Modulation of macrophage C3b receptor function by cytochalasin-sensitive structures

A quantitative rosette assay was employed in order to determine if through pharmacologic probes we could gain an insight into the nature of the interaction between C3b-coated particles and the macrophage C3b receptor. Rabbit alveolar macrophage monolayers were challenged with chromium-labeled, complement-coated (via cold agglutinin) human erythrocytes (HEC3b) and the per cent of bound counts determined in the distilled water lysate. With this assay system in which ingestion is negligible, the cytochalasins (A greater than E greater than D greater than B) produced the most marked inhibition of rosette formation compared to control treated monolayers. No agent examined produced consistent augmentation. Cytochalasin A at 10(-5), 10(-6), and 10(-7) M inhibited rosette formation by 77+/- 2, 44 +/- 4 and 15 +/- 7 (S.E.), per cent, respectively. Cytochalasin E was also markedly inhibitory, Cytochalasins B and D produced approximately 30% inhibition at 10(-5) M. The cytochalasin effect was not secondary to an interaction between these agents and complement-sensitized erythrocytes, although cytochalasin E was also able to reduce erythrocyte-bouund C3b reactivity. Cytochalasin A and E modulation of the macrophage C3b reactivity occurred within a few minutes and was only slightly reversible. Cytochalasins A and E could also disrupt performed rosettes but the effect was not as pronounced as when these agents were present before and/or during the actual adherence phenomenon. Vinblastine and colchicine (10(-5) and 10(-6) M) also produced significant inhibition of rosette formation, although the magnitude of the effect was less than that for cytochalasins A and E. Further characterization of the vinblastine and colchicine effect demonstrated that the inhibition was rapid, irreversible over a 60-min incubation, and not explained by an alteration in macrophage attachment or in HEC3b reactivity. Agents producing insignificant inhibition of rosette formation included the following: dibutyryl cAMP and cAMP agonists (PGE1, theophylline), 8-bromo cGMP and cGMP agonists (carbachol, asorbic acid), dimethylsulfoxide, heparin, ethanol, dextran sulfate, DEAE-dextran, and poly-L-lysine. The data suggest that cytochalasin, vinblastine and colchicine sensitive membrane structures, most likely microfilaments and microtubules, are important in the interaction of C3b-coated particles with the macrophage C3b receptor.     

Inhibition of T-lymphocyte rosetting by HL-A alloantisera and complement.

The relationship between HL-A antigens and rosetting of sheep red blood cells (SRBC) with peripheral human lymphocytes has been investigated by incubating them with HL-A antibodies. Although sensitizing the lymphocytes with HL-A alloantisera had no effect on their ability to form rosettes with SRBC, further sensitization with C6 deficient rabbit serum as a source of early complement components inhibited the formation of rosettes with SRBC. The involvement of HL-A alloantibodies in the inhibition of rosette formation was shown first by correlating the HL-A phenotype of the lymphocytes and the HL-A specificity of the alloantisera and, second, by specifically absorbing the HL-A alloantibodies from the alloantisera. Complement was needed to inhibit rosette formation since this effect was lost when rabbit serum was treated to inactivate complement. The participation of complement's classical pathway in rosette inhibition was shown by chelating the Ca2+ ions by EGTA treatment of the C6 deficient rabbit serum. Perhaps, binding of HL-A antibodies and early complement components to the lymphocyte surface disturbs the distribution of the receptors or affects the charge of the cell membrane, thus inhibiting the rosette formation with SRBC.     

Cytochalasin binding in lymphocytes and polymorphonuclear leukocytes

The binding of [³H]cytochalasin B (CB) to intact cells was compared in lymphocytes, polymorphonuclear leukocytes (PMNLs) and erythrocytes over a broad range of cytochalasin concentrations. Binding curves consistent with the presence of high and low affinity binding sites were demonstrated in all three cell types. However, in contrast to observations in erythrocytes, in lymphocytes and PMNLs CB binding was unaffected by d-glucose. p-Hydroxymercuribenzoate and p-hydroxymercurisulfonate were only partially inhibitory and unlabeled cytochalasins E, D and A (CE, CD, CA) inhibited [³H]CB binding more effectively than unlabeled CB. While attempts to demonstrate that plasma membrane-rich subcellular fractions from lymphocytes selectively bind [³H]CB were inconclusive, radioautographic studies on unbroken cells indicated that most or all of the high affinity CB-binding sites in lymphocytes and PMNLs were in close proximity to the cell surface.     

