ARTICLE |
Correspondence to: Dominique Gaillard, Unité INSERM U314, Université de Reims Champagne-Ardenne, CHU Maison Blanche, 45 rue Cognacq-Jay, 51092 Reims Cedex, France.
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Summary |
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Most pathological conditions resulting from infection with the human malaria parasite Plasmodium falciparum occur as a consequence of the sequestration by several adhesion molecules of parasite-infected red blood cells (IRBCs). Recent reports have provided evidence that placental vascular endothelial ligands for IRBCs were mostly restricted to chondroitin sulfate A (CSA). The expression of CSA in malaria-infected placentas was investigated in a prospective case-control study in a hypoendemic area (Dakar, Senegal). The tissue distribution of CSA was measured in the terminal villi by immunostaining combined with image processing in 20 infected and 20 noninfected frozen sections of placenta. The villous surface immunostained by anti-CSA antibody was higher in infected than in noninfected placentas (p<0.03), in placentas with active infection than in those with past chronic infection (p<0.05), and in infected placentas with positive imprints than in those with negative imprints (not significant; p=0.06). Labeling was found in the extracellular matrix and in endothelial and stromal cells of all the placentas. Syncytiotrophoblast immunostaining was detected in all placentas associated with active or active chronic infection (n=7) but in only 4/13 placentas with past chronic infection (p<0.01). The presence of P. falciparum in the imprint was significantly correlated with immunostaining of CSA in syncytiotrophoblasts (p=0.003). These results suggest that CSA can play an important role in the sequestration of P. falciparum in human placentas during the acute phase of infection. (J Histochem Cytochem 47:751756, 1999)
Key Words: image analysis, chondroitin sulfate A, placenta, Plasmodium falciparum, infection
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Introduction |
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During pregnancy, Plasmodium falciparum malaria is associated with a number of maternofetal complications such as pre-eclampsia (
The availability of in vitro parasite cultures and assays to detect and measure cytoadherence of IRBCs on target cells has enabled the identification of several host proteins that are putative receptors to which IRBCs bind (
The aim of this study was to analyze and quantify expression of CSA in P. falciparum-infected or noninfected human placenta by immunohistochemistry and image analysis, to determine if (a) CSA is more strongly expressed in the villi of infected placentas and (b) CSA is found in syncytiotrophoblasts.
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Materials and Methods |
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A prospective case-control study was carried out in two maternity centers in Dakar (Senegal) where malaria is hypoendemic with a low and seasonal transmission: the Hôpital Principal de Dakar and the Public Health Center in Ouakam. Placentas were collected with informed consent of the parturients during a 5-month period (July to November, the rainy season), when malarial transmission was maximal (
Pathological Examination
All fixed samples were embedded in paraffin. Paraffin sections 5 µm thick were stained with hematoxylin, eosin, and saffron (HES). Sections were examined under light microscopy and polarized light to assess the deposition of malarial pigment. To confirm the presence of malarial pigment and to eliminate the artifact due to formalin pigment, cryostat sections of positive samples were taken and stained with HES.
Malarial infection of the placenta was classified into four groups, using the histological classification of
Placentas were deemed infected when they showed histological lesions of P. falciparum infection and belonged to Category 1, 2, or 3, irrespective of the positivity or the negativity of the imprint. Placentas without such histological lesions were considered noninfected.
Immunohistochemical Study
For each placental sample, 5-µm cryostat sections were cut. The slides were rinsed for 10 min with PBS at room temperature (RT). Blocking nonimmune serum was applied for 10 min and the tissue section was incubated for 1 hr at RT with a mouse IgG1 primary MAb against CSA (1:200; Chemicon International-Euromedex, Souffelweyersheim, France). Secondary incubation was performed with an avidinbiotin immunoperoxidase kit (Universal, LSAB Kit; Dako, Glostrup, Denmark) and the sections were stained at RT for 5 min with diaminobenzidine (DAB; Immunotech, Marseille, France). The sections were counterstained with ethyl green (CAS; Elmhurst, NY) which actually gives a nuclear stain (Figure 1A and Figure 2).
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Negative control slides were processed after incubation with normal nonimmune mouse serum instead of primary antibody. To test the specificity of reactivity of the antibody used, control slides were treated with chondroitinase AC. Slides of infected and uninfected placentas were incubated with 0.1 U of chondroitinase AC (Chondroitinase AC; Sigma-Aldrich, St Quentin Falavier, France) for 30 min at 37C, after which the slides were rinsed several times with PBS and the normal immunohistochemical technique was performed with anti-CSA antibody as primary antibody. Positive control slides were performed from normal frozen skin biopsy specimens with anti-KL-1 antibody (1:50, Immunotech) as primary antibody.
Quantitative Image Analysis
The CSA staining was quantified by image cytometry using a multiparameter image analysis research station (Discovery; Becton-Dickinson, Richmond, CA) equipped with three CDD cameras, one black-and-white camera for low magnification, one for high magnification, and one color CDD camera. For each slide, the fields to be measured were selected by microscopic examination under low magnification using a x10 objective lens, and included terminal villi.
