Plasmodium falciparum gametocyte adhesion to C32 cells via CD36 is inhibited by antibodies to modified band 3.

Plasmodium falciparum gametocyte-infected erythrocytes are characterized by their ability to sequester in the microvasculature of various organs, primarily the spleen and bone marrow. This phenomenon is thought to play a critical role in the development and survival of the sexual stages. Little is known, however, about ligands on the gametocyte-infected erythrocyte. Infection of erythrocytes with mature asexual stages of P. falciparum (trophozoites and schizonts) has been shown to induce modification of the erythrocyte anion transporter, band 3, and this has been linked to the acquisition of an adherent phenotype. Here, we demonstrate for the first time that immature gametocyte-infected erythrocytes also express modified band 3. In vitro binding assays demonstrate that gametocyte-infected erythrocytes of the 3D7 strain utilize this surface receptor for adhesion to C32 amelanotic melanoma cells via the host cell receptor CD36 (platelet glycoprotein IIIb). Adhesion of gametocyte-infected erythrocytes to CD36-transfected CHO cells is also dependent on modified band 3. However, modified band 3 does not mediate adhesion of gametocyte-infected erythrocytes to intercellular adhesion molecule 1, a second host receptor for gametocytes expressed on C32 cells.     

The C-terminal segment of the cysteine-rich interdomain of Plasmodium falciparum erythrocyte membrane protein 1 determines CD36 binding and elicits antibodies that inhibit adhesion of parasite-infected erythrocytes

Attachment of erythrocytes infected by Plasmodium falciparum to receptors of the microvasculature is a major contributor to the pathology and morbidity associated with malaria. Adhesion is mediated by the P. falciparum erythrocyte membrane protein 1 (PfEMP-1), which is expressed at the surface of infected erythrocytes and is linked to both antigenic variation and cytoadherence. PfEMP-1 contains multiple adhesive modules, including the Duffy binding-like domain and the cysteine-rich interdomain region (CIDR). The interaction between CIDRalpha and CD36 promotes stable adherence of parasitized erythrocytes to endothelial cells. Here we show that a segment within the C-terminal region of CIDRalpha determines CD36 binding specificity. Antibodies raised against this segment can specifically block the adhesion to CD36 of erythrocytes infected with various parasite strains. Thus, small regions of PfEMP-1 that determine binding specificity could form suitable components of an antisequestration malaria vaccine effective against different parasite strains.     

Phosphatidylserine-positive erythrocytes bind to immobilized and soluble thrombospondin-1 via its heparin-binding domain

Phosphatidylserine (PS)-dependent erythrocyte adhesion to endothelium and subendothelial matrix components is mediated in part via thrombospondin (TSP). Although TSP exhibits multiple cell-binding domains, the PS-binding site on TSP is unknown. Because a cell-binding domain for anionic heparin is located at the amino-terminus, we hypothesized that PS-positive red blood cells (PS(+ve)-RBCs) bind to this domain. We demonstrate that both heparin and its low-molecular-weight derivative enoxaparin (0.5-50 u/mL) inhibited PS(+ve)-RBC adhesion to immobilized TSP in a concentration-dependent manner (21% to 77% inhibition, P < 0.05). Preincubation of immobilized TSP with an antibody against the heparin-binding domain blocked PS(+ve)-RBC adhesion to TSP. Antibodies that recognize the collagen- and the carboxy-terminal CD47-binding domain on TSP had no effect on this process. Although preincubation of PS(+ve)-RBCs with TSP peptides from the heparin-binding domain that contained the specific heparin-binding motif KKTRG inhibited PS(+ve)-erythrocyte adhesion to matrix TSP (P < 0.001), these peptides in the immobilized form supported PS-mediated erythrocyte adhesion. A TSP-peptide that lacks the binding motif neither inhibited nor supported PS(+ve)-RBC adhesion. Additional experiments show that soluble TSP also interacted with PS(+ve)-RBCs via its heparin-binding domain. Our results demonstrate that PS-positive erythrocytes bind to both immobilized and soluble TSP via its heparin-binding domain and that both heparin and enoxaparin, at clinically relevant concentrations, block this interaction. Other studies have shown that heparin inhibited P-selectin- and soluble-TSP-mediated sickle erythrocyte adhesion to endothelial cells. Our results, taken together with the previously documented findings, provide a rational basis for clinical use of heparin or its low-molecular-weight derivatives as therapeutic agents in treating vaso-occlusive pain in patients with sickle cell disease.     