Kinetic and morphological changes induced in human blood leucocytes by cytochalasin D and E

The effect of increasing concentrations of cytochalasin D and E, up to toxicity, on the velocity of blood leucocytes from normal subjects was measured in vitro using a high-resolution objective and phase-contrast time-lapse photography. The dose-response effect for the two different cytochalasins differed in accordance with the different cell specificity of their membrane binding. The average velocity of granulocytes was reduced at cytochalasin D concentrations above 5 x 10(-7)M and cytochalasin E concentrations above 5 x 10(-5)M. The effect on monocytes and eosinophils was similar. In contrast the velocity of lymphocytes was not affected until cytotoxic concentrations were reached. The concentration ranges which inhibited locomotion corresponded well with the concentration ranges of the cytochalasins which have an in vitro effect on microfilaments. The concentrations which induced additional morphological changes in lymphocytes also correlate well with the concentrations found to inhibit cross-linking in vitro, as well as those known to induce morphological changes in, for example, fibroblasts in vivo. Cytotoxic effects were first observed with ten-fold higher concentrations of cytochalasin E than of cytochalasin D.     

人和猴T淋巴细胞表面TRBC受体及其配体不同于E2分子和CD2的配体

CD2 (E receptor, LFA-3 receptor) and E2 molecules (Bernard, 1988) on human T lymphocytes, CD58 (LFA-3, lymphocyte function associated antigen 3) on human erythrocytes and S14,S42,S110-220 molecules (Bernard, 1987) of sheep erythrocytes are involved in rosette formation of human T lymphocytes with human or sheep erythrocytes. Rosette formation of human and macaque pan-T lymphocytes with tree shrew (Tupaia belangeri) red blood cells (TRBC) (TRBC rosette) has shown different physicochemical properties from that of rosette formation with sheep red blood cells (E rosette) (Ben, 1985). CD2, CD3/TCR complex, CD5, CD6, and CD7 are not involved in TRBC rosette formation (Zheng, 1990). In order to know whether E2, LFA-3,S14,S42 and S110-220 molecules are involved in TRBC rosette formation or human and macaque T lymphocytes, rosette inhibition and antigenic modulation or co-modulation were performed with relevant monoclonal antibodies (McAbs), and hemolytic assay and slide agglutination were also conducted. TRBC rosette formation of human and rhesus monkey PBL was not blocked by E2 McAb (inhibition rate 2.8% and 2.1%, respectively). In contrast, human E rosette formation was obviously blocked at inhibition rate of 49.8% and macaque E rosette formation was slightly inhibited (13.3%). The modulation or co-modulation of E2 molecule with E2 McAb did not affect human TRBC rosette formation. Similar results were shown in rosette formation inhibition of Jurkat cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochalasin inhibition of hexose transport by platelets

Previously we described a two-transporter model (T1, T2) for galactose uptake by platelets (Horne, M.K. and Hart, J.S. (1986) Biochim. Biophys. Acta 856, 448-456). In the current work we have sought corroborative evidence for this model by studying the effects of cytochalasins on this transport system. Of the various cytochalasins tested, cytochalasin B was the most potent inhibitor (I) of galactose transport, whereas cytochalasin A was less inhibitory and dihydrocytochalasin B and cytochalasin E had no inhibitory effect. The same order of potency was observed for the inhibition of L-glucose diffusion into platelets. The mechanism of cytochalasin B inhibition was investigated in detail. Inhibition of T1 was competitive and required a higher concentration of cytochalasin B (Ki1 approximately 1.7 microM) than inhibition of T2, which was of a mixed type (Ki2 approximately 0.8 microM). The effect of cytochalasin B on T2 could be accounted for by a membrane alteration which enhanced the affinity of the transporter for galactose while simultaneously preventing passage of the TSI complex into the cell. Since a similar effect on membrane permeability would also explain cytochalasin B inhibition of L-glucose diffusion, it is hypothesized that cytochalasin B binds to a membrane structure shared by T2 and the passage for L-glucose. The differences in cytochalasin B sensitivity and mechanism of inhibition manifested by T1 and T2 support our original hypothesis that galactose is indeed transported by kinetically distinct agencies and suggest that these may be physically distinct as well.     

