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This paper presents a method for the isolation of highly enriched antigen-specific B lymphocytes from peripheral blood mononuclear cells (PBMC) by means of an immunomagnetic procedure. The method is illustrated by the isolation of B cells positive for rheumatoid factors (RF) from patients with rheumatoid arthritis and healthy blood donors. After Epstein-Barr virus transformation of isolated cells, more than 90% of the generated immunoglobulin-secreting cells produced RF in several experiments. Compared to the low frequency of B cells positive for RF in PBMC, this represents a 10³- to 104-fold enrichment of antigen-specific B cells. [ABSTRACT FROM AUTHOR]

The regulation of IgE synthesis in vitro and in viva by schistosomula-released products (SRP) has been shown to be dependent on the presence of serine proteases. The present paper concerns the characterization of the enzymes involved. The labelling of SRP with [3H]diisopropyl phosphofluoridate revealed two molecules, one major with an MW of 27,500 and one minor with an MW of 29,000. The same pattern was obtained by labelling of schistosomula or cercariae surfaces as well as of the total schistosomulum homogenate. The properties of these enzymes were studied by means of various specific substrates or inhibitors of serine proteases. The specificity was relatively narrow, but had some similarity with trypsin. When added to lymphoid rat cells in culture, SRP induced an increased expression of receptors for the Fc fragment of IgE (FcεRII). This suggests that the IgE-enhancing property of SRP was due to serine protease activity which may act by enhancing the lymphocyte FcεRII. [ABSTRACT FROM AUTHOR]

X-irradiated (250 rad), cyclophosphamide-treated or Atx A mice injected with syngeneic trinitrophenylated spleen cells (TNP-SC) and footpad challenged with syngeneic lymphoblasts generated delayed-type hypersensitivity (DTH) responses 24, 48 and 72 h after challenge. The syngeneic-DTH (syn-DTH) response was mediated by Lyt-1+ cells and suppressed with Lyt-1+2+3+, I-Jk+ cells. The suppressor cells were obtained from spleens or thymuses of normal syngeneic mice. Suppressor factor (SF) was extracted or released from Lyt-1+2+3+, I-Jk+ cells obtained from normal A mice (but not from X-irradiated A mice). The factor blocked the DTH responses of X-irradiated mice injected with syngeneic TNP-SC and challenged with syngeneic lymphoblasts when injected into the mice both at the induction phase and the elicitation phase of the DTH. The factor failed to abrogate allogeneic and xenogeneic DTH. However, allogeneic factor (derived from C57BL/6 mice) abolished the syn-DTH response of mice injected with syngeneic TNP-SC and challenged with syngeneic lymphoblasts. The SF was produced by Lyt-1+2+3+, I-Jk+ T cells or by thymocytes. The combined extracted product of Lyt-1+ and Lyt-2+ cells did not abrogate the syn-DTH response. Normal spleen cells depleted of phagocytes by a magnetic procedure also produced the SF. These findings indicate, therefore, that suppressive factor (or factors; see Discussion of the accompanying paper, Ref.17) controls the immunological autoreactivity against syngeneic TNP-SC. [ABSTRACT FROM AUTHOR]

We have developed a new assay for quantitative detection of all helper T cells that can induce normal B lymphocytes to proliferation and Ig secretion. To establish the optima] assay conditions, we have used cloned I helper cells of defined specificities that had previously been shown to activate normal B lymphocytes expressing the specific antigen(s) on direct cellular interactions. As shown in this paper, "irrelevant" B lymphocytes-that is, those that do not express either antigen or restriction elements recognized by the effector helper T cells-can also be induced in the presence of appropriate concentrations of pokeweed mitogen which are not mitogenic for the "target" B lymphocytes. 'Nonspecific" plaque-forming cell responses are of the same magnitude as those provided to specifically triggered targets and equal or better than those induced by lipopolysaecharide. The assay is highly sensitive and enables "semiquantitative' detection of less than 20-30 effector T cells per culture. Since effeetor helper T cells can be detected regardless of the clonal specificity, the assay appears useful for quantitative studies of various populations of T helper cells displaying mixed specificities and for the classification of cells with unknown functions. [ABSTRACT FROM AUTHOR]

This paper describes the results obtained using an indirect protein A plaque-forming-cell (PFC) assay applied to human peripheral blood lymphocytes (PBL) activated with pokeweed mitogen (PWM). A maximal-response was obtained after 6 days of culture with regard to the three major classes of immunoglobulin (Ig) investigated. No difference was found between females and males. T and B lymphocytes mixed in ratios varying from 1:8 to 8:1 and unfractionated cells were investigated. A maximal PFC response of isolated lymphocytes was found in cultures of 1:4 T/B reconstituted suspensions of untreated cells, whereas the maximal response of untreated B and 2000-rad-irradialed T lymphocytes was found in cultures with a 4:1 T/B ratio. The number of plaques developed in cultures of unfractionated cells exceeded the response of 4:1 T/B reconstituted untreated lymphocytes but was far below the number of PFC developed in cultures of untreated B and 2000-rad-irradiated T lymphocytes. Normal donors developed approximately 70 × 10³ PFC/106 cells. The viability was 85% alter the incubation period. No difference was found in the PFC response of cultures of separated, reconstituted T/B lymphocytes set up in autologous and allogeneic combinations. [ABSTRACT FROM AUTHOR]

