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Original Article |
christian.bogdan{at}mikrobio.med.uni-erlangen.de
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Key Words: Leishmania major • fibroblasts • persistent infection • nitric oxide • macrophages
After spontaneous or chemotherapy-mediated healing of the infection, both mice and humans continue to harbor small numbers of alive Leishmania parasites in the lymphoid tissue 111213. This was demonstrated most convincingly by the recrudescence of the disease after treatment with immunosuppressive drugs, depletion of CD4+ T cells, or inhibition of NOS2 activity 14151617. Although the components of the immune system that are responsible for the resolution of acute Leishmania infection are well defined, little is known about the mechanisms that allow the parasites to survive lifelong in the host. In genetically resistant mice that had resolved a skin infection with L. major, we found that NOS2 activity was indispensable for the long-term control of the remaining parasites. 30–40% of the parasites persisting in the draining lymph node colocalized with NOS2-positive macrophages or dendritic cells, whereas 60–70% of the parasites were located in NOS2-negative areas that could not be stained with known markers for macrophages, dendritic cells, granulocytes, or endothelial cells and thus remained undefined 17. As fibroblasts had been reported to be susceptible to infection with various Leishmania species in vitro 181920, and lymph nodes from chronically infected mice contained strongly increased amounts of fibrous tissue, we considered the possibility that Leishmania might also reside in fibroblasts in vivo. In this study, we identify reticular fibroblasts in lymph nodes as major host cells for L. major during latent disease and provide evidence that these cells might function as "safe targets" 21 for the parasites.
Mice.
Macrophages.
Isolation of Fibroblasts.
Reticular fibroblasts were obtained from popliteal lymph nodes (
For infection or phenotypic characterization, skin or lymph node fibroblasts were harvested with a rubber policeman, seeded, and used as confluent monolayers. To minimize growth, the serum concentration was reduced to 1–2.5%.
Infection of Macrophages and Fibroblasts with L. major Parasites.
The infection rate and the number of intracellular parasites per infected cell were determined by immunoenzymatic or immunofluorescence staining of the monolayers. At least 300 cells were evaluated. The total number of intracellular parasites per culture was determined by two different methods that were previously shown to yield comparable results 22. In brief, infected macrophage or fibroblast monolayers were lysed in SDS (0.01% in serum-free RPMI) to release intracellular parasites. After addition of a twofold volume of modified Schneider's Drosophila medium (mSDM) 25, the cell lysates were spun and the pellets containing the amastigotes were resuspended in 500 µl modified mSDM. The parasite suspensions were seeded in triplicates into 96-well flat-bottomed plates for further incubation (24–48 h) and pulsed with 1 µCi (37 kBq)/well of [3H]desoxy-thymidine (25 Ci/mmol; Amersham Pharmacia Biotech) for the last 12–18 h. Alternatively, the parasite suspensions were subjected to limiting dilution analysis using serial 2-fold dilutions and 12–24 individual wells per dilution step. After 7 d of culture, the number of wells negative for parasites was determined for each dilution and the number of viable L. major parasites per culture condition was calculated by applying Poisson statistics and the
Cocultures of Macrophages and Fibroblasts.
Introduction
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Introduction
Materials and Methods
Results
Discussion
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A hallmark of infections with certain viruses (e.g., herpesviruses), intracellular bacteria (e.g., Mycobacteria, Coxiella, Chlamydiae), or protozoa (e.g., Trypanosoma cruzi, Leishmania) is the long-term persistence of the pathogen after clinical cure of the disease. Based on in vitro results, modulation of host cell antimicrobial activities, synthesis of inhibitory cytokines, impairment of T cell activation, or retreat of the pathogen into cells that do not elicit an immune response have been proposed as viral or microbial survival strategies, but the mechanisms of persistence in vivo remain ill defined 12. One example are infections with Leishmania parasites. Leishmania promastigotes are transmitted by sand flies to mammalian hosts, where they infect macrophages, granulocytes, and dendritic cells, transform into amastigotes, and cause cutaneous, mucocutaneous, or progressive visceral disease. In most strains of mice as well as in humans, infections with Leishmania major usually elicit skin swellings or single ulcers that are ultimately controlled by a CD4+ T cell response involving the production of IFN-
and the activation of antileishmanial effector mechanisms in macrophages 3456. In mice, IFN-
/β and IFN- induced the production of nitric oxide (NO) by the inducible (or type 2) NO synthase (iNOS or NOS2), which was shown to be indispensable for the healing of acute cutaneous lesions 78910.