A first surface (Surface 1) (Figure 1B) was delineated under low magnification (x125) by illuminating specimens with monochromatic light (620 nm) suitable to analyze the counterstaining ethyl green, the brown staining of DAB, and the very light background staining in the cytoplasm and the extracellular matrix detectable by image analysis. In the same field, a second surface (Surface 2) (Figure 1C) was delineated with monochromatic light (500 nm), corresponding to the absorption peak of the DAB wavelength (
To ensure the reproducibility of the immunoperoxidase staining, the samples were processed twice with 10 slides of infected placentas and 10 slides of noninfected placentas, using identical parameters. To eliminate nonspecific or background immunostaining, a measurement was done in terminal villi. The computer of the multiparameter image analysis research station evaluated the intensity of labeling in this entire area and established a histogram of this intensity. In this histogram, the threshold of intensity was adjusted in Surface 1 so that the light brown stain present across the tissue was eliminated, and in Surface 2 so that intensity of labeling was similar to that of the epidermal area in our control slides.
Statistical Analysis
The relationship between the expression of CSA and the other parameters (infection of the placenta, presence of P. falciparum in imprints, histologic category) was evaluated with the Fisher exact test or the chi-square test. p values less than 0.05 were considered statistically significant. These analyses were performed using the SAS statistical software package (SAS Institute; Carey, NC).
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Results |
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Six (30%) positive imprints (Figure 3) were detected out of the 20 infected placentas (Figure 4). Among these 20 parturients, two (10%) had a positive QBC test but no positive QBC test was recorded for cord blood samples.
Quantitative Image Analysis
Surface 1 assessed the total surface of the villi, including cells and the extracellular matrix except for the capillary lumen (Figure 1A and Figure 1B). Surface 2 corresponds to a cellular or an extracellular area stained by the chromogen (Figure 1A and Figure 1C). The percentage of the surface immunostained for CSA was significantly higher in infected placentas than in noninfected placentas (Fisher test p<0.03) (Table 1; Figure 5). The percentage of the villous area immunostained for CSA was higher in placentas with positive imprints than in those with negative imprints, although nonsignificantly (Fisher test p=0.06). However, this percentage was significantly higher in Categories 1 and 2 than in Category N (Fisher test p<0.05). No significant difference could be found between Category N and Category 3. Considering Categories 1 and 2 as a single entity because both refer to stages of acute infection, it appears that placentas in this 1+2 category have larger CSA surface immunostaining than placentas belonging to Categories N or 3 (Fisher test p<0.05).
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Immunohistochemical Localization of CSA
CSA was detected in the extracellular matrix, in a few endothelial cells, and in stromal cells of the terminal villi in all placentas, whether or not they were infected (Figure 1A and Figure 2). In 11 infected placentas, immunohistochemical examination showed cytoplasmic expression of CSA in syncytiotrophoblasts, and sometimes this expression was membranous (Figure 1A). There was a significant correlation between the positivity of the imprints and the presence of CSA in syncytiotrophoblasts (chi-square test p=0.003). Placentas with active infection (Category 1) and with active chronic infection (Category 2) showed syncytiotrophoblast immunostaining of CSA more frequently than placentas with past chronic infection (Category 3) (Fisher test p<0.02) (Table 1).
The two types of negative control slides, after incubation with normal nonimmune mouse serum instead of primary antibody and after incubation with chondroitinase AC, were both negative.
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Discussion |
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Histological lesions of P. falciparum-induced placentitis are well known (
If the amount of the immunoreactivity (i.e., the "antigen"antibody immune complex formation) in a tissue section is proportional to the amount of antigen in the section, immunohistochemistry is therefore applicable to assess the antigen content in this section (
We reported two important results which can be helpful in understanding the action of CSA in placentas infected with P. falciparum. First, the expression of CSA in the terminal villi was higher in infected placentas than in noninfected placentas, and in placentas with acute infection than in those with past chronic infection. Two hypotheses can be formulated on the basis of these results: (a) P. falciparum alone can induce the expression of CSA in vivo, as , can directly induce the expression of CSA and then increase the expression of CSA in placentas from Categories 1 and 2. However, in vitro studies of syncytiotrophoblasts revealed that TNF
did not modify the expression of CSA (
The second major result of the present study is that CSA was detected in the cytoplasm and in plasma membrane of syncytiotrophoblasts in most infected placentas, and especially in placentas with active inflammation. This suggests that the CSA adhesion molecule detected on the surface of syncytiotrophoblasts, may play a major role in the sequestration of IRBCs in placenta. In fact, the increased surface immunostained for CSA in infected placentas corresponded to an increase in the number of immunostained cells, particularly syncytiotrophoblasts, and was dependent on the intensity and the acuteness of the inflammation, as well as on the presence of IRBCs in the intervillous space.
CSA appears to be directly involved in both the sequestration of IRBCs in the placenta and, particularly, in the acute phase of malarial infection during pregnancy. This research strongly suggests that prevention of maternal malaria can be achieved by means of inhibitors of CSA/IRBC interactions, which could be potentially mediated by either antibody-based therapy or subunit vaccines, as has already been suggested (
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Acknowledgments |
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We would like to thank the midwives of the Public Health Center of Ouakam and of the Hôpital Principal de Dakar, and particularly Ms Coumba Diop and Ms Carole Todote. We thank Dr G. Michel for support in this work and J.M. Zahm for technical assistance. We also thank Effie Gournis (Yale University; New Haven, CT) for helpful comments and revision of the manuscript.
Received for publication December 1, 1998; accepted January 19, 1999.
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