Cellular cytotoxicity reactions can be studied in a manner analogous to that used lo measure enzyme activity. This approach yields two parameters : Vmax, the maximal rate of target cell lysis that can be achieved by the lymphocyte preparation tested, and KMapp, the apparent Michaelis constant. By analogy to many enzyme-catalysed reactions, KMapp values for cytotoxicity reactions have generally been interpreted in terms of dissociation constants for the interaction of receptor sites on effector cells with antigens on the target cells. In this paper we demonstrate that experimentally determined KMapp values for natural or antibody dependent cytotoxicity reactions arc approximately equal to the concentration of NK or K effector cells in the lymphocyte preparation tested. This result makes possible the simultaneous determination of both effector cell frequency and lytic activity in a given lymphocyte preparation. [ABSTRACT FROM AUTHOR]

This paper deals with the correlation of clinical scoring and serologic markers of inflammation in atopic dermatitis and psoriasis. Serum eosinophil cationic protein (ECP), soluble interleukin-2 receptor (sIL-2R), total serum IgE, IgG and IgM anti-IgE antibodies, and IgE immune complexes were evaluated in monitoring inflammatory sin diseases such as atopic dermatitis and psoriasis. Well-established clinical activity scores were used as standards in recording skin improvement under treatment in a clinical setting. Serum ECP was found to be increased in both atopic dermatitis and psoriasis patients compared to normal controls; sIL-2R and IgE immune complexes were increased only in atopics with increased only in atopics with increased serum IgE. Anti-IgE antibodies did not show any deviation in both groups of patients. There was a significant elevation of sIL-2R and IgE immune complexes and a nonsignificant elevation of ECP in high-IgE atopics in comparison to those with normal serum IgE. In both groups of patients there was a significant reduction of ECP and sIL-2R accompanying the improving skin condition. Serum IgE and the other immune parameters failed to respond. In contrast to other studies, serum ECP failed to correspond significantly with disease activity in our study. Our results showed measurable changes of ECP and sIL-2R for atopic dermatitis and/or psoriasis under treatment, but comparison to clinical scores remains difficult due to the different basis of the two systems. The only significant correlation was established for relative changes in sIL-2R and psoriasis area and intensity (PASI), a correlation which might be a useful approach in psoriasis. [ABSTRACT FROM AUTHOR]

An assay, the paper radioallergosorbent test (PRAST), for the measurement of specific serum IgE antibody in the ral is described in detail. This assay has been used, in conjunction with a modified PRAST for the determination of relative specific serum IgG antibody and the PRIST assay for total serum IgE [13], lo measure specific IgG and IgG and total IgE immune responses in normal parasite infected rats immunized using various protocols. The results indicate that there is a relationship between the basic IgE level and the immune response, i,e. a rat strain with a low constitutive IgE level demonstrates a weak response whereas a high level strain reacts strongly. When PRAST and passive cutaneous anaphylaxis (PCA) were compared, using standardized IgE antibody containing sera, the results were in good agreement. However, PRAST is the preferable assay as it shows less intrinsic variation, is more sensitive than PCA, and is not influenced by high serum IgE levels in the recipient animal. [ABSTRACT FROM AUTHOR]

This paper describes lymphocyte binding of aggregated immunoglobulin, which appears to bind to B lymphocytes and the third lymphocyte-like cell population. The applications of aggregated Ig binding are also discussed, and the relation to Fc receptors and Ia-like antigens. [ABSTRACT FROM AUTHOR]

This paper describes column fractionation techniques for separating different lymphocyte subpopulations. For this purpose columns can bc coupled with antigen, specific antibodies to membrane determinants on lymphocytes, or immune complexes or aggregated immunoglobulins, which interact with Fc receptors. [ABSTRACT FROM AUTHOR]

This paper deals with recent developments in the fractionation of immunocornpetent cells with different physical properties, using preparative free flow cell electrophoresis, fractionation of cells according to their size and density. [ABSTRACT FROM AUTHOR]

This paper describes the identification of important allergens from the granary weevil (Sitophilus granarius) (Sg). Sera from Danish bakers whose skin prick tests were positive to extracts of Sg were screened for IgE against Sg extracts. We found that 54% (n = 66) had elevated levels of IgE (RAST classes 1-3, by luminescent immunoassay) against whole-body extracts of Sg. The specificity of patient IgE was investigated in an inhibition-dot immunoblotting assay. IgE binding was inhibited in all sera but two, thus indicating that the patients' IgE was indeed specific for the Sg extract. In crossed immunoelectrophoresis, 23 different proteins were identified. All RAST-positive sera were investigated in crossed radioimmuno-electrophoresis. At least 11 proteins in the Sg extract were capable of binding IgE. All individual sera reacted with at least four different proteins. The two most prominent allergens bound IgE from 88% and 100%, respectively, of the patients. These two are considered to be the most important allergens from Sg, and will be useful as markers in environmental immunochemical assays to detect allergens in samples from bakeries, grain stores, etc. [ABSTRACT FROM AUTHOR]

