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The Journal of Immunology, 1999, 162: 995-1002.
Copyright © 1999 by The American Association of Immunologists

Development of an Animal Model for Neurocysticercosis: Immune Response in the Central Nervous System Is Characterized by a Predominance of {gamma}{delta} T Cells1

Astrid E. Cardona*, Blanca I. Restrepo*,{dagger}, Juan M. Jaramillo*,{dagger} and Judy M. Teale2,*

* Department of Microbiology, University of Texas Health Science Center, San Antonio, TX 78284; and {dagger} Corporación para Investigaciones Biológicas, Medellín, Colombia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neurocysticercosis is the most common parasitic disease of the central nervous system worldwide. It is caused by the metacestode form of the helminth Taenia solium. Study of the immune response in the human brain has been limited by the chronic progression of the disease, the influence of corticosteroid treatment, and the scarcity of patients who undergo surgical intervention. To better understand the immune response and associated pathology in neurocysticercosis, a mouse model was developed by intracranial infection of BALB/c mice with Mesocestoides corti, a cestode organism related to T. solium. The immune response reveals the presence of abundant inflammatory infiltrates appearing as early as 2 days postinfection in extraparenchymal regions. In contrast, infiltration of immune cells into parenchymal tissue is significantly delayed. There is a natural progression of innate (neutrophils and macrophages), early induced (NK cells and {gamma}{delta} T cells), and adaptive immune responses ({alpha}ß T cells and B cells) in infected mice. {gamma}{delta} T cells are the predominant T cell population. A cell-mediated Th1 pathway of cytokine expression is evident in contrast to the previously described Th2 phenotype induced in the periphery.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Infection of the human central nervous system (CNS)3 with metacestodes (tissue larvae) of the parasite Taenia solium causes neurocysticercosis (NCC) (1). NCC is likely the most common parasitic disease of the CNS (2). The highest prevalence rates are in Mexico, Central and South America, India, Africa, and Asia, with a minimum estimate of 50 million infected persons worldwide (3, 4, 5, 6). With immigration, NCC is becoming increasingly more common in the United States. Although most of the cases in the United States occur in immigrants, there is clear evidence for locally acquired cases (7, 8, 9, 10, 11). Epidemiological studies indicate that NCC is the most common cause of late-onset epilepsy in areas where Taenia is endemic (12). Other symptoms include chronic headaches and, in severe cases, hydroencephaly (1, 2).

Much of the pathology of NCC is thought to be due to the host immune response to the metacestode in the brain (13, 14). Therefore, a careful characterization of the immune response is critical for understanding the disease process. This has been hampered by the difficulty in studying CNS immune responses in patients and the complexity of the associated biology. The immune response is thought to vary depending on the incubation period, the number and location of cysts in the brain, and the stage of the cyst (alive or in any of various stages of disintegration) (13, 14, 15). Because of the number of variables, we sought to develop a mouse model in which a systematic study of the immune response could be performed.

T. solium is not infectious in mice. Therefore, a related cestode parasite, Mesocestoides corti, was used (16, 17, 18, 19, 20). In nature, M. corti ova are thought to be ingested by terrestrial arthropods (20). An intermediate host, e.g., lizard or mouse, then consumes the arthropod, whereby the oncosphere develops into a mature larva or metacestode. Upon ingestion of the intermediate host by a carnivorous mammal such as a dog, cat, or skunk, a mature intestinal tapeworm develops, releasing eggs and perpetuating the life cycle. A similar life cycle is observed with T. solium in cysticercosis (2). In NCC, however, individuals consume the ova, which appears to result in an immature larva migrating to the brain and developing into a metacestode. An immune response to the metacestode is then elicited, causing much of the pathology (1, 2).

To parallel the human disease of NCC as much as possible, metacestodes from M. corti were injected intracranially, avoiding penetration of the brain tissue. Similar to T. solium larvae, they were found to be highly invasive, infiltrating within days ventricular and subarachnoid spaces as well as the parenchyma. Animals were killed at various times after infection, and the immune cells and cytokines were analyzed by in situ immunohistochemistry. One of the most interesting aspects of the response was an extensive accumulation of {gamma}{delta} T cells that remained throughout the course of the study. Importantly, the infection-induced CNS immune response proceeded through a typical innate response, followed by an early induced response of NK cells and {gamma}{delta} T cells and, eventually, an adaptive immune response. Similar to our analyses of brain specimens from NCC patients (21), the cytokine response was typical of a Th1 or inflammatory response. The study shows that this animal model represents a prototype for NCC and will be invaluable for further analysis of the immune response and the associated pathology.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Female BALB/c mice were purchased from the National Cancer Institute Animal Program (Bethesda, MD). Animal experiments were conducted under the guidelines of the University of Texas System, The United States Department of Agriculture, and the National Institutes of Health.

Parasites and inoculations

M. corti metacestodes were used in all experiments. Parasites were maintained by serial i.p. inoculations of 8- to 12-wk-old female BALB/c mice with 50–75 metacestodes in 0.5 ml of HBSS (16, 22).

For intracranial inoculations, parasites were aseptically collected from the i.p. cavity of mice that had been infected for about 6 mo. Harvested parasites were extensively washed in HBSS. Analysis of number and viability of organisms was determined by direct stereoscopic examination. The metacestodes (~40 parasites) were then suspended in 60 µl of sterile HBSS and injected intracranially into 3–5-wk-old female BALB/c mice using 1-ml syringes and a 25-gauge needle (23). The needle was inserted to a 2-mm depth at the junction of the superior sagittal and the transverse sutures. This allows insertion of the needle into a protective cuff avoiding penetration of the brain tissue (23). Control mice were injected with 60 µl of sterile HBSS using the same protocol. Before intracranial inoculation, mice were anesthetized with a mixture of phenobarbital (1.2 mg) and xylazine (0.16 mg) in 100 µl. At several times after inoculation, animals were sacrificed and assessed for extent of infection and various immune parameters (see below). Before sacrifice, animals were anesthetized with a mixture of ketamine (6 mg) and xylazine (0.8 mg) and perfused through the left ventricle with 20 ml of cold PBS.

