Evaluation of IgM,IgG, IgA,IgD, and IgE secretion by human peripheral blood lymphocytes in cultures stimulated with pokeweed mitogen and Staphylococcus aureus Cowan I

Sheep erythrocytes coated with staphylococcal protein A were used as target cells in a reverse hemolytic plaque assay. Monospecific antisera to human immunoglobulins G, M, A, D, and E were used to detect each of these classes in cultures of human peripheral blood mononuclear cells stimulated with the polyclonal B-cell activators pokeweed mitogen and Staphylococcus aureus (Cowan I). Both mitogens induced substantial increases in the numbers of cells actively secreting immunoglobulins; in time-course experiments, the peak response was seen on Day 5. The numbers of cell secreting IgM, IgE, and IgD were usually higher in cultures stimulated with S. aureus than with pokeweed mitogen.     

Serum immunoglobulins in patients with carcinoma of the oral cavity,uterine cervix and breast

Summary Serum immunoglobulin levels (IgG, IgA, IgM, IgD and IgE) were estimated in 196 patients with carcinoma of the oral cavity, 172 patients with cervical cancer and 166 patients with breast cancer. The values were compared with those of 50 patients with benign lesions of the breast and cervix and 100 healthy adult controls. Only the serum IgE levels were found to be elevated in the benign group. Serum IgA, IgD and IgE levels were found to be elevated in all the three types of cancers and the levels were found to increase with clinical stage. In carcinoma of uterine cervix IgG levels were also found to be elevated. Immunoglobulins A and D returned to normal after clinical cure whereas IgE remained slightly elevated. IgD and IgE remained high in patients who had residual cancer.     

In vitro and in vivo B lymphocyte-activating properties of monoclonal anti-delta antibodies. I. Determinants of B lymphocyte-activating properties

To appreciate better the mechanisms by which B lymphocytes are activated by anti-Ig antibodies, we characterized seven monoclonal mouse allo-antibodies to IgD of the a allotype for their isotypes, fine specificities, IgD-cross-linking abilities, avidities, and abilities to activate B cells in vitro and in vivo. Three of the monoclonal antibodies tested bound to the Fc fragment of IgD with relatively high avidity and were effective at cross-linking IgD, since they precipitated soluble IgD and rapidly capped B cell membrane IgD. These were the only antibodies tested that induced B cell DNA synthesis in vitro and were the most effective antibodies at inducing in vivo increases in B cell size and DNA synthesis and in vitro and in vivo increases in B cell surface Ia expression. Two antibodies bound to the Fd fragment of IgD with relatively high avidity but could not rapidly cap cell membrane IgD or precipitate soluble IgD even in the presence of 2% polyethylene glycol. These high-avidity, poorly cross-linking antibodies were unable to stimulate B cell DNA synthesis in vitro and were much less effective than the first group of anti-delta antibodies at stimulating in vivo increases in B cell DNA synthesis, size, or surface Ia expression or in vitro increases in surface Ia expression. One antibody, which bound to the Fc fragment of IgD with an intermediate avidity, was unable to rapidly cap B cell membrane IgD or precipitate soluble IgD in saline, but could precipitate soluble IgD in the presence of 2% polyethylene glycol. This antibody failed to induce B cell DNA synthesis in vitro and was as effective as the higher-avidity, poorly cross-linking antibodies at stimulating increases in B cell size, surface Ia expression, and DNA synthesis in vivo, and surface Ia expression in vitro. One antibody, which bound to the Fd fragment of IgD with low avidity and was unable to precipitate soluble IgD or to cap cell membrane IgD, had little ability to activate B cells by any of the parameters studied. Each of the monoclonal anti-delta antibodies, regardless of isotype or fine specificity, when bound to agarose to increase its ability to cross-link IgD, was mitogenic for B cells in vitro. None of the monoclonal antibodies to IgD of the a allotype stimulated B cells from b allotype mice to increase their size, surface Ia expression, or synthesis of DNA in vitro or in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

The determination of allergen-specific IgE and IgG antibodies in patients sensitized to the house dust mite Dermatophagoides pteronyssinus

45 patients, hypersensitive to house-dust mites, were examined by the method of skin tests to D. pteronyssinus allergen. Besides, in their blood sera the levels of allergen-specific IgE antibodies were determined in the radioallergosorbent test and allergen-specific IgG antibodies, in the enzyme immunoassay. These tests revealed that in 91% of the patients the results of skin tests were positive, in 68% an elevated level of specific IgE antibodies and in 93% of the patients an elevated level of specific IgG antibodies were detected. All patients showed the positive result in one of the above-mentioned tests. The largest group of the patients (55%) included persons showing the positive result of the skin test and having elevated levels of allergen-specific IgG and IgE antibodies. Thus, in cases of hypersensitivity to house-dust mites the levels of allergen-specific IgG and IgE antibodies in the patients' blood sera should be determined.     