ELISA is increasingly used to measure antibodies in new circumstances. Recently, it has been applied to the measurement of IgG subclass antibodies against common antigens in early childhood. These studies have raised concerns about the specificity of some of these assays. This paper details the results of experiments which have assessed the specificity of IgG1 binding to allergens of dust mite (Dermatophagoides pteronyssinus) and ryegrass (Lolium perenne) pollen by inhibition ELISA in the sera of 2-year-old children of atopic parents. Six sera which showed binding of IgG1 to D. pteronyssinus and six to L. perenne were used. All had IgG1 antibody against ovalbumin. In the children's sera, binding to D. pteronyssinus was substantially inhibited by preincubation with the homologous antigen, but not with ovalbumin, thereby confirming the specificity of the assay. However, suppression of IgG 1 binding to L. perenne with the homologous antigen was conparatively small, and ovalbumin could cause an equivalent inhibition, indicating poor specificity. Furthermore, the level of IgG1 binding to L. perenne was closely correlated to the level of IgG1 binding to ovalbumin (r=0.98; P <0.001). When the assay was reversed, IgG1 binding to ovalbumin was only slightly inhibited by L. perenne, indicating that most antibody binding to ovalbumin was specific. Thus, binding IgG1 in both adult and child sera to D. pteronyssinus appeared to be specific, while child, but not adult, IgG1 binding to L. perenne showed poor specificity. This disparity may be due to differences in the affinities of the respective antibodies, and it illustrates the importance of determining assay specificity when making measurements in early childhood. [ABSTRACT FROM AUTHOR]

In this paper, we have determined the levels of IgG1 and IgG4 anti-IgE in the sera of 66 asthma patients suffering from mild (n= 24), moderate (n= 23), or severe (n= 19) symptoms, and 20 nonatopic, healthy subjects. The study has revealed that although asthma patients have significantly elevated levels of IgG1 and IgG4 anti-IgE antibodies, the concentration of these autoantibodies is not related to the severity of asthma. This conclusion may be related to the known heterogeneity of autoanti-IgE antibodies in terms of their ability to trigger basophil histamine release. [ABSTRACT FROM AUTHOR]

The article presents abstracts of various research papers to be presented in a conference to be held on September 14, 1993. One of the abstracts is on atopic allergy, which is causally related with selective activation of allergen-specific T helper type 2 (Th2) cells, which produce high amounts of interleukin (IL)-4 and IL-5. The aim of the study is to identify the factors, present during the differentiation of T helper cells that lead to selective outgrowth of Th2 cells. Another abstract is on neuroendocrine peptides which are involved in the regulation of effector cells of allergy. These peptides can also inhibit immunoglobuline synthesis but no data are available concerning human immunoglobulin (Ig) E. Neuroendocrine peptides endogenously modulate human in vitro IgE synthesis and represent eventually the postulated neuroendocrine arm in diseases like atopic dermatitis.

Serum IgE levels in healthy blood donors who had no history of atopy were measured by a paper-disc RIA and analyzed according to the donors' smoking habits. the IgE geometric mean for regular smokers was 41.7 IU/ml, which was significantly higher than that for nonsmokers (19.3 IU/ml) or rare smokers (22.7 IU/ml). Whereas 28% of smokers had IgE levels greater than 200 IU/ml none of the rare smokers or nonsmokers did. IgE levels in smokers showed a moderate inverse correlation with the degree of smoking. The mean IgE level was 189.8 IU/ml in those who smoked 1-9 cigarettes/day but only 32.8 IU/ml in those who smoked 10-19 cigarettes per day and 11.1 IU/ml in those who smoked 20 or more cigarettes/day. The number of years a person smoked did not seem to significantly influence the IgE level. The mean IgE level in ex-smokers (50.5 IU/ml) was much lower than in current light smokers but was still higher than in nonsmokers. There was a moderate inverse correlation between IgE levels and duration of cessation of smoking. Our data suggest a characteristic pattern for the influence of cigarette smoking on serum IgE level, namely, a striking rise associated with light smoking and a remarkable drop in heavy smokers, and such changes seemed reversible after the habit was stopped. Smoking status, therefore, appears to be an important consideration in interpreting serum IgE levels and in revising the "norms" of IgE levels. [ABSTRACT FROM AUTHOR]

The article focuses on the naturally occurring allergens that elicit immediate hypersensitivity reactions when combining with homologous IgE antibodies in human individuals. Data from a large number of investigations have been utilized in this presentation but reference is made only to a few selected papers summarizing the most important information in this field. The definition as well as the demonstration of the existence of an allergen depends on an immune reaction occurring in a living organism. One prominent characteristic of IgE is its property to bind to specific IgE receptors on mast cells and basophil granulocytes of the same or closely related species.

A 27 kDa Chlamydia trachomatis Mip-like protein with homology of a 175-amino-acid C-terminal fragment to the surface-exposed Legionella pneumophila mip-gene product has previously been described. In this paper the entire chlamydia Mip-like sequence of C. trachomatis serovar L2 (lymphogranuloma venereum (LGV) biovar) is presented. The sequence shows high similarity to the legionella Mip protein and its C-terminal region, like that of the legionella Mip, has high amino acid similarity to eukaryotic and prokaryotic FK506-binding proteins. The chlamydial mip-like gene was detected by polymerase chain reaction (PCR) in other C. trachomatis serovars and by sequencing of the mip-like genes of serovars B and E (trachoma biovar) was shown to be highly conserved within the two major biovars of C. trachomatis. Monoclonal and polyclonal antibodies raised against the recombinant Mip-like protein tailed to demonstrate surface-exposed epitopes on infectious elementary bodies or reproductive reticulate body forms either by immunofluorescence or immuno-gold electron microscopy. However, a complement-dependent inhibition of up to 91% of infectivity for cell cultures was observed with antibodies to the N-terminal fragment of the Mip-like protein suggesting that antibody-accessible epitopes are present on infectious EBs. [ABSTRACT FROM AUTHOR]