Correlation between effects of 24 different cytochalasins on cellular structures and cellular events and those on actin in vitro

To compare the effects of cytochalasins on the cellular level with those on the molecular level, 24 cytochalasins, 20 natural compounds and 4 derivatives, were used. The following effects were tested for each of 24 cytochalasins; (a) four high dose (2-20 muM) effects on the cellular level: rounding up of fibroblastic cells, contraction of actin cables, formation of hairy filaments containing actin, and inhibition of lymphocyte capping; (b) a low dose (0.2-2 muM) effect: inhibition of membrane ruffling; and (c) two in vitro effects: an inhibition of actin filament elongation (the high affinity effect [low dose effect] in vitro) and an effect on viscosity of actin filaments(the low affinity effect [high dose effect] in vitro). These results indicated that there are almost the same hierarchic orders of relative effectiveness of different cytochalasins between low and high dose effects and between cellular and molecular effects. From the data obtained with the 24 cytochalasins, we have calculated correlation coefficients of 0.87 and 0.79 between an effect in vivo, inhibition of capping, and an effect in vitro, inhibition of actin filament elongation, as well as between inhibition of capping and another effect in vitro, effect on viscosity of actin filaments, respectively. Furthermore, a correlation coefficient between the high affinity effect and the low affinity effect determined in vitro was calculated to be 0.90 from the data obtained in this study. The strong positive correlation among low and high dose effects in vivo and those in vitro suggests that most of the effects caused by a cytochalasin, irrespective of doses or affected phenomena, might be attributed to the interaction between the drug and the common target protein, actin. In the course of the immunofluorescence microscope study on cytochalasin-treated cells using actin antibody, we have found that aspochalasin D, a 10-isopropylcytochalasin, strongly induced the formation of rodlets containing actin in the cytoplasm of the treated fibroblasts. In contrast, the other cytochalasins, including cytochalasin B, cytochalasin C, cytochalasin D, and cytochalasin H, were found to induce the formation of nuclear rodlets. Both cytoplasmic and nuclear rodlets found in the cytochalasin-treated cells were similar in ultrastructures to those induced by 5 to 10 percent (vol/vol) dimethyl sulfoxide in the same type of cells.     

Histidine-rich glycoprotein blocks T cell rosette formation and modulates both T cell activation and immunoregulation

Histidine-rich glycoprotein (HRGP) is a plasma and platelet protein with undefined function in vivo. It has been reported to inhibit rosette formation between murine T cells and erythrocytes. We have shown that HRGP binds specifically to human T lymphocytes but not sheep erythrocytes and have demonstrated a 56-kDa HRGP-binding protein on the T cell surface which is distinct from the CD2 sheep erythrocyte receptor. We have now investigated whether HRGP can inhibit human T cell-sheep erythrocyte rosette formation and whether HRGP can modulate T cell activation. HRGP at physiologic concentrations specifically inhibited rosette formation between human T lymphocytes and sheep erythrocytes. HRGP suppressed proliferation of antigen receptor (CD3)-triggered T cells induced by interleukin 2; this suppression was specifically reversed by prior incubation of HRGP with affinity-purified anti-HRGP IgG. Addition of HRGP 12-24 h after CD3 triggering no longer suppressed T cell proliferation, suggesting HRGP suppressed T cell division by interfering with one or more early events in the process of T cell activation. Human serum (containing 100-150 micrograms/ml HRGP) was also capable of suppressing T cell proliferation; serum which had been immunodepleted of HRGP no longer inhibited T cell proliferation. Furthermore, HRGP inhibited interleukin 2 receptor expression on activated T cells, causing decreased T cell interferon-gamma release and altered T cell-dependent inhibition of erythropoiesis. HRGP is thus capable of modulating T cell activation and T cell immunoregulation; HRGP may function as a natural suppressive regulator of human T lymphocyte activation.     

