Immunoglobulins in bovine mammary secretions: Quantitative changes in early lactation and absorption by the neonatal calf

Antibodies against Escherichia coli 08 in bovine colostrum and serum have been studied by gel filtration chromatography and the red cell linked antigen—antiglobulin reaction. Immunoglobulins were assayed by radial immunodiffusion.

In bovine colostrum anti-E. coli activity was attributable to IgA and the level of activity was comparable with that detected in IgM and IgG fractions. The immunoglobulin profile and distribution of E. coli antibodies in post-colostral calf serum was similar to that in colostrum, thus providing an unusual occurrence of high levels of secretory IgA in serum. However the immunoglobulin disappeared rapidly from the serum with an apparent half life of approximately 2 days; the value for IgM was 4 days.

During the first 3 days of lactation the levels of immunoglobulins fall rapidly and milk is subsequently secreted with uniformly low levels of antibodies.

     

Synthesis of immunoglobulins by biopsied tissues and cell lines from Burkitt's lymphoma

Cell suspensions of Burkitt''s lymphomas and cell lines derived from the same tumours were compared for immunoglobulin synthesis by analysis of the culture fluid. Thirty-one out of fifty tumour cell suspensions (i.e. twenty-one out of thirty-five patients) synthesized IgG (γ-chains) with type κ and/or type λ light chains; IgM synthesis was found in only five cases. Of the Burkitt''s lymphoma cell lines, twelve out of nineteen synthesized IgG (γ-chains); type κ light chains were produced by ten of these cell lines and type λ light chains by three. Only three cell lines synthesized μ chains. IgA synthesis was not detected in any of the biopsied tissues or cell lines.Comparison of the immunoglobulin synthesis by the cells of the biopsied tissue and the derived cell line showed very good agreement. This leads to the conclusion that the pattern of immunoglobulins synthesized by Burkitt''s lymphoma cell lines is representative of the original tumour.Investigation of cells from repeat biopsies, and serial testing of the derived cell lines showed that the capacity to synthesize particular immunoglobulins and chains remained constant.The fact that many of the biopsied tumour tissues and cell lines synthesized more than one immunoglobulin, or different classes of heavy chains and types of light chains, raises the question whether these immunoglobulin-producing cells originate from one or more cells.     

Porcine colostral IgA and IgM antibodies to Escherichia coli and their intestinal absorption by the neonatal piglet

Antibodies against Escherichia coli 0141 and Escherichia coli 08 have been studied in porcine colostrum and serum using gel filtration and DEAE-cellulose chromatography. Immunoglobulins were assayed by radial immunodiffusion.

Immune inhibition studies with a rabbit anti-IgA-globulin serum showed that a high proportion of antibodies in colostrum measured by the antiglobulin haemagglutination test were associated with IgA. The IgA antibody could not be detected in sow serum where the antibody was almost entirely confined to IgM. Thus it appears that colostral IgA antibody was synthesized in the mammary gland.

Studies of the absorption of immunoglobulins and antibodies from colostrum in six litters of piglets showed that although IgA was absorbed from the colostrum, colostral IgA antibody to E. coli was not acquired as part of the passive immunity of the neonatal piglet.

The absorbed antibody was associated with IgM; another high molecular weight immunoglobulin 18S IgG was also absorbed.

It is suggested that structures on secretory IgA which correspond to cellular receptors for intestinal transmission may be blocked by the integration of secretory `piece'.

     

The formation of immunoglobulins by human tissues in vitro: III. Spleen, lymph nodes, bone marrow and thymus

The site of immunoglobulin formation in human tissues was studied with three techniques: incubation of tissue fragments with radioactive amino acids followed by autoradiographic analysis of the synthesized immunoglobulins, immunofluorescent staining of tissue sections and cell suspensions, and morphological examination.

These studies showed that the spleen, lymph nodes and bone marrow synthesize IgG, IgA and IgM. In agammaglobