Chondroitin-4-sulfate impairs in vitro and in vivo cytoadherence of Plasmodium falciparum infected erythrocytes.

BACKGROUND: Chondroitin-4-sulfate (CSA) was recently described as a Plasmodium falciparum cytoadherence receptor present on Saimiri brain microvascular and human lung endothelial cells. MATERIALS AND METHODS: To specifically study chondroitin-4-sulfate-mediated cytoadherence, a parasite population was selected through panning of the Palo-Alto (FUP) 1 P. falciparum isolate on monolayers of Saimiri brain microvascular endothelial cells (SBEC). Immunofluorescence showed this SBEC cell line to be unique for its expression of CSA-proteoglycans, namely CD44 and thrombomodulin, in the absence of CD36 and ICAM-1. RESULTS: The selected parasite population was used to monitor cytoadherence inhibition/dissociating activities in Saimiri sera collected at different times after intramuscular injection of 50 mg CSA/kg of body weight. Serum inhibitory activity was detectable 30 min after injection and persisted for 8 hr. Furthermore, when chondroitin-4-sulfate was injected into monkeys infected with Palo-Alto (FUP) 1 P. falciparum, erythrocytes containing P. falciparum mature forms were released into the circulation. The cytoadherence phenotype of circulating infected red blood cells (IRBC) was determined before and 8 hr after inoculation of CSA. Before inoculation, in vitro cytoadherence of IRBCs was not inhibited by CSA. In contrast, in vitro cytoadherence of circulating infected erythrocytes obtained 8 hr after CSA inoculation was inhibited by more than 90% by CSA. CONCLUSIONS: In the squirrel monkey model for infection with P. falciparum, chondroitin-4-sulfate impairs in vitro and in vivo cytoadherence of parasitized erythrocytes.     

Plasmodium falciparum: cytoadherence of a knobless clone

Sequestration of Plasmodium falciparum-infected erythrocytes is crucial to parasite survival as it prevents destruction in the liver and spleen. Knobs have been considered necessary but not sufficient for cytoadherence to vascular endothelial cells in vivo and to melanoma or umbilical vein endothelial cells in vitro. We describe here a knobless clone that cytoadheres strongly to C32 melanoma cells. This clone cannot express the knob-associated histidine-rich protein (KAHRP) due to the deletion of the KAHRP gene. Our results raise the possibility of an alternative mechanism for in vitro cytoadherence and suggest that the use of long term cultured isolates and melanoma cells as a model for cytoadherence in vivo may be misleading.     

The binding site on ICAM-1 for Plasmodium falciparum-infected erythrocytes overlaps, but is distinct from, the LFA-1-binding site.

The intercellular adhesion molecule-1 (ICAM-1, CD54) is one of three putative endothelial receptors that mediate in vitro cytoadherence of P. falciparum-infected erythrocytes. Since cytoadherence to postcapillary venular endothelium is thought to be a major factor in the virulence of P. falciparum malaria, we have examined the interaction between ICAM-1 and the P. falciparum-infected cell, and have compared it with the interaction to the physiological counter receptor, the leukocyte integrin LFA-1. Our results demonstrate that the malaria-binding site resides in the first two domains of the ICAM-1 molecule and overlaps, but is distinct from, the LFA-1 site.     

The role of variant surface antigens on malaria-infected red blood cells.

It has been proposed that the primary role of variant antigens appearing on the surface of red blood cells infected with malaria parasites is to mediate cytoadherence, and that the antigenic variation they display is an adaptation to avoid immune attack. Here, Allan Saul proposes that their role is the opposite: that their primary purpose is to generate an immune response, which regulates their growth and thereby establishes a chronic infection, and that the role of cytoadherence is to ensure that parasites failing to express this flag to the immune system are destroyed by the spleen.     

Characteristics of immune interferon produced by human lymphocyte cultures compared to other human interferons.