Antigenic and functional differences in adhesion of Plasmodium falciparum-infected erythrocytes to human and bovine CD36.

Cytoadherence by Plasmodium falciparum-infected erythrocytes (PRBC) to microvascular endothelium is, in part, mediated by the specific interaction between a parasite-derived erythrocyte surface ligand and a specific binding site on human CD36. We describe the selection for increased adhesion of PRBC to bovine CD36 and demonstrate that the molecular interaction between PRBC and bovine CD36 is independent of and distinct from the OKM5/8 monoclonal antibody epitopes which block PRBC-human CD36 binding.     

Regulation of T‐lymphocyte motility,adhesion and de‐adhesion by a cell surface mechanism directed by low density lipoprotein receptor‐related protein 1 and endogenous thrombospondin‐1

T lymphocytes are highly motile and constantly reposition themselves between a free‐floating vascular state, transient adhesion and migration in tissues. The regulation behind this unique dynamic behaviour remains unclear. Here we show that T cells have a cell surface mechanism for integrated regulation of motility and adhesion and that integrin ligands and CXCL12/SDF‐1 influence motility and adhesion through this mechanism. Targeting cell surface‐expressed low‐density lipoprotein receptor‐related protein 1 (LRP1) with an antibody, or blocking transport of LRP1 to the cell surface, perturbed the cell surface distribution of endogenous thrombospondin‐1 (TSP‐1) while inhibiting motility and potentiating cytoplasmic spreading on intercellular adhesion molecule 1 (ICAM‐1) and fibronectin. Integrin ligands and CXCL12 stimulated motility and enhanced cell surface expression of LRP1, intact TSP‐1 and a 130 000 MW TSP‐1 fragment while preventing formation of a de‐adhesion‐coupled 110 000 MW TSP‐1 fragment. The appearance of the 130 000 MW TSP‐1 fragment was inhibited by the antibody that targeted LRP1 expression, inhibited motility and enhanced spreading. The TSP‐1 binding site in the LRP1‐associated protein, calreticulin, stimulated adhesion to ICAM‐1 through intact TSP‐1 and CD47. Shear flow enhanced cell surface expression of intact TSP‐1. Hence, chemokines and integrin ligands up‐regulate a dominant motogenic pathway through LRP1 and TSP‐1 cleavage and activate an associated adhesion pathway through the LRP1–calreticulin complex, intact TSP‐1 and CD47. This regulation of T‐cell motility and adhesion makes pro‐adhesive stimuli favour motile responses, which may explain why T cells prioritize movement before permanent adhesion.     

A cytokine‐controlled mechanism for integrated regulation of T‐lymphocyte motility,adhesion and activation

The co‐ordination of T‐cell motility, adhesion and activation remains poorly understood. It is also unclear how these functions are co‐ordinated with external stimuli. Here we unveil a series of molecular interactions in cis at the surface of T lymphocytes with potent effects on motility and adhesion in these cells, and communicating with proliferative responses. These interactions were controlled by the signature cytokines of T helper subsets interleukin‐2 (IL‐2) and IL‐4. Low‐density lipoprotein receptor‐related protein 1 (LRP1) was found to play a key role for T‐cell motility by promoting development of polarized cell shape and cell movement. Endogenous thrombospondin‐1 (TSP‐1) enhanced cell surface expression of LRP1 through CD47. Cell surface expressed LRP1 induced motility and processing of TSP‐1 while inhibiting adhesion to intercellular adhesion molecule 1 and fibronectin. Interleukin‐2, but not IL‐4, stimulated synthesis of TSP‐1 and motility through TSP‐1 and LRP1. Stimulation of the T‐cell receptor (TCR)/CD3 complex inhibited TSP‐1 expression. Inhibitor studies indicated that LRP1 regulated TSP‐1 expression and promoted motility through JAK signalling. This LRP1‐mediated motogenic signalling was connected to CD47/Gi protein signalling and IL‐2‐induced signalling through TSP‐1. The motogenic TSP‐1/LRP1 mechanism antagonized TCR/CD3‐induced T‐cell proliferation. These results indicate that LRP1 in collaboration with TSP‐1 directs a counter‐adhesive and counter‐proliferative motogenic cascade. T cells seem programmed to prioritize movement before adhesion through this cascade. In conclusion, vital decision‐making in T lymphocytes regulating motility, adhesive interactions and proliferation, are integrated through a molecular mechanism connecting different cell surface receptors and their signalling pathways.     