Sheep erythrocyte rosetting induces multiple alterations in T lymphocyte function: inhibition of T cell receptor activity and stimulation of T11/CD2

When T lymphocytes from human blood or lymphoid organs are prepared by the sheep red blood cell (SRBC) rosetting procedure, glycoproteins of the SRBC membrane interact intimately with the CD2 (T11) molecule on the T cell surface. We now show that rosette formation has measurable short- and long-term effects upon the T cells. First, for a period of 24-48 hr after rosetting, the signal transducing and activation functions of the T3/Ti T cell antigen receptor complex is paralyzed for anti-T3-induced calcium mobilization, with a concomitant decrease in proliferative response to mitogens or stimulatory anti-T3 antibodies. Calcium mobilization through the alternate pathway of T cell activation, the T11/CD2 SRBC receptor, was also inhibited by rosetting. Second, rosetting appears to confer a partial stimulatory signal through the T11/CD2 pathway. Thus, 72 hr or more after rosetting, there was increased expression of the T11(3) activation epitope, and rosetted T cells were stimulated to proliferate in the presence of anti-T11(3) antibodies alone. These results provide further details on the effects of SRBC-T cell interactions, with important methodological implications. Moreover, they suggest a hitherto unrecognized down-regulatory effect of engaging the CD2 molecule, and provide further evidence that the T cell receptor is functionally interconnected to the CD2 activation pathway.     

Human autologous rosette-forming cells. III. Binding of erythrocytes from different species to the T-cell receptors for autologous red blood cells

Spontaneous rosette formation in humans is restricted to a subpopulation of the circulating T cells. We have previously shown that the interaction between lymphocytes and autologous red blood cells (auto-RBC) is not mediated by a self-recognition mechanism, since allogeneic (allo-) RBC bind to T cells through the same receptors. In this work, we have extended these observations to thymocytes. Using a mixed-rosette assay in which one type of erythrocyte was identified by FITC labeling, we have shown that almost all the thymocytes which attached auto-RBC could also fix allo-RBC. However, as for the peripheral blood lymphocytes (PBL), binding of human RBC to thymocytes occurred with varying affinities according to the erythrocyte's origin. In order to further study the specificity of the erythrocyte to lymphocyte binding in rosette formation, PBL were mixed with auto-RBC and erythrocytes of xenogeneic (xeno-) origin. Although very disparate incidences of rosettes were found according to the species from which the RBC were derived, most of the autorosetting lymphocytes also had receptors for xeno-RBC. In addition, preincubation of PBL with monoclonal antibody OKT11A (directed against the sheep RBC receptors on T cells) completely abrogated rosette formation with all the erythrocytes tested (human auto- and allo-, sheep, pig, and rabbit) except mouse RBC. Taken together these data strongly suggest that human auto- or allo-, as well as sheep or some other xeno-RBC, bind to T lymphocytes by a single receptor and that the combining sites are expressed with different densities or varying affinities depending upon the RBC's origin. Therefore, spontaneous autorosettes may represent T lymphocytes having high-affinity receptors for sheep RBC.     

Natural cytotoxic reactivity of human lymphocytes against a myeloid cell line: characterization of effector cells.

Using a series of techniques to identify and deplete various peripheral blood lymphocyte subpopulations, we studied the cytotoxic reactivity of normal individuals against the myeloid cell line K-562 in a 4-hr 51chromium-release assay. Depletion of lymphocytes bearing complement receptors had a variable, usually negligible effect on cytotoxicity. In contrast, depletion of lymphocytes bearing Fc receptors abrogated target cell lysis. Separation of lymphocytes with high-affinity binding of sheep red blood cells (SRBC) evidenced by rosette formation at 29 degrees C yielded a population of rosette-forming cells containing few cytotoxic cells, whereas separation of total E-RFC under optimal rosetting conditions produced a rosette fraction containing a major proportion of the effector cells. These data indicate that the cytotoxic lymphocyte in this system is Fc receptor positive, largely complement receptor negative, and may possess low density or low affinity receptors for SRBC.     

Mechanism of the effect of sera from rats subjected to high external temperature on rosette formation by splenic lymphocytes from non-immune animals]

Serum of rats subjected to the action of moderately high external temperature increased the immunogenicity of heterologous erythrocytes, but became inactive at a temperature of 2 degrees C and after the treatment with a polyvalent proteinase inhibitor--trasilol. Incubation of sheep erythrocytes with the serum of heated rats intensified the reaction of rosette formation by the lymphocytes of the spleen of intact rats. Incubation of the spleen cells with the sera of intact and heated rats failed to influence the rosette formation. After trasilol treatment the serum of heated rats completely lost the property to intensify the reaction of rosette formation. Phenomenon of intensification of rosette formation was revealed in case of treatment of erythrocytes with the sera at a temperature of 37 degrees C and was not reproduced at a temperature of 2 degrees C. Investigations carried out indicated that the increase in the immunogenicity of sheep erythrocytes and the intensification of their binding with the lymphocyte receptors of intact rats under the effect of the serum of heated animals were caused by the action of the same factor.     