A factor with antiviral activity has been produced in vitro by combined macrophage-lymphocyte cultures from patients with recent herpes labialis in response to HSV antigen stimulation. It has been designated "immune interferon" and characterized in comparison to several other human interferons. It was shown to be relatively unstable at pH 2 and at 56 degrees C. Rabbit anti-human leukocyte interferon serum was shown to be less active against immune interferon than against diploid cell interferon or against vesicle fluid interferon. The possibility of immune interferon being a totally different anti-viral protein or a protein with certain shared antigen determinants or structures with classical viral interferon is discussed. A simplified method for the assay of anti-interferon sera with microtiter plates is also described.     

Sensitization of circulating basophils in guinea pig recipients of passive transfer of cutaneous basophil hypersensitivity (CBH) with immune serum: antigen-specific histamine release in vitro

An in vitro histamine release assay was used to test the hypothesis that passive sensitization of circulating basophils is associated with the activity of immune serum that transfer the ability to elicit cutaneous basophil hypersensitivity (CBH) reactions. Systemic i.v. transfer of several types of immune sera that mediate CBH also led to passive sensitization of circulating basophils for antigen-specific release of histamine in vitro. In addition, we found that immune serum passively sensitizes basophils in vitro. Thus immune sera had three activities that are probably interconnected: sera will 1) passively transfer CBH in vivo, 2) passively sensitize basophils in vivo, and 3) passively sensitize basophils in vitro. These results suggest that passive sensitization of circulating basophils by immune serum contributes to the mechanism by which antibodies transfer the ability to elicit CBH reactions.     

Inhibition of in vitro fertilization by mouse anti-mouse sperm sera and preliminary antigen identification

Antisperm antibodies are implicated as one causative factor of infertility, but the target antigens have not been identified. Immune responses to sperm antigens are qualitatively variable even within a single mouse strain. We took advantage of this variability and immunized individual female mice to allogeneic sperm to reflect their natural exposure during mating. We determined the ability of the individual sera to inhibit in vitro fertilization and to bind to sperm antigens separated by electrophoresis. Compared to preimmune sera, four of five immune sera significantly inhibited in vitro fertilization. The serum from individual mice bound variable panels of sperm antigens. By comparing the panels, we identified two polypeptides with molecular weights of 40,000 and 44,000 that were bound by all sera. We propose that these molecules may be good candidates for further investigation of the immunoprophylaxis of pregnancy.     

Selection of Babesia bovis-infected erythrocytes for adhesion to endothelial cells coselects for altered variant erythrocyte surface antigen isoforms

Sequestration of Babesia bovis-infected erythrocytes (IRBCs) in the host microvasculature is thought to constitute an important mechanism of immune evasion. Since Ig is considered to be important for protection from disease, an in vitro assay of B. bovis sequestration was used to explore the ability of anti-B. bovis Ig to interfere with IRBC cytoadhesion, and to identify IRBC surface Ags acting as endothelial cell receptors. Bovine infection sera reactive with the IRBC surface inhibited and even reversed the binding of IRBCs to bovine brain capillary endothelial cells (BBECs). This activity is at least partially attributable to serum IgG. IgG isolated from inhibitory serum captured the variant erythrocyte surface ag 1 (VESA1) in surface-specific immunoprecipitations of B. bovis-IRBCs. Selection for the cytoadhesive phenotype concurrently selected for antigenic and structural changes in the VESA1 Ag. In addition, the anti-VESA1 mAb, 4D9.1G1, proved capable of effectively inhibiting and reversing binding of adhesive, mAb-reactive parasites to BBECs, and by immunoelectron microscopy localized VESA1 to the external tips of the IRBC membrane knobs. These data are consistent with a link between antigenic variation and cytoadherence in B. bovis and suggest that the VESA1 Ag acts as an endothelial cell ligand on the B. bovis-IRBC.     