Band 3 clustering promotes the exposure of neoantigens in Plasmodium falciparum-infected erythrocytes

Erythrocytes infected with the human malaria parasite Plasmodium falciparum become structurally and antigenically modified as a consequence of intracellular parasite development. The new antigens that appear on the surface of the infected erythrocyte originate from parasite-encoded proteins and by modification of the erythrocyte membrane protein band 3. Here, we show that anti-peptide antibodies generated against an amino acid sequence (YETFSKLIKIFQDH) of human band 3, and previously identified as mediating adhesion of infected erythrocytes to CD36, recognized P. falciparum-infected erythrocytes. In addition, sera from individuals living in a malaria endemic area (and who are presumably immune) contained immunoglobulins specific for this region of band 3. The anti-peptide antibodies reacted with the surface excrescences (knobs) on falciparum-infected erythrocytes. In uninfected erythrocytes, the band 3 region was cryptic and its exposure on the falciparum-infected erythrocyte surface required clustering of band 3 protein. Thus, a parasite-induced modification of band 3 promotes adhesion and induces antigenic changes in the P. falciparum-infected erythrocyte.     

Cytoadherence characteristics of rosette-forming Plasmodium falciparum.

Sequestration of Plasmodium falciparum-infected erythrocytes to the capillary endothelium can cause obstruction and localized tissue damage. Occlusion of vessels in falciparum malaria infection has been related to two properties of the parasite: adhesion to endothelial cells and rosette formation. Our study on P. falciparum isolates from Thailand producing variable numbers of rosettes suggests the involvement of rosettes in capillary blockage caused by direct adhesion of the rosette-forming infected erythrocytes to various target cells, e.g., live human umbilical vein endothelial cells, monocytes, and platelets. These rosettes did not bind Formalin-fixed target cells, nor did they bind to live or fixed C32 or G361 melanoma cells. Classification of the receptors involved in cytoadherence of endothelial cells and monocytes by specific antibody blocking and flow cytometry indicated that CD36 was involved in the adherence of monocytes but that other receptors besides CD36 may be involved in parasite adherence to endothelial cells. The cytoadherence of infected erythrocytes to monocytes was also associated with CD54 (ICAM-1). Further, differentiation of adherent monocytes resulted in an inversion of CD36 and CD54 levels on the cell surface which correlated with a decrease in surface binding of infected erythrocytes. This observation suggests that the state of cell activation and differentiation may also contribute to sequestration of parasites and to the pathogenesis of malaria.     

Carrageenans inhibit the in vitro growth of Plasmodium falciparum and cytoadhesion to CD36

Carbohydrates are implicated in many of the invasive and adhesive interactions that occur between Plasmodium falciparum malaria parasites and human host cells, including invasion of sporozoites into hepatocytes, entry of merozoites into new host erythrocytes during asexual blood-stage replication, adhesion of infected erythrocytes to uninfected erythrocytes (rosetting) and to a number of host endothelial receptors including ICAM-1, CD36 and chondroitin-4-sulphate. In addition to increasing our understanding of host–parasite interactions, the investigation of carbohydrates with differing levels and patterns of sulphation as inhibitors may contribute to the development of novel therapeutics targeting malaria. Here we show that three polysaccharides derived from seaweed (carrageenans) with differing sulphation levels and patterns can inhibit the in vitro erythrocytic invasion and growth of both drug sensitive and drug resistant P. falciparum lines and the adhesion of parasitized erythrocytes to the human glycoprotein CD36.     