Materials and Methods
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Introduction
Materials and Methods
Results
Discussion
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Parasites.
The L. major strain MHOM/IL/81/FE/BNI 17 was propagated in vitro in RPMI 1640 plus 10% FCS on Novy-Nicolle-MacNeal blood agar slants for a maximum of six passages. Fresh L. major promastigotes were derived from amastigotes that were isolated from the ulcerated skin lesions or the spleens of BALB/c mice as described 22.
Female C57BL/6 mice, weighing 16–18 g, were purchased from Charles River, housed in our own facilities, and used at 8–12 wk of age. C57BL/6 mice were inoculated into the right hind footpad with 3 x 106 stationary phase L. major promastigotes. In some experiments, mice were infected bilaterally into both hind footpads. The footpad swelling was measured with a metric caliper. The mice resolved their skin lesions usually within 60–70 d after infection. For immunohistological analyses, the popliteal lymph nodes draining the site of infection were removed from mice that had been infected for at least 100 d.
Thioglycollate-elicited peritoneal exudate macrophages (PEMs) were prepared from the peritoneal cavity of C57BL/6 mice as described 23. Resident peritoneal macrophages (RPMs) were obtained from C57BL/6 mice by flushing the peritoneal cavity twice with 10 ml ice-cold PBS. The cells were resuspended in RPMI 1640 culture medium (supplemented as described [23] plus 1 or 2.5% fetal bovine serum [Sigma-Aldrich]), seeded into 24-well plates (106 cells/well in 500 µl) or 8-well LabTek® chamber slides (Permanox®; Nalge Nunc International), and cultured at 37°C in 5% CO2/95% humidified air. After 90–120 min, nonadherent cells were washed off and the macrophage monolayers were further incubated as indicated.
Murine skin fibroblasts were established from ear skin explants of C57BL/6 mice as described 24. In brief, after removal of the epidermis, the dermis was cut into 1 x 2 mm pieces, which then were allowed to attach to tissue culture petri dishes before addition of complete RPMI 1640 medium (see above) supplemented with 10–20% FCS. Nonadherent cells were removed by weekly medium exchanges. After 2–4 wk, fibroblasts grew out from the explants that were passaged after treatment with 0.25% trypsin-EDTA solution and further propagated in RPMI 1640 supplemented with 5% FCS by weekly 1:3 splitting. 
30–90 mg) of chronically infected mice (>200 d after infection with L. major). Collagenase D (100 U/ml in PBS; Boehringer) was injected into the lymph nodes that had been carefully prepared and freed of extracapsular tissue. The lymph nodes were opened and the cell suspension was harvested in PBS with 100 U/ml collagenase D. Finally, the lymph nodes were teased into small fragments that were further digested in PBS plus 400 U/ml collagenase D at 37°C for 60–90 min. Undigested (capsular) fragments were removed and the cells were pooled with the primary suspension, centrifuged, washed, and seeded in small tissue culture flasks (Nalge Nunc International) in RPMI 1640 medium plus 10–20% FCS. Nonadherent cells were removed by weekly medium exchanges. After 2–4 wk, fibroblasts were growing in the cultures.
Monolayers of macrophages or fibroblasts in 24-well plates were incubated with a 3–10-fold excess of L. major promastigotes or amastigotes, either continuously (permanent infection) or for 4–14 h after which nonphagocytosed parasites were washed off (pulse infection). The cells were activated with recombinant murine (rm)IFN- (provided by Dr. G. Adolf, Ernst Boehringer Institut, Vienna, Austria) with or without rmTNF- (R&D Systems) or LPS (Escherichia coli O111:B; Sigma-Aldrich). Culture medium was replaced every 24 h. 
2 minimization method 822.
