters, under a controlled photoperiod (14L:10D) with free access to commercial rodent food and tap water. The rats were divided into four experimental groups (n = 10/group in three different experiments): 1) intact animals (IN group); 2) acute bacterial-induced prostatitis (BP group); 3) orchidectomized rats (OX group); and 4) testosterone-treated group (TT group). All animal experiments were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction.
Bacterial Prostatitis Model (BP group)
A strain of uropathogenic E. coli (kindly provided by Dr. Oscar Pessah, Department of Microbiology, National University of Córdoba) isolated from a patient with a complicated urinary tract infection was stored at –20°C before being grown overnight in tryptic soy broth at 37°C and used for inoculations. The bacterial cells were pelleted, washed three times in sterile PBS, and resuspended at a concentration of 108 cells/ml.
The rats were anesthetized with inspired ether, and subjected to laparotomy to expose the ventral prostate. Acute prostatitis was induced by injection of 200 µl of the E. coli suspension. The bacterial solution was injected with a 30-G needle directly beneath the capsules of both ventral lobes. The peritoneum, abdominal muscles, and skin were closed using a simple continuous suture with a chromic surgical filament. The animals were killed 24 or 48 h after bacterial inoculation. As controls, sham-operated rats were subjected to laparotomy but were not injected with the bacteria. The ventral prostate was harvested and processed for biochemical studies.
Orchidectomy and Testosterone Treatment
The influence of androgens on TLR4 expression in the prostate gland was studied in male rats (OX group) that were orchidectomized via the scrotal route under ether anesthesia; the epididymis and epididymal fat were also removed during this operation. Control rats for the OX group were submitted to sham surgery, with the animals being killed 10 days postsurgery. In the TT group, castrated rats were injected subcutaneously with testosterone (10 mg/kg body weight; Sustanon; Organon) suspended in sunflower oil, each day for 10 days beginning 1 day after castration. As controls for this group, castrated rats were injected with the vehicle alone. The animals were killed 24 h after the last injection. Samples of the ventral prostate were obtained and processed for biochemical studies.
Serum Testosterone Levels
Prior to killing, blood was obtained by intracardiac puncture from the IT, OX, and TT groups and their controls. The serum total testosterone levels of individual rats were determined by electrochemiluminescence (ECL) immunoassay using the Roche Elecsys E170 immunoassay analyzer (Roche Diagnostics).
Microbiological Studies
For bacterial culture, pieces of prostate from each rat were weighed and cultured qualitatively by plating them on McConkey agar (Sigma Chemical Co., St. Louis, MO) and incubating overnight at 37°C. After 24 h, bacterial isolates were picked and identified using conventional Gram staining. The samples were considered to be sterile if no microorganisms were detected after 48 h of culture.
Light Microscopy and Electron Microscopy
Rats were fixed by perfusion with 4% formaldehyde, and the ventral prostate blocks were embedded in paraffin, cut into 4–µm-thick sections and stained with hematoxylin-eosin or immunostained with specific antibodies.
Other prostate blocks were fixed in Karnovsky mixture that contained 1.5% (v/v) glutaraldehyde and 4% (w/v) formaldehyde in 0.1 M cacodylate buffer, and then treated with 1% osmium tetroxide, dehydrated, and embedded in Araldite. For light microscopy, 1-µm-thick sections were cut serially and stained by the silver technique, following the methodology outlined below. For ultrastructural studies, thin sections were cut with a diamond knife on Porter-Blum MT2 and JEOL JUM-7 ultramicrotomes and examined using the Zeiss LEO 906E electron microscope
For ultrastructural immunocytochemistry, prostate tissue blocks were embedded in acrylic resin (LR-White; London Resin Corp.) omitting osmium fixation.
Silver Staining
The silver methenamine technique for polysaccharides [13] was performed on Araldite semithin sections. This procedure applied to plastic-embedded sections provided similar results to periodic acid-Schiff (PAS) staining of paraffin sections but with a better resolution, simplifying the identification of tissue structures, such as collagen fibers, basement membranes, and glycoprotein components.