AIDS vaccination studies using an ex vivo feline immunodeficiency virus model: reevaluation of neutralizing antibody levels elicited by a protective and a nonprotective vaccine after removal of antisubstrate cell antibodies

In the feline immunodeficiency virus system, immunization with a fixed-infected-cell vaccine conferred protection against virulent homologous challenge but the immune effectors involved remained elusive. In particular, few or no neutralizing antibodies were detected in sera from vaccinated cats. Here we show that, when preadsorbed with selected feline cells, the same sera revealed clearly evident virus-neutralizing activity. Because high titers of neutralizing antibody in cell-adsorbed sera from 23 cats immunized with fixed-infected-cell or whole-inactivated-virus vaccines correlated with protection, it is likely that they were more important for protection than formerly realized. In vitro, the fixed-cell vaccine efficiently removed neutralizing antibody from immune sera while the whole-inactivated-virus vaccine was much less effective.     

Animal and human antibodies reactive with the outer surface protein A and B of Borrelia burgdorferi are borreliacidal, in vitro, in the presence of complement

Abstract Polyspecific antibodies present in ascitic fluids of mice (pMIAFs) immunized with whole Borrelia burgdorferi cells exerted borreliacidal activity in vitro when tested with complement and homologous antigen but not with heterologous B. hermsii . Similarly, monospecific mouse antibodies obtained by immunizing mice with purified preparations of outer surface protein A and B of B. burgdorferi were borreliacidal. On the contrary, mouse monospecific antibodies raised against the 41-kDa flagellar protein of B. burgdorferi did not kill borreliae in the presence of complement. A complement-mediated, in vitro, borreliacidal activity was observed in human sera from patients with Lyme disease when antibodies against OspA and/or OspB were detectable in sera by the Western blotting technique. The in vitro borreliacidal activity of human sera was evident after 14 h incubation with live B. burgdorferi spirochaetes and complement, whereas antibodies present in mouse immune ascitic fluids killed borreliae after 1 h incubation.     

Heat-Labile Factor Necessary for Hemagglutination-Inhibition Testing of Horse Sera

Normal and immune sera were obtained from horses immunized with either aqueous, alum, or adjuvant bivalent vaccines containing Milford equine 2 virus. Upon heating at 56 C for 30 min, a factor, required for hemagglutination-inhibition but not complement fixation or neutralization testing, was destroyed. This factor which is present in normal sera does not appear to be complement.     

Antibody and immune complexes induce tissue factor production by human endothelial cells

Patients with systemic lupus erythematosus (SLE) have an increased incidence of arterial and venous thromboses. The mechanism by which thromboses develop in these patients is unknown. We had previously observed that the sera of patients with SLE contain antibodies and immune complexes that can bind to endothelial cells. Because endothelial cells can synthesize tissue factor, a potent activator of coagulation, we studied the effect of IgG complexes and sera from patients with SLE on the production of tissue factor by these cells. Human umbilical venous endothelial cells incubated with heat-aggregated IgG (HA-IgG) (0.5 to 4.0 mg) elaborate procoagulant activity in a dose-dependent manner. All procoagulant activity was found in the particulate cell fraction, and none was secreted into the medium. Maximum expression of procoagulant activity required 6 to 8 hr, and its production was totally inhibited by the addition of cyclohexamide or actinomycin D. The presence of gel-filtered platelets augmented production of procoagulant activity by endothelial cells stimulated by HA-IgG. Endothelial cell procoagulant activity was not inactivated by diisofluoropropylphosphate, required the presence of Factor VII for its expression, and was neutralized by a specific anti-tissue factor antibody. Endothelial cells incubated with sera from 14 of 16 patients with SLE produced increased amounts of tissue factor compared with 21 normal sera (p less than 0.025). Fractions of two SLE sera containing monomeric IgG, IgA, or IgM, as well as fractions containing IgG complexes, each stimulated endothelial cells to produce more tissue factor than similar fractions prepared from two normal sera. These studies demonstrate that endothelial cells will produce the procoagulant tissue factor after exposure to anti-endothelial cell antibodies or IgG-containing immune complexes. The production of tissue factor by endothelial cells at sites of immune vascular injury may play a role in the development of thromboses in patients with SLE.     