PEMs were allowed to adhere (2 h) to the outer side of the membrane (pore size 0.45 µM) of cell culture insets (Costar). The insets were then immerged in culture wells containing complete RPMI 1640 medium with 2.5% FCS. Reticular fibroblasts were added and, after adherence to the inner side of the membrane (6 h), were pulse-infected with L. major amastigotes (ratio 4–10:1) for 12 h. Thereafter, the insets were transferred to new wells containing culture medium with or without stimuli (see also Fig. 3 D).
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Transmission Electron Microscopy.
Incubation of fibroblasts with L. major parasites was stopped by adding an excess amount of cold Ito's fixative to the cells 26. After overnight fixation at 4°C, the samples were further processed according to established protocols 27. In brief, washes with 0.1 M cacodylate buffer, pH 7.4, were followed by postfixation in ferricyanide-reduced 1% osmium tetroxide, washes with 0.9% saline solution, encapsulation in 2% agar, en bloc staining with an alcoholic mixture of 0.5% phosphotungstic acid and 0.25% uranyl acetate, physical dehydration with a graded series of ethanolic solutions ending with pure acetone, and embedding in Epon 812 resin. Ultrathin sections were placed onto 200-mesh standard square copper grids, stained with a mixture of 10% uranyl acetate and 2.8% lead citrate, and viewed with a Zeiss type 906 transmission electron microscope.
Antibody Reagents.
Rabbit antisera against human fibronectin, human laminin-1, human collagen VI (recombinant N9-N2 domain), mouse fibulin-2, or mouse perlecan (recombinant III-3 domain) were as described 28293031. The human or rabbit antiserum against L. major, the rabbit antiserum against mouse NOS2 peptide, the rat mAbs against macrophages (Mac-1, F4/80, BM-8, MOMA-2, and ER-MP-23), granulocytes (GR-1), dendritic cells (NLDC-145), or endothelial cells (MECA-32) were the same as used previously 17. The rat mAbs M5/114.15.2 32 and ER-TR7 33 were used for the detection of MHC class II antigens and mouse reticular fibroblasts, respectively. All rat mAbs as well as all of the secondary antibodies (affinity-purified biotin-conjugated donkey anti–rabbit IgG, mouse anti–rat IgG or goat anti–human IgG F[ab']2 fragments; affinity-purified Cy5- or lissamine rhodamine sulfochloride [LRSC]-conjugated donkey anti–rabbit IgG, Cy5- or dichlorotriazinyl aminofluorescein [DTAF]-conjugated donkey anti–rat IgG, or DTAF-conjugated goat anti–human IgG F[ab']2 fragments) were purchased from Dianova.
Immunoenzymatic Staining of Frozen Tissue Sections and of Fibroblasts and Macrophages.
5–6-µM tissue sections from embedded lymph nodes were prepared with a cryostat microtome (model HM 500 OM; Fa. Microm International GmbH), thawed onto slides coated with Fro-Marker® (Science Services), surrounded with PAP PEN® (Science Services), air-dried, fixed in acetone (for 10 min, at –20°C), and briefly washed in PBS/0.05% Tween 20. Monolayers of macrophages or fibroblasts on Permanox® chamber slides (Nalge Nunc International) were washed with PBS and fixed in acetone without prior air-drying. Nonspecific binding sites were blocked for 30 min with PBS/0.1% saponin/1% BSA/20% FCS. Immunoperoxidase staining (with 3-amino-9-ethyl-carbazole as a substrate), alkaline phosphatase immunoenzymatic labeling (with Fast Blue BB salt as a substrate), and hematoxylin counterstaining were performed as described previously 1723.
Immunofluorescence and Laser Scanning Confocal Microscopy.