In contrast to MN receptors, Pr1 and Pr2 antigens of isolated glycoprotein of erythrocytes are not inactivated by acylation of free amino groups. By introduction of easily leaving groups, e.g. trifluoroacetic residues, inactivation of the MN activity can be made reversible. No differences between Pr1 and Pr2 antigens could be found with the reactions described in this paper. Acylation m aqueous phase (buffer/anhydrous acetic acid) and m organic phase (pyridine/ anhydrous acetic acid) gave identical results qualitatively and quantitatively. It was not possible to split the N-acetic binding neither under mild alkaline conditions nor with aminoacylase. 4-5 amino groups out of 11 could be acylated, whereas succinylation in aquous phase (buffer/ anhydrous succinic acid) resulted in modification of only one free amino group, the calculations being based on a molecular weight of 30 000. Following succinylation MN activity was also lost, whereas the antigenic activity of Pr1 and Pr2 remained unchanged. The appearance of a negative charge instead of the positive charge of the free amino group on the glycoprotein did not affect Pr1 or Pr2 activity m any way. If glycoprotein was esterified under conditions which cause esterification of free sialic acid to a wide extent neither MN activity nor Pr1/Pr2 activity was altered. [ABSTRACT FROM AUTHOR]

Specific antibodies to two plant, hormones, namely indole-3-acetic acid and gibberellic acid, were produced in rabbits which were immunized with conjugates of either hormone to hemocyanin. The specific antibodies obtained were used for assaying the hormones by several immunological assays. The most sensitive assay reported in this paper, for the determination of indole-3-acetic acid and gibberellic acid, is the inhibition of inactivation of modified bacteriophage. For this assay conjugates of either hormone with bacteriophage T4 were prepared and characterized. Such chemically modified bacteriophages were completely inactivated by the respective specific antiserum and this inactivation was inhibited by the free hormone. The specificity of the antibodies produced was tested by a series of cross-reactions. There was no immunological cross-reaction between the two hormones which can, therefore, be determined independently. However, antibodies to indole-3-acetic acid cross-reacted with chemically related compounds such as indole-acetyl-ε-amino caproic acid, tryptamine and tryptophan. The immunological specificity of different gibberellins tested was found to be in good correlation with the biological specificity. [ABSTRACT FROM AUTHOR]

An immunoglobulin M from Waldenström's disease (IgM Ga) was hydrolysed by papain and a glycopeptide fraction was isolated by gel filtration on Sephadex G-100. This fraction was then successively resolved into fourteen glycopeptides by gel filtration on Sephadex G-50 and by preparative paper electrophoresis at pH 1.9. The amino acid and carbohydrate content of these glycopeptides was determined by ionexchange chromatography in an autoanalyzer. All of them contain aspartic acid and serine, but they are quite different from one another, in amino acid composition and sequence about, the carbohydrate-peptide linkage, especially with respect to histidine, tyrosine, threonine, valine, glutamic acid, lysine and proline. Furthermore, a number of glycopeptides contain only mannose and glucosamine, while others contain in addition, galactose, fucose, and N-acetylneuraminic acid, in variable amounts. These results suggest the presence of several linkage sites of the oligosaccharide chains on the H-chain and the true heterogeneity of these oligosaccharide chains. The peptide sequence of a few glyeopeptides was reported, especially the following: r-Asn(carbohydrates)-Ser-Thr, also present, in the Fc fragment of Immunoglobulin G. [ABSTRACT FROM AUTHOR]

Vertebrates develop a highly specialized system of defense mechanisms as a result of continuous stimulation by foreign materials from the environment. Whether free in body fluids or attached to cell surfaces, antibodies play the key role in the recognition of, and protection of the body against, foreign substances. Important characteristics of anitbodies are their adaptive production as a result of antigenic stimulation and the specificity they exhibit in their reaction with the antigen that induced their production. Antibodies are present in the globulin fraction of the body fluids and are summarily called immunoglobulins. Knowledge pertaining to antibody structure has been acquired principally from research of the last two decades. During this period many important findings have been made that are applicable to other proteins; in fact, anitbodies are presently the most completely characterized of the complex proteins. This paper will encompass a brief review of the structure of antibodies. The rapid development in this field makes it difficult to ensure the inclusion of all appropriate publications, and therefore we shall refer the reader principally to the most recent review articles and books which can be examined for more detailed information. Of these, four deserve special attention: Kochwa & Kunkel (1971), Amos (1971), Merler (1970), and the Cold Spring Harbor Symposia on Quantitative Biology (1967). [ABSTRACT FROM AUTHOR]

It has previously been shown that rats do not expel a primary infection of Nippostronglus brasiliensis during lactation. In the present paper, three aspects of the immune response of the lactating rat to the parasite were examined and compared with the response of non-lactating rats. Protective and reaginic antibodies were present in the circulation of rats first infected during lactation, in titres as high or higher than those in non-lactating controls. The intestinal mast cell response was similar both in timing and magnitude and lactating rats became susceptible to shock with the i.v. injection of antigen. Attention was drawn to the similarity between the response of the lactating rat and that of the neonate to this infection. [ABSTRACT FROM AUTHOR]

Ficoll, a polymer of sucrose, and bovine serum albumin have been compared as media for preparation of density gradients for separation of lymphoid cells. Direct vapour pressure osmometer measurements demonstrated that Ficoll at high concentrations has a marked effect on the osmolarity of the medium in which it is dissolved. Albumin, prepared in the manner we have described in earlier papers of this series, has no effect, and density gradients prepared with this albumin do not contain gradients of osmolarity. Antibody forming calls give basically similar density distributions after separation in both media, apart from a slight overall shift to the dense end due to shrinkage in Ficoll. Heterogeneous, multiple peak distribution profiles, or relatively simple, homogeneous profiles may be obtained in both media, depending on the source of antibody-forming cells, the stage of differentiation of the population, and the degree of resolution of the gradient. Antibody-forming cell density heterogeneity is a property of the cell population, rather than an artifact of the separation system. Different animal species have a wide variation in the osmolarity of their sera. Albumin-gradients of osmolarity which were not iso-osmotic for the mouse caused a general shift in the density of mouse cell populations but no marked change in the relative distribution of cells. However, a change from iso-osmotic conditions did affect the ability of cells to reband on a second gradient. The use of gradients iso-osmotic for the particular species under study is recommended as a general procedure. [ABSTRACT FROM AUTHOR]