Dye exclusion assay

Integrity of the blood-brain barrier (BBB) was assessed 10 min and 24 h after intracranial inoculation. Ten minutes before sacrifice, mice were injected in the tail vein with 100 µl of 4% Evans blue dye in PBS, pH 7.4 (24). Blood samples were taken by cardiac puncture using 3% sodium citrate in H2O as an anticoagulant. Then mice were perfused through the left ventricle. Brains were removed, homogenized for 10 s in 3 ml of PBS using an Omni 2000 homogenizer (Omni International, Waterbury, CT), and centrifuged for 20 min at 750 x g. Supernatants were again centrifuged for 30 min at 2500 x g, and optical densities were read at 610 nm. For blood samples, 100 µl of plasma was used. A standard curve was used to quantify the amount of dye present in the brain and plasma as previously described (24, 25, 26).

Tissue processing

The brain was immediately removed from perfused animals, embedded in optimal cutting temperature compound, and snap frozen. Serial horizontal cryosections 10 µm in thickness were placed on silane preparation slides (Sigma, St. Louis, MO). One in every four slides was fixed in formalin for 12 min at room temperature and stained with hematoxylin and eosin (H&E). The remainder of the slides was air dried overnight and fixed in fresh acetone for 20 s at room temperature. Acetone-fixed sections were wrapped in aluminum foil and stored at -80°C or processed immediately for immunohistochemistry.

H&E staining

After formalin fixation, slides were washed twice in deionized water, stained 30 s in hematoxylin, and washed in distilled water for 1.5 min. Slides were submerged in 0.5% HCl and then in 0.1% NH4(OH)2. Tissues were dehydrated in 95% ethanol for 1 min and stained in eosin for 15 s followed by a 2-min treatment with 95% and 100% ethanol each. Slides were allowed to air dry. They were then submerged in xylene for 3 min and mounted using Pro-Texx mounting medium (Baxter Diagnostics, Deerfield, IL). By light microscopy, the number and location of parasites were assessed, as well as the presence or absence of mononuclear infiltrates.

Monoclonal Abs

Biotinylated Abs were used to identify particular murine leukocytes and cytokines by immunohistochemistry. Biotinylated Abs purchased from PharMingen (San Diego, CA) include 145-2C11 (anti-CD3{epsilon}), RM4-5 (anti-CD4), 53-6.7 (anti-CD8), GL3 (pan-anti-{gamma}{delta}), H57-597 (pan-anti-{alpha}ß), 5E6 (anti-NK), M1/70 (anti-Mac1), 1D3 (CD19 and B cells), 25-9-17 (which reacts with both I-Ad and I-Ab), 536 (anti-V{gamma}5 (V{gamma}3 according to nomenclature developed by Garman et al. (27)), UC3–10A6 (anti-V{gamma}4, or V{gamma}2 by Garman et al. (27) nomenclature), GL2 (anti-V{delta}4), XMG1.2 (anti-IFN-{gamma}), SXC-1 (anti-IL-10), BVD6-24G2 (anti-IL-4), MP5-32C11 (anti-IL-6), JES6-5H4 (anti-IL-2), G297-289 (anti-IL-12), and G277-3960 (anti-IL-15). The 1400-24.17 mAb (anti-IL-1ß) was purchased from Endogen (Woburn, MA). The mAb 17C (anti-{delta}6.3) was kindly donated by Dr. Simon Carding (University of Pennsylvania, Philadelphia, PA). The G297-289 purified mAb (anti-IL-12), which was obtained from PharMingen, and the purified anti-mouse polyclonal Ab against the IL-13 from R&D Systems (Minneapolis, MN) were biotinylated using the protocol previously described (21). Each of the mAbs was initially titrated on spleen sections of infected mice. A dilution was chosen to give maximum sensitivity with no background staining. Some Abs were completely negative on brain specimens but known to be active by positive staining on spleen sections.

Immunohistochemistry

Frozen sections were thawed for 60 min at room temperature and postfixed in acetone for 10 min at room temperature. Sections were then hydrated in 0.1% BSA in PBS for 6 min. To block binding by endogenous avidin and biotin, sections were treated with an avidin-biotin blocking kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. Sections were also blocked for nonspecific Ig binding by incubation for 30 min with a 1:10 dilution of normal serum from the appropriate host species. Sections were then incubated for 1 h at 37°C, with specific Abs diluted in 3% host normal serum in HBSS. This was followed by a 30-min incubation with the streptavidin-alkaline phosphatase conjugate (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Between each incubation step of the protocol, the slides were rinsed three times in PBS for 3 min each. The reaction product was developed using the alkaline phosphatase substrate kit (Vector Laboratories) according to the manufacturer’s instructions, which produced a red stain.

Sections were counterstained with an alcian blue-methyl green solution (0.25% alcian blue and 0.25% methyl green in 1 mM sodium acetate, pH 5.2) for 1 min and then dehydrated in solutions of increasing ethanol concentrations (75%, 95%, and 100%, 1 min per solution). After 3 min in xylene, sections were mounted with Pro-Texx mounting medium (Baxter Diagnostics).

