Characterisation of renal antigens on distinct parts of the human nephron by monoclonal antibodies

Summary Ten monoclonal antibodies (TN 1-TN 10) directed against different renal antigens of distinct sites of the human nephron were derived from a fusion between P3-NS1/1-Ag4-1 mouse myeloma and spleen cells of a mouse hyperimmunized against isolated human kidney cells. Two of these reagents (TN 1, TN 10) were shown by immunoperoxidase labelling on frozen sections of five normal kidneys and of other selected human organs, as well as by immunofluorescence studies on normal peripheral blood cells and selected lymphohematopoietic cell lines, to detect antigens exclusively expressed on visceral glomerular or proximal tubular epithelial cells. The other eight antibodies were found to react with different determinants shared between renal structures, muscle cells, different epithelia, B-lymphocytes and granulocytes. In heterogeneous cultures of isolated kidney cells these monoclonal reagents could be used to identify distinct cell types of tubular origin. Thus such hybridoma-derived antibodies provide new tools to correlate structural characteristics of various renal epithelial cells with their functional properties and will contribute to the study of their influence on immunologically mediated kidney injuries in different forms of glomerulonephritis in man.This study was supported by two grants from the Deutsche Forschungsgemeinschaft, DFG, Mu 523/3-1 and SFB 120, Leukämieforschung und Immungenetik, Project A2 and A3     

Metabolism of glutamine in erythrocytes infected with the human malaria parasite: Plasmodium falciparum

The metabolism of glutamine was studied in erythrocytes infected with Plasmodium falciparum, comparatively to normal cells, in presence or not of DON (6-diazo-5-oxo-L-norleucine) or acivicin, two glutamine antagonists which have been shown to inhibit the growth of P. falciparum in vitro. Extracellular glutamine was partially converted into glutamate using two routes corresponding to gamma-glutamyl transpeptidase (GGT) and glutaminase activities. In cells infected with mature trophozoites, the observed enhancement of the glutamine influx and of the glutamate formation was consistent with the enhancement of GGT and glutaminase activities.     

Water and urea transport in human erythrocytes infected with the malaria parasite Plasmodium falciparum

The permeability properties of the human red cell membrane to various solutes are altered by malarial infection. In the present work we show that the permeability of the red cell membrane to water is also affected by the intraerythrocytic growth of the malaria parasite Plasmodium falciparum, whereas urea permeability appears unchanged. The data from infected cells show decreases in membrane surface area, cell volume, the osmotically active water fraction (W_eff), and osmotic water permeability (P_f) as measured by stopped-flow spectroscopy. On the other hand, the data suggest an increase in diffusive water permeability (P_d) in infected cells with no change in urea permeability when measured by the continuous flow method. The decreased P_f/P_d ratio of infected cell membranes and its implications in the geometry of the red cell membrane water channel or pore are discussed.     

Human Trophoblast-Specific Surface Antigens Identified Using Monoclonal Antibodies*

ABSTRACT: Mouse monoclonal antibodies have been produced against syncytiotrophoblast plasma membrane preparations isolated from term human placentas. Of 20 positive clones, two antibodies (H309, H318) were directed against the C_γ2 domain of IgG, one (H312) was directed against albumin, and another (H303) was directed against a further normal human serum component (not transferrin). One monoclonal antibody (H310) recognized an antigenic epitope restricted to trophoblast and lymphocytes: this antibody did not inhibit mitogenic or allogeneic stimulation of lymphocytes. Two monoclonal antibodies (H315 and H317) reacted with trophoblast-specific antigenic determinants. The H315 antigen was present on first trimester syncytiotrophoblast, unlike the H317 antigen.     