This paper describes a new method for the morphological study of individual antibody-forming cells (AFC) on cell smears of the quality of normal haematological preparations. The early AFC response to polymerized flagellin of S. adelaide was studied in vivo using C57BL mice, which have very low background levels of AFC and in vitro using dispersed spleen cell cultures from CBA mice. AFC, arising as a result of in vivo or in vitro stimulation were found to comprise a heterogeneous population, including basophilic mononuclear cells, lymphocytes of most sizes, immature blast cells and occasional plasma cells. The earliest AFC detected comprised a high percentage (28 per cent in vivo, 31 per cent in vitro) of small lymphocyte-like cells. Studies of the incorporation of [³H]thymidine showed that most AFC arose by proliferation but that a proportion of AFC, the small lymphocyte-like cells, arose by differentiation of precursor cells not involving cell division. The effects of antigen concentration on the kinetics of AFC were investigated in vitro. Subtolerogenic antigen doses caused a delayed and decreased AFC response. [ABSTRACT FROM AUTHOR]

Data presented in this paper indicate that by column chromatography using an O acetylated galactan, a considerable degree of purification of specific antibody to O antigen 5 may be achieved. [ABSTRACT FROM AUTHOR]

When lymphoid cells from hypersensitive rabbits are incubated with antigen, biologically active substances are formed and released which are capable of inhibiting the migration of normal non-sensitized mesenchymal cells. In the present paper some basic parameters of their production were determined. These substances were regularly obtained after 6 and 18 hours incubation, but not after 2 hours. Under more favourable cultivation conditions (lower density of lymphocyte suspension) an increased activity in the cell extracts as compared with the supernatants was observed. Another critical factor in the production of these substances is the quantity of antigen added. Ten micrograms of PPD leads to the production and liberation of a highly effective substance. A lower dose of antigen results in the liberation of a substance into the supernatant which by itself is almost inactive, but becomes more active when more antigen is added. The efficiency of the released substances was determined by serial dilution. The inhibiting activity was maintained at 1:5 and 1:20 and sometimes at 1:100 dilutions. [ABSTRACT FROM AUTHOR]

Rat liver microsomes were separated into three subfractions by means of ultracentrifugation through sucrose media of different densities and in the presence of different cations. Detergent extracts of these preparations were analysed by double diffusion in agar gel and immunoelectrophoresis with rabbit antisera against each of the subfractions. One subfraction was derived from the ‘rough’ and another from the ‘smooth’ part of the endoplasmic reticulum of the liver parenchymal cells. Their extracts contained at least thirteen soluble antigens in common. However, fraction-specific antigens were also present. Thus, the extracts of the rough (R) membranes contained at least one typical antigen, not found in any of the other fractions. An antiserum against this component also precipitated a non-migrating antigen present only in the extracts of smooth (Sa) membranes. These two antigens may represent different molecular forms of a substance involved in binding of the ribosomes or in the assembly of membrane subunits. At least eleven of the antigens common for the two fractions exhibited enzymatic activities when assayed after precipitation with antibody in the immunoelectro- phoretic plates. Six immunologically and electrophoretically distinct antigens had esterase activity with α-naphthyl propionate as substrate. Two of these esterases also split indoxyl acetate. Three other antigens with acid phosphatase activity split both α-naphthyl acid phosphate and β-glycerophosphate. Three antigens had nucleoside diphosphatase activity when tested with uridine- or inosine-diphosphate. Preliminary experiments also suggested that two additional antigens possessed NADH-diaphorase activity and thus could belong to the microsomal electron trans- port systems. The third subfraction consisted of electron-microscopically smooth membranes (Sb), enzymatically different from those of the endoplasmic reticulum. The antigens typical for the latter were either absent or present only in minor and variable concentrations. Its extracts contained at least two typical antigens. One of these was identified as contaminating ferritin. The nature and origin of the other antigen has not yet been established. All antigens described in this paper were immunologically different from the common rat serum proteins. [ABSTRACT FROM AUTHOR]

The present paper deals with some of the quantitative aspects of immunoglobulin formation in vitro. The results of this study show that isolated spleen cells synthesize about 1.25 μg IgG per 108 lymphoid cells during the first 6 hours of incubation. The initial rate of synthesis was not maintained throughout the 48 hours of incubation, but decreased markedly after 6 hours. Comparison of the intensity of the autoradiographic line with the amount of radioactive immunoglobulin in the precipitation line revealed that a very distinct line (gradation + + +) corresponds to about 0.0125 μg IgG. The limit of visibility (+) is reached at approximately 0.0002/μg radioactive IgG. [ABSTRACT FROM AUTHOR]