Immunocytochemistry
Slides from paraffin-embedded prostates were cleared with xylene and rehydrated in a descending concentration series of ethanol. Microwave pretreatment (antigen retrieval method) was performed. To block the endogenous peroxidase activity, slides were treated with H2O2 in methanol for 15 min. Sections were incubated for 30 min in 10% normal rabbit serum (Sigma), to block nonspecific binding, followed by overnight incubation with a 1/400 dilution of polyclonal goat antibody to TLR4 (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C in a humidified chamber. The sections were then incubated with a biotinylated secondary antibody (Santa Cruz Biotechnology) and ABC complex (Vector Laboratories, Burlingame, CA). Diaminobenzidine (DAB; Sigma) was used as the chromogenic substrate for 10 min at RT, and the sections were rinsed in running water. Harris hematoxylin was used as the counterstain. The expression of two markers was evaluated by immunohistochemistry, to characterize the stromal cellular phenotype; the procedure was similar to the above-mentioned technique for TLR4, using monoclonal antibodies to vimentin (VIM) and ACTA2 (Novocastra Laboratories, Ltd.), and applying a goat anti-mouse biotinylated IgG (Amersham Pharmacia Biotech) secondary antibody.
Ultrastructural Immunocytochemistry
LR-White thin sections mounted on 250-mesh nickel grids were incubated overnight on a drop of goat anti-TLR4 (Santa Cruz) diluted 1/500, and immunoreactive sites were labeled with 16-nm colloidal gold/anti-goat IgG complex (Pelco International). For the controls, the primary antibody was replaced with goat normal serum (Sigma), purified goat IgG (Santa Cruz Biotechnology) or PBS–BSA.
Western Blotting
For immunoblotting, prostate tissues were minced and homogenized on ice with a teflon-glass Potter-Elvehjem tissue grinder in 2-ml cold PBS that contained 1.25% Igepal CA-630, 1 mM EDTA, 2 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The lysate was centrifuged at 14,000 x g for 20 min at 4°C to pellet the Igepal CA-630-insoluble material, and the supernatant was withdrawn and stored in aliquots frozen at –70°C until required. Prostatic lysates from duplicate experimental conditions were pooled before loading onto electrophoresis gels. Total protein concentration was measured with the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). Denatured protein samples (30 µg/lane) were then separated on a 12% SDS polyacrylamide gel and blotted to a Hybond-C membrane (Amersham Pharmacia). To assess the corresponding molecular masses, the Full Range Rainbow Molecular Weight Marker (Amersham Pharmacia) was used. Incubation steps were performed in 5% defatted dry milk in PBS/0.1% Tween-20. The blots were incubated with a 1/250 dilution of goat anti-TLR4 IgG (Santa Cruz Biotechnology) and peroxidase-conjugated bovine anti-goat antibody (Jackson ImmunoResearch Laboratories, Inc.), and visualized by the chemiluminescence technique. The expression of ß-actin (ACTB) (1/5000; monoclonal anti ß-actin; Sigma) was used as an internal control to confirm equivalent loading of total protein. Semiquantitative signals were derived by densitometric analysis using the Scion Image software (version beta 4.0.2; Scion Corp.) and data displayed as area units per mg protein.
Statistical Analysis
Data from more than two groups were examined using analysis of variance with Tukey as a post test. Statistical testing and calculation of the Western blot data were performed using the InStat V2.05 program from GraphPad Inc..
RESULTS
Characterization of Experimental Models
In the rat prostates from the IN group, the epithelium lining the glandular alveoli was comprised mainly of cylindrical secretory cells; the stroma was scarce and composed mainly of a thin periacinar layer of smooth muscle cells and interstitial fibroblasts (Fig. 1A). The periacinar stromal layer exhibited a continuous pattern of ACTA2 reactivity, specifically of smooth muscle cells (Fig. 1B), and a few VIM-positive fibroblastic cells (Fig. 1C).
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All of the prostate samples from the BP group tested positive for E. coli at 24 h and 48 h after inoculation; while the prostates from the controls and IT group were sterile.
All of the prostates from the BP group showed acute inflammation, which was distributed throughout the gland. At 24 h after bacterial inoculation, the epithelium was hypertrophied, and displayed an undulating contour. Furthermore, in the stroma, which was infiltrated by numerous inflammatory cells, there was massive development of the ACTA2-positive smooth muscle periacinar layer (Fig. 1, D and E). VIM immunoreactivity formed two interrupted layers that flanked the smooth muscle cells (Fig. 1F). Infiltrating inflammatory cells in the prostatic stroma also showed VIM-immunoreactivity. Similar results were observed at 48 h after bacterial inoculation.
In the OX group, the prostatic epithelium exhibited an important reduction of the nucleus/cytoplasm ratio as a result of androgen deprivation. In contrast, smooth muscle cells in the periacinar layer were significantly enlarged (Fig. 1G); ACTA2 staining showed that the periacinar smooth muscle layer was larger in the OX group than in the BP group (Fig. 1H). Isolated fusiform hypertrophied VIM-positive cells appeared under the epithelial basal membrane as well as outside the smooth cell layer (Fig. 1I).