An alternative pathway of B cell activation: stilbene disulfonates interact with a Cl- binding motif on AEn-related proteins to stimulate mitogenesis.

Stilbene disulfonates are known to competitively inhibit Cl-/HCO3- flux through Band 3-related anion exchange (AE) proteins. To study the role of AE in lymphocyte activation, stilbene disulfonates were added to cultures of rat splenocytes (SPL). Four different stilbene derivatives were tested and each directly stimulated mitogenic proliferative responses of SPL. The mitogenic activity of these analogs paralleled their known patterns of interaction with Band 3-related AE proteins, as measured by; (a) their effective mitogenic concentrations, (b) their rank order of mitogenic potency [DIDS greater than SITS greater than DNDS congruent to DAzDS], (c) their patterns of nonreversible binding to the mitogenic receptor [DIDS much greater than SITS, DNDS], and (d) the specific, noncompetitive inhibition of their activity by the antagonist niflumic acid. Stilbene disulfonates directly activated purified B cell populations but not isolated T cells and furthermore, acted in synergy with anti-IgM to stimulate proliferation of SPL. These findings show that stilbene disulfonates represent a novel class of mitogens that interact with AEn-related proteins to stimulate an alternative activation pathway in B cells. These studies also indicate that immunomodulating activities of nonsteroidal anti-inflammatory drugs such as niflumic acid may be mediated, in part, by their interactions with AEn-related proteins.     

Thrombospondin induces glomerular mesangial cell adhesion and migration.

BACKGROUND: Extracellular matrix components are known to modulate mesangial cell functions as adhesion, motility and proliferation. Among other extracellular matrix components, mesangial cells have been recently described to secrete thrombospondin (TSP), a high molecular weight glycoprotein, produced by several cell types, and known to play a role in embryogenesis, wound healing, angiogenesis, and tumorigenesis. The aim of this work was the functional and molecular characterization of TSP interactions with mesangial cells. EXPERIMENTAL DESIGN: Adhesion of mesangial cells to TSP-coated plastic, and chemotaxis in the Boyden chamber assay were tested. In order to identify TSP active domains, heparin, known to bind to the amino-terminal region of TSP, four monoclonal antibodies directed against specific domains of the molecule, and TSP fragments obtained by enzymatic digestion were used. RESULTS: We found that TSP induces mesangial cell adhesion and chemotaxis, in a dose dependent manner. Adhesion was inhibited by antiserum against TSP, and by an anti-CD36 monoclonal antibody tested in the presence of heparin, but not by the peptide Gly-Arg-Gly-Asp-Ser. We have also found that only the carboxy-terminal end of TSP retains the adhesive properties of the molecule, while all the fragments tested showed some degree of chemotactic activity. CONCLUSIONS: We conclude that TSP modulates mesangial cell adhesion and motility, thus acting as a potential autocrine and paracrine regulator of mesangial cell functions in normal and pathologic conditions.     

Molecular analysis of the cytoadherence phenotype of a Plasmodium falciparum field isolate that binds intercellular adhesion molecule-1

The ability of Plasmodium falciparum-infected erythrocytes to adhere to endothelial receptors and sequester in diverse host organs is an important pathogenic mechanism. Cytoadherence is mediated by variant surface antigens, which are referred to as PfEMP-1 and are encoded by var genes. The extracellular regions of PfEMP-1 contain multiple conserved cysteine-rich domains that are referred to as Duffy-binding-like (DBL) domains. Here, we analyze the adhesive phenotype of an Indian P. falciparum field isolate, JDP8, which binds ICAM-1 but does not bind CD36. This is a unique cytoadherence phenotype because P. falciparum strains that bind ICAM-1 described thus far usually also bind CD36. Moreover, binding to both receptors is thought to be important for static adhesion under flow. The ICAM-1 binding population of P. falciparum JDP8 adheres to endothelial cells under flow despite poor binding to CD36. We have also identified an expressed var gene, JDP8Icvar, which mediates the ICAM-1 binding phenotype of JDP8. Expression of different regions of JDP8Icvar on the surface of COS-7 cells followed by binding assays demonstrates that the ICAM-1 binding domain maps to the DBL2betaC2 domain of JDP8Icvar. Sequence comparison with two previously identified ICAM-1 binding domains of PfEMP-1, which also map to DBLbetaC2 domains, suggests that diverse P. falciparum isolates use a structurally conserved domain to bind ICAM-1. It thus appears that functional constraints may place limits on the extent of sequence diversity in receptor-binding domains of PfEMP-1.     