The formation of immunoglobulins by human tissues was studied by three techniques: (1) Autoradiography of the immunoelectrophoretic pattern of culture fluids of tissue fragments or cell suspensions in a medium with radio- active amino acids; (2) immunofluorescent staining of the tissue sections or cell suspensions, before incubation, with antisera specific for one immunogtobulin; and (3) histological and cytological study of the samples. The paper gives a detailed description of the techniques applied and evaluates the specificity of the immunological methods used. Various control experiments were performed: Incubation of dead tissues or normal serum in the radioactive culture medium excluded the adsorption of [14C]amino acids on to immunoglobulins. Cultures of tissue fragments in the presence of puromycin showed an inhibition of the synthesis of [14C]immunoglobulins. Autoradiography of a ‘fingerprint’ of radioactive IgG demonstrated radioactive amino acids present in different peptides of the synthesized molecule. From these control experiments it can be concluded that the labelling of the immunoglobulins is based on the incorporation of [14C]amino acids during incubation in vitro. The specificity of the immunofluorescent staining was evaluated by immuno- diffusion of the antisera and conjugates, by blocking procedures and by specific staining of bone-marrow samples from patients with various immunoglobulin abnormalities. [ABSTRACT FROM AUTHOR]

Immunofluorescent and isotope coprecipitation techniques provided methods of studying in vitro production of antibody by chicken spleen cells. In some culture preparations precipitating antibody was produced in sufficient quantity to be measured by quantitative techniques. The in vitro production of globulins by chicken spleen cells has been demonstrated by paper and immunoelectrophoresis of tissue culture medium. The antibody in the globulin produced in the in vitro tissue culture preparations may be shown by an autoradiograph of the immunoelectrophoresis after the addition of antigen trace labelled with radioactive iodine. [ABSTRACT FROM AUTHOR]

Red cells sensitized with naturally occurring or 'immune' fowl antibody were lysed by fresh fowl serum. This fowl complement could not be replaced by that of human, rabbit or guinea pig. Both naturally occurring and 'immune' antibodies to red cells in the mammalian species tested did not lyse with fowl complement. Lysis by fresh fowl serum of sheep red cells sensitized with mammalian antibody is due to the presence of naturally occurrent antibody to these cells in the fowl serum; the enhancement of this lysis by the mammalian antibody will be described more fully in the next paper. Naturally occurring antibody to sheep red cells present in turkey serum and 'immune' antibody to fowl red cells were not lytic with fresh turkey serum, with fowl complement, with guinea-pig complement or with any combination of these. Some properties of fowl complement are given. [ABSTRACT FROM AUTHOR]

Heat and pH stability studies and experiments with organic solvents show that the A-antigens discussed in the preceding paper (Augustin, 1959c) are much more labile than the I-(‘inner ring’) antigens. Breakdown products and/or aggregates are produced which no longer precipitate with antisera to the original extracts, but act as inhibitors. Solutions of pollen allergens, on the other hand, are found to withstand even autoclaving for 15 min. at 20 atm. and vigorous boiling over the naked flame of a bunsen burner. None of the carbohydrates tested has a demonstrable effect on skin reactivity which is, however, destroyed by crystalline pepsin, crystalline trypsin, a crystalline mould protease and a tissue protease (a partially purified extract from rabbit spleen). It follows that the bulk of the allergens—if not all—are proteins. The relation of skin reactivity, immuno-electrophoretic patterns, carbohydrate and protein reactions to the selective destruction of the pollen antigens is investigated. Pollen components prove to have a somewhat wider range of electrophoretic mobilities than serum proteins and are probably as complicated a mixture. The most and least highly negatively charged components are without skin reactivity in allergic subjects. The skin reactive allergens appear to have the mobilities of α- and β-globulins. Not all the hay fever subjects react equally to all the components, and Cocksfoot and Timothy activity patterns vary in different subjects. [ABSTRACT FROM AUTHOR]

Whilst the ability of isoantibodies to agglutinate mouse red cells in saline medium does depend in part upon the properties of the antibody molecules, the concentration of antigen upon the red cell and certain genetically determined properties of the cell surface are of even greater importance. The red cells of mice of any strain will give positive results in the human serum:dextran system whilst reactions in a saline medium are unusual. In this paper the term ‘incomplete antibody’ is used to denote antibodies that can only be detected in conjunction with other antibodies in the human serum:dextran system. The time of appearance of antibodies to homografts of an ascites sarcoma, an ascites leukosis and a solid mammary carcinoma has been studied. Incomplete antibody as defined above has been detected by two methods. In the blocking test incomplete antibody is allowed to react with red cells before contact with complete antibody, a positive result being observed as a fall in titre when the treated red cells are subsequently exposed to complete antibody. In the second type of test, the synergic test, incomplete antibody is mixed with suitably diluted complete antibody and fitrated against suitable red cells, a positive result being a significant rise in titre as compared with complete antibody mixed with normal serum. Antibodies produced by the antigen donors did not give significant blocking or synergic effects. Incomplete antibodies are sometimes detectible on the third day and with regularity on the fourth day. Antibodies ‘complete’ for A strain red cells may appear at any time from the fifth day onwards. There is sometimes a drop in fitre of varying duration on the sixth day. This corresponds with the commencement of marked inflammatory changes on the graft bed. The maximum titre is attained at about two weeks. Antibodies may disappear in under three months or persist as long as a year. The function of antibodies in homograft reactions varies with the target tissue. However, they appear before the onset of anatomical signs of homograff response regardless of the type of target cell. [ABSTRACT FROM AUTHOR]

Summary: The study of cellular and molecular immunology is almost completely dependent on monoclonal antibody technology. Despite the relatively long history of monoclonal antibodies, there is still huge potential for developing novel and transformative uses for this technology. In this issue of Immunology, we present two such papers, one focussing on anti‐complement (C5) antibodies, the other on anti‐CD6‐specific antibodies. Both manuscripts describe novel ways that monoclonal antibodies may be used for scientific and potentially for therapeutic benefit. [ABSTRACT FROM AUTHOR]

Comments on the paper 'Rapid Detection of Antibodies to Immunoglobulin A Molecules by Using the Particle Gel Immunoassay,' published in a 2002 issue of 'Vox Sanguinis.' Limitations of the method proposed in the paper; Effective methods for the detection of antibodies against immunoglobulin.