An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies

We sought to develop and optimize a hybridoma-based technology for generating human hybridomas that secrete virus-specific monoclonal antibodies for clinical diagnosis and therapy. We developed a novel electrofusion protocol for efficiently fusing Epstein-Barr virus (EBV)-transformed human B cells with myeloma partners. We tested seven myeloma cell lines and achieved highest efficiency when the HMMA 2.5 line was used. We optimized the electrofusion process by improving cell treatments before and after electrofusion as well as varying cell ratios, fusion medium and other experimental parameters. Our fusion efficiency increased remarkably to 0.43%, a significant improvement over the efficiency of previous PEG-based or other electrofusion methods. Using the optimized protocol, we obtained human hybridomas that secrete fully human monoclonal antibodies against two major human respiratory pathogens: respiratory syncytial virus (RSV) and an influenza H3N2 vaccine virus strain. In conclusion, we have developed an efficient and routine approach for the generation of human hybridomas secreting functional human virus-specific monoclonal antibodies.     

Biosynthesis of glycosphingolipids de-novo by the human malaria parasite Plasmodium falciparum

Glycolipids are important components of cellular membranes involved in various biological functions. In this report we describe the identification of the de-novo synthesis of glycosphingolipids by intraerythrocytic, asexual stages of the malaria parasite, Plasmodium falciparum. Parasite-specific glycolipids were identified in organic solvent extracts of parasites metabolically labeled with tritiated serine and glucosamine and characterised as sphingolipids based on their insensitivity towards alkaline treatment. While the de-novo synthesis of parasite glycosphingolipids was affected by fumonisin B1, threo-PPMP, cyclo-serine and myriocin, these well established inhibitors of de-novo ceramide biosynthesis were unable to arrest the intraerythrocytic development of the parasites in culture.     

抗人载脂蛋白E单克隆抗体制备及免疫学特性的研究

应用人血清纯化的ApoE免疫BALB/C小鼠,其脾细胞与NS-1骨髓瘤细胞融合,获得3株分泌抗人ApoE单克隆抗体的杂交瘤细胞株(E6C3,E4C2和E3C3),对其免疫学特性进行了研究。腹水效价为8×10~(-5)～2×10~(-6),与其它载脂蛋白没有交叉反应,制备免疫亲和层析柱纯化了ApoE,单抗相加试验和双抗体夹心试验证明,两株MeAb识别ApoE的两个不同抗原决定簇,抗体亚型均为IgG_1型。应用ELISA双抗体夹心法测定了人血清ApoE含量。     

Polyreactivity of human monoclonal antibodies: Human IgM anti-rhesus monoclonal antibodies are frequently polyreactive

The aim of this study was to characterize human anti-Rhesus monoclonal antibodies cross-reacting with tissue antigens. Of the 155 monoclonal alloantibodies tested, 49 also reacted with intracellular antigens, as demonstrated by immunofluorescence assay on cryostat sections of animal and human tissues. This cross-reactivity was mainly a property of monoclonal alloantibodies belonging to the IgM isotype (among the 49 cross-reacting Mabs, 37 were IgM). The results confirm that during an immune response against a foreign antigen (alloantigen), B cells that produce polyreactive antibodies are not excluded from the pool of responding cells.     

Circumsporozoite protein of the human malaria parasite Plasmodium ovale identified with monoclonal antibodies.

Monoclonal antibodies (MAbs) have been produced against Plasmodium ovale sporozoites and used to characterize the circumsporozoite (CS) protein. Six MAbs were produced, and all were species specific. By using Western blot (immunoblot) analysis, three polypeptides were detected: a predominant 51,000-Mr polypeptide and two presumed precursor 57,000- and 67,000-Mr molecules. The presence of a repeating epitope in the CS protein of P. ovale was demonstrated by using one of the MAbs in a single-antibody two-site enzyme immunoassay. Three MAbs recognized epitopes on the surfaces of sporozoites; the presence of at least one other epitope within the CS protein, but not on the surfaces of P. ovale sporozoites, was also demonstrated.     