For double and triple immunofluorescence, acetone-fixed fibroblast or macrophage monolayers or cryostat sections were blocked (see above) and simultaneously incubated (45 min, room temperature) with two or three different primary antibodies diluted in PBS/0.1% BSA/0.1% saponin. After washing with PBS/0.05% Tween 20, the fluorochrome (Cy5, DTAF, or LRSC)-conjugated secondary antibody reagents (diluted in PBS/0.1% BSA/0.1% saponin) were added sequentially for 30 min each, followed by extensive washing steps in between. In cases where two of the primary antibodies were derived from the same species, complexes of the first and secondary antibody were formed and free binding sites of the secondary antibody were saturated with the respective nonimmune serum (10%, 30 min on ice) before adding the complexes to the sections. The slides were finally mounted with Mowiol (Hoechst) containing 1,4-diazabicyclo-2,2,2-octane (DABCO; Sigma-Aldrich) as an antifading reagent. DTAF (green) was excited at 488 nm and collected using a 515–545-nm band pass filter. LRSC (red) was excited at 574 nm and collected with a 590-nm long pass filter. Cy5 (red) was excited at 651 nm and collected with a 665-nm long pass filter. Nuclei were visualized with the DNA stain TOTO-3 (red; Molecular Probes), which was excited at 642 nm and collected with a 665-nm long pass filter. The slides were examined with a Leica laser confocal microscope equipped with an argon/krypton laser (laser lines of 488, 568, and 647 nm) using the Leica TCS NT software (v1.6.551). For the three-color presentation of the images, the far red emission of Cy5 or of TOTO-3 was turned into blue.
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Fibroblasts Have a Reduced Capacity to Produce NO and to Kill Leishmania Parasites.
As NOS2-derived NO has been shown to be indispensable for the control of Leishmania in phagocytes and in mice 893435, we analyzed the production of NO by fibroblasts in response to cytokines or microbial products. IFN- alone or IFN- plus TNF were sufficient to activate resting or inflammatory macrophages for high production of NO. In contrast, skin or lymph node fibroblasts required a microbial costimulus such as LPS (Fig. 3 A) or infection with L. major pro- or amastigotes (Fig. 3 B; see also Fig. 3 D). However, whereas >90% of infected macrophages stimulated with IFN-/LPS were positive for NOS2 protein by immunofluorescence analysis, the expression of NOS2 in L. major–infected and IFN-/LPS–stimulated CHF-1 or NOBO-1 fibroblasts was restricted to 33 (± 13) or 55 (± 14)% of the cells, respectively (mean ± SD of five culture wells from two experiments). Furthermore, in the same experiments, only 34 (± 14) or 62 (± 9)% of the intracellular parasites were localized in NOS2-positive fibroblasts.
In accordance with previous reports 2234, stimulation of amastigote-infected macrophages with IFN- or IFN-/TNF for 48–72 h caused a reduction of the total number of intracellular parasites by 20- to several hundredfold. In contrast, the average reduction of intracellular amastigotes was 1.2 (± 0.2) or 4.0 (± 1.8)-fold in reticular fibroblasts (NOBO-1) and 2.6 (± 1.1) or 5.65 (± 2.2)-fold in dermal fibroblasts (CHF-1) after stimulation with IFN- or IFN-/TNF, respectively (mean ± SEM of three to seven experiments; Fig. 3 C). However, coculture of infected fibroblasts with uninfected macrophages that were separated by a membrane significantly increased the killing of the intracellular amastigotes compared with cultures of fibroblasts alone (Fig. 3 D).
Reticular Fibroblasts as Host Cells for Persisting Leishmania In Vivo.
Next, we addressed the question whether fibroblasts represent (part of) the hitherto undefined NOS2-negative host cell population that harbors 60–70% of all parasites persisting in the lymph nodes of clinically cured mice 17. Initial experiments using immunoperoxidase/alkaline phosphatase double labeling revealed parasites closely associated with the reticular fibroblast marker ER-TR7 (Fig. 4 A) or various matrix proteins (not shown). Subsequently, >200 sections of 5 lymph nodes derived from 5 independent time course experiments (day 145–530 after infection) were analyzed by confocal laser microscopy. In a first series, 332 L. major amastigotes were analyzed for their colocalization with NOS2 and extracellular matrix proteins (fibulin-2, perlecan, laminin, or collagen VI). 256 (77%) of all parasites were NOS2-negative, confirming our previous findings 17. 102 of the NOS2-negative Leishmania (i.e., 30% of the total parasites) were tightly embedded in or closely surrounded by matrix. Only 8% of the total parasites colocalized with matrix and NOS2. Similar results were obtained in a second series of sections, in which 824 L. major amastigotes were assessed for their association with the ER-TR7 marker, NOS2, and host cell nuclei. 549 (66%) of all parasites were found within NOS2-negative areas, of which 358 (i.e., 43% of all parasites and 65% of the NOS2-negative parasites) colocalized with ER-TR7 (Fig. 4 C). Only 20% of all parasites colocalized with ER-TR7 and NOS2 (Fig. 4 D). At least 50% of the ER-TR7–positive amastigotes were located in the close vicinity of host cell nuclei, which further demonstrates that fibroblasts harbor Leishmania in vivo (Fig. 4 B). By similar triple immunofluorescence labeling, we found parasites not only within macrophages (Fig. 4 E) and dendritic cells (not shown), but also in areas devoid of host cell nuclei and fibroblasts (Fig. 4 F). Thus, fibroblasts are host cells for persisting L. major along with other cell types, but Leishmania can also be detected in necrotic areas of the chronically infected lymph nodes.
