Regulation of naturally occurring autoantibody secretion by a radiosensitive lymphocyte: initial characterization and ontogeny

Low level irradiation (400-500 R) of normal mice or of murine spleen cells resulted in the detection of an enhanced number of plaque-forming cells against bromelain-treated autologous red cells (Br MRBC) 1 day later. The mechanism responsible for the increased numbers of plaques is apparently the elimination of a suppressor T cell since the addition of thymocytes or of Lyt 1+2+ splenic cells to cultures of irradiated cells reversed the radiation-induced increase. Studies on the ontogeny of the phenomenon indicate that anti-Br MRBC plaques can be formed by spleen cells taken from mice shortly after birth although adult levels are not reached until after 3 weeks of age. Radiation-induced increases in the number of plaques were not seen until 3 weeks of age, thus, suggesting a temporal developmental sequence of the ability to produce autoantibodies and to regulate such production.     

Plasmodium vivax invasion of human erythrocytes inhibited by antibodies directed against the Duffy binding protein

Background

Plasmodium vivax invasion requires interaction between the human Duffy antigen on the surface of erythrocytes and the P. vivax Duffy binding protein (PvDBP) expressed by the parasite. Given that Duffy-negative individuals are resistant and that Duffy-negative heterozygotes show reduced susceptibility to blood-stage infection, we hypothesized that antibodies directed against region two of P. vivax Duffy binding protein (PvDBPII) would inhibit P. vivax invasion of human erythrocytes.

Methods and Findings

Using a recombinant region two of the P. vivax Duffy binding protein (rPvDBPII), polyclonal antibodies were generated from immunized rabbits and affinity purified from the pooled sera of 14 P. vivax–exposed Papua New Guineans. It was determined by ELISA and by flow cytometry, respectively, that both rabbit and human antibodies inhibited binding of rPvDBPII to the Duffy antigen N-terminal region and to Duffy-positive human erythrocytes. Additionally, using immunofluorescent microscopy, the antibodies were shown to attach to native PvDBP on the apical end of the P. vivax merozoite. In vitro invasion assays, using blood isolates from individuals in the Mae Sot district of Thailand, showed that addition of rabbit anti-PvDBPII Ab or serum (antibodies against, or serum containing antibodies against, region two of the Plasmodium vivax Duffy binding protein) (1:100) reduced the number of parasite invasions by up to 64%, while pooled PvDBPII antisera from P. vivax–exposed people reduced P. vivax invasion by up to 54%.

Conclusions

These results show, for what we believe to be the first time, that both rabbit and human antibodies directed against PvDBPII reduce invasion efficiency of wild P. vivax isolated from infected patients, and suggest that a PvDBP-based vaccine may reduce human blood-stage P. vivax infection.     

Characterization and detection of naturally occurring antibodies against IL-1 alpha and IL-1 beta in normal human plasma.

During the development and testing of a radioreceptor assay (RRA) for human IL-1, we have detected and identified the presence of auto-antibodies to IL-1 in normal human plasma (NHP). The RRA is based on the competition between human 125I-labeled rIL-1 alpha and standard or unknown quantities of IL-1 alpha or IL-1 beta for binding to a limited amounts of IL-1 receptor (IL-1R) isolated from the EL4 mouse thymoma cell line. NHP from 20 out of 100 unselected blood donors were found to completely inhibit the binding of 125I-labeled IL-1 alpha to its receptor, suggesting the presence in these NHP samples of either abnormal amounts of IL-1 or of a factor binding to the 125I-labeled IL-1 alpha. Special care was taken to ascertain that the inhibitory factors were antibodies and not soluble IL-1 receptor antagonist. When plasma samples with inhibiting activity were incubated with labeled IL-1 alpha and chromatographed on a Sephadex G200 column, they were found to contain 125I-labeled complexes with an apparent molecular weight of 150-200kD. The IL-1 binding factor could be eliminated from plasma by incubation with protein A-Sepharose, suggesting that it consisted in IgG antibodies directed against IL-1. Furthermore, the antibody nature of the inhibiting factor was confirmed by its binding to purified rIL-1 coupled to Sepharose. Screening of 200 NHP samples by incubation with 100 pg of 125I-labeled IL-1 followed by precipitation with 12% of polyethylene glycol (PEG) confirmed that about 25% of NHP contain detectable IgG antibodies to IL-1 alpha, while only 2% of NHP contain antibodies to IL-1 beta. No correlation between the presence of these anti-IL-1 antibodies and any particular major histocompatibility complex or any pathological conditions was detected. We suggest that all serum samples assayed for IL-1 alpha or IL-1 beta content should be pretested with the PEG precipitation assay described here.     