The glycosylation of human serum IgD and IgE and the accessibility of identified oligomannose structures for interaction with mannan-binding lectin

Analysis of the glycosylation of human serum IgD and IgE indicated that oligomannose structures are present on both Igs. The relative proportion of the oligomannose glycans is consistent with the occupation of one N-linked site on each heavy chain. We evaluated the accessibility of the oligomannose glycans on serum IgD and IgE to mannan-binding lectin (MBL). MBL is a member of the collectin family of proteins, which binds to oligomannose sugars. It has already been established that MBL binds to other members of the Ig family, such as agalactosylated glycoforms of IgG and polymeric IgA. Despite the presence of potential ligands, MBL does not bind to immobilized IgD and IgE. Molecular modeling of glycosylated human IgD Fc suggests that the oligomannose glycans located at Asn(354) are inaccessible because the complex glycans at Asn(445) block access to the site. On IgE, the additional C(H)2 hinge domain blocks access to the oligomannose glycans at Asn(394) on one H chain by adopting an asymmetrically bent conformation. IgE contains 8.3% Man(5)GlcNAc(2) glycans, which are the trimmed products of the Glc(3)Man(9)GlcNAc(2) oligomannose precursor. The presence of these structures suggests that the C(H)2 domain flips between two bent quaternary conformations so that the oligomannose glycans on each chain become accessible for limited trimming to Man(5)GlcNAc(2) during glycan biosynthesis. This is the first study of the glycosylation of human serum IgD and IgE from nonmyeloma proteins.     

Serum immunoglobulin levels in human hydatidosis

Serum IgG, IgM, IgA and IgD levels of 83 patients with hepatic or pulmonary hydatidosis and 15 postoperative individuals with no cysts were compared with the mean values of 80 healthy Lebanese controls. A significant increase of IgG was present in all patients. An increase in the mean IgM and IgA levels was significant only in pulmonary cases. There was no significant correlation between the titres of antibodies as detected by haemagglutination and complement-fixation tests and the respective serum IgG and IgM immunoglobin levels. Mean IgD levels were not different between patients and controls. Elevated IgE levels were detected in 77% of 21 hydatid patients. A persistent hyperglobulinemia was observed in postoperative follow-ups.     

The appearance of an IgD-like molecule on pig lymphocytes during ontogeny

An IgD-like molecule was found on the surface of pig fetal lymphocytes using guinea pig anti-human IgD antisera and indirect immunofluorescence. The binding of antiIgD antisera could be specifically inhibited with human IgD. Surface IgD-like molecules were found later in ontogeny than surface IgM (after about 70 d of gestation in the spleen). There is evidence for their endogenous origin. The number of lymphocytes bearing IgD-like molecules rapidly increased during ontogeny.     

草鱼IgM、IgD 和IgZ 的抗体制备与组织表达分析

根据已报道的草鱼免疫球蛋白IgM、IgZ 和IgD 的序列设计表达引物进行PCR 扩增, 将扩增片段克隆至表达载体pET-32a, 并在大肠杆菌Rosetta-gami (DE3)中进行诱导表达。利用亲和层析法纯化表达的重组蛋白, 然后免疫日本大耳白兔, 获得兔抗IgM、IgZ 和IgD 的抗血清。经免疫印迹检测, 表明IgM、IgZ 和IgD的表达产物能够被兔多克隆抗体特异性识别。应用兔抗草鱼IgM 和IgZ 的多克隆抗体, 对草鱼多种器官、组织提取的总蛋白进行免疫印迹检测, 在肠、头肾、中肾、皮肤、脾脏、脑、鳃和血液中都检测到IgM 和IgZ的表达。       