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60–70% of the total parasites [17]) are tightly associated with reticular fibroblasts. Thus, fibroblasts represent a hitherto unrecognized important host cell for Leishmania persisting in vivo.
Fibroblasts as Safe Targets for Leishmania.
Previous reports on the interaction of unstimulated human skin fibroblasts with Leishmania species other than L. major provided evidence that the parasites can persist within these cells in vitro for prolonged periods of time (3–7 d), although replication of the parasites did not occur 181920. Our present analysis shows that cytokine-stimulated fibroblasts have a limited capacity to kill intracellular L. major parasites in vitro. This is not due to a principal inability to produce NO. However, adherent fibroblasts stimulated with IFN- (with or without TNF-) released much less NO than macrophage monolayers, and even after stimulation with IFN- plus LPS the expression of NOS2 protein was restricted to 30–50% of the fibroblasts (depending on the cell line). Similar observations were previously made with embryonic fibroblasts 50. After infection, production of NO was enhanced but still considerably lower than in macrophages. Furthermore, only 35–60% of the parasites were localized in NOS2-positive fibroblasts. Both factors might support the survival of L. major parasites within fibroblasts. Importantly, parasites residing in fibroblasts remained susceptible to the NO produced by neighboring macrophages (Fig. 3 D). Thus, macrophage-derived NO is able to control the Leishmania in nearby NOS2-negative fibroblasts, which might help to maintain a stable balance of parasite killing and evasion in the chronically infected lymph nodes.
We considered the possibility that the localization of Leishmania in fibroblasts might also protect the parasites against the activity of standard antileishmanial drugs and thereby account for the persistence of Leishmania after chemotherapy. When we treated long-term–infected, clinically cured C57BL/6 mice with a combination of pentostam and amphotericin B (Ambisome®), there was a strong (900-fold) reduction of the parasite burden. However, we did not observe an increase in the number of parasites associated with fibroblast markers. Thus, to date we do not have evidence for a preferential survival of L. major parasites after chemotherapy in fibroblasts.
Extracellular Matrix and Leishmania.
Although our confocal laser microscopy analysis strongly suggests that L. major parasites are located within fibroblasts in vivo (Fig. 4 B), we cannot exclude that some of the persisting Leishmania amastigotes are located extracellularly tightly embedded by matrix (Fig. 4a and Fig. c, and data not shown). In addition, in the chronically infected lymph nodes, Leishmania amastigotes were also seen in necrotic areas lacking nucleated cells (Fig. 4 F). In this context, it is important to note that L. mexicana amastigotes express surface molecules that were shown to function as ligands for certain (extracellular) proteoglycans 51. Extracellular amastigotes, surrounded by connective matrix, have also been seen in acute leishmanial skin lesions of humans infected with L. braziliensis guyanensis, but the finding was not discussed 52. Whether a possible extracellular localization of the parasite in a connective tissue matrix contributes to the control (i.e., killing) of the parasite or to its long-term survival in the host is unknown to date.
In conclusion, we have demonstrated that ingestion of Leishmania by fibroblasts is a frequent event during latent disease. Our data suggest that fibroblasts might form a less hostile environment for L. major than macrophages and thereby allow for the persistence of the parasites. Parasite survival and replication, however, are subject to control by neighboring macrophages that are effective against Leishmania residing in fibroblasts and thereby help to maintain a stable host–parasite relationship. Whether such a mechanism also operates in latently infected humans remains to be established.
