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(Journal of Leukocyte Biology. 2001;69:685-690.)
© 2001 by Society for Leukocyte Biology

Analysis of macrophage activation in African trypanosomiasis

Donna M. Paulnock and Susan P. Coller

Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, Wisconsin

Correspondence: Donna M. Paulnock, Ph.D., Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706-1532. E-mail: paulnock{at}facstaff.wisc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
African trypanosomes cause a fatal disease of man and animals that is characterized by extensive functional, histological, and pathological changes in the lymphoid tissues of infected hosts, including an increase in the numbers and activation state of macrophages. Macrophage activation during infection is the result of exposure of these cells to parasite components and host-derived IFN-{gamma}, produced in response to parasite antigens. The balance of these different activation signals may determine the outcome of infection. In the experiments described here, we assessed the ability of the variant surface glycoprotein (VSG) of the organism Trypanosoma brucei rhodesiense (T.b. rhodesiense) to activate macrophages directly. Our results demonstrate that macrophages bind and are activated by the VSG molecule. The resulting profile of activation differs from that stimulated by IFN-{gamma}. These results suggest that the interaction of host macrophages with VSG released during parasite infection may be a key component of trypanosomiasis.

Key Words: host defense • innate immunity • trypanosomes • gene expression • variant surface glycoprotein


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
African trypanosomiasis is a disease of medical and veterinary importance caused by extracellular hemoflagellates of the genus Trypanosoma [1 ]. Trypanosome-infected hosts are exposed to many parasite antigens during the course of infection. These antigens include invariant membrane, cytoplasmic, and nuclear antigens as well as a series of distinct, variable surface glycoprotein (VSG) molecules associated classically with this infection [1 , 2 ]. VSG-specific, B-cell responses are associated with temporal immunity to the trypanosome variant antigenic types (VATs) arising during chronic infection, and T-cell-dependent immune responses to invariant components of the VSG molecule are associated with the development of a polarized T helper 1-type response in resistant mice [2 , 3 ]. The sum and interaction of these immune responses are thought to determine the relative resistance and susceptibility of the host and the outcome of infection.

Macrophage activation is also one of the hallmarks of infection with the African trypanosomes [4 5 6 7 8 ]. There is extensive evidence that the numbers and activity of macrophages increase dramatically in the tissues of trypanosome-infected animals. Within the first 2 weeks of experimental Trypanosoma brucei rhodesiense (T.b. rhodesiense) infection, a large percentage of cells in the enlarged spleen exhibit membrane and functional characteristics associated with activated macrophages. These include increases in the release of interleukin (IL)-12 and up-regulation of mRNA and protein expression for tumor necrosis factor (TNF)-{alpha} and inducible nitric oxide synthase (iNOS), among other markers [3 , 9 ]. The expression of these activation markers is associated with modulation of host immunity and resistance.

The source(s) and modes of action of the factors delivered to macrophages during trypanosome infection that result in this activation response are understood only partially. One major antigen of parasite origin is the trypanosome VSG molecule, which is anchored to the parasite membrane by a glycosylphosphatidylinositol (GPI) anchor (Fig. 1 ). The precursor of the GPI anchor of the trypanosome VSG is synthesized in the endoplasmic reticulum and subsequently, is covalently attached to newly synthesized VSGs after proteolytic cleavage of a VSG C-terminal, GPI attachment-signal sequence [10 11 12 ]. After further modifications in the glycoprotein and GPI anchor residue, the mature VSG is transported to and anchored in the trypanosome plasma membrane as membrane-form VSG (GPI-mfVSG). During the course of infection, a trypanosome-associated phospholipase C (GPI-PLC) becomes activated and cleaves the GPI anchor as shown in Figure 1 , releasing soluble VSG, which retains only the glycosylphosphatidyinositol phosphate (GIP) substitutent of the original GPI anchor (GIP-sVSG), and leaving the dimyristoylglycerol (DMG) lipid component remaining in the parasite membrane [13 , 14 ]. Because trypanosome numbers approach 108–109 organisms per ml of blood during peak parasitemia, and there are approximately 107 molecules of VSG per cell, the amount of GIP-sVSG saturating host tissues during infection is likely to be quite substantial.



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  		Figure 1. GPI membrane-anchor substituents of the trypanosome VSG molecule. The GPI anchor of the T.b. rhodesiense, LouTat1, VSG molecule is depicted here, showing GIP substituents associated with sVSG after cleavage from membrane-anchored GPI membrane from VSG by GPI-PLC (adapted from [10 11 12 ]).