Activation of human complement by mouse and mouse/human chimeric monoclonal antibodies

The complement (C)-activating capabilities in human serum of 32 mouse and 10 mouse/human chimeric MoAbs of different isotypes, and their fragments, were tested in vitro. Activation of C via the classical pathway (CP) was performed in 1% factor D-deficient serum in gelatin containing Veronal buffer in the presence of calcium and magnesium (GVB⁺⁺), while activation of the alternative pathway of C (AP) was assessed in 10% Clq-depleted serum in the presence of 5 mm MgCl₂ in GVB⁺⁺. The C-activating ability of MoAbs was expressed relative to the degree of activation of complement by aggregated IgG for the CP and relative to mouse IgG 1 for the AP. All of seven mouse IgG2a MoAbs were potent activators of the CP. The results of CP activation by IgG1, IgG2b and IgG3 isotypes were different for individual MoAbs. Only three (two IgG 1 and one IgG3) of 32 mouse MoAbs were potent activators of the AP. IgG2a and IgG2b were relatively poor AP activators. There were a few MoAbs which activated both the AP and CP. Of 10 chimeric MoAbs, two IgG1, one IgG2 and one IgG4 were poor or non-activators of the CP. On the other hand, IgG2 and IgG4 were good AP activators. IgG3 was the most potent AP activator. Most of the F(ab')₂ fragments were activators of the AP and displayed no activation of the CP. Fc fragments only activated the CP. whereas Fab'did not activate the CP or the AP. These studies suggest that the route of complement activation by class and subclass MoAbs can not always be predicted in advance and based only on their subclass identity.     

Enzymes of purine and pyrimidine metabolism from the human malaria parasite, Plasmodium falciparum

Plasmodium falciparum trophozoites were isolated by mechanical rupture of infected human erythrocytes followed by a series of differential centrifugation steps. After lysis with sonication, the 100 000 x g supernatant of parasites and uninfected host cells was used to determine the specific activities of a number of enzymes involved in purine and pyrimidine metabolism. P. falciparum possessed the purine salvage enzymes: adenosine deaminase, purine nucleoside phosphorylase, hypoxanthine-guanine phosphoribosyltransferase (PRTase), xanthine PRTase, adenine PRTase, adenosine kinase. The last two enzymes, however, were present at much lower activity levels. Hypoxanthine was converted (presumably via IMP) into adenine and guanine nucleotides only in the presence both of supernatant and membrane fractions of P. falciparum. Two enzymes involved in the de novo synthesis of pyrimidines, orotic acid PRTase, and orotidine 5'-phosphate decarboxylase, were present in parasite extracts as were the enzymes for pyrimidine nucleotide phosphorylation: UMP-CMP kinase, dTMP kinase, nucleoside diphosphate kinase. Xanthine oxidase, CTP synthetase, cytidine deaminase and several kinases for the salvage of pyrimidine nucleosides were not detected in the parasites. Both phosphoribosyl pyrophosphate synthetase and uracil PRTase were present but at low activity levels. Human erythrocytes displayed similar but not identical enzyme patterns. Enzyme specific activities, however, were generally much lower than those of the corresponding parasite enzymes.     

Expression of the antigen detected by the monoclonal antibody Ca 19.9 in human breast tissues

Summary The incidence and significance of the expression of the antigen defined by the monoclonal antibody Ca 19.9 (Sialyl Le^a) has been assessed in human breast tissue. Frozen and formalin-fixed, paraffin embedded specimens of normal, hyperplastic, pregnant breast and carcinomas were examined using an immunoperoxidase technique.Ductal and acinar epithelium of normal and hyperplastic tissues showed variable reactivity in frozen sections but there was a reduction in staining in comparable samples after fixation and processing, such that in many instances only focal ductal epithelium reacted. A distinctive feature in the pregnant breast was the absence of staining in acini showing differentiated secretory activity, despite a reaction in adjacent non-secretory acini and ducts.The overall incidence of detection of the Ca 19.9 antigen in breast carcinomas was 62%, but in half of these only a small number of cells stained. A significant relationship between expression of Sialyl Le^a and poor differentiation of carcinomas was identified, but there was no correlation with local lymph node status. In contrast to the non-malignant tissue fixation and processing had little effect on the reactivity of carcinomas. It is suggested that this difference may be quantitative in nature, with malignant breast showing much greater expression, or be related to organisation of the antigen.The observations concerning carcinomas and pregnant breast indicate that the synthesis of the Ca 19.9 antigen is related to the state of differentiation and functional activity of human breast.     