Characteristics of spontaneous rosette formation by mouse lymphocytes with autologous erythrocytes

A substantial proportion of mouse spleen and thymus cells form spontaneous rosettes with syngeneic and allogeneic erythrocytes. Rosette formation is independent of the metabolic state of the cell: Inhibitors of ATP formation (NaCN, NaN₃) and of cell motility (cytochalasin B, colchicine, vinblastine) have little or no effect. Erythrocytes can be bound by living or dead lymphocytes. The stability of rosettes against high temperature and shearing forces is weaker than that of immune rosettes. The increase or decrease of electrostatic repulsion between cells will enhance or reduce rosette formation. Membrane proteins are involved in cell-to-cell contacts, since several proteases destroy relevant structures on the cell surfaces of lymphocytes, erythrocytes, or both.     

Studies of the sheep red blood cell receptor using anti-lymphocyte antibodies

Human thymus derived lymphocytes (T cells) interact with sheep red blood cells (SRBC) to form rosettes. We wanted to determine whether the lymphocyte's receptor for SRBC is associated with serologically detectable cell surface antigens. Antisera were prepared by immunizing horses with either fresh human thymus (ATG) or with B lymphocytes from an established lymphoid cell line in culture (ALG). ATG, ALG or Concanavalin A (Con A) were added to lymphocyte preparations to determine their effect on rosetting. The results showed that ATG inhibited rosettes in a dose dependent manner. In contrast, both the Con A and ALG had no effect. By immunofluorescence, Con A and ALG staining cells were able to form rosettes. ATG staining cells were unable to form rosettes. Removal of the ATG receptor by capping could not restore the rosette forming capacity suggesting that inhibition was not due to steric hindrance. We conclude that antibody directed against T cells but not B cells binds to surface antigens which appear to be identical with or in close proximity to the specific SRBC receptor.     

Cytochalasins induce actin polymerization in human leukocytes.

We studied the effect of cytochalasins (B, D, and E) on the F-actin content in human neutrophils and lymphocytes using NBD-phallacidin labeling followed by flow cytometry. All three cytochalasins induced a concentration- and time-dependent increase in the F-actin content in both cell types. The order of potency was cytochalasin D greater than E greater than B. The increase in F-actin content was accompanied by a decrease in the G-actin content as measured by DNase I inhibition assay. These observations suggest that in intact cells cytochalasins may function differently compared to purified and semipurified systems, and their effects may be modified through other actin-binding or sequestering proteins. 2-deoxyglucose (20 mM) caused a decrease in the basal F-actin content and significantly reduced the change induced by the cytochalasins. These results suggest that the state of actin in intact cells is regulated by cytosolic ATP levels, primarily by the integrity of the glycolytic pathway. Based on these observations, we conclude that the mechanism of action of cytochalasins in intact cells is more complex than current models suggest.     

Unmasking by antimetabolites of receptors for sheep red blood cells on human lymphocytes

The action of antimetabolites (puromycin, cycloheximide) and cold was studied in the human rosette system. We found that the number of detectable receptors for sheep red blood cells on peripheral blood lymphocytes was increased in presence of some concentration of these drugs. A similar finding was noted when the blood lymphocytes were left at 4 °C. The possibility that both cold and antimetabolites, by modifying the cell membrane mobility, increase the receptor affinity and thus the number of detectable receptors is discussed. Another attractive possibility is also presented. We propose that the unmasking effect by antimetabolites is due to inhibition of protein synthesis which is necessary to better express the receptors for sheep red blood cells on human lymphocytes. This concept of decreased protein synthesis affecting the expression of surface receptors may be a more general phenomenon.     

Receptors for IgM-coated erythrocytes on chronic lymphatic leukemia cells.