Cloning and expression of partial Japanese flounder (Paralichthys olivaceus) IgD

The cDNA sequence of the Japanese flounder (Paralychthys olivaceus) IgD has been previously reported (GenBank accession no. AB052658) and this was followed by the detection of IgD mRNA expression in some flounder organ tissues. However, it has not been determined whether the flounder IgD gene is virtually expressed into IgD protein. To characterize the flounder immunoglobulins utilized in elucidating the mechanism, evolution and diversity of the flounder immune system, antibodies specific to IgD and IgM were necessary. In the present study, partial flounder recombinant IgD (rIgD), IgM (rIgM) and the conserved regions of IgD and IgM (rCIg) were produced by cloning the cDNA sequence using isotype specific primers which were designed to produce unique fragments of IgD and IgM specific amino acid sequences. The production of recombinant Igs was ascertained by SDS-gel electrophoresis and immunoblot analysis using anti-T7 d Taq antibody. The produced recombinant Igs were purified using affinity columns, and used as immunogens. Antibodies specific to the isotype of flounder Igs were generated by immunizing rabbits with rfIgs and the antibodies produced were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Specificities of the generated antibodies were evaluated by testing cross-reactivity between recombinant IgM and IgD. By ELISA, rabbit antibodies against the rfIgD fragment (anti-rfIgD) failed to recognize any kind of flounder serum Igs, whereas respective antibodies against rfCIg (anti-rfCIg) and rfIgM fragments (anti-rfIgM) reacted with serum Igs. Likewise, in immunoblot assays, though anti-rfIgD did not, both anti-rfCIg and anti-rfIgM bound with the ~85 kd flounder IgM heavy chain. By flow cytometry analysis, anti-rfCIg, anti-rfIgD and anti-rfIgM reacted with 6%, 3% and 6.5% of cells, respectively, suggesting that flounder IgD is not secreted in serum but expressed on flounder B-like cell surfaces as in mammals. Antibodies produced against recombinant flounder Igs could be used to develop sandwich assay systems for detecting flounder Igs and for further investigating the flounder immune system.     

Lack of pokeweed mitogen-induced IgE formation in vitro by human peripheral blood mononuclear cells: detection of cross-reacting idiotypic determinants on polyclonal Ig by antibodies to a single IgE myeloma protein

The ability of human peripheral blood mononuclear cells to synthesize IgE in vitro in response to pokeweed mitogen (PWM) is controversial. To determine whether the conflicting results obtained by different laboratories could be due to inherent qualitative differences in the anti-IgE antibodies used to measure low concentrations of IgE in culture supernatants, we compared the specificities of anti-IgE reagents prepared by various methods. Immunoadsorbent-purified antibodies were isolated from a goat antiserum to the lambda, IgE myeloma protein PS and a rabbit antiserum to the kappa, IgE protein Bed in three ways: 1) antibodies to IgE PS (anti-PS) were isolated from the goat antiserum by affinity chromatography with PS coupled to Sepharose 4B; these antibodies consisted of anti-epsilon chain-specific and anti-idiotypic antibodies to protein PS; 2) antibodies specific for the epsilon-chain (anti-epsilon) were purified by affinity chromatography with IgE myeloma proteins that were not used for immunization; and 3) antibodies to idiotypic determinants of proteins PS (anti-id PS) and Bed (anti-id Bed) were isolated on affinity columns with the respective myeloma proteins after absorption of the epsilon-chain-specific antibodies. These three types of antibodies were then used in a solid phase radioimmunosorbent test to quantitate the amount of "IgE" synthesized by peripheral blood mononuclear cells from nonatopic and atopic donors cultured for 7 days in the presence and absence of PWM. ANTI-PS antibodies detected a PWM-induced "IgE formation" in cell culture supernatants of both non-atopic and atopic donors. (ABSTRACT TRUNCATED AT 250 WORDS)     

Serological and genetic identification of a bovine B lymphocyte alloantigen system

A two-colour fluorescence micro cytotoxicity test was used to screen antisera for antibodies specific for bovine B lymphocytes. A total of 114 cattle alloantisera were screened against peripheral blood lymphocytes from 100 unrelated individuals. Anti-B lymphocyte activity was detected in 47 antisera. Cytotoxic antibodies to antigens other than B lymphocyte specific antigens were removed by absorbing the antisera with buffy coat cells or platelets isolated from whole blood. Selected antisera were used to type paternal half-sib families. The presence of a polymorphic, MHS-linked antigen system on B lymphocytes was demonstrated. The tissue distribution and MHS linkage of these antigens suggests this system is analogous to the class II or Ia antigens of other species.     