Please cite this paper as: Isolation of human eosinophils: microbead method has no impact on IL-5 sustained viability. Experimental Dermatology 2009; 19: 467–469. Background/purpose: In a recent issue of Experimental Dermatology (18, 2009, 654), Schefzyk et al. concluded that multi-antibody eosinophil isolation (Miltenyi) should be abandoned, as differential purity was minimal, and eosinophils underwent accelerated apoptosis when compared with those isolated with traditional anti-CD16 microbeads. Our intent was to investigate the universality of these findings. Methods: We isolated eosinophils from normal donor granulocyte packs using two methods, and evaluated purity, viability and annexin-V/propidium-iodide staining. Results: Purity was substantially greater when multi-antibody isolation was used for eosinophil isolation from granulocyte packs (98% vs 69%). No differential survival was detected when eosinophils were maintained in culture with or without interleukin-5. Conclusions: Multi-antibody eosinophil isolation represents a substantial advantage over anti-CD-16 microbeads when isolating large numbers of eosinophils from concentrated leucocyte preparations. No differential survival was observed. While appropriate consideration of methods is always crucial, multi-antibody eosinophil isolation should not be abandoned completely. [ABSTRACT FROM AUTHOR]

The author reflects findings of the paper on allergen-independent immunomodulatory activities of immunoglobulin E (IgE). An overview of the correlation of the level of total IgE in a subject's serum with the level of high affinity IgE receptor (FceRI) expression on their basophils in the 1970s is offered. The author suggests that ongoing and future investigations will probably lead to further comprehensive understanding of IgE's antigen-independent bioactivities.

The mission of the US Food and Drug Administration (FDA) is to protect public health by ensuring the safety, efficacy, and quality of drugs. In drug development, animal studies play a key role in assessing the safety (toxicology), pharmacokinetics (PK), and proof-of-concept efficacy of a new product. When clinical studies are neither ethical nor feasible to conduct, the Animal Rule (67 FR 37988, 2002) introduces the potential for drug (e.g., countermeasure) approval based on efficacy studies in animals, and appropriate human safety and pharmacokinetic information. The Center for Drug Evaluation and Research is responsible for the review of drug and therapeutic biologic applications. The use of the word “drugs” in this paper will henceforth refer to both drugs and therapeutic biologics (e.g., monoclonal antibodies and small protein molecules). Information about vaccines and other biologics, such as antisera and blood products, should be obtained from the Center for Biologic Evaluation and Research.Clinical Pharmacology & Therapeutics (2008); 83, 6, 918–920 doi:10.1038/clpt.2008.23 [ABSTRACT FROM AUTHOR]

Introduction: Despite the remarkable progress achieved in the research on Alzheimer's disease (AD), its exact pathogenesis is not fully understood and effective therapies do not currently exist. In order to find effective therapy for AD, I ranged extensively over the literature and found an important paper by Tiffany and colleagues. Results and Conclusion: Neuroinflammation has been proposed as a possible cause or driving force of AD. The discovery by Tiffany et al. that amyloid β (Aβ) is a formylpeptide receptor 2 agonist indicated that Aβ is a potent chemoattractant for phagocytic leukocytes. Therefore, in all likelihood Aβ attracts peripheral blood neutrophils, monocytes, as well as microglia cells in brain parenchyma, and activates them. However, the role of microglia cells and their precursor monocytes in AD pathogenesis remains elusive. Recently, neutrophils were found to be present in areas with Aβ deposits in AD brain and in transgenic AD model mice. Because brain is vulnerable to the effects of reactive oxygen species (ROS) and neutrophils secrete a large amount of ROS, neutrophils look like a driving force of AD. Therefore, a possibility arises that anti‐IL‐17A and anti‐IL‐23 antibodies are effective against AD, because these antibodies can be thought to interfere with neutrophil trafficking from the bone marrow to the blood circulation and thus inhibit neutrophil infiltration into AD brain. Clinical studies using anti‐IL‐17A and anti‐IL‐23 antibodies in patients with AD are required. [ABSTRACT FROM AUTHOR]

Linked Content This article is linked to Nakase and Nakase et al papers. To view these articles visit https://doi.org/10.1111/apt.14365 and https://doi.org/10.1111/apt.14318. [ABSTRACT FROM AUTHOR]

This article is linked to Fang et al and Fang and Murray papers. To view these articles visit https://doi.org/10.1111/apt.14250 and https://doi.org/10.1111/apt.14333. [ABSTRACT FROM AUTHOR]

This article presents abstracts of several manuscripts related to allergic diseases and medicine. According to one abstract, the paper describes a quantitative radioimmunoassay for IgE antibodies. According to another abstract, the paper studies the efficacy of preventing the development of serious otitis media by using an oral decongestant. In association with this article, in a randomized double-blind study, 190 children were treated for acute otitis media with antibiotics and either pseudoephedrine hydrochloride or placebo.