In animals treated with testosterone (TT group), the enlarged prostate gland exhibited striking hypertrophy of the glandular alveoli, resulting in a large lumen and an increased secretory volume, while the stroma was comparable to that of intact animals (Fig. 1J). Antibodies against ACTA2 and VIM provided a staining pattern with similar characteristics to that of the IN group (Fig. 1, K and L).
Analysis of TLR4 Expression
To establish whether TLR4 is expressed in rat ventral prostate cells, and to characterize its response to Gram-negative bacterial infection, the expression of TLR4 protein was analyzed by Western blotting and immunocytochemistry. As shown in Figure 2, the prostate glands from intact rats expressed TLR4 constitutively. Western blotting revealed a positive band of about 90 kDa for all the experimental conditions (Fig. 2).
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As expected, in the BP group, there were significant increases in TLR4 expression both 24 h and 48 h after inoculation as compared with their controls and IN rats (Fig. 3). The increment was time-dependent, with a greater increase in TLR4 expression at 48 h after bacterial inoculation compared with 24 h after infection.
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The experimental groups exposed to different testosterone levels, i.e., the IN, OX, and TT groups (Fig. 4A), were used to investigate the influence of androgens on TLR4 expression. Surprisingly, the TLR4 levels in the ventral prostate increased significantly in OX animals relative to their respective controls and IN rats. In contrast, the TT group exhibited no significant differences relative to the IN group (Fig. 4B).
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Immunocytochemistry of TLR4 Expression
The localization of TLR4 in the prostate gland and the changes in TLR4 expression detected by Western blotting were examined by immunocytochemistry using light and electron microscopy.
In the IN group, TLR4 immunolabeling was faint in the epithelial cells (Fig. 5A) and stroma. Stronger staining was noted in the walls of the arterioles. The ultrastructural immunogold technique revealed labeling that was associated mainly with rough endoplasmic membranes (Fig. 6B), although the apical plasma membranes were negative (Fig. 6A). In addition, TLR4 was localized in periacinar smooth muscle cells (Fig. 6C and Fig. 7A). When specific anti-TLR4 antibody was replaced by goat normal serum or purified goat IgG, no labeling was detected (data not shown).
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In the BP group, the increased TLR4 level found by Western blotting correlated well with the strong immunostaining observed in the epithelial cells and periacinar layer (Fig. 5B); some inflammatory cells infiltrating the gland were also positive. The immunogold labeled not only intracytoplasmic membranes, as in the IN group (Fig. 6G), but also appeared to be clearly polarized to the apical cytoplasm (Fig. 6F), intercellular junctional complexes, and microvilli (Fig. 6, D and E). Abundant secretory granules, which were increased in number by the infection, were negative for TLR4 (Fig. 6, D, E, and F). Hypertrophied smooth muscle cells also exhibited strong, specific TLR4 immunoreactivity for this technique (Fig. 7B).
In the OX group, supporting the increased TLR4 expression found in Western blots, intense immunoreactivity was detected in the epithelial and stromal cells (Fig. 5C). Surprisingly, TLR4 was strikingly expressed in the scarce cytoplasmic remnants of the epithelial cells (Fig. 5C). Under the electron microscope, TLR4 exhibited heterogeneous localization in the cytoplasm (Fig. 6H) and was often associated with poorly-developed rough endoplasmic reticulum, distributed along the lateral membranes (Fig. 6, I and J) and the basal cytoplasm, where the gold particles appeared to delineate a thin cytoplasmic region in contact with the basement membrane (Fig. 6K). In the stromal compartment, intense TLR4 staining was observed in the periacinar layer (Fig. 5C), with immunogold labeling being constrained to an area that contained predominantly smooth muscle cells (Fig. 7C), which frequently exhibited intense labeling of the plasmalemma (Fig. 7C, inset).
In the TT group, TLR4 immunoreactivity was weak in the epithelial cytoplasm and stromal cells, and also in the IN group (Fig. 5D).
DISCUSSION
Host defense proteins in myeloid cells and in the respiratory and digestive tracts have aroused a great deal of interest. Over the last few years, several groups have demonstrated the importance of innate immune system components in the urogenital tract [1, 11, 14–17]. In the male reproductive system, investigations have focused on the epididymis [16, 17] and testis [18, 19]. The epididymis has been found to secrete antimicrobial proteins, such as defensins [16], and the testis expresses galectins [18], which are key regulators of the immune system. However, little is known about the innate immune response within the prostate gland.