A semiquantitative analyses of the cellular infiltrates was done by counting the number of cells of a given cell type/cytokine per section. An analysis of all of the markers required many sections. Serial sections can vary in the extent of exposed infiltrate. Therefore, comparison of actual numbers of cells staining with different markers is only semiquantitative. This problem was alleviated somewhat by testing all markers on two infected animals. Positive cells were counted on an entire section. The area of the sections ranged from 50 to 78 mm2. The results were scored, in positive cells per section, as follows: +, 1–100; ++, 100–300; +++, 300–500; and ++++, >500. The results shown represent the average of two mice.

Transmission electron microscopy

A mouse brain infected for 3 wk was processed for analysis by electron microscopy. The brain was perfused with 20 ml of cold PBS and then fixed in a standard phosphate-buffered mixture (84 mM NaH2PO4, 68 mM NaOH) of 4% formaldehyde (v/v) and 1% glutaraldehyde (v/v). From 1-mm2 pieces, ultrathin 75-nm sections were collected on a 150-mesh copper grid and stained with saturated aqueous uranyl acetate and Reynolds lead citrate (Electron Microscopy Sciences, Fort Washington, PA). Photographs were taken using a JEOL 100CX electron microscope (JEOL, Peabody, MA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distribution of larvae in the brain with time postinfection

Mice were inoculated with approximately 40 metacestodes, since this number of organisms resulted in infection of essentially all inoculated animals. With this dose, animals usually began to show neurological symptoms including staggering and abnormal vestibular functions (e.g., abnormal landing foot splay). Even though symptoms started by 3–5 wk postinoculation (p.i.), mice did not usually succumb until about 13 wk. We are currently experimenting with lower numbers of organisms so that animals infected for longer periods can be analyzed.

Serial horizontal sections spaced every 10 µm were stained with H&E to assess the number of larvae, location, and presence of cellular infiltrate. Table IGo shows the number and location of organisms. Within the first few days of infection, many of the organisms remained outside the brain and were lost when the brain was removed for analysis. By 1 wk p.i., the majority of parasites were extraparenchymal (in the ventricles, leptomeninges, and subarachnoid spaces). However, by 3 wk p.i., approximately half of the organisms had penetrated the parenchyma. Fig. 1GoA shows a larva invading brain parenchyma. The percentage of organisms located in parenchymal tissue continued to increase with time postinfection.


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  		Table I. Distribution of M. corti larvae in brain1

 


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  		FIGURE 1. Brain sections were processed for H&E staining or immunohistochemistry as described. A, A larva invading the brain is shown in an H&E-stained section. The meningeal membranes are penetrated, and the tegument that covers the parasite is shown intact. B, Infiltrating {gamma}{delta} T cells detected by in situ immunohistochemistry are associated with two parasites in the subarachnoid space; positive cells are shown in red. C, Parenchymal parasite shown by H&E staining with no evidence of associated inflammatory cells detected by immunohistochemistry. D, Inflammatory infiltrate in ventricle analyzed by electron microscopy PMN (small arrow) and plasma cell (large arrow) are shown. E, {gamma}{delta} T cells detected using a specific Ab against the V{delta}4 chain in a ventricular infiltrate. F, IFN-{gamma}-producing cells detected by immunohistochemistry within a ventricle. Magnifications: A and C, x150; B, x100; D, x7250; E and F, x600.

 
The vast majority of extraparenchymal organisms were associated with a mononuclear infiltrate as early as 2 days p.i., indicating a rapid immune response. This was not due to damage to the BBB by the inoculation protocol, since control mice that were injected with HBSS did not show any cellular infiltrate. One control animal of two showed the presence of a few macrophages, but there was also indication that the injection had penetrated the brain tissue. Moreover, dye exclusion experiments performed to assess the effect of intracranial injections on the integrity of the BBB showed that the BBB was intact by 24 h after injection. Thus, no significant difference was found among control mice not exposed to the inoculation procedure and HBSS- or M. corti-injected animals in terms of the amount of Evans blue dye detected in the brain and serum sample (data not shown). Early after infection (2–5 days p.i.), the mononuclear infiltrate was dramatic in the extraparenchymal spaces, and one could often locate the organism by following the trail of cells (Fig. 1GoB). In contrast, the infiltration of mononuclear cells into the parenchyma was slower, and at the earliest times postinfection many parenchymal parasites showed no evidence of associated infiltrate (Fig. 1GoC). This was confirmed by immunohistochemistry (data not shown).

Temporal appearance of specific cell types p.i. in extraparenchymal spaces

To characterize the nature of the cellular infiltrate, immunohistochemistry was performed on brain specimens at various times after infection. Control HBSS-injected mice were also analyzed, but since no infiltrate was observed, except in the animal described above, these negative data are not included in any of the tables. Because of the more rapid infiltration of cells into the extraparenchymal spaces compared with the parenchyma, these two areas were analyzed separately for the appearance of distinct cell types. The data provided in Table IIGo indicate that by 2–3 days p.i. the CNS immune response consisted of neutrophils (detected by H&E staining), macrophages (Mac-1+), {gamma}{delta} T cells, and NK cells. Most dramatic was the infiltration of {gamma}{delta} T cells (Fig. 1GoB). The cellular infiltrate was also analyzed by electron microscopy. Polymorphonuclear neutrophils (PMN), macrophages, and plasma cells were present in ventricular (Fig. 1GoD) and parenchymal infiltrates. MHC class II expression (I-Ad-positive cells) was also observed consistent with the presence of macrophages. The absence of a direct correlation between the frequency of CD3+ cells and that of {gamma}{delta} T cells has been noted by other investigators and is likely due to sensitivity/accessibility issues (28). Initially, the number of mononuclear cells was found to be relatively small, but by 1 wk of infection, the accumulation of cells surrounding organisms was substantial (>300–500 cells/brain section). Moreover, by 5–7 days p.i., {alpha}ß T cells were detectable, as well as relatively small numbers of CD19+ B cells. Although both CD4+ and CD8+ T cells were detected, CD4+ cells consistently outnumbered CD8+ cells by approximately 2:1. Interestingly, {gamma}{delta} T cells continued to be a predominant cell type throughout the course of the 13-wk study.