Highly potent,naturally acquired human monoclonal antibodies against Pfs48/45 block Plasmodium falciparum transmission to mosquitoes

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Potent transmission-blocking monoclonal antibodies from naturally exposed individuals target a conserved epitope on Plasmodium falciparum Pfs230

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Analysis of antibodies directed against merozoite surface protein 1 of the human malaria parasite Plasmodium falciparum

The 190-kDa merozoite surface protein 1 (MSP-1) of Plasmodium falciparum, an essential component in the parasite's life cycle, is a primary candidate for a malaria vaccine. Rabbit antibodies elicited by the heterologously produced MSP-1 processing products p83, p30, p38, and p42, derived from strain 3D7, were analyzed for the potential to inhibit in vitro erythrocyte invasion by the parasite and parasite growth. Our data show that (i) epitopes recognized by antibodies, which inhibit parasite replication, are distributed throughout the entire MSP-1 molecule; (ii) when combined, antibodies specific for different regions of MSP-1 inhibit in a strictly additive manner; (iii) anti-MSP-1 antibodies interfere with erythrocyte invasion as well as with the intraerythrocytic growth of the parasite; and (iv) antibodies raised against MSP-1 of strain 3D7 strongly cross-inhibit replication of the heterologous strain FCB-1. Accordingly, anti-MSP-1 antibodies appear to be capable of interfering with parasite multiplication at more than one level. Since the overall immunogenicity profile of MSP-1 in rabbits closely resembles that found in sera of Aotus monkeys immunized with parasite-derived MSP-1 and of humans semi-immune to malaria from whom highly inhibiting antigen-specific antibodies were recovered, we consider the findings reported here to be relevant for the development of MSP-1-based vaccines against malaria.     

Characterization of murine monoclonal antibodies against a repetitive synthetic peptide from the circumsporozoite protein of the human malaria parasite, Plasmodium falciparum

Monoclonal antibodies (mAbs) were raised in mice against the synthetic peptide (NANP)40, consisting of 40 (NANP) repeats of the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium falciparum, and characterized. (i) Five of these mAbs recognized the P. falciparum CS protein in western blot experiments and in immunofluorescence assays using different preparations of sporozoites. The remaining two mAbs (CT3.2 and CT3.3, both IgG1) gave negative results by both techniques. (ii) When the anti-(NANP)40 peptide mAbs were functionally tested in vitro to assess their ability to inhibit the attachment and penetration of the parasites into cultured human liver cells, six of them exhibited inhibitory activities ranging between 66 and 90%. CT3.2 mAbs, also, inhibited sporozoite attachment and penetration, despite the negative results by immunofluorescence and western blot experiments. However, when immunofluorescence was repeated in the presence of calcium, CT3.2 did reveal a positive recognition of P. falciparum sporozoites, suggesting that this mAb could recognize the (NANP) sequence when calcium was bound to the repetitive peptide. (iii) Furthermore, the binding of an anti-(NANP)40 IgM mAb (CT1) to the solid-phase peptide was not inhibited by preincubation of the peptide with a mAb against the P. falciparum CS protein. (iv) Finally, one anti-(NANP)40 IgG1 mAb (CT3.1) was unable to bind to the shorter (NANP)3 peptide, although it recognized the (NANP)40 peptide and the P. falciparum CS protein. The results presented here suggest that heterogeneous antibody populations are produced upon immunization of mice with (NANP)40 synthetic peptide and that epitopes different from those simply related to the linear (NANP) amino acid sequence are likely to be present in long (NANP)n constructs as well as in the repetitive domain of the P. falciparum CS protein.     

恶性疟原虫红内期保护性抗原的分析

本文用7株单克隆抗体(McAb),疟疾流行区感染病人免疫血清和BALB/c小鼠免疫血清对用~(35)S—蛋氨酸和~(125)Ⅰ—表面标记的恶性疟原虫无性红内期保护性抗原进行了分析。实验结果表明,抑制性McAbs和不同来源的多价免疫血清巳鉴定出10余种功能性靶抗原。两种不同来源的多价免疫血清不但能识别由这7株McAbs所识别的靶抗原而且还能识别140和100KDa以及某些较小分子量的多肽。同时还用抗原竞争试验证实了免疫沉淀靶抗原的特异性。