TLR4, which was originally detected in mammalian immune cells, is critically involved in the innate immune response as a membrane receptor for Gram-negative bacteria, the activation of which triggers an inflammatory cascade that is mediated by NF-B [3]. Furthermore, TLR4 is expressed by epithelia that interface with the external environment, as in the cornea [20], oral cavity [21], respiratory tract [22], intestine [23], and urinary tract [11]. In this study, we observed constitutive in vivo expression of TLR4 in the rat ventral prostate, and the upregulation of TLR4 by E. coli or castration, not only in epithelial cells but also in the stromal compartment.
Previous studies have reported that prostatic epithelial cells and their secretory products may actively participate as local modulators in response to bacteria [24, 25]. However, the molecules involved in this process have not yet been fully characterized. Recently, Takeyama et al. [26] and Gatti et al. [27] have reported that prostate cells secrete inflammatory cytokines in response to M. hominis and LPS in vitro through a TLR2- and TLR4- mediated mechanism, which suggests that epithelial cells can act in the first line of host defense in the prostate gland. We found that TLR4 is localized in vivo in the prostatic epithelium, with weak intracytoplasmic staining that is mainly associated with the rough endoplasmic reticulum. This finding contrasts with the classical localization of TLR4 on the surfaces of macrophages and other immune cells [7]. However, Hornef et al. [23] have also localized TLR4 to the cytoplasm of epithelial cells in the intestinal mucosa. This localization seems to be related to cell function in that immune cells should be ready to initiate an immune response, whereas epithelial cells that are exposed to the normal microflora must try to avoid interactions between normal commensal bacteria and TLR4 and the consequent activation of inflammatory signals; although the bacterial flora of the prostate is not well defined, the occurrence of a normal microflora has been suggested [28]. In addition to the LPS from Gram-negative bacteria, other products, such as hyaluronan, heparin, and fibrinogen, have been shown to activate the TLR4 system [29]. Some of these molecules, the so-called endogenous ligands of TLRs, are normally present in the seminal plasma [30–32] and could trigger unwanted inflammatory reactions, since semen is often in contact with the prostate epithelium surface [33]. Consequently, the intracellular distribution of TLR4 could serve to prevent permanent activation of TLR4 cascades in prostatic epithelial cells.
In view of the increments in epithelial TLR4 contents that have been described in the digestive and respiratory tracts subjected to bacterial infection [3], we were interested in determining if E. coli could achieve the same effect in the prostate. Towards this goal, in the present study, we applied a novel experimental model of bacterial prostatitis induced by direct injection of E. coli into the ventral prostate. In contrast to other bacterial prostatitis models in which E. coli is introduced through transurethral instillation, in our model, the delivery of the bacteria to the prostate is controlled, and contamination with urine or pathogens from the bladder and the urethra is avoided. These experimental conditions induce a strong local immune response. Interestingly, in our model, prostatic epithelial cells exhibit early hypertrophy, an increase in TLR4 cytoplasmic content, and migration of this receptor to the apical plasma membrane. Thus, the prostatic epithelium mimics the cells of the innate immune system. Moreover, we noted an increase in the number of epithelial secretory granules that contained antimicrobial proteins, such as surfactant protein-D, after bacterial infection (unpublished data). This modulation of innate immunity within the prostate gland could be a key mechanism in guaranteeing effective clearance of microorganisms, thus avoiding the progression of infections towards the restricted sites of the male reproductive tract, such as the epididymis and testis.
It is well established that testosterone is required for the structural and functional integrity of the prostate. Androgen deprivation caused by castration leads to a marked involution of this gland, with severe epithelial alterations that include a decrease in secretion activity and loss of epithelial cells by apoptosis. The expression of TLR4 is modulated by a variety of environmental factors, such as microbial invasion, microbial components, and cytokines [3]. However, little information is available regarding the influence of sexual hormones on TLR expression. In our experimental models, prostatic TLR4 was significantly increased in the castrated group, as quantified by Western blotting. From the immunocytochemistry, this increase could be ascribed to two different sources, the epithelium and the stroma. Epithelial cells not only maintain their TLR4 expression after castration, but often show more intense immunostaining in comparison to intact rats. The TLR4 molecules appeared to be associated with the basal and basolateral plasma membranes, in addition to their cytoplasmic localization. It has been described that remnants of the epithelial population are mostly comprised of basal cells after castration [34, 35]. Therefore, the cells that were strongly stained for TLR4 may correspond to basal cells that decrease TLR4 expression once they differentiate into mature secretory cells after testosterone replacement therapy. On the other hand, testosterone deprivation caused hypertrophy of the stromal compartment, contributing significantly to increase TLR4 content in the prostatic gland.