 
Although limited in vitro studies by several laboratories have begun to characterize the activating effects of GPI substituents on macrophages [12 , 15 , 16 ], it is not known yet how molecules containing GPIs interact with the macrophage membrane. Specifically, the full extent of the macrophage-activating capabilities of the GIP-sVSG component of the VSG molecule remains to be determined. In the work presented here, we confirmed first that detectable levels of GIP-sVSG were released during infection of mice with the LouTat1 strain of T.b. rhodesiense. Using primary spleen macrophages and the RAW 264.7 macrophage cell line as target cells, we then determined that the GIP-sVSG molecule prepared from this parasite could bind to macrophages in vitro and that this interaction induced changes in the expression of selected genes. These preliminary results demonstrate that the released trypanosome VSG molecule has a potent macrophage-activating capability that may be the result of interaction of the GPI membrane anchor with these cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Female C57BL/6J mice (6–8 weeks of age) were obtained from the Jackson Laboratory (Bar Harbor, ME) and used for trypanosome infection and serum isolation. Outbred Swiss mice also obtained from Jackson Laboratory were used for expanding trypanosome stabilates, as previously described [9 , 17 ]. All animals were housed in university-approved facilities and were handled strictly according to National Institutes of Health and University of Wisconsin-Madison Research Animal Resource Center guidelines.

Cells and cell cultures
The RAW 264.7 macrophage cell line (obtained from American Type Culture Collection, Manassas, VA), was used as one target cell for in vitro stimulation with GIP-sVSG. Mycoplasma-free cells were maintained in complete medium as previously described: RPMI 1640 medium (Life Technologies, Grand Island, NY), supplemented with 2 mM glutamine, 1 mM pyruvate, 50 U/ml penicillin, 50 mg/ml streptomycin, and 2 g/L sodium bicarbonate (all from Sigma Chemical Co., St. Louis, MO), plus 10% fetal bovine serum (FBS; Life Technologies), as previously described [18 ].

For cell stimulation, RAW 264.7 monolayers were established in tissue culture dishes (Corning/Costar, Corning, NY) and stimulated with GIP-sVSG for 24 h at 37°C, 7% CO2. At the completion of the experiment, cells were harvested by scraping with a rubber policeman and processed for reverse transcriptase-polymerase chain reaction (RT-PCR) or prepared for flow cytometric analysis (described below). Selected experiments were performed using mouse spleen cells. Single-cell suspensions were prepared from the spleens of infected or control C57BL/6 mice by our routine methods and resuspended in complete medium [4 ]. Red blood cells were lysed by hypotonic shock, washed several times with medium, and used for flow cytometry or RT-PCR analysis. Cell viability was routinely greater than 95% viable cells as monitored by trypan blue exclusion.

Trypanosomes
Stabilates of T.b. rhodesiense, clone LouTat1, were used for infection and for preparation of GIP-sVSG as previously described [9 , 17 ]. To expand trypanosome stabilates for establishing experimental infections, Swiss mice were immunosuppressed with cyclophosphamide (300 mg/kg body weight) before infection to permit unrestricted trypanosome growth. The trypanosomes were isolated subsequently from the blood of cyclophosphamide-treated, infected mice by exsanguinations from the retrobulbar sinus. The blood was diluted with an equal volume of ice-cold, phosphate-buffered saline containing 1% (w/v) glucose (PBSG) and passed over a (diethylamino)ethyl (DEAE)-cellulose column equilibrated with PBSG. Under these conditions, cellular blood components adhere to the column matrix, and trypanosomes pass through and are found in the void volume [9 , 17 ]. Trypanosomes isolated in this manner were washed subsequently with PBSG by centrifugation at 1000 g for 10 min at 4°C and counted in a hemacytometer. For experimental infections, all mice received an intraperitoneal (i.p.) injection of 1 x 105 trypanosomes.

Preparation of GIP-sVSG
Monomeric GIP-sVSG for use in in vitro studies was prepared as described previously [9 , 17 ]. Briefly, washed trypanosomes in PBSG were concentrated by centrifugation. Trypanosomes were resuspended to 109 cells/ml in 0.3 mM zinc acetate containing 0.1 mM tosyl lysine chloromethyl ketone. Cells were incubated in siliconized test tubes on ice at 4°C for 15 min and then were centrifuged at 3000 g for 10 min at 4°C. Supernatant fluid was removed and reserved; the pellet was resuspended in an equal volume of 10 mM phosphate buffer containing 0.1 mM tosyl lysine chloromethyl ketone. After an incubation period of 20 min at 37°C, the suspension was cooled to 4°C and centrifuged at 10,000 g for 15 min; the supernatant fluid from this and the previous step was combined and centrifuged at 300,000 g for 1 h at 4°C. The resultant supernatant fluid was concentrated in a Centriprep-30 tube (Amicon Corp., Danvers, MA) by centrifugation. Subsequently, the concentrate was passed over a DEAE-cellulose column equilibrated with 10 mM phosphate buffer, pH 8.0; purified VSG was detected in the first peak eluted from the DEAE-cellulose column. All VSG samples were assessed for purity by electrophoretic analysis. VSG purified in this manner appeared as a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gels run under reducing condition with an apparent molecular mass of 62 kDa. Confirmation of VSG purification and identity was made by Western blot analysis with VSG antibody as we have described [14 , 15 ].

For analysis of binding to macrophages, GIP-sVSG prepared by the above procedure was labeled with fluorescein isothiocyanate (FITC-VSG), as we have previously described [19 ].

RNA isolation and RT-PCR
Isolation of RNA, reverse transcription, and PCR assays were performed as described previously [18 , 20 , 21 ]. Briefly, total RNA was isolated using RNA STAT-60 (Tel-test "B", Friendswood, TX), according to the manufacturer’s instructions. Synthesis of cDNA from purified RNA was done by priming with oligo(dT) (Boehringer-Mannheim, Indianapolis, IN), and each cDNA sample was used as a template for gene-specific, PCR amplification. PCR amplifications were performed in a 96-well thermocycler (MJR Research, Watertown, MA). To verify that equal amounts of cDNA were added to each PCR reaction, G3PDH gene expression was assessed. PCR products were separated in a 1% agarose gel and visualized by ethidium-bromide staining. G3PDH primers were purchased from Clontech Laboratories (Palo Alto, CA); all other PCR primers were designed in our laboratory using the Oligo 4.0 Macintosh program (National Biosciences, Plymouth, MN) and have been described previously [18 , 20 , 21 ].

Analysis of cells by flow cytometry
Flow cytometry was performed as we have previously described [18 ]. For these studies, 2 x 106 RAW 264.7 cells or purified mouse spleen cells were analyzed by two-color immunofluorescence for simultaneous binding of FITC-VSG, prepared as described above, and of the macrophage marker, F4/80, labeled with phycoerythrin (PE; PharMingen, San Diego, CA). Control reagents used were PE-labeled immunoglobulin (Ig; PharMingen) and FITC-labeled 5D4, a control glycoprotein preparation prepared in our laboratory. Macrophages in spleen preparations were identified first by forward-angle light scatter; subsequently, fluorescence of the PE- and FITC-labeled molecules was monitored on this gated population. Cells were fixed in 2% paraformaldehyde (Kodak Chemicals, Rochester, NY) before analysis and analyzed on a FACScan flow cytometer with a logarithmic scale (Becton-Dickinson, Bedford, MA).