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Acknowledgments |
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This study was supported by the Deutsche Forschungsgemeinschaft (SFB263, project A5).
Submitted: 31 March 2000
Accepted: 14 April 2000
A. Diefenbach's present address is Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720.
M.G. Rittig's present address is Institut National de la Santé et de la Recherche Médicale, U431, University Montpellier II, Montpellier Cedex 5, France.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Murray P.J.. Defining the requirements for immunological control of mycobacterial infections, Trends Microbiol., 7, 1999, 366–372.[Medline]
Bogdan C. & Röllinghoff M.. How do protozoan parasites survive inside macrophages?, Parasitol. Today., 15, 1999, 22–28.[Medline]
Titus R.G., Theodos C.M., Shankar A. & Hall L.R.. Interactions between Leishmania major and macrophages, Zwilling T. & Eisenstein T., Macrophage–Pathogen Interactions, 1993, 437–459, Marcel Dekker, Inc, New York.
Pearson R.D. & de Queiroz Sousa A.. Clinical spectrum of leishmaniasis, Clin. Infect. Dis., 22, 1996, 1–13.[Medline]
Reiner S.L. & Locksley R.M.. The regulation of immunity to Leishmania major, Annu. Rev. Immunol., 13, 1995, 151–177.[Medline]
Solbach W. & Laskay T.. The host response to Leishmania infection, Adv. Immunol., 74, 2000, 275–317.[Medline]
MacMicking J., Xie Q.-w. & Nathan C.. Nitric oxide and macrophage function, Annu. Rev. Immunol., 15, 1997, 323–350.[Medline]
Diefenbach A., Schindler H., Donhauser N., Lorenz E., Laskay T., MacMicking J., Röllinghoff M., Gresser I. & Bogdan C.. Type 1 interferon (IFN-/β) and type 2 nitric oxide synthase regulate the innate immune response to a protozoan parasite, Immunity., 8, 1998, 77–87.[Medline]
Niedbala W., Wei X.-Q., Piedrafita D., Xu D. & Liew F.Y.. Effects of nitric oxide on the induction and differentiation of Th1 cells, Eur. J. Immunol., 29, 1999, 2498–2505.[Medline]
Diefenbach A., Schindler H., Röllinghoff M., Yokoyama W. & Bogdan C.. Requirement for type 2 NO-synthase for IL-12 responsiveness in innate immunity, Science., 284, 1999, 951–955.
Aebischer T.. Recurrent cutaneous leishmaniasisa role for persistent parasites, Parasitol. Today., 10, 1994, 25–28.[Medline]
Ramirez J.L. & Guevara P.. Persistent infection by Leishmania (Viannia) braziliensis, Mem. Inst. Oswaldo Cruz., 92, 1997, 333–338.[Medline]
Schubach A., Haddad F., Neto M.P.-O., Degrave W., Pirmez C., Grimaldi G. & Fernandes O.. Detection of Leishmania DNA by polymerase chain reaction in scars of treated human patients, J. Infect. Dis., 178, 1998, 911–914.[Medline]
Ma D.F.F., Concannon A.J. & Hayes J.. Fatal leishmaniasis in renal-transplant patient, Lancet., 2, 1979, 311–312.[Medline]
Badaró R., Carvalho E.M., Rocha H., Queiroz A.C. & Jones T.C.. Leishmania donovanian opportunistic microbe associated with progressive disease in three immunocompromised patients, Lancet., 1, 1986, 647–649.[Medline]
Müller I., Garcia-Sanz J.A., Titus R., Behin R. & Louis J.. Analysis of the cellular parameters of the immune responses contributing to resistance and susceptibility of mice to infection with the intracellular parasite, Leishmania major, Immunol. Rev., 112, 1989, 95–113.[Medline]
Stenger S., Donhauser N., Thüring H., Röllinghoff M. & Bogdan C.. Reactivation of latent leishmaniasis by inhibition of inducible nitric oxide synthase, J. Exp. Med., 183, 1996, 1501–1514.