TLRs are considered to be key factors in the stimulation of the immune system. Therefore, the increment of TLR4 in the prostate gland after castration described in the present study supports the concept that testosterone has suppressive effects on immune responses [36]. Androgens appear to be responsible for the immunosuppressive profile of normal prostatic cells, which results in the low-level expression of proinflammatory compounds and the generation of high levels of immunosuppressive factors. In this regard, Desai et al. [37] have reported that several genes that encode cytokines involved in the immune response, such as IL-15 and IL-18, are specifically upregulated in the ventral prostate after androgen deprivation. This regulation by testosterone contributes to the immune privileged status of the prostate gland, in which harmful inflammatory responses are usually suppressed [38].
The prostatic stroma is generally implicated in paracrine regulation of the epithelial structure and function, producing critical regulatory factors that are responsible for organ homeostasis and mutual crosstalk [39]. In castrated rats, increased concentrations of TLR4 were detected in the basal aspect of the basement lamina and on the plasmalemma of smooth muscle cells. This observation appears to implicate TLR4 as a crucial player in epithelial-stromal interactions following androgen deprivation.
The smooth muscle cells of the periacinar layer not only underwent hypertrophy, but also became a massive TLR4-positive mass, which probably acts as a pivotal component in the response to the two different stimuli evaluated here. It is well recognized that smooth muscle cells are responsible for the stromal reorganization that occurs after androgen withdrawal [40]. However, these are the first data that define a specific stromal reaction in response to bacterial infection, which appears to be an active component of the innate immune system. Much evidence suggests that smooth muscle cells are metabolically dynamic cells with the potential to express and secrete numerous highly active signaling proteins [41]. In the prostate gland, the enhancement of TLR4 expression could provide the smooth muscle cells with the necessary sensors to detect a wide range of harmful signals from the environment, and thereby produce quickly an innate immune response that is similar to that produced by the cells of the immune system.
The expression of TLRs in stromal cells has been reported at some sites, including the female genital tract [42], respiratory system [43], and in vascular smooth muscle cells [44]. At these locations, the stromal compartment plays an important role in inflammatory diseases, and it is possible that the TLR4 system contributes greatly by amplifying inflammatory signals under certain conditions, such as atherosclerosis and asthma. In the prostate gland, modifications of the fibromuscular stroma are involved in the development of benign and malignant cell growth. Alternatively, an emerging body of evidence supports a possible link between chronic intraprostatic inflammation and prostate cancer. Therefore, it is conceivable that the TLR4 expressed in modified prostatic stromal cells participates in the extensive signaling pathway involved in prostate carcinogenesis. In fact, an association between polymorphisms of the TLR4 gene and prostate cancer risk has been reported [45].
The data presented here have a wide range of implications for the pathophysiology of the prostate gland. In part, prostatic cells appear to provide the most important membrane safeguard in sensing the microenvironment of the gland. Simultaneously, the intracellular localization of TLR4 emerges as a defensive mechanism that could protect the gland from unnecessary activation of TLR4 signaling. Finally, these results provide strong new evidence that the smooth muscle plays a major role in controlling situations that could compromise the homeostasis of the prostate gland.
ACKNOWLEDGMENTS
The authors thank Mrs. Elena Pereyra, Mrs. Lucia Artino, Ms. Mercedes Guevara, and Mr. Cristian Giacomelli for expert technical assistance, and Gabriel Balabanian for valuable assistance with the electron microscope. We are indebted to the entire Team-18 for help with the biochemical analyses and for advice. We thank Dr. Paul Hobson for revising the manuscript.
FOOTNOTES
2 Correspondence: Cristina A. Maldonado, Centro de Microscopía Electrónica, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Pab. Biología Celular 1° piso, Haya de La Torre esq. Enrique Barros, Ciudad Universitaria, X5000HRA Córdoba, Argentina. FAX: 54 0351 4333021; cmaldon{at}cmefcm.uncor.edu 
1 Supported by research grants from Consejo National de Investigaciones Científicas y Técnicas (CONICET PEI 1923/03), Grant PICT from FONCyT-ANPCyT, and by a fellowship to A.A.Q. from Fundacion Florencio Fiorini.
Received: 18 May 2006.
First decision: 9 June 2006.
Accepted: 18 July 2006.
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