Eimeria acervulina: DNA cloning and characterization of recombinant sporozoite and merozoite antigens

Genes encoding antigens of Eimeria acervulina were cloned from cDNA expression libraries prepared from the sporozoite and merozoite stages in order to examine humoral and cellular immune responses to this protozoan parasite. Two clones expressing surface antigens were characterized by DNA hybridization studies to identify homologous genomic DNA fragments. The proteins they encode were identified by 125I-labeling, immunoblotting, immunofluorescence, and T-cell activation experiments. One, designated cSZ-1, encodes a 130-kDa beta-galactosidase fusion protein which represents a portion of a p240/p160 immunodominant sporozoite surface antigen. Immunofluorescence studies using anti-cSZ-1 sera and live or 1% paraformaldehyde-fixed E. acervulina sporozoites have confirmed this surface locale. Purified cSZ-1 fusion protein, which is not recognized by sera from E. acervulina-infected chickens, induced the activation of immune T lymphocytes in vitro. Another cDNA clone, designated cMZ-8, gives rise to a 150-kDa fusion protein and encodes a portion of a p250 immunodominant merozoite surface antigen. This was established by immunoblotting of 125I-labeled merozoite proteins with anti-cMZ-8 sera and immunofluorescence staining of live and 1% paraformaldehyde-fixed E. acervulina merozoites. Purified cMZ-8 is recognized by sera from E. acervulina-infected chickens and induces a significant activation of immune T lymphocytes in vitro.     

Pathogenic complement activation in collagen antibody-induced arthritis in mice requires amplification by the alternative pathway

Immune complex-induced inflammation can be mediated by the classical pathway of complement. However, using mice genetically deficient in factor B or C4, we have shown that the collagen Ab-induced model of arthritis requires the alternative pathway of complement and is not dependent on the classical pathway. We now demonstrate that collagen Ab-induced arthritis is not altered in mice genetically deficient in either C1q or mannose-binding lectins A and C, or in both C1q and mannose-binding lectins. These in vivo results prove the ability of the alternative pathway to carry out pathologic complement activation in the combined absence of intact classical and lectin pathways. C3 activation was also examined in vitro by adherent collagen-anti-collagen immune complexes using sera from normal or complement-deficient mice. These results confirm the ability of the alternative pathway to mediate immune complex-induced C3 activation when C4 or C1q, or both C1q and mannose-binding lectins, are absent. However, when all three activation pathways of complement are intact, initiation by immune complexes occurs primarily by the classical pathway. These results indicate that the alternative pathway amplification loop, with its ability to greatly enhance C3 activation, is necessary to mediate inflammatory arthritis induced by adherent immune complexes.     

“Weak” histocompatibility antigens generate functionally “strong” humoral immunity

The kinetics and complexity of the immune responses generated by weak histocompatibility antigens on transplants of normal tissues were investigated by analyzing (1) the survival times of test skin grafts on mice that had been initially immunized with skin, liver, or kidney implants, (2) the survival times of test skin grafts on mice that had received putatively immune sera or lymphocytes harvested from an allografted host, and (3) the cytotoxic potential of these cells and sera in vitro. The data indicate that despite chronic rejection, weak antigens rapidly immunize hosts, and that the immune response includes a major humoral component that can either accelerate graft rejection or prolong graft survival.     