Chang K.-P.. Leishmania infection of human skin fibroblasts in vitroabsence of phagolysosomal fusion after induced phagocytosis of promastigotes, and their intracellular transformation, Am. J. Trop. Med. Hyg., 27, 1978, 1084–1096.
Dedet J.P., Ryter A., Vogt E., Hosli P. & L. Pereira da Silva. Uptake and killing of Leishmania mexicana amazonensis amastigotes by human skin fibroblasts, Ann. Trop. Med. Parasitol., 77, 1983, 35–44.[Medline]
Schwartzman J.D.. The interaction of Leishmania donovani promastigotes and human fibroblasts in vitro, Am. J. Trop. Med. Hyg., 34, 1985, 850–855.
Mirkovich A.M., Galelli A., Allison A.C. & Modabber F.Z.. Increased myelopoiesis during Leishmania major infection in micegeneration of "safe targets," a possible way to evade the effector immune mechanism, Clin. Exp. Immunol., 64, 1986, 1–7.[Medline]
Bogdan C., Stenger S., Röllinghoff M. & Solbach W.. Cytokine interactions in experimental cutaneous leishmaniasis. Interleukin 4 synergizes with interferon- to activate murine macrophages for killing of Leishmania major amastigotes, Eur. J. Immunol., 21, 1991, 327–333.[Medline]
Stenger S., Thüring H., Röllinghoff M. & Bogdan C.. Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major, J. Exp. Med., 180, 1994, 783–793.
Werner-Felmayer G., Werner E.R., Fuchs D., Hausen A., Reibnegger G. & Wachter H.. Tetrahydrobiopterin-dependent formation of nitrite and nitrate in murine fibroblasts, J. Exp. Med., 172, 1990, 1599–1607.
Lima H.C., Bleyenberg J.A. & Titus R.G.. A simple method for quantifying Leishmania in tissues of infected animals, Parasitol. Today., 13, 1997, 80–82.[Medline]
Ito S. & Karnovsky M.J.. Formaldehyde-glutaraldehyde fixatives containing trinitro compounds, J. Cell Biol., 39, 1968, 168a–169a.
Glauert A.M.. Fixation, dehydration, and embedding of biological specimens, Glauert A.M.. Practical Methods in Electron Microscopy. Vol. 3, Part I, 1975, 133–162, North Holland Publishing Co, Amsterdam.
Timpl R., Rohde H., Gehron-Robey P., Rennard S.I., Foidart J.M. & Martin G.R.. Laminin—a glycoprotein from basement membranes, J. Biol. Chem., 254, 1979, 9933–9937.
Specks I., Mayer U., Nischt R., Spissinger T., Mann K., Timpl R., Engel J. & Chu M.-L.. Structure of recombinant N-terminal globule of type VI collagen 3 chain and its binding to heparin and hyaluronan, EMBO (Eur. Mol. Biol. Organ.) J., 11, 1992, 4281–4290.[Medline]
Pan T.-C., Sasaki T., Zhang R.-Z., Fässler R., Timpl R. & Chu M.-L.. Structure and expression of fibulin-2, a novel extracellular matrix protein with multiple EGF-like repeats and consensus motifs for calcium-binding, J. Cell Biol., 123, 1993, 1269–1277.
Schulze B., Mann K., Battistutta R., Wiedemann H. & Timpl R.. Structural properties of recombinant domain III-3 of perlecan containing a globular domain inserted into an epidermal-growth-factor-like motif, Eur. J. Biochem., 231, 1995, 551–556.[Medline]
Battacharya A., Dorf M.E. & Springer T.A.. A shared alloantigenic determinant on Ia antigens encoded by the I-A and I-E subregionsevidence for I region gene duplication, J. Immunol., 127, 1981, 2488–2495.[Abstract]
van Vliet E., Melis M., Foidart J.M. & van Ewijk W.. Reticular fibroblasts in peripheral lymphoid organs identified by a monoclonal antibody, J. Histochem. Cytochem., 34, 1986, 883–890.[Abstract]
Green S.J., Crawford R.M., Hockmeyer J.T., Meltzer M.S. & Nacy C.N.. Leishmania major amastigotes initiate the L-arginine-dependent killing mechanism in IFN- stimulated macrophages by induction of tumor necrosis factor-, J. Immunol., 145, 1990, 4290–4297.[Abstract]
Murray H.W. & Nathan C.F.. Macrophage microbicidal mechanisms in vivoreactive nitrogen versus oxygen intermediates in the killing of intracellular visceral Leishmania donovani, J. Exp. Med., 189, 1999, 741–746.