Serological and genetic identification of a bovine B lymphocyte alloantigen system

A two-colour fluorescence microcytotoxicity test was used to screen antisera for antibodies specific for bovine B lymphocytes. A total of 114 cattle alloantisera were screened against peripheral blood lymphocytes from 100 unrelated individuals. Anti-B lymphocyte activity was detected in 47 antisera. Cytotoxic antibodies to antigens other than B lymphocyte specific antigens were removed by absorbing the antisera with buffy coat cells or platelets isolated from whole blood. Selected antisera were used to type paternal half-sib families. The presence of a polymorphic, MHS-linked antigen system on B lymphocytes was demonstrated. The tissue distribution and MHS linkage of these antigens suggests this system is analogous to the class II or Ia antigens of other species.     

Protein D of Haemophilus influenzae. A novel bacterial surface protein with affinity for human IgD

Protein D, a novel surface protein of the bacterial species Haemophilus influenzae with affinity for human IgD, was isolated after solubilization with sonication and Sarcosyl-extraction by a single SDS-PAGE step. From 1 ml of packed bacteria was prepared 0.25 mg of purified protein D. The apparent m.w. of protein D was estimated to 42,000 by SDS-PAGE and gel chromatography. Edman degradation cycles of protein D produced no amino acid phenylthiohydantoin derivatives and the amino-terminal end of the single protein D polypeptide chain is thus probably blocked. Protein D differs from all previously described outer membrane proteins (protein 1 to 6) of H. influenzae. Thus, protein D did not react with antibodies against protein 1 or protein 2 and the latter proteins did not bind IgD. Protein D was found to exhibit unique Ig-binding properties. Thus, in dot blots protein D bound four different human IgD myeloma proteins but not IgG, IgM, IgA, IgE, or some additional proteins. On the IgD molecule, constant parts of the H chains both in the Fab and Fc fragments appear responsible for the interaction with protein D. This novel Ig-binding reagent promises to be of theoretical and practical interest in immunologic and microbiologic research.     

Bovine lymphocyte antigens: serological relationships with erythrocyte and spermatozoan antigens

Bovine lymphocytotoxicity tests with 20 unabsorbed bovine blood group sera revealed extensive reactivity which in the majority of cases had no indication of blood group relationship. Six of these sera were absorbed with selected lymphocytes to produce eleven antisera of reduced specificity. Again, most of the sera had reaction patterns which could not be related to the hemolytic patterns of any known blood group antibodies. However, five comparisons involving unabsorbed antisera and two comparisons involving absorbed antisera provided statistical evidence of similarities between their lymphocytotoxic reaction patterns and the hemolytic reaction patterns of certain blood group antibodies. Several of the sera appeared to contain related cytotoxic specificities, and three such absorbed sera may have contained an anti-J specificity. All six examined monospecific isoimmune blood group antisera contained lymphocytic reactivities not related to their hemolytic specificites. Two normal sera containing naturally occurring anti-J had no cytotoxic activity. Anti-semen sera likewise were devoid of lymphocytotoxic activity.     

Extracellular release of rat eosinophil peroxidase (EPO) I. Role of anaphylactic immunoglobulins

The release of intracellular peroxidase (EPO) was investigated in order to evaluate rat eosinophil activation by various immunoglobulin (Ig) isotypes. After successive incubations with purified rat IgG1, IgG2a, IgG2b, IgG2c, IgE, or IgM and their respective anti-Ig antisera, eosinophils released significant amounts of EPO (up to 26% of the intracellular content) only in the case of Ig with anaphylactic activities (IgG2a and IgE). Other classes and subclasses were unable to induce EPO exocytosis. Selective depletion and reconstitution experiments suggested that mast cells were not required in this process. Similar levels of EPO could be released after interaction of eosinophils with antigen-antibody complexes (IgG2a monoclonal antibody and Schistosoma mansoni antigen) immobilized on nonphagocytosable surfaces. These results indicate that EPO exocytosis can be obtained after cell activation with specific antibodies, and that this mechanism is independent of phagocytosis. A kinetic study of eosinophils from S. mansoni-infected rats revealed that IgG2a and IgE cytophilic antibodies induced EPO release after incubation with either specific antisera or specific antigen, which suggests the in vivo relevance of such findings. The present work underlines the parallelism of interaction of anaphylactic-type Ig with eosinophils and with mast cells. Moreover, EPO release seems to represent an interesting marker of eosinophil activation, because close relationships were established between the present findings and previous work on the effector function of rat eosinophils.     