The frequency of BDCA3-positive dendritic cells is increased in the peripheral circulation of Kenyan children with severe malaria

The ability of Plasmodium falciparum-infected erythrocytes to adhere to host endothelial cells via receptor molecules such as ICAM-1 and CD36 is considered a hallmark for the development of severe malaria syndromes. These molecules are also expressed on leukocytes such as dendritic cells. Dendritic cells are antigen-presenting cells that are crucial for the initiation of adaptive immune responses. In many human diseases, their frequency and function is perturbed. We analyzed the frequency of peripheral blood dendritic cell subsets and the plasma concentrations of interleukin-10 (IL-10) and IL-12 in Kenyan children with severe malaria and during convalescence and related these parameters to the adhesion phenotype of the acute parasite isolates. The frequency of CD1c(+) dendritic cells in children with acute malaria was comparable to that in healthy controls, but the frequency of BDCA3(+) dendritic cells was significantly increased. Analysis of the adhesion phenotypes of parasite isolates revealed that adhesion to ICAM-1 was associated with the frequency of peripheral blood CD1c(+) dendritic cells, whereas the adhesion of infected erythrocytes to CD36 correlated with high concentrations of IL-10 and low concentrations of IL-12 in plasma.     

Fresh isolates from children with severe Plasmodium falciparum malaria bind to multiple receptors

The sequestration of Plasmodium falciparum-infected erythrocytes (pRBC) away from the peripheral circulation is a property of all field isolates. Here we have examined the pRBC of 111 fresh clinical isolates from children with malaria for a number of adhesive features in order to study their possible coexpression and association with severity of disease. A large number of adhesion assays were performed studying rosetting, giant rosetting, and binding to CD36, intercellular adhesion molecule 1, platelet endothelial cell adhesion molecule 1, thrombospondin, heparin, blood group A, and immunoglobulins. Suspension assays were performed at the actual parasitemia of the isolate, while all the static adhesion assays were carried out at an equal adjusted parasitemia. The ability to bind to multiple receptors, as well as the ability to form rosettes and giant rosettes, was found to be more frequent among isolates from children with severe versus mild malaria (P = 0.0015). Rosettes and giant rosettes were more frequent for children with severe malaria, and the cell aggregates were larger and tighter, than for those with mild disease (P = 0.0023). Binding of immunoglobulins (97% of isolates) and of heparin (81% of isolates) to infected erythrocytes was common, and binding to heparin and blood group A was associated with severity of disease (P = 0.011 and P = 0.031, respectively). These results support the idea that isolates that bind to multiple receptors are involved in the causation of severe malaria and that several receptor-ligand interactions work synergistically in bringing about severe disease.     

T‐cell regulation through a basic suppressive mechanism targeting low‐density lipoprotein receptor‐related protein 1

Cell adhesion is generally considered to depend on positive regulation through ligation of integrins and cytokine receptors. However, here we show that T‐cell adhesion, and notably also T‐cell receptor (TCR) ‐induced activation, are subject to constant suppression through shedding of low‐density lipoprotein receptor‐related protein 1 (LRP1). The broad‐spectrum metalloprotease inhibitor GM6001 abrogated shedding, so inducing prominent cell surface expression of LRP1 while enhancing TCR‐induced activation and adhesion to β₁ and β₂ integrin ligands, hence arresting the cells. Integrin ligands also inhibited shedding but the effect was less potent than that of GM6001. Unlike GM6001, integrin ligands also induced cell surface expression of full‐length thrombospondin‐1 (TSP170) and TSP130, which associated with LRP1, and TSP110, which did not associate with LRP1. Cell surface expression of LRP1 and TSP130 were induced exclusively in adhering cells, expression of TSP110 preferentially in non‐adhering cells and expression of TSP170 correlated with T‐cell motility. The pro‐adhesive chemokine CXCL12 also inhibited LRP1 shedding and induced surface expression of TSP170 and TSP130 while inhibiting TSP110. Exogenous TSP‐1 and ligation of CD28 inhibited shedding although less effectively than GM6001, and the inhibition through CD28 was independent of TSP‐1. Small interfering RNA silencing experiments confirmed involvement of LRP1 and TSP‐1 in integrin‐dependent adhesion and TCR‐induced activation. Hence, the poor LRP1 expression in T cells depends on shedding. Integrin ligands and CXCL12 antagonize shedding through a TSP‐1‐dependent pathway and ligation of CD28 antagonizes shedding independent of TSP‐1. The disappearance of LRP1 from the cell surface may provide basic immunosuppression at the T‐cell level.     