Chang K.P.. Leishmanicidal mechanisms of human polymorphonuclear phagocytes, Exp. Parasitol., 55, 1981, 377–385.
Pearson R.D. & Steigbigel R.T.. Phagocytosis and killing of the protozoan Leishmania donovani by human polymorphonuclear leukocytes, J. Immunol., 127, 1981, 1438–1443.[Abstract]
Pearson R.D., Sullivan J.A., Roberts D., Romito R. & Mandell G.L.. Interaction of Leishmania donovani promastigotes with human phagocytes, Infect. Immun., 40, 1983, 411–416.
Grimaldi G. Jr., Soares M.J. & Moriearty P.L.. Tissue eosinophilia and Leishmania mexicana mexicana eosinophil interactions in murine cutaneous leishmaniosis, Parasite Immunol., 6, 1984, 735–739.
Pearson R.D., Uydess I.L., Chapman S.W. & Steigbigel R.T.. Interaction of human eosinophils with Leishmania donovani, Ann. Trop. Med. Parasitol., 81, 1987, 735–739.[Medline]
Oliveira S.H.P., Fonseca S.G., Romao P.R.T., Figueiredo F., Ferreira S.H. & Cunha F.Q.. Microbicidal activity of eosinophils is associated with activation of the arginine-NO pathway, Parasite Immunol., 20, 1998, 405–412.[Medline]
Williams R.O.. Invasion of murine dendritic cells by Leishmania major and L. mexicana mexicana, J. Parasitol., 74, 1988, 186–187.[Medline]
Moll H., Flohé S. & Röllinghoff M.. Dendritic cells in Leishmania major-immune mice harbor persistent parasites and mediate an antigen-specific T cell immune response, Eur. J. Immunol., 25, 1995, 693–699.[Medline]
Belle E.A.. Cultivation of Leishmania donovani in human amnion epithelial cell tissue culturesa preliminary report, Can. Med. Assoc. J., 79, 1958, 726–728.
Adler S.. Attempts to transmit visceral leishmaniasis to man. Remarks on the histopathology of leishmaniasis, Trans. R. Soc. Trop. Med. Hyg., 33, 1940, 419–437.
Pai H.C. & Hu C.H.. Attempts to grow Leishmania donovani in tissue cultures, Proc. Soc. Exp. Biol. Med., 46, 1941, 606–608.
Hawking F.. Growth of protozoa in tissue culture. V. Leishmania donovani, Trans. R. Soc. Trop. Med. Hyg., 41, 1948, 545–554.[Medline]
Zuckerman A.. Initial reaction to the subcutaneous inoculation of cultures of Leishmania tropica in the hamster, Acta Medica Orientalia., 12, 1953, 238–240.[Medline]
El-Shoura S.M., Sheikha A.K., Bahamdan K.A., Tallab T.M. & Hassounah O.A.. Visceral and cutaneous leishmaniasiscomparative ultrastructure of host-parasite interactions, J. Egypt. Soc. Parasitol., 25, 1995, 861–876.[Medline]
Lavnikova N. & Laskin D.L.. Unique patterns of regulation of nitric oxide production in fibroblasts, J. Leukoc. Biol., 58, 1995, 451–458.[Abstract]
Love D.C., Esko J.D. & Mosser D.M.. A heparin-binding activity on Leishmania amastigotes which mediates adhesion to cellular amastigotes, J. Cell Biol., 123, 1993, 759–766.
Esterre P., Dedet J.P., Guerret S., Chevallier M., Frenay C. & Grimaud J.A.. Matrix remodelling and fibroblast phenotype in early lesions of human cutaneous leishmaniasis, Pathol. Res. Pract., 187, 1991, 924–930.[Medline]
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