7-Dehydrobrefeldin A, a naturally occurring brefeldin A derivative, inhibits secretion and causes a cis-to-trans breakdown of Golgi stacks in plant cells.

7-Dehydrobrefeldin A (7-oxo-BFA) is a brefeldin A (BFA) analog that, like BFA, is a potent phytotoxin of Alternaria carthami, a fungal pathogen of safflower (Carthamus tinctorius L.) plants. Both BFA and 7-oxo-BFA have been shown to be causal agents of the leaf spot disease of these plants. We have investigated the effects of 7-oxo-BFA on the secretion and the structure of the Golgi stacks of sycamore maple (Acer pseudoplatanus) suspension-cultured cells to determine whether 7-oxo-BFA affects these cells in the same manner as BFA. When applied at 10 micrograms/mL for 1 h, 7-oxo-BFA inhibits secretion of proteins by approximately 80%, the same value obtained for BFA. However, electron micrographs of high-pressure frozen/freeze-substituted cells demonstrated that 7-oxo-BFA is a more potent disrupter of the Golgi stacks of sycamore maple cells than BFA. In cells treated for 1 h with 10 micrograms/mL 7-oxo-BFA, very few Golgi stacks can be discerned. Most of those that are left consist of fewer than three cisternae, all of which stain like trans-Golgi cisternae. They are surrounded by clusters of large (150-300 nm in diameter), darkly staining vesicles that are embedded in a fine-filamentous, ribosome-excluding matrix. Similarly sized and stained vesicles are seen budding from the rims of the residual trans-Golgi cisternae. Both the large vesicles and the residual Golgi stack buds stain with anti-xyloglucan polysaccharide antibodies. Recovery of Golgi stacks after removal of 7-oxo-BFA from 1-h-treated cells takes 2 to 6 h, compared with 1 to 2 h for cells treated with BFA. In contrast to 7-oxo-BFA, the BFA breakdown product BFA acid had no effect either on secretion or on the secretory apparatus. This is the first report, to our knowledge of a BFA analog inhibiting secretion in a eukaryotic cell system.     

Expression of a cross-reactive idiotope on naturally occurring murine antibodies against 3-fucosyllactosamine, levan, and dextran

We recently identified a cross-reactive Id (6C4) that is expressed on the H chain of many BALB/c mAb against the 3-fucosyllactosamine (3-FL) determinant, Gal(beta 1-4) (Fuc(alpha 1-3] GlcNAc-R. The VH segments of seven mAb that we recently sequenced are encoded by VH441, which also encodes VH segments of antibodies against galactan, levan, and dextran. To analyze the expression of the 6C4 Id on naturally occurring anti-carbohydrate antibodies, we isolated 6C4+ antibodies by affinity chromatography from pools of normal BALB/c serum. Approximately 20 to 30% of antibodies against 3-FL and levan, and all antibodies against dextran, were removed from the sera by passage over a column containing mAb 6C4. Absorption of the eluate with 3-FL beads removed anti-3-FL antibodies but not anti-dextran or anti-levan. The expression of a cross-reactive Id on naturally occurring antibodies against several carbohydrate Ag suggests that these antibodies may participate in an Id network. We also reported previously that BALB/c mice have naturally occurring anti-3-FL antibodies and respond well to immunization against this determinant, whereas C57BL/6 mice do neither. To examine the role of the Igh-C allotype in the regulation of the anti-3-FL response, we studied congenic strains of BALB/c and C57BL/6 mice. Both congenic strains produced anti-3-FL antibodies in response to immunization, but only C.B-20 mice exhibited naturally occurring antibodies. These data suggest that the naturally occurring and elicited antibody responses against 3-FL are differentially regulated.     

Comparison of the suppressive effects of antisense oligonucleotides and siRNAs directed against the same targets in mammalian cells

RNA interference appears to be a potentially powerful tool for studies of genes of unknown function. However, differences in efficacy at different target sites remain problematic when small interfering RNA (siRNA) is used as an effector. Similar problems are associated with attempts at gene inactivation using antisense oligonucleotides (ODNs) and ribozymes. We performed a comparative analysis of the suppressive effects of three knockdown methods, namely, methods based on RNA interference (RNAi), antisense ODNs, and ribozymes, using a luciferase reporter system. Dose-response experiments revealed that the IC50 value for the siRNA was about 100-fold lower than that of the antisense ODN. Our results provide useful information about the positional effects in RNAi, which might help to improve the design of effective siRNAs.     