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  		Table II. Immune cells detected in the extraparenchymal regions of brains of infected mice1

 
Slower appearance of cells and fewer mononuclear cells in brain parenchyma

During the first week of infection, very few mononuclear cells were found in the parenchyma despite the presence of metacestodes (Table IIIGo). Only granulocytes and macrophages were present and in substantially fewer numbers compared with the extraparenchyma spaces. {gamma}{delta} T cells were detected by 3 wk and continued to be found for the duration of the study. CD4+ and CD8+ cells were not detected until 5 wk p.i. Although there were a few NK cells detected in the parenchyma in the absence of an associated organism, no NK cells were detected in the direct vicinity of a metacestode. Macrophages and T cells could also be detected in such areas devoid of parasites. The mechanism for the presence of immune cells in the absence of an organism is not clear to us. Because entire brains were serially sectioned and the location of all metacestodes was determined, we are confident that the immune cells observed were not in the vicinity of any parasite, even the ones out of the plane of the section being observed. It is possible, though, that migrating organisms leave parasite Ags behind that can elicit an immune response.


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  		Table III. Immune cells detected in the brain parenchyma of infected mice

 
Variable genes used by predominant {gamma}{delta} T cell infiltrate

To ensure that our pan-anti-{gamma}{delta} T cell reagent was not picking up some cross-reactive cell type and to determine the diversity associated with the {gamma}{delta} T cell response, various Abs to {gamma}{delta} variable regions were used in immunohistochemistry. The results (Table IVGo) indicate that the V{delta}4-positive T cells (Fig. 1GoE) and V{gamma}4-positive T cells substantially contributed to the {gamma}{delta} T cell population. V{gamma}5 cells and V{delta}6.3 {gamma}{delta} T cells were detected less frequently.


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  		Table IV. {gamma}{delta}TCR repertoire in inflammatory infiltrates in extraparenchymal regions

 
Production of cytokines in the CNS is consistent with a Th1-type inflammatory response

To further characterize the cellular response due to CNS infection by M. corti metacestodes, the production of several cytokines was assessed by in situ immunohistochemistry. Cytokines detected in ventricles and subarachnoid space were predominantly IL-2, IL-12, IL-15, and IFN-{gamma} (Table VGo). IL-2 and IL-12 appeared after 2 days p.i., IL-15 by day 5 p.i., and IFN-{gamma} by 1 wk p.i. (Fig. 1GoF). All four cytokines colocalized (adjacent serial sections) to areas where {gamma}{delta} TCR+ cells and macrophages were present. These four cytokines appeared to be the predominant cytokines produced throughout the course of infection. IL-4 was detected after 1 wk p.i. in low levels. There was sporadic appearance of a few TNF-{alpha}- and IL-10-producing cells later in infection. However, several cytokines were not detected, including IL-1, IL-6, and IL-13. The absence of key proinflammatory cytokines, including IL-1 and IL-6, is of interest. Moreover, the Th2 cytokines, IL-4 and IL-10, highly induced during helminth infection were not predominantly expressed, and IL-13, which is also an important cytokine in Ab-mediated immune responses, was undetectable at all time points tested.


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  		Table V. Cytokine-producing cells detected in the extraparenchymal regions of brains of infected mice1

 
Essentially the same cellular infiltrates and Th1 cytokines were also found in the parenchymal sites (Tables III and VI). However, the cellularity was diminished, and the cytokines IL-2, IL-12, IL-15, and IFN-{gamma} were present at certain time points and in low abundance (Table VIGo).


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  		Table VI. Cytokine-producing cells detected in the brain parenchyma of infected mice1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous attempts to develop an appropriate animal model for NCC, including hamsters, monkeys, and wild boars, have been limited because of the complex life cycle of T. solium (29, 30, 31). The normal course of infection for the intermediate host, as well as infection of humans leading to NCC, is ingestion of the ovum form of the organism. It has been very difficult to obtain ova from humans, since the eggs are not passed regularly in the feces. In addition, animal models of the immediate host have resulted in sterile tapeworms. Therefore, in the animal models studied to date, the metacestode form of the organism has been injected and invasion of the liver and peritoneum has been mostly studied (2, 32, 33, 34, 35, 36). While not a perfect model, it is still valid, since it is the metacestode that encysts the liver and other body tissues, eliciting an immune response. However, these animal models lack CNS involvement, thus preventing an analysis of the immune response to metacestodes in the brain.

The purpose of this study was to develop an animal model for NCC, one of the most common parasitic infections of the CNS. M. corti metacestode was chosen as the infecting organism since it is a cestode like T. solium, is amenable to intracranial injections, and is the form of cestodes that encysts the brain in NCC. We are also in the process of testing a murine Taenia species, T. crassiceps. The metacestode form of this organism, however, is large and appears to be less invasive compared with M. corti. Nevertheless, studies with T. crassiceps will help determine the generality of the immune response to platyhelminths in the CNS.

The results indicate that within 2–3 days M. corti larvae have already invaded both parenchymal and extraparenchymal areas of the brain. The majority of the invading organisms are first found in the extraparenchymal spaces. This seems logical, since invasion of the parenchyma probably involves induction of the enzymatic machinery (e.g., proteolytic enzymes) necessary to penetrate the brain tissue. By 3–5 wk of infection, there is an equal distribution of organisms in parenchyma and extraparenchymal spaces, but eventually the majority of the organisms appear to enter the parenchyma. This parallels the results found in humans with T. solium, most of whom present with parenchymal cysts (2).

An interesting aspect of these studies is that there appears to be a natural progression of innate, early induced, and adaptive immune responses. {alpha}ß T cells are delayed by several days, particularly in the parenchyma, compared with cells ascribed to the innate and early induced responses. This is important, because it has been proposed that only memory T cells pass through the BBB and remain in the CNS, and only if the relevant Ags are present (37). It has been further proposed that Ag-activated memory T cells in the brain then initiate/amplify the inflammatory response as a result of CNS infections or autoimmune diseases such as multiple sclerosis or experimental allergic encephalitis (38, 39, 40). In the model described here, the first encounter with the Ag (organism) is in the CNS, and the results suggest that under these circumstances the CNS response begins with innate immunity similar to a peripheral infection. Given the extent of neurotropism associated with T. solium, especially with the organisms endemic to Mexico and Central and South America (4, 12), it is possible that a similar situation can occur in NCC. To address this further, we plan to first infect mice with M. corti i.p. and follow the infection with an i.c. inoculation at a later time point to see whether this changes the course of the CNS immune response. It will also be important to examine cell activation markers. Also of potential relevance, these types of organisms are known to produce excretory/secretory molecules (41, 42, 43). The extent to which these molecules enter the periphery and induce a systemic immune response with possible migration of activated immune cells back to the CNS is not known.

The cytokines produced by infiltrating cells in the CNS during M. corti infection are indicative of a cell-mediated, Th1 type of response (44, 45). Thus, the cytokines detected include IL-2, IL-12, IL-15, IFN-{gamma}, and TNF-{alpha}. IL-4, the major indicator of the Th2 Ab/eosinophilia immune pathway (45), was detected in low levels during the first weeks of infection. This correlates with the presence of low numbers of B cells present after the first week postinfection. Therefore, a complete dichotomy in the Th1 and Th2 types of responses was not observed. This is of interest since M. corti is a platyhelminth that typically induces a strong Th2 response replete with an IgG1, IgE hypergammaglobulinemia, and eosinophilia when the infection is a peripheral one (16, 22). In a previous study, we analyzed human brain specimens from four NCC patients for the cell types and cytokines present (21). Importantly, the results were similar in that a Th1-type, cell-mediated response predominated. Thus, it is possible that with helminth infections the microenvironment of the CNS favors a Th1 response, whereas the systemic environment favors a Th2 response.

IL-12 is considered to be a pivotal cytokine for inducing the cell-mediated, Th1-type pathway (44). Recently, it has been shown that IL-12 is protective in vesicular stomatitis virus CNS infections (46). IL-12 appears to directly activate neurons as well as astrocytes and microglia to produce nitric oxide synthase. Therefore, it will be very important to determine in our studies whether IL-12 is involved in immunoprotection and the role of brain-resident cells that may be influenced by the infection-induced cytokine microenvironment.

There are several factors that contribute to the immune privilege status of the normal brain, especially the BBB. Even when the BBB was breached during infection, there was a substantial delay in the appearance of immune cells and associated cytokines in the parenchyma compared with the extraparenchymal spaces. Granuloma formation was also delayed. When M. corti metacestodes are injected i.p., the liver is the main organ that is encysted (16). Under these circumstances, evidence of mature granuloma formation is found by the first 2–3 wk of infection. However, in this study granuloma formation was not observed. Interestingly, mature granulomas were not found in the human specimens either (21), although magnetic resonance imaging scans of NCC patients often exhibit calcifications (1, 2, 47), suggesting that granulomas eventually form (48). Therefore, it will be important to examine mice injected with fewer organisms and reexamine the cytokine response and the potential appearance of granulomas after 6 mo to 1 year of infection.

One of the most interesting aspects of this CNS infection is the dramatic accumulation of {gamma}{delta} T cells. A {gamma}{delta} T cell response has been described in a number of different types of infections including bacteria, viruses, and parasites (49). Although there is evidence for a late dominance of {gamma}{delta} T cells in murine influenza (50), {gamma}{delta} T cells typically appear early in the infectious process (51, 52). Because of their restricted repertoire, they are thought to provide a first line of defense and respond to a limited number of Ags. However, little is known regarding effector functions. Earlier studies suggested that {gamma}{delta} T cells might respond to a common stress response (53). This is of interest, since our previous work demonstrated that M. corti metacestodes actively secrete a number of molecules, including at least two heat shock proteins (42, 43). More recently, it has been shown that {gamma}{delta} T cells can respond to nonpeptide Ags such as alkyl phosphates (54) and that the crystalline structure of the {gamma}{delta} TCR combining site more closely resembles an Ab (55). Thus, it will be important to determine the nature of the antigenic stimulus in this infection.

Based on the V region analysis, it does not appear that the infection is inducing a single subset of {gamma}{delta} T cells. However, the predominance of V{delta}4, V{gamma}4, and V{delta}6.3 suggests that the source of {gamma}{delta} T cells be mainly from lymphoid tissue and not the epithelium (49). The role of {gamma}{delta} T cells in the CNS defense will also be important to assess. Although {gamma}{delta} T cells have been described as a minor population in multiple sclerosis lesions (56), to our knowledge, they have not been described as a major cell type in inflammatory infiltrates in the brain. It is possible that the early response in the CNS helps to establish a cytokine microenvironment that promotes the Th1 pathway. Consistent with this, immunocytochemistry of serial sections indicate that {gamma}{delta} T cells colocalized to IL-2, IFN-{gamma}, and IL-12. Since other cell types are present, we are in the process of performing double labeling experiments. {gamma}{delta} T cells have been described previously to produce IL-2 and IFN-{gamma} (57) and are a likely source of these cytokines early in the infection. Also of interest is the up-regulation of IL-15 in the infection, since this cytokine has been shown to cause proliferation and prevent apoptosis of {gamma}{delta} T cells (58). IL-15 has also been shown to increase the cytotoxicity of granular lymphocytes in patients with lymphoproliferative disease of granular lymphocytes (59). Since the {gamma}{delta} T cells often associate with the organism, we are currently looking for the presence of cytotoxic molecules (e.g., perforins) that would be indicative of a cytotoxic role.