Our experiments show that lymphocytes of CLL patients, having typical B cell characteristics, form rosettes with IgM-coated bovine erythrocytes. Of 18 investigated patients, 3 to 78% (mean 29%) of the isolated lymphocytes reacted with EA-IgM. With mixed rosette assays. EA-IgM bound to cells bearing receptors for IgG as well, but not receptor-bearing lymphocytes. Rosette formation could be completely blocked by addition of IgM at concentrations as low as 0.17 mg/ml. Ten milligrams per milliliter of aggregated human IgG had no effect on the rosette formation with EA-IgM but completely abolished the binding of EA-IgG. Adult human or rabbit serum blocked the EA-IgM binding, whereas cord blood serum and FCS had no effect. These inhibition data indicate that EA-IgM binding does not occur via a somewhat altered IgG-Fc receptor but reacts with different membrane structures. That EA-IgM receptor can be cleaved off with trypsin and can be reconstituted after overnight cultivation, also supports this viewpoint. In contrast to the situation in normal subjects, in CLL patients the IgM receptors are demonstrable before overnight cultivation and are found on cells with B cell characteristics.     

Effects of Multidrug Resistance-Related ATP-Binding-Cassette Transporter Proteins on the Cytoskeletal Activity of Cytochalasins

Cytochalasins are microfilament-active mould metabolites, widely utilized to study the involvement of the actin cytoskeleton in cellular processes as well as in genotoxicity and cell kinetic research. In this study we have investigated whether multidrug-resistance phenotypes, caused by overexpression of the ATP-binding-cassette transporter proteins P-glycoprotein (P-gp) or multidrug-resistance-associated protein (MRP), influence the microfilament-depolymerizing effect of cytochalasins. Using four well-characterized multidrug-resistance cell models, we have shown that both the microfilament-disrupting (phalloidine staining) and the cytotoxic (MTT-assay) activity of cytochalasins are reduced in parallel with increased P-gp expression and restorable by P-gp-modulating agents. This also applied to the cytochalasin D-mediated induction of polykaryons (microscopic evaluation) which arise as a consequence of impaired cytokinesis but unaffected karyokinesis. The reduced cellular activity of cytochalasins in P-gp-positive cell lines was correlated with decreased intracellular accumulation ([³H]cytochalasin B accumulation) which was also restorable by P-gp modulators. Moreover, the dose-dependent inhibition of P-gp photoaffinity labeling ([³H]azidopine) suggested cytochalasins as P-gp-binding agents. In contrast, MRP overexpression had no effect on either cytochalasin microfilament activity or cytotoxicity. In conclusion, data indicate that the microfilament-destructive effects of cytochalasins are impaired due to a reduction of the intracellular cytochalasin accumulation by P-gp but not by MRP. Results are discussed with regard to P-gp as a resistance factor when cytochalasins are utilized to study microfilament dynamics, cell cycle kinetics or chromosomal damage. Moreover, the polykaryon-inducing activity of cytochalasin D is suggested as a specific indicator for a P-gp-mediated multidrug-resistance phenotype and the reversing potency of chemosensitizers.     

Mapping of the region of complement receptor (CR) 1 required for Plasmodium falciparum rosetting and demonstration of the importance of CR1 in rosetting in field isolates

The malaria parasite Plasmodium falciparum induces a number of novel adhesion properties in the erythrocytes that it infects. One of these properties, the ability of infected erythrocytes to bind uninfected erythrocytes to form rosettes, is associated with severe malaria and may play a direct role in the pathogenesis of disease. Previous work has shown that erythrocytes deficient in complement receptor (CR) 1 (CR1, CD35; C3b/C4b receptor) have greatly reduced rosetting capacity, indicating an essential role for CR1 in rosette formation. Using deletion mutants and mAbs, we have localized the region of CR1 required for the formation of P. falciparum rosettes to the area of long homologous repeat regions B and C that also acts as the binding site for the activated complement component C3b. This result raises the possibility that C3b could be an intermediary in rosetting, bridging between the infected erythrocyte and CR1. We were able to exclude this hypothesis, however, as parasites grown in C3-deficient human serum formed rosettes normally. We have also shown in this report that rosettes can be reversed by mAb J3B11 that recognizes the C3b binding site of CR1. This rosette-reversing activity was demonstrated in a range of laboratory-adapted parasite strains and field isolates from Kenya and Malawi. Thus, we have mapped the region of CR1 required for rosetting and demonstrated that the CR1-dependent rosetting mechanism occurs commonly in P. falciparum isolates, and could therefore be a potential target for future therapeutic interventions to treat severe malaria.