The immunoglobulin D-binding part of the outer membrane protein MID from Moraxella catarrhalis comprises 238 amino acids and a tetrameric structure

Moraxella catarrhalis IgD-binding protein (MID), a 200-kDa outer membrane protein comprising 2,139 amino acids, has recently been isolated and shown to display a unique and specific affinity for human IgD. To identify the IgD-binding region, MID was digested with proteases. In addition, a series of truncated fragments of MID were manufactured and expressed in Escherichia coli followed by analysis for IgD binding in Western and dot blots. The smallest fragment with essentially preserved IgD binding was comprised of 238 amino acid residues (MID(962-1200)). Shorter recombinant proteins gradually lost IgD-binding capacity, and the shortest IgD-binding fragment comprising 157 amino acids (MID(985-1142)) displayed a 1,000-fold reduced IgD binding compared with the full-length molecule. The truncated MID(962-1200) was efficiently attracted to a standard IgD serum and to purified myeloma IgD(kappa) and IgD(lambda) sera but not to IgG, IgM, or IgA myeloma sera. Furthermore, the fragment specifically bound to peripheral blood B lymphocytes, and the binding was inhibited by preincubation with anti-IgD-Fab polyclonal antibodies. Results obtained by introducing five amino acids randomly into MID(962-1200) using transposons suggested that alpha-helix structures were important for IgD binding. Ultracentrifugation experiments and gel electrophoresis revealed that native MID(962-1200) was a tetramer. Interestingly, tetrameric MID(962-1200) attracted IgD more than 20-fold more efficiently than the monomeric form. Thus, a tetrameric structure of MID(962-1200) is crucial for optimal IgD-binding capacity.     

Bovine lymphocyte antigens: serological relationships with erythrocyte and spermatozoan antigens.

Bovine lymphocytotoxicity tests with 20 unabsorbed bovine blood group sera revealed extensive reactivity which in the majority of cases had no indication of blood group relationship. Six of these sera were absorbed with selected lymphocytes to produce eleven antisera of reduced specificity. Again, most of the sera had reaction patterns which could not be related to the hemolytic patterns of any known blood group antibodies. However, five comparisons involving unabsorbed antisera and two comparisons involving absorbed antisera provided statistical evidence of similarities between their lymphocytotoxic reaction patterns and the hemolytic reaction patterns of certain blood group antibodies. Several of the sera appeared to contain related cytotoxic specificities, and three such absorbed sera may have contained an anti-J specificity. All six examined monospecific isoimmune blood group antisera contained lymphocytic reactivities not related to their hemolytic specificities. Two normal sera containing naturally occurring anti-J had no cytotoxic activity. Anti-semen sera likewise were devoid of lymphocytotoxic activity.     

Age of appearance of IgG, IgM, and IgE antibodies specific for Loa loa in Gabonese children

IgG, IgM, and IgE antibodies against the filaria Loa loa were measured in umbilical cord blood and in blood from young Gabonese children by an ELISA technique using a homologous metabolic antigen. For children in eight consecutive age groups and adults the percentage of the population positive for each of the antibody classes was determined. The number of children with maternal IgG decreased until one year of age when new synthesis began to become apparent. IgM antibodies were detected only after six months, probably indicating an early infancy as opposed to a fetal infection. The percentage of individuals positive for IgM or IgE reached a peak between two and three years old, followed by a slight decline. Over half of the individuals over one year of age had IgM antibody against L. loa, indicating long-term synthesis of this class of immunoglobulin in many people. In the first two years of life, IgE antibodies were usually accompanied by L. loa-specific IgM. This specific IgE did not appear to trigger the synthesis of nonspecific IgE. By the age of two, 95% of the population had some antibodies against L. loa and by five the percentage of individuals positive for each antibody class had reached adult levels.     

Cross-reactivity of human and a putative pig IgD. pig IgD-like molecules as serum and lymphocyte components

Proteins of pig serum and splenocytes which were isolated on immobilized guinea pig antibodies against human IgD were characterized by SDS-PAGE. In both cases the main isolated components possessed several properties nearly identical with those of human IgD: molar mass of the components and their chains, and susceptibility of the heavy chains, light chains, the chymotryptic peptide maps of the chains, and susceptibility of the heavy chains, the chymotryptic peptide maps of the chains, and susceptibility of the heavy chains in the native molecule to trypsin digestion. These IgD-like components not adsorbed on immobilized normal guinea-pig IgG. We suggest that the components of pig serum and splenocytes cross-reacting with highly specific antibodies against human IgD represent candidates for pig IgD. The percentage of the IgD-like molecules on the surface of pig splenocytes labeled with the antibody against human IgD was similar to the percentage of splenocytes bearing all immunoglobulins. Therefore, we suggest that IgD-like molecules were occurring on the surface of cells mostly together with molecules of another immunoglobulin class.