In summary, this study describes a viable model for the study of the human disease NCC. M. corti metacestodes are shown to invade the CNS, resulting in a multifaceted immune response that includes innate, early induced, and adaptive immunity. Although an analysis of the kinetics of the immune response in NCC patients is not possible, the cell types identified in surgical specimens are essentially the same as shown here (21). Heterogeneity in cell types was observed in the different immune lesions, but among the patients analyzed, the cell types identified included macrophages, neutrophils, NK cells, CD4+ and CD8+ T cells, and plasma cells. Thus, the results were consistent with cells characteristic of early and late immune responses (21). A prominent feature of the animal model is a striking {gamma}{delta} T cell response. It is not known yet whether human NCC exhibits a similar {gamma}{delta} T cell response, since our human studies were completed before our knowledge of the {gamma}{delta} T cell response in mice. We plan to determine this as soon as we obtain human surgical specimens from Dr. Antonio Enciso (Instituto Mexicano del Seguro Social, Mexico City, Mexico). Another important characteristic of the animal model is that the predominant immune response follows a Th1 pathway. This provides another parallel to NCC, in which the cells and cytokines identified in the human specimens were most characteristic of a Th1 pathway. Thus, IL-12 was found in all immune lesions analyzed and IL-2 and IFN-{gamma} were also prominent. In contrast, IL-4 was not detected in any of the inflammatory infiltrates of human specimens analyzed (21). The mice also develop neurological complications, as do many NCC patients, and it will be important to correlate neurological symptoms with the immunopathology. Taken together, the animal model described here should provide a unique opportunity for delineating immunoregulatory and pathogenic mechanisms in NCC.


   Acknowledgments
 
We thank Dr. Sally Atherton and her group for advice on immunocytochemistry in the brain and Dr. Simon Carding for providing the anti-V{delta}6.3 Ab.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants NS35974, AI3321, and AI19896. Back

2 Address correspondence and reprint requests to Dr. Judy M. Teale, Department of Microbiology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284. E-mail address: Back

3 Abbreviations used in this paper: CNS, central nervous system; NCC, neurocysticercosis; H&E, hematoxylin and eosin; p.i., postinoculation; BBB, blood-brain barrier; PMN, polymorphonuclear neutrophils. Back

Received for publication June 26, 1998. Accepted for publication September 25, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Davis, L. E., M. Kornfeld. 1991. Neurocysticercosis: neurologic, pathogenic, diagnostic and therapeutic aspects. Eur. Neurol. 31:229.[Medline]
  2. Jr White, C. A.. 1997. Neurocysticercosis: a major cause of neurological disease worldwide. Clin. Infect. Dis. 24:101.[Medline]
  3. Del Brutto, S.. 1988. Neurocysticercosis: an update. Rev. Infect. Dis. 10:1075.[Medline]
  4. Schantz, P. M.. 1989. Surveillance and control programs for cestodes diseases. M. J. Miller, and E. J. Love, eds. Parasitic Diseases: Treatment and Control 275.-290. CRC Press, Boca Raton, FL.
  5. Mahajan, R. C.. 1982. Geographical distribution of human cysticercosis. A. Flisser, and K. Willims, and J. P. LaClette, eds. Cysticercosis: Present State of Knowledge and Perspectives 39.-46. Academy Press, New York.
  6. Tsang, V. C. W., M. Wilson. 1995. Taenia solium cysticercosis: an under-recognized but serious public health problem. Parasitol. Today 11:124.
  7. Schantz, P. M., A. C. Moore, J. L. Muñoz, B. J. Hartman, J. A. Schaefer, D. Persaud, E. Sarti, M. Wilson, A. Flisser. 1992. Neurocysticercosis in an orthodox Jewish community in New York City. N. Engl. J. Med. 327:692.[Abstract]
  8. Sorvillo, F. J., S. H. Waterman, F. O. Richards, P. M. Schantz. 1992. Cysticercosis surveillance: locally acquired and travel-related infection and detection of intestinal tapeworm carriers in Los Angeles. Am. J. Trop. Med. Hyg. 47:365.
  9. Stamos, J. K., E. G. Chadwick, P. M. Schantz, M. Wilson, A. H. Rowley. 1994. New-onset seizures due to cysticercosis in low-risk children in Chicago. Abstracts of the 34th Interscience Conference on Antimicrobial Agents and Chemotherapy 198. American Society for Microbiology, Orlando, FL.
  10. Moore, A. C., L. I. Lutwick, P. M. Schantz, J. B. Pilcher, M. Wilson, A. W. Hightower, E. K. Chapnick, E. I. M. Abster, J. R. Grossman, J. A. Fried, D. A. Ware, X. Haichou, S. S. Hyon, R. L. Barbour, R. Antar, A. Hakim. 1995. Seroprevalence of cysticercosis in an orthodox Jewish community. Am. J. Trop. Med. Hyg. 53:439.
  11. Centers for Disease Control: locally acquired neurocysticercosis, North Carolina, Massachusetts and South Carolina, 1989–1991. 1992. Morbid. Mortal. Wkly. Rep. 41:1.
  12. Pittella, J. E.. 1997. Neurocysticercosis. Brain Pathol. 7:681.[Medline]
  13. Escobar, A.. 1983. The pathology of neurocysticercosis. E. Palacios, and J. Rodriguez-Carbajal, and J. M. Taveras, eds. Cysticercosis of the Central Nervous System 27.-54. Thomas, Springfield, IL.
  14. Rabiela-Cervantes, M. T., A. Rivas-Hernandez, J. Rodriguez-Ibarra, S. Castillo-Medina, F. M. Cancion. 1982. Anatomopathological aspects of human brain cysticercosis. A. Flisser, and K. Willms, and J. P. LaClette, and C. Larralde, and C. Ridaura, and F. Beltran, eds. Cysticercosis: Present State of the Knowledge and Perspectives 179.-200. Academic Press, New York.
  15. Flisser, A., B. Espinoza, A. Tovar, A. Plancarte, D. Correa. 1986. Host-parasite relationship in cysticercosis: immunologic study in different compartments of the host. Vet. Parasitol. 20:95.[Medline]
  16. Mitchell, G. F., J. J. Machalonis, P. M. Smith, W. L. Nicholas, N. L. Warner. 1977. Studies on immune responses to larval cestodes in mice: immunoglobulins associated with the larvae of Mesocestoides corti. AJEBAK 55:187.
  17. Eckert, J., T. Von Brand, M. Voge. 1969. Asexual multiplication of Mesocestoides corti (Cestoda) in the intestine of dogs and skunks. J. Parasitol. 55:241.[Medline]
  18. Specht, D., M. Voge. 1965. Asexual multiplication of Mesocestoides corti tetrathyridia in laboratory animals. J. Parasitol. 51:268.[Medline]
  19. Webster, J.. 1948. Fragmentary studies on the life history of the cestode Mesocestoides latus. J. Parasitol. 34:83.
  20. Novak, M.. 1972. Quantitative studies on the growth and multiplication of tetrathyridia of Mesocestoides corti Hoeppli, 1925 (Cestoda:Cyclophyllidia) in rodents. Can. J. Zool. 50:1189.[Medline]
  21. Restrepo, B. I., P. Llaguno, M. A. Sandoval, J. A. Enciso, J. M. Teale. 1998. Analysis of immune lesions in neurocysticercosis: central nervous system response to helminth appears Th1-like instead of Th2. J. Neuroimmunol. 89:64.[Medline]
  22. Abraham, K. M., J. M. Teale. 1987. The contribution of parasite-specific T cells to isotype restriction in Mesocestoides corti. J. Immunol. 139:2530.[Abstract]
  23. Ding, J. C., M. Bauer, D. M. Diamond, M. A. Leal, D. Johnson, B. K. Williams, A. M. Thomas, L. Najvar, J. R. Graybill, R. A. Larsen. 1997. Effect of severity of meningitis on fungicidal activity of flucytosine combined with fluconazole in a murine model for cryptoccocal meningitis. Antimicrob. Agents Chemother. 41:1589.[Abstract]
  24. Neill, A. L., N. H. Hunt. 1995. Effects of endotoxin and dexamethasone on cerebral malaria in mice. Parasitology 111:443.
  25. Uyama, O., N. Okamura, M. Yanase, M. Narita, K. Kawabata, M. Sugita. 1988. Quantitative evaluation of vascular permeability in the gerbil brain after transient ischemia using Evans blue fluorescence. J. Cereb. Blood Flow Metab. 8:282.[Medline]
  26. Wolman, M., I. Klatzo, E. Chui, F. Wilmes, K. Nishimoto, K. Fujiwara, M. Spatz. 1981. Evaluation of the dye-protein tracers in pathophysiology of the blood-brain barrier. Acta Neuropathol. 54:55.[Medline]
  27. Garman, R. D., P. J. Doherty, D. H. Raulet. 1986. Diversity, rearrangement and expression of murine T cell {gamma} genes. Cell 45:733.[Medline]
  28. Fidel, P. L., N. A. Wolf, M. KuKuruga. 1996. T lymphocytes in the murine vaginal mucosa are phenotypically distinct from those in the periphery. Infect. Immun. 64:3793.[Abstract]
  29. Ito, A., J. D. Smyth. 1987. Adult cestodes: immunology of the lumen dwelling cestode infections. E. J. L. Soulsby, ed. Immune Response in Parasitic Infections: Immunology, Immunopathology and Immunoprophylaxis 115.-163. CRC Press, Boca Raton, FL.
  30. Fan, P. C.. 1988. Taiwan Taenia and taeniasis. Parasitol. Today 4:86.[Medline]
  31. Fan, C., W. C. Chung, C. H. Chan, M. M. Wong, C. C. Wu, M. Hsu, S. H. Huang, and Y. A. Chen. 1987. Studies of taeniasis in Taiwan. III. Preliminary report of experimental infection of Taiwan Taenia in domestic animals. Proceedings of the First Sino-American Symposium on Biotechnology and Parasitic Diseases 1:119.
  32. Kunz, J., B. Kalinna, V. Watscke, and E. Geyer. 1989. Taenia crassiceps, metacestode vesicular fluid antigens shared with the Taenia larval stage and reactive with serum antibodies from patients with neurocysticercosis. Zentralbl. Bakteriol. Mikrobiol. Hyg. Abt. 1 Orig. C271:510.
  33. Larralde, C., R. M. Montoya, J. Sotelo, J. Hayunga, E. Scuitto, G. Palencia, A. Padilla, T. Govezensky, M. L. Diaz. 1990. Murine T. crassiceps antigens in immunodiagnosis of T. solium human cysticercosis, T. saginata bovine cysticercosis, and human E. granulosus hydatidosis. Bull. Soc. Fr. Parasitol 8:58B.
  34. Scuitto, E., G. Fragoso, M. Baca, V. de la Cruz, L. Lemus, E. Lamoyi. 1995. Depressed T-cell proliferation associated with susceptibility to experimental infection with Taenia crassiceps infection. Infect. Immun. 63:2277.[Abstract]
  35. Villa, O., R. E. Kuhn. 1991. Antigenic and immunogenic analysis of Taenia crassiceps and Taenia solium using sera from their natural intermediate hosts. ASM Bull. 39:99.
  36. Villa, O., R. E. Kuhn. 1996. Mice infected with the larvae of Taenia crassiceps exhibit a Th2-like immune response with concomitant anergy and downregulation of Th1-associated phenomena. Parasitology 112:561.
  37. Hickey, W. F., B. L. Hsu, H. Kimura. 1991. T-lymphocyte entry into the central nervous system. J. Neurosci. Res. 28:254.[Medline]
  38. Bradl, M.. 1996. Immune control of the brain. Springer Semin. Immunopathol. 18:35.[Medline]
  39. Merril, J. E., E. N. Benveniste. 1996. Cytokines in inflammatory brain lesions: helpful and harmful. Trends Neurosci. 19:331.[Medline]
  40. Shrinkant, P., E. N. Benveniste. 1996. The central nervous system as an immunocompetent organ: role of glial cells in antigen presentation. J. Immunol. 157:1819.[Abstract]
  41. Jr White, C. A., P. Robinson, R. Kuhn. 1997. Taenia solium cysticercosis: host-parasite interactions and the immune response. Chem. Immunol. 66:209.[Medline]
  42. Ernani, F. P., J. M. Teale. 1993. Release of stress proteins from Mesocestoides corti is a brefeldin A-inhibitable process: evidence for active export of stress proteins. Infect. Immun. 61:2596.[Abstract/Free Full Text]
  43. Estes, D. M., J. M. Teale. 1991. Biochemical and functional analysis of extracellular stress proteins of Mesocestoides corti. J. Immunol. 147:3926.[Abstract]
  44. Mosmann, T. R., R. L. Coffman. 1989. Heterogeneity of cytokine secretion patterns and functions of helper T cells. Adv. Immunol. 46:111.[Medline]
  45. Sher, A., R. L. Coffman. 1992. Regulation of immunity to parasites by T cells and T cell-derived cytokines. Annu. Rev. Immunol. 10:385.[Medline]
  46. Komatsu, T., C. S. Reiss. 1997. IFN-{gamma} is not required in the IL-12 response to vesicular stomatitis virus infection of the olfactory bulb. J. Immunol. 159:3444.[Abstract]
  47. Cook, M. A., O. Cosme, D. Nedunchezian, M. Stern. 1994. Neurocysticercosis: an old disease with new questions. J. Fam. Pract. 39:583.[Medline]
  48. Rajshekar, V., M. Chandy. 1994. Enlarging solitary cysticercus granulomas. J. Neurosurg. 80:843.
  49. Haas, W., P. Pereira, S. Tonegawa. 1993. {gamma}/{delta} Cells. Annu. Rev. Immunol. 11:637.[Medline]
  50. Carding, S. R., W. Allan, S. Kyes, A. Hayday, K. Bottomly, P. C. Doherty. 1990. Late dominance of the inflammatory process in murine influenza by {gamma}{delta}+ T cells in vivo. J. Exp. Med. 172:1225.[Abstract/Free Full Text]
  51. Ohga, S., Y. Yoshikai, Y. Takeda, K. Hiromatse, K. Nomoto. 1994. Sequential appearance of {gamma}{delta} and {alpha}ß bearing T cells in the peritoneal cavity during an intraperitoneal infection with Leisteria monocytogenes. J. Immunol. 20:533.
  52. Ferrick, D., M. Schrenzel, T. Mulvania, B. Hsieh, W. Ferlin, H. Lepper. 1995. Differential production of interferon-{gamma} and interleukin-4 in response to Th1- and Th2-stimulating pathogens by {gamma}{delta} T cells in vivo. Nature 373:255.[Medline]
  53. Groh, V., A. Steinle, S. Bauer, S. Thomas. 1998. Recognition of stress-induced MHC molecules by intestinal epithelial {gamma}{delta} T cells. Science 279:1737.[Abstract/Free Full Text]
  54. Kaufmann, S. H. E.. 1997. The roles of conventional and unconventional T cells in antibacterial immunity. ASM News 63:251.
  55. Li, H., M. I. Lebedeva, A. S. Llera, B. A. Fields, M. B. Brenner, R. A. Mariuzza. 1998. Structure of the V{delta} domain of the human {gamma}{delta} T-cell receptor. Nature 391:502.[Medline]
  56. Hvas, J., J. R. Oksenberg, R. Fernando, L. Steinman, C. Bernard. 1993. {gamma}{delta} T cell receptor repertoire in brain lesions of patients with multiple sclerosis. J. Neuroimmunol. 46:225.[Medline]
  57. Christmas, S.. 1992. Cytokine production by T lymphocytes bearing the {gamma}-{delta} T cell antigen receptor. Chem. Immunol. 53:32.[Medline]
  58. Inagaki-Ohara, K., H. Nishimura, A. Mitani, Y. Yoshikai. 1997. Interleukin-15 preferentially promotes the growth of intestinal intraepithelial lymphocytes bearing {gamma}-{delta} T cell receptor in mice. Eur. J. Immunol. 27:2885.[Medline]
  59. Zambello, R., M. Facco, L. Trentin, R. Sancetta, C. Tassinari, A. Perin, A. Milani, G. Pizzolo, F. Rodeghiero, C. Agostini, R. Meazza, S. Ferrini, G. Semenzato. 1997. Interleukin-15 triggers the proliferation and cytotoxicity of granular lymphocytes in patients with lymphoproliferative disease of granular lymphocytes. Blood 89:201.[Abstract/Free Full Text]



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