Novel fibrillar structure confers adhesive property to malaria–infected erythrocytes

Scholander, Carin; Treutiger, Carl Johan; Hultenby, Kjell; Wahlgren, Mats

doi:10.1038/nm0296-204

Cited by 99 publications

(109 citation statements)

References 18 publications

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“…After incubation for 30 min at room temperature and protected from light (gentle resuspension after 15 min), the pRBCs were washed three times with RPMI 1640 medium, pH 6.8. Parasites were stained with ethidium bromide as described previously 22 and the fluorescence rate was assessed by counting the number of surface fluorescent pRBCs per total number of pRBCs in a Nikon (Tokyo, Japan) Optiphot-2 UV microscope, using a 10ϫ ocular and a 40ϫ lens.…”

Section: Methodsmentioning

confidence: 99%

Binding of Plasmodium falciparum-infected erythrocytes to soluble platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): frequent recognition by clinical isolates.

Heddini

Chen

Obiero

et al. 2001

The American Journal of Tropical Medicine and Hygiene

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Abstract. Platelet-endothelial cell adhesion molecule-1 or CD31 (PECAM-1/CD31) is a receptor recognized by Plasmodium falciparum-parasitized erythrocytes (pRBCs). Fluorescence-labeled soluble recombinant PECAM-1/ CD31 (sPECAM-1/CD31) is shown to bind to the surface of P. falciparum-infected erythrocytes on up to 70% of the cells. Binding is blocked by the addition of the unlabeled receptor in a dose-dependent fashion, but not by unrelated receptor-proteins. A significant correlation was found between the binding of sPECAM-1/CD31 to pRBCs and the binding to transfected L cells expressing the receptor as seen with six different P. falciparum lines or clones. Panning of cultures on PECAM-1/CD31 transfected L cells was paralleled by an increase in the binding of sPECAM-1/CD31. The pRBCs of 54% of fresh patient-isolates bound sPECAM-1/CD31 with a mean rate of 12.9% (range ϭ 1.1-44%). The data suggest that PECAM-1/CD31 is a common receptor recognized by wild isolates and that the soluble PECAM-1/CD31 suspension assay is a sensitive and reliable way to study PECAM-1/CD31 binding.

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Section: Methodsmentioning

confidence: 99%

Binding of Plasmodium falciparum-infected erythrocytes to soluble platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): frequent recognition by clinical isolates.

Heddini

Chen

Obiero

et al. 2001

The American Journal of Tropical Medicine and Hygiene

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“…We were able to obtain mAbs against the surface of B. ovataPRBCs, but not against the surface of T. sergenti-PRBCs. Various antigenic modifications are known to occur on the surface of PRBCs, including expression of parasite-derived antigens on the erythrocyte surface [2,6], alteration of selfantigens in the erythrocyte membrane [5] and deposition of serum components on the erythrocytes [7,14]. Such modifications appeared to be common to many Babesia [2,15] and Plasmodium species [3].…”

mentioning

confidence: 99%

Preparation of Antibodies Directed to the Babesia ovata- or Theileria sergenti-parasitized Erythrocytes.

Tsuji

Arai

Nakamura

et al. 1999

The Journal of Veterinary Medical Science

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ABSTRACT. To investigate the surface antigens of the bovine red blood cells (RBCs) parasitized by Babesia ovata or Theileria sergenti, attempts were made to produce monoclonal antibodies (mAbs) with BALB/c mice. Comparable numbers of hybridomas producing antipiroplasm mAbs, as well as anti-bovine RBC mAbs, were obtained from the mice immunized with B. ovata-or T. sergenti-PRBCs. However, mAbs directed to the surface of parasitized RBCs (PRBCs) were obtained only from the mice immunized with B. ovata-PRBCs, but not from those immunized with T. sergenti-PRBCs. When serum samples from the immunized mice and the infected cattle were examined, antibodies recognizing B. ovata-PRBC surface were detected in the sera against B. ovata, but analogous antibodies were undetectable in the sera against T. sergenti, despite that the sera showed substantial antibody titers to T. sergenti piroplasms. The results suggest that significant antigenic modifications occur on the surface of B. ovata-PRBCs, but not on the surface of T. [12,19]. Blood samples containing 30-60% parasitemia were harvested from the Bo-RBC-SCID mice and immunized into BALB/c mice (SLC Inc.). For each parasite antigen, six mice were intravenously injected 3 times at 3 days intervals with 100 µl of 20% suspension of infected RBCs in 0.85% NaCl. Four weeks after the last injection, the mice were boosted intraperitoneally with an equal amount of infected RBCs. Three days later, they were euthanatized by CO 2 asphyxiation, and the spleens were removed to obtain lymphocytes. Serum samples were prepared from all the mice, and they were absorbed with an excess amount of uninfected bovine RBCs prior to being tested. The method to produce mAbs was described previously [12]. Briefly, the spleen cells from two immunized mice were pooled, and 1.5 × 10 8 of the cells were fused with 1.5 × 10 7 of mouse myeloma P3-X63-Ag8-U1 cells using polyethylene glycol 4000 (Merck). Cells were plated 3.5 × 10 6 /well in 96-well plates, and hybridomas were selected in Dulbecco's modified Eagle medium (D-MEM; Flow laboratories) containing 16 µM hypoxanthine, 0.4 µM aminopterin, 100 µM thymidine and 10% fetal bovine serum (FBS). Cultured supernatants were examined for antibody production by the two immunofluorescence assays (IFA) described below. Cells in the antibody-positive wells were cloned twice by limiting dilution.Fixed-cell IFA: Approximately 30% suspension of B. ovata-or T. sergenti-infected RBCs, which have 10-40% parasitemia, was prepared in phosphate buffered saline (PBS), pH 7.2, containing 1% bovine serum albumin (BSA). Roughly 0.3 µl aliquots were spread and dried out in each well of 24-well HT Coating Slide (MS342BL; Bokusui Brown). The slides were fixed in acetone for 5 min, and immediately transferred into PBS. Following removal of solution by blotting with a filter paper, 15 µl of cultured supernatant was added to each well. After 1 hr incubation