Effect of mouse antiserum against isologous aggregated immunoglobulins on the accumulation of rosette- and antibody-forming cells in mice immunized with ram erythrocytes

The paper describes the effect of mouse antiserum against isologous aggregated immunoglobulins (termed MAAS) on the kinetics of rosette-forming and antibody-forming cells (RFC and AFC, respectively) in mice immunized with SRBC. MAAS effect was assessed in vivo by injecting this serum for 5 days to mice CBA, combining the first injection with the injection of 5.10(7) SRBC. MAAS administration to mice immunized with SRBC induced a marked reduction of RFC in the spleen on the 5th and 9th days after the immunization. At the same periods MAAS produced no significant effect on the proliferation of AFC producing IgM-hemagglutinins. At the same time MAAS intensified the IgG-AFC proliferation in the period of the maximal content of these cells in the spleen of the immunized mice. After the MAAS absorption with the immune complexes formed by the mouse IgG-antibodies this serum largely lost its capacity to block RFC in vivo. On the basis of the data obtained it is suggested that the property of MAAS to influence the accumulation of RFC and AFC producing IgG-hemagglutinins is caused by the factor reacting with the immune complex formed by mouse IgG-antibodies. Possibly this factor represented antibodies against the aggregated immunoglobulins of this class.     

Early regenerative responses induced by monoclonal antibodies directed against a surface glycoprotein of goldfish retinal ganglion cells.

Monoclonal antibodies directed primarily against antigenic determinants associated with the goldfish optic nerve were prepared and characterized. One selected clone 23-4-C(IgG2a), detected antigenic determinants of glycoprotein nature with an apparent mol. wt. of 140 000. Following injury the level of these molecules increased with a peak at 5-7 days after the lesion (2- to 3-fold higher than the basal level). The results strongly suggest that the increase derives, at least partially, from a real increment in the level of these molecules in the retinal ganglion cells rather than from changes in accessibility. Immunofluorescence studies indicate that the retinal ganglion cells carry the antigenicity. Intraocular injection of the monoclonal antibodies, concomitantly with crush injury, resulted in an earlier and higher regenerative response, reflected by sprouting capacity, protein synthesis and accumulation of radiolabeled material in the tectum contralateral to the side of injury. This may indicate that the antibodies directly activate retinal cells via interaction with surface molecules. Alternatively, the antibodies may be directed against surface molecules which are associated with axonal growth inhibitors, and may therefore mask these surface antigens from further interaction with their native substrate.     

Monoclonal antibodies directed against protoplasts of soybean cells: analysis of the lateral mobility of plasma membrane-bound antibody MVS- 1

A monoclonal antibody (MVS-1) was used to monitor the lateral mobility of a defined component (Mr approximately 400,000) of the plasma membrane of soybean protoplasts prepared from suspension cultures of Glycine max (SB-1 cell line). The diffusion coefficient (D) of antibody MVS-1 bound to its target was determined (D = 3.2 X 10(-10) cm2/s) by fluorescence redistribution after photobleaching. Pretreatment of the protoplasts with soybean agglutinin (SBA) resulted in a 10-fold reduction of the lateral mobility of antibody MVS-1 (D = 4.1 X 10(-11) cm2/s). This lectin-induced modulation could be partially reversed by prior treatment of the protoplasts with either colchicine or cytochalasin B. When used together, these drugs completely reversed the modulation effect induced by SBA. These results have refined our previous analysis of the effect of SBA on receptor mobility to the level of a defined receptor and suggest that the binding of SBA to the plasma membrane results in alterations in the plasma membrane such that the lateral diffusion of other receptors is restricted. These effects are most likely mediated by the cytoskeletal components of the plant cell.     