NCO-sP(EO-stat-PO) surface coatings preserve biochemical properties of RGD peptides

We have previously reported that star shaped poly(ethylene oxide-stat-propylene oxide) macromers with 80% EO content and isocyanate functional groups at the distal ends [NCO-sP(EO-stat-PO)] can be used to generate coatings that are non-adhesive but easily functionalized for specific cell adhesion. In the present study, we investigated whether the NCO-sP(EO-stat-PO) surfaces maintain peptide configuration-specific cell-surface interactions or if differences between dissimilar binding molecules are concealed by the coating. To this end, we have covalently immobilized both linear-RGD peptides (gRGDsc) and cyclic-RGD (RGDfK) peptides in such coatings. Subsequently, SaOS-2 or human multipotent mesenchymal stromal cells (MSC) were seeded on these substrates. Cell adhesion, spreading and survival was observed for up to 30 days. The time span for cell adherence was not different on linear and cyclic RGD peptides, but was shorter in comparison to the unmodified glass surface. MSC proliferation on cyclic RGDfK modified coatings was 4 times higher than on films functionalized by linear gRGDsc sequences, underlining that the NCO-sP(EO-stat-PO) film preserves the configuration-specific biochemical peptide properties. Under basal conditions, MSC expressed osteogenic marker genes after 14 days on cyclic RGD peptides, but not on linear RGD peptides or the unmodified glass surfaces. Our results indicate specific effects of these adhesion peptides on MSC biology and show that this coating system is useful for selective testing of cellular interactions with adhesive ligands.     

Ectophosphorylation of CD36 regulates cytoadherence of Plasmodium falciparum to microvascular endothelium under flow conditions

The adhesion of Plasmodium falciparum-infected erythrocytes (IRBCs) to human dermal microvascular endothelial cells (HDMECs) under flow conditions is regulated by a Src family kinase- and alkaline phosphatase (AP)-dependent mechanism. In this study, we showed that the target of the phosphatase activity is the ectodomain of CD36 at threonine-92 (Thr92). Mouse fibroblasts (NIH 3T3 cells) transfected with wild-type CD36 or a mutant protein in which Thr92 was substituted by Ala supported the rolling and adhesion of IRBCs. However, while the Src family kinase inhibitors PP1 and PP2 and the specific AP inhibitor levamisole significantly reduced IRBC adhesion to wild-type CD36 transfectants as with HDMECs, the inhibitors had no effect on IRBC adhesion to the mutant cells. Using a phosphospecific antibody directed at a 12-amino-acid peptide spanning Thr92, we demonstrated directly that CD36 was constitutively phosphorylated and could be dephosphorylated by exogenous AP. Endothelial CD36 was likewise constitutively phosphorylated. The phosphospecific antibody inhibited IRBC adhesion to HDMECs that could be reversed by preincubating the antibody with the phosphorylated but not the nonphosphorylated peptide. Pretreatment of HDMECs with AP abrogated the effect of PP1 on IRBC adhesion. Collectively, these results are consistent with a critical role for CD36 dephosphorylation through Src family kinase activation in regulating IRBC adhesion to vascular endothelium.     