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“…The TM284 strain, which forms rosettes and binds both IgG and IgM on the surface of the pRBC, was isolated in 1990 from a Thai patient with cerebral malaria. 3 The P. falciparum strain 3D7.2 is a rosette-forming, non-immunoglobulin-binding clone, which originally had a very low rate of rosette formation, but was enriched for rosette formation using micromanipulation in which single rosettes were isolated with a micropipette (Sundström A, Fernandez V, and others, unpublished data).Estimation of rosette formation rate. The parasites were stained with one drop (10 g/ml) of acridine orange to determine parasitemia, and counted in a Nikon (Tokyo, Japan) Optiphot-2 UV microscope using a 10ϫ ocular and a 40ϫ lens.…”

mentioning

confidence: 99%

“…3 Based on this knowledge, we have developed a novel method for the enrichment of immunoglobulin-binding, infected erythrocytes using antiimmunoglobulin-coated magnetic beads.…”

mentioning

confidence: 99%

“…The fluorescence rate was assessed by counting the number of fluorescent late-stage pRBC per total number of late-stage pRBC in a Nikon Optiphot-2 UV microscope, using a 10ϫ ocular and an oil immersion lens with a magnification of 100ϫ. 3 Coating of Dynabeads with immunoglobulin. Three milliliters of Dynabeads suspension (Dynabeads M-450, 4.5m diameter, Tosylactivated; Dynal A.S., Oslo, Norway) were coated with sheep anti-human immunoglobulin antibodies (5 g/ml beads; SBL, Sweden) according to the protocol provided by Dynal A.S.…”

mentioning

confidence: 99%

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Enrichment of immunoglobulin binding Plasmodium falciparum-infected erythrocytes using anti-immunoglobulin-coated magnetic beads.

Heddini

Treutiger²,

Wahlgren³

1998

The American Journal of Tropical Medicine and Hygiene

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Abstract. It has been shown that nonimmune, human immunoglobulins are bound to the surface of certain strains of Plasmodium falciparum-infected erythrocytes. We describe a novel way of enriching parasitized red blood cells (pRBC) for immunoglobulin binding/rosette formation using Dynabeads coated with antibodies raised against human immunoglobulins. Whole P. falciparum cultures were mixed with the precoated beads for approximately 120 min at room temperature, and the bound pRBC were isolated by magnetic force. The nonbound cell fraction contained ringinfected pRBC, immunoglobulin-negative, trophozoite-infected pRBC, and uninfected erythrocytes. A consistent elevation in the immunofluorescence and rosette formation rates of 100% and 86% respectively, was detected after the first enrichment and subcultivation. Protein A or G were also found to support binding of pRBC through surfaceexpressed immunoglobulin. The Dynabead technique is a novel way of enriching pRBC based on the immunoglobulinbinding capacity of the infected erythrocyte.Severe malaria is the cause of death of several million individuals each year, primarily due to cerebral malaria and to anemia. It is generally agreed that excessive sequestration of infected and uninfected erythrocytes (cytoadherence and rosette formation) is the primary reason for the vascular obstruction seen at autopsy in these patients.1-3 Minute electron-dense excrescences on the infected erythrocyte surface (knobs) have been found to mediate the attachment of parasitized erythrocytes to endothelial cells and to uninfected erythrocytes.3 An electron-dense fibrillar structure, which is involved in the binding and contains immunoglobulins M or M and G, is found at the knobs. 3 Based on this knowledge, we have developed a novel method for the enrichment of immunoglobulin-binding, infected erythrocytes using antiimmunoglobulin-coated magnetic beads. MATERIALS AND METHODS Parasites.The Plasmodium falciparum strains TM284 and the 3D7.2 were cultured according to standard procedures with 10% AB ϩ Rh ϩ serum added to the buffered medium (malaria incomplete medium; RPMI 1640 medium supplemented with HEPES (Gibco, Paisley, Scotland), gentamycin (Sigma, St. Louis, MO), and sodium bicarbonate (Merck, Darmstadt, Germany). The TM284 strain, which forms rosettes and binds both IgG and IgM on the surface of the pRBC, was isolated in 1990 from a Thai patient with cerebral malaria. 3 The P. falciparum strain 3D7.2 is a rosette-forming, non-immunoglobulin-binding clone, which originally had a very low rate of rosette formation, but was enriched for rosette formation using micromanipulation in which single rosettes were isolated with a micropipette (Sundström A, Fernandez V, and others, unpublished data).Estimation of rosette formation rate. The parasites were stained with one drop (10 g/ml) of acridine orange to determine parasitemia, and counted in a Nikon (Tokyo, Japan) Optiphot-2 UV microscope using a 10ϫ ocular and a 40ϫ lens. The rosette formation rate was calculated as the number of rosette-f...

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Novel fibrillar structure confers adhesive property to malaria–infected erythrocytes

Cited by 99 publications

References 18 publications

Binding of Plasmodium falciparum-infected erythrocytes to soluble platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): frequent recognition by clinical isolates.

Binding of Plasmodium falciparum-infected erythrocytes to soluble platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): frequent recognition by clinical isolates.

Preparation of Antibodies Directed to the Babesia ovata- or Theileria sergenti-parasitized Erythrocytes.

Enrichment of immunoglobulin binding Plasmodium falciparum-infected erythrocytes using anti-immunoglobulin-coated magnetic beads.

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