Monoclonal antibodies against human myelomonocytic cells: detection of certain lineage-specific antigens on CFU-GM progenitor cells

A series of monoclonal antibodies (mAb) were raised against nonlymphoid leukemic cell lines. Three of them have been characterized in detail. mAb H8 (IgG2), mAB U2 (IgG1), and mAb ML143 (IgM) were established with HEL, an erythroleukemia cell line, U937, a monocytoid (histiocytic) line, and ML-1, a myeloid cell line as immunogen, respectively. A 65 to 75 KD polypeptide was precipitated from monocytes by mAb H8, a 160 KD protein from monocytes by mAb U2, and two broad bands in the regions of 150 and 195 KD from granulocytes by mAb ML143. All three mAb stained peripheral blood monocytes and granulocytes, but not lymphocytes, platelets, and erythrocytes. The mAb reacted with immature myeloid cells in bone marrow, ranging from myeloblasts to mature myelomonocytic cells. They also were reactive with various nonlymphoid cell lines and leukemia of myelomonocytic origin. They did not react with B cell lines and B cell CLL cells. By complement-mediated cytolysis and/or an immune rosette method, antigens H8 and U2 were found to be expressed on the vast majority of CFU-GM (14 days) progenitors but not on BFU-E. Antigen ML143 was not expressed by either progenitor. Furthermore, ML143 antigen was found on T leukemia cell lines, a subpopulation of mitogen-activated T cells, and certain non-T/non-B ALL cells. This reactivity was not found with mAb H8 and U2. The relationship between these mAb and those reported are discussed. The possibility of using these mAb to obtain a markedly enriched CFU-GM progenitor population is also raised.     

Isolation from human erythrocytes of a new membrane protein which inhibits the formation of complement transmembrane channels

A protein which inhibited complement channel formation was isolated from extracts of papain-digested human erythrocyte membranes using DEAE-Sephacel, Bio-Gel A0.5m column chromatographies, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose paper and elution with 2% NP-40 solution. The purified protein showed a molecular weight of 18 kDa, and efficiently inhibited hemolysis of EC5-7 cells with C8 and C9, but did not show any decay-accelerating activity to C5 convertase. Immunochemical analysis of native membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the antibody against this protein gave a single band having the same mobility as this protein; papain did not eliminate a significant portion of this protein.     

Monoclonal antibodies directed against extracellular matrix proteins reduce the adherence of Candida albicans to HEp-2 cells

The presence of the extracellular matrix (ECM) proteins collagen types I and IV, laminin and fibronectin on the surface of HEp-2 cells was confirmed by flow cytometry using monoclonal antibodies. Monoclonal antibodies directed against these ECM proteins reduced the adherence of C. albicans ATCC 44990 to HEp-2 cells, the greatest reductions being evident in assays which incorporated anti-collagen type IV monoclonal antibody. The ability of sugaramines to inhibit the adherence of C. albicans to a variety of cell types has been demonstrated previously and the most significant reduction in C. albicans – HEp-2 adherence was in assays which incorporated 0.2M galactosamine. The combination of anti-collagen IV monoclonal antibody and galactosamine reduced the adherence of C. albicans to HEp-2 cells by approximately 70% (p < 0.05). This revised version was published online in June 2006 with corrections to the Cover Date.     

Exofacial regions of the glucose transporter of human erythrocytes: detection with polyclonal antibodies

The glucose transporter of human erythrocytes is a glycoprotein of 492 amino acids with a Mr of 55,000. From hydrophobicity plots based on the transporter's amino acid sequence, it has been proposed that exofacially, there are only a segment of 34 residues and the glycosylating carbohydrate branch. To detect changes in the number of glucose transporters during metabolic regulation in intact cells, one should obtain antibodies directed to exofacial sites of the transporter. Antibodies to the purified glucose transporter (Band 4.5), intact or deglycosylated with endoglycosidase F, were raised in rabbits. These antibodies, when purified by column chromatography on protein A-Sepharose and by adsorption onto erythrocyte membranes, cross-reacted with the glycosylated glucose transporter on Western blots. The reactivity of the polyclonal antibodies with intact cells was tested by incubating these cells with the antibody, followed by a centrifugation and a subsequent reaction with 125I-labelled goat-antirabbit immunoglobulin G. Intact human erythrocytes reacted positively with the anti-Band 4.5 antibodies but not with nonimmune sera. Reaction with human erythrocytes was about 10 times greater than with pig erythrocytes, which lack glucose transporters. The reaction with intact cells was not due to contamination with broken cells since under the conditions used, broken (freeze-thawed) cells or membranes did not sediment. Reaction with human erythrocyte membranes was more than fivefold higher than with pig erythrocyte membranes. Rat L6 muscle cells reacted with anti-Band 4.5 antibodies; there were about 10 times more binding sites in any one cell in L6 cells than in human erythrocytes, roughly paralleling their relative content of glucose transporters.(ABSTRACT TRUNCATED AT 250 WORDS)