Immunoblot analysis
Immunoblot analysis of VSG present in serum samples was performed essentially as described previously [21 ]. Briefly, whole-blood samples collected at various time-points before and after infection of C57BL/6 mice with T.b. rhodesiense LouTat1 were separated by centrifugation and serum samples stored at -70°C until use. Serum proteins were separated by SDS-PAGE electrophoresis and transferred electrophoretically onto nitrocellulose (Micron Separations, Westborough, MA) using a Bio-Rad transfer apparatus (Hercules, CA). Following protein transfer, the nitrocellulose was washed and incubated with a polyclonal antibody directed against the LouTat1 strain, developed in the laboratory of Dr. John Mansfield (University of Wisconsin-Madison). Bound antibody was detected using an anti-rabbit, IgG secondary antibody conjugated to horseradish peroxidase (Bio-Rad) developed with LumiGLO reagents according to the manufacturer’s instructions (Kirkegaard & Perry Laboratories, Gaithersburg, MD). In some cases, the VSG detectable in serum was also probed with a cross-reacting determinant (CRD)-specific antibody ({alpha}CRD) recognizing an epitope exposed on intact GIP-sVSG from multiple trypanosome strains after cleavage of the molecules by GPI-phospholipase C (PLC; the kind gift of Dr. James Bangs, University of Wisconsin Medical School, Madison, WI) [22 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
GIP-sVSG is released into the blood during trypanosome infection
For these experiments, blood was harvested from uninfected and infected mice by heart puncture and allowed to clot at room temperature. The resulting serum was retrieved by centrifugation and separated by electrophoresis on a 12% polyacrylamide gel. Separated serum components were then blotted onto nitrocellulose and subjected to Western blot analysis with a VSG-specific polyclonal antibody. As shown in Figure 2 , immunoblot analysis of sequential serum samples revealed that released GIP-sVSG could be detected readily in sera taken from infected, but not uninfected, mice. Pre-infection serum did not demonstrate reactivity with the anti-VSG-specific antibody (day 0), and serum VSG of the appropriate molecular weight was detected 3, 5, and 7 days after initial infection (Fig. 2) . The polyclonal serum also detected many background, or nonspecific, proteins in the serum samples, but these were present in samples from infected and uninfected mice [e.g., in Fig. 2 , compare day 0 sample with that of normal serum (NS)+VSG]. Thus, GIP-sVSG, released from bloodstream parasites through the action of GPI-PLC, can be found in the serum of infected mice within the first few days after infection and over the period of peak parasitemia (days 5–7 after infection) [1 , 2 ]. Our conservative calculations, based on the levels of parasitemia reached during the period of VSG release, predict that the infected host is exposed to 15–20 µM VSG with each wave of parasitemia (calculations not shown).



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  		Figure 2. GIP-sVSG is released into serum during trypanosome infection. Serum samples were harvested from uninfected mice (day 0) and infected mice on days 3, 5, and 7 post-infection. Serum proteins were separated by electrophoresis and analyzed for the presence of GIP-sVSG by immunoblotting using a VSG-specific polyclonal antibody. Controls included normal serum "spiked" with 5 µg VSG (NS+VSG) and 1 µg-purified GIP-sVSG (VSG). Selected samples also were probed with a CRD-specific antibody ({alpha}CRD), recognizing an epitope exposed on intact GIP-sVSG from multiple trypanosome strains after cleavage of the molecules by GPI-PLC (nt=not tested with the CRD reagent).

 
GIP-sVSG binds to and activates macrophages
GIP-sVSG-FITC (25 nM) was used to stain primary mouse spleen cells, as described in Materials and Methods, followed by staining with the macrophage-specific antibody F4/80 conjugated with phycoerythrin (F4/80-PE). Cells were then fixed and analyzed for two-color membrane fluorescence. As shown in Figure 3A , spleen macrophages showed little or no binding of the two control reagents labeled with FITC (5D4) or PE (control IgG). However, F4/80-positive macrophages showed readily detectable binding of FITC-GIP-sVSG, with all F4/80-positive cells binding the labeled GIP-sVSG molecule (Fig. 3B) . This binding was inhibited by preincubation of the cells with unlabeled GIP-sVSG but not by incubation with the control glycoprotein bovine serum albumin (unpublished results).



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  		Figure 3. GIP-sVSG binds to the macrophage membrane and activates cells. Flow cytometry: GIP-sVSG was prepared from T.b. rhodesiense, clone LouTat1, and labeled with FITC, as described in Materials and Methods. Mouse spleen cells were incubated with PE- and FITC-labeled control molecules (A) or with GIP-sVSG-FITC (25 nM) and anti-F4/80-PE (B) for 30 min at 4°C. Then, spleen macrophages (identified by forward-angle, light-scatter properties) were analyzed by flow cytometry for two-color membrane fluorescence. RT-PCR: RAW 264.7 cells were stimulated with recombinant IFN-{gamma} (20 U/ml) or GIP-sVSG (100 µg/ml) for 24 h in the presence of polymyxin B. Then, stimulated cells were lysed by phenol-choloroform extraction and analyzed for the expression of selected genes by RT-PCR. Amplified products were separated by gel electrophoresis and visualized by ethidium-bromide staining. The same activation effects were seen following stimulation of primary peritoneal macrophages (unpublished results). _art>

 
In separate experiments, the exposure of RAW 264.7 cells to GIP-sVSG (100 µg/ml, 1.67 µM) in the presence of polymyxin B resulted in the induction of mRNA expression (Fig. 3C) and secreted protein (unpublished results) for selected genes, including TNF-{alpha} and IL-6. This pattern of activation-induced gene expression was largely distinct from that induced by the prototypical macrophage-activating factor interferon (IFN)-{gamma}; Fig. 3C ). The same activation events were seen using primary mouse spleen cells (unpublished results). Thus, GIP-sVSG released from bloodstream trypanosomes binds to macrophage membranes and induces new gene expression.