Abnormal PfEMP1/knob display on Plasmodium falciparum-infected erythrocytes containing hemoglobin variants: fresh insights into malaria pathogenesis and protection

Hemoglobin (Hb) variants are associated with reduced risk of life-threatening Plasmodium falciparum malaria syndromes, including cerebral malaria and severe malarial anemia. Despite decades of research, the mechanisms by which common Hb variants - sickle HbS, HbC, α-thalassemia, fetal HbF - protect African children against severe and fatal malaria have not been fully elucidated. In vitro experimental and epidemiological data have long suggested that Hb variants do not confer malaria protection by restricting the growth of parasites in red blood cells (RBCs). Recently, four Hb variants were found to impair cytoadherence, the binding of P. falciparum-infected RBCs (PfRBCs) to microvascular endothelial cells (MVECs), a centrally important event in both parasite survival and malaria pathogenesis in humans. Impaired cytoadherence is associated with abnormal display of P. falciparum erythrocyte membrane protein 1 (PfEMP1), the parasite's major cytoadherence ligand and virulence factor, on the surface of host RBCs. We propose a model in which Hb variants allow parasites to display relatively low levels of PfEMP1, sufficient for sequestering PfRBCs in microvessels and avoiding their clearance from the bloodstream by the spleen. By preventing the display of high levels of PfEMP1, Hb variants may weaken the binding of PfRBCs to MVECs, compromising their ability to activate endothelium and initiate the downstream microvascular events that drive the pathogenesis of malaria.     

Effect of sera against aggregated mouse immunoglobulins on a population of lymphoid and hematopoietic cells]

The in vitro treatment of the mouse spleen cells immunized by the ram erythrocytes with the rabbit and mouse sera against the thermoaggregated mouse immunoglobulins resulted in the inhibition of antigen binding receptors of rosette forming cells. The mouse serum, unlike the rabbit one, induced the inactivation of receptors in rosette forming lymphocytes both in the non-immune and immune mice on the 8th day after the antigenic stimulation. The treatment of bone marrow cells from the intact mice with these sera increased insignificantly the number of hemopoietic colonies in the spleens of lethally irradiated syngenic recipients and stimulated markedly the migration of spleen cells. This may be due both to the direct effect of these sera and to their mediated (through the humoral factor) influence. The inactivation of antigen binding receptors in the spleen rosette forming cells suggests the presence of immunoglobulins on the membrane of B-lymphocytes in the aggregated state or in the form of antigen--antibody complexes.     

Inhibition of the immune response by membrane-bound antibody.

Cells from the spleens of "normal" swine, which were pretreated with pronase to remove surface membrane-bound immunoglobulin, gave an enhanced hemolytic plaque-forming cell response to sheep red blood cells in vitro in comparison with untreated controls. The enhancement could be abrogated by preincubating pronase-treated spleeen cells in preparations containing antibody to sheep red blood cells. This effect was demonstrated by autologous sera, immune sera, and all three known classes of porcine serum immunoglobulins, including IgM, IgA, and IgG and could be removed by absorption with sheep red blood cells. Surface membrane-bound antibody exerted its effect by binding to the nonadherent cell population. The response of normal spleen cells was unaffected by antibody treatment. Pronase-treatment was not mitogenic, did not function as a polyclonal B cell activator, and did not selectively eliminate T or B cells. The results indicate that removal of antibody from the surface of lymphoid cells enhanced the humoral immune response invitro and confirm that membrane-bound antibody can inhibit response to antigen.     

小鼠中性粒细胞在抗体及补体参与下对日本血吸虫童虫杀伤作用的体外实验

用光镜及电镜观察小鼠中性粒细胞及中性粒细胞依赖抗体及补体对体外培养的日本血吸虫童虫 的作用。结果表明：单纯中性粒细胞很少粘附到童虫表面,仅个别十分疏松地粘附在童虫表面,被粘附 的童虫结构正常。提示：单纯中性粒细胞对童虫无明显作用,在抗体及补体协同下,中性粒细胞成群且 紧密地粘附在童虫体表,在细胞集聚的周围,虫体体被出现隧道样及火山口样变化,紧贴童虫的中性 粒细胞伸出伪足,虫体体棘紊乱,皮层变平,体被剥脱,虫体变形,说明中性粒细胞在抗体及补体协同 下,对童虫有杀伤作用、文中对杀伤机制进行了扼要的讨论。