Erythrocyte adhesion to the vascular endothelium]

Blood cells are in continuous contact with the vascular endothelium. Endothelial cell culture, intravital videomicroscopy allowed the investigation of blood cell-endothelium interactions in dynamic conditions. In the various diseases, diabetes mellitus, sickle cell anemia and malaria, erythrocytes have an increased adhesion to endothelial cells. The presence of advanced glycation end products (AGE) on erythrocytes of diabetics is responsible for their binding to the receptor RAGE present on the endothelium. The AGE-RAGE binding provokes an oxidant stress and induces the expression of the adhesion molecule. Furthermore, erythrocyte AGE induce an increase in vascular permeability. In sickle cell anemia, the increased adhesiveness and the sickling of red blood cells are responsible for thrombosis. Plasmodium falciparum infestation of erythrocytes induces knob formation at the cell surface and the P. falciparum protein binding to CD36, ICAM-1 and thrombospondin present on the endothelium, and facilitates the parasite dissemination.     

Evaluation of the effects of defensins on neutrophil functions

Defensins are known to be the microbicidal components of neutrophil granules, which contribute to oxygen-independent antimicrobial mechanisms. In this study, we have examined the effect of defensins on neutrophil functions, such as adhesion, superoxide anion generation, phagocytosis and chemotaxis. Guinea-pig defensins increased the expression of CD11b, CD11c and CD54 (intercellular adhesion molecule ICAM-1) on human neutrophils, and induced adhesion of guinea-pig and human neutrophils. When the effect of guinea-pig defensins on superoxide anion generation was examined, defensins inhibited superoxide anion generation during phagocytosis of complement-opsonized particles. Furthermore, defensins inhibited complement-dependent phagocytosis. However, they did not inhibit the binding of complement-opsonized particles to neutrophils, suggesting that defensins possibly inhibit complement-dependent phagocytosis by affecting the ingestion step but not the binding step. Defensins exhibited neither chemotactic nor chemokine activity. Interestingly, 10–20% of total defensins were released extracellularly from phagocytosing neutrophils. Together these observations indicate that, in addition to their antimicrobial activity, defensins may have the ability to modulate the functions of neutrophils at sites of infection or inflammation.accepted by M. J. Parnham     

Ex vivo desequestration of Plasmodium falciparum-infected erythrocytes from human placenta by chondroitin sulfate A

We performed ex vivo experiments with Plasmodium falciparum-infected human placentas from primi- and multigravida women from Cameroon. All women, independent of their gravida status, had anti-chondroitin sulfate A (CSA) adhesion antibodies which cross-reacted with heterologous strains, such as FCR3 and Palo-Alto(FUP)1, which were selected for CSA binding. These antibodies, directed against the surface of infected erythrocytes obtained by flushing with CSA (IRBC(CSA)), were restricted to the immunoglobulin G3 isotypes. Massive desequestration of parasites was achieved with soluble CSA but not with anti-ICAM-1 and anti-CD36 monoclonal antibodies. All of the CSA-flushed parasites were analyzed immediately by using in vitro assays of binding to Saimiri brain endothelial cells (SBEC) expressing various adhesion receptors. Parasites derived from all six placentas displayed the CSA adhesion phenotype. However, only partial inhibition of adhesion was observed in the presence of soluble CSA or when Sc1D SBEC were treated with chondroitinase ABC. These results suggest that an additional adhesive molecule of IRBC(CSA) which binds to an unidentified receptor is present in the placenta. This new phenotype was lost once the parasites adapted to in vitro culture. We observed additional differences in the CSA adhesion phenotype between placental parasites and in vitro-cultured parasites panned on endothelial cells carrying CSA. The minimum size of fractionated CSA required for a significant inhibition of placental IRBC(CSA) adhesion to Sc1D cells was 1 to 2 kDa, which contrasts with the 4-kDa size necessary to reach equivalent levels of inhibition with panned IRBC(CSA) of this phenotype. All placental IRBC(CSA) cytoadhered to Sc17 SBEC, which express only the CSA receptor. Panning of IRBC(CSA) on these cells resulted in a significant quantitative increase of IRBC cytoadhering to the CSA of Sc1D cells but did not change their capacity for adhesion to CSA on normal placenta cryosections. Our results indicate that the CSA binding phenotype is heterogeneous and that several distinct genes may encode P. falciparum-CSA ligands with distinct binding properties.