GIP-sVSG induces the expression of a selected profile of genes in a dose-dependent manner
Subsequent experiments assessed the genes induced by GIP-sVSG in vitro through the use of a range of RT-PCR primers designed to detect mRNA for diverse pro- and anti-inflammatory genes, as well as genes associated with T-cell activation. As shown in Figure 4 , treatment of RAW 264.7 cells with 500 µg/ml GIP-sVSG (8.35 µM) induced the expression of mRNA for a specific array of genes, including TNF-{alpha}, IL-6, IL-12, iNOS, and granulocyte-macrophage colony-stimulating factor (GM-CSF; Figs. 3 and 4 ). The macrophage-activating effect occurred in a dose-dependent manner, with 100 µg/ml (1.67 µ) GIP-sVSG affecting little or no gene induction (Fig. 4 ; modest changes in GM-CSF and TNF-{alpha} mRNA levels only) compared with treatment with 500 µg/ml, which had the maximal effect. Full induction of mRNA expression was observed after 24 h but not after 6 h of stimulation with GIP-sVSG (unpublished results). Unexpectedly, only a very select panel of genes was the target of VSG-mediated activation, because additional genes associated with the development of inflammation, including IL-1ß, IFN-ß, and IL-10, were not induced (Fig. 4) . Enzyme-linked immunosorbent assay (ELISA) analyses of supernatant fluids collected from RAW 264.7 cells activated with 500 µg/ml GIP-sVSG revealed that IL-6 and IL-12 could be detected in medium harvested from activated, but not control-treated, cell cultures, along with dramatic increases in the amount of TNF-{alpha} protein over basal levels (unpublished results).



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  		Figure 4. The macrophage-activating effects of GIP-sVSG occur in a dose-dependent manner. RAW 264.7 cells were stimulated with GIP-sVSG at 100 µg/ml (1.67 µM) or 500 µg/ml (8.35 µM) for 24 h, as described in Materials and Methods. RNA from stimulated cells was isolated and analyzed for changes in mRNA expression as described in the legend to Figure 3 .

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It has long been known that multiple, parasite-derived molecules interact with host cells and tissues during the development of African trypanosomiasis. However, the sources and modes of action of potential activating factors delivered to macrophages during trypanosome infection are understood only partially. The results presented here demonstrate that one major activation factor of parasite origin is the trypanosome VSG, presumably the GPI membrane anchor of this molecule (GIP-sVSG). Our initial studies have determined that this molecule binds readily to primary macrophages and the RAW 264.7 cell line. Further, the outcome of this interaction is the up-regulation of selected genes and their protein products that have multiple roles in the immune response to this organism. Thus, TNF-{alpha} and NO (the product of iNOS activity) have been implicated in the suppressor cell activity exhibited by macrophages at different time-points of infection [9 , 12 ]. Similarly, an increase in IL-12 production is known to be important for the development of polarized Th1 cell responses that occur to trypanosome antigens [9 ]. Finally, TNF-{alpha} and presumably IL-6 contribute to immunopathology through the development of a dramatic, inflammatory response during infection; the role of TNF-{alpha} in disease-associated cachexia also has long been observed [1 , 2 ]. Thus, although we have not done an exhaustive study of induced gene expression, our results suggest that direct interaction of GIP-sVSG with macrophages contributes to the development of the major immune responses to this organism, including potentiation of a Th1 response, development of macrophages with suppressive activity, and inflammation through modulation of macrophage gene expression. These results confirm previous studies of Magez et al. [12 ] that demonstrated a role for GIP-sVSG in TNF-{alpha}, IL-6, and IL-12 induction in a macrophage hybridoma by the AnTat1 variant of the T.b. brucei species of trypanosome and extend them to demonstrate that these activating effects result in additional, selected gene induction in a macrophage cell line and primary mouse spleen cells. In both experimental systems, a similar level of GIP-sVSG was required to stimulate macrophages effectively, suggesting that a specific threshold concentration of soluble VSG is required to achieve macrophage activation. The full spectrum of induced genes and the mechanism(s) leading to the particular changes in gene expression observed in response to GIP-sVSG stimulation, as well as the specific component(s) of the VSG molecule responsible for such changes, remain to be determined.