An assay for immune complexes [ICs] in gingival crevicular fluid [GCF] could be potentially useful to ascertain the risk of periodontal disease activity at specific oral sites. This paper describes and ELISA-based method sufficiently sensitive to measure ICs in GCF samples at levels below 1 μg/ml. Static GCF samples taken from 5 adults showed IC levels ranging from 5 to 166 μg/ml. Additional tests of 10 GCF samples from 1 adult were conducted with complement components and indicated that either C1q or rabbit anti-human C3d was suitable as a capture agent in the assay procedure. Further application of this assay may help to assess the usefulness of IC levels in GCF samples as possible diagnostic indicators of periodontal disease activity. [ABSTRACT FROM AUTHOR]

This paper describes how specific hybridoma clones can be rapidly selected using paramagnetic beads coated with the antigen used for immunization. Spleen cells from a mouse immunized with fragment D dimer (DD) from plasminolysed fibrin were first fused with X-63 mouse myeloma cells. Paramagnetic monodisperse beads (precoated with sheep anti-mouse antibodies) were then coated with S4, a monoclonal antibody to DD, and subsequently with DD. Mixing such beads with the fused cells allowed selective harvesting of cells with membrane-expressed anti-DD gammaglobulins using a magnetic particle concentrator. Within 24 h, the cells spontaneously detached from the beads and were plated out on 96-well plates. Supernatants from the clones obtained were tested by the ELISA technique. Antibodies specific for DD were produced by 40-79% of the tested clones. It is concluded that it is possible to use antigen-coated paramagnetic beads to select, prior to cloning, hybridomas that produce specific antibodies. Implementation of this technique has significantly reduced costs and time in our efforts to obtain hybridoma clones of interest. [ABSTRACT FROM AUTHOR]

The article focuses on purification and characterization of atopic allergens. In the past two decades, several atopic allergens, which induce IgE antibody-mediated immediate hypersensitivity in man have been purified to near homogeneity and studied for their immunochemical properties. In this paper will be reviewed some of the general findings drawn from such studies. Allergen characterization studies with extracts from these sources have confirmed the views of allergists that all extracts are multi-allergen systems and that individuals can have varying patterns of sensitivity to different allergens.

Linked Content This article is linked to Strik et al and Bar‐Yoseph, Chowers papers. To view these articles visit https://doi.org/10.1111/apt.13994 and https://doi.org/10.1111/apt.14047. [ABSTRACT FROM AUTHOR]

Click here to view the accompanying paper in this issue. [ABSTRACT FROM AUTHOR]

The antigen‐antibody interaction determines the sensitivity and specificity of competitive immunoassay for hapten detection. In this paper, the specificity of a monoclonal antibody against alternariol‐like compounds was evaluated through indirect competitive ELISA. The results showed that the antibody had cross‐reactivity with 33 compounds with the binding affinity (expressed by IC50) ranging from 9.4 ng/mL to 12.0 μg/mL. All the 33 compounds contained a common moiety and similar substituents. To understand how this common moiety and substituents affected the recognition ability of the antibody, a three‐dimensional quantitative structure‐activity relationship (3D‐QSAR) between the antibody and the 33 alternariol‐like compounds was constructed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods. The q2 values of the CoMFA and CoMSIA models were 0.785 and 0.782, respectively, and the r2 values were 0.911 and 0.988, respectively, indicating that the models had good predictive ability. The results of 3D‐QSAR showed that the most important factor affecting antibody recognition was the hydrogen bond mainly formed by the hydroxyl group of alternariol, followed by the hydrophobic force mainly formed by the methyl group. This study provides a reference for the design of new hapten and the mechanisms for antibody recognition. [ABSTRACT FROM AUTHOR]

Problem: Endometriosis (EM) is a chronic immunoinflammatory disease associated with an abnormal immunotolerant microenvironment. Myeloid‐derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that play a major role in immunosuppression in cancer, inflammation and other diseases. This paper aims to elucidate whether or not MDSCs are involved in regulating this microenvironment in EM and how this regulation occurs. Method of Study: Immunochemistry (IHC) and qPCR were conducted to measure CD11b and ARG1 expression in the ectopic endometrium samples from EM patients. CCL25 levels in EM PF and the expression of CCR9 on M‐MDSCs were measured by ELISA. M‐MDSC migration was determined towards rhCCL25, α‐CCR9, α‐CCL25 and EM PF through in vitro chemotaxis assay. CD33+CD14+CD11b+HLA‐DR‐ M‐MDSCs isolated from EM PBMCs were added to CD8+ T cells stimulated with α‐CD3/α‐CD28 antibody. After 72 hours of co‐culture, proliferation was measured to rate the immunosuppressive function of M‐MDSCs. Finally, levels of IL‐10, GM‐CSF and arginase activity in the cultured supernatants were detected. Results: IHC and qPCR results revealed higher CD11b and ARG1 expression in EM endometrium than normal endometrium. MDSCs accumulated in the EM microenvironment, in which M‐MDSCs were the predominant type. CD33+CD14+CD11b+HLA‐DR− M‐MDSCs expressed high CCR9 levels and were recruited through CCL25. M‐MDSCs from EM PBMCs inhibited proliferation and activity in autologous T cells. rhCCL25 promoted IL‐10 and GM‐CSF secretion and arginase enzymatic activity in CD33+CD14+CD11b+HLA‐DR− M‐MDSCs. Conclusion: CD33+CD14+CD11b+HLA‐DR− M‐MDSCs recruited and activated by CCR9/CCL25 play a crucial role in the pathogenic progression of endometriosis, thus providing a potential target for EM treatment. [ABSTRACT FROM AUTHOR]