The nature of the macrophage-sVSG interaction demonstrated in these studies also remains to be established. Our observation that GIP-sVSG binds specifically to F4/80-positive primary spleen cells suggests a specific, rather than nonspecific, membrane interaction with cells of the mononuclear phagocyte lineage. Studies from other laboratories of trypanosome and other parasite systems have suggested that GPI substituents exhibit signaling activities when used to treat diverse cells, suggesting a specific, membrane-ligand interaction [12 , 15 , 16 ]. The dose- and time-dependence of the activation process we observed also supports the hypothesis that this interaction may be receptor-mediated. That specific signals are delivered to the cell is apparent from recent in vitro studies showing that GIP substituents (specifically the core glycan sequence) activate a specific tyrosine kinase in stimulated cells [16 ]; however, it is not yet known how GPIs interact with the macrophage membrane or what receptors may be important in delivering GPI-mediated activation signals to the cell nucleus. These will be critical targets for future studies.

Previous studies have demonstrated that macrophage activation can be detected within the first week of infection of mice with African trypanosomes [9 , 17 ]. The studies demonstrated here extend those observations in vitro to demonstrate that GPI substituents of VSG bind to the macrophage membrane. This binding results in a dose-dependent induction of the expression of selected genes associated previously with aspects of the immune response to this organism. Our data also provide clear evidence that host cells are exposed to biologically active levels of GIP-sVSG during infection through its release into the serum, and presumably, this exposure will occur with each wave of parasitemia. However, the impact of cyclical, tissue saturation with this activating agent in vivo during infection has not been defined yet or fully appreciated, particularly with respect to its macrophage-activating effects. In fact, it is tempting to speculate why trypanosomes may induce such effects on macrophages. One idea is that it may be important for trypanosomes to induce early, temporal protection against infection, regardless of the genetically based resistance status of the host, to allow disease transmission before the host is killed by the infection. Alternatively, activation might be linked to the early generation of suppressor-macrophage activity, designed to depress host T-cell responses to avoid parasite elimination. In addition, it remains unclear exactly how the interplay between parasite-derived, activating factors like GIP-sVSG and host-derived, macrophage-activating factors such as IFN-{gamma}, the earliest activating agent to be detected during infection, modulates host resistance to this organism. The system described here should provide an ideal model for further exploration of these unanswered questions.


   ACKNOWLEDGEMENTS
 
The authors gratefully acknowledge the advice and encouragement provided by Dr. John Mansfield (University of Wisconsin-Madison) and the members of his laboratory during the completion of these studies. We also thank Ms. Karen Demick for her excellent technical efforts in diverse aspects of this work.

Received December 2, 2000; revised February 21, 2001; accepted February 22, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Cooperative Role of Macrophages and Neutrophils in Host Antiprotozoan Resistance in Mice Acutely Infected with Cryptosporidium parvum
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B. Stijlemans, T. N. Baral, M. Guilliams, L. Brys, J. Korf, M. Drennan, J. Van Den Abbeele, P. De Baetselier, and S. Magez
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B. J. Leppert, J. M. Mansfield, and D. M. Paulnock
The Soluble Variant Surface Glycoprotein of African Trypanosomes Is Recognized by a Macrophage Scavenger Receptor and Induces I{kappa}B{alpha} Degradation Independently of TRAF6-Mediated TLR Signaling
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T. H. Harris, N. M. Cooney, J. M. Mansfield, and D. M. Paulnock
Signal transduction, gene transcription, and cytokine production triggered in macrophages by exposure to trypanosome DNA.
Infect. Immun., August 1, 2006; 74(8): 4530 - 4537.
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M. Shi, G. Wei, W. Pan, and H. Tabel
Trypanosoma congolense infections: antibody-mediated phagocytosis by Kupffer cells
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N. Maina, J. M. Ngotho, T. Were, J. K. Thuita, D. M. Mwangangi, J. M. Kagira, J. M. Ndung'u, and J. Sternberg
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S. P. Coller, J. M. Mansfield, and D. M. Paulnock
Glycosylinositolphosphate Soluble Variant Surface Glycoprotein Inhibits IFN-{gamma}-Induced Nitric Oxide Production Via Reduction in STAT1 Phosphorylation in African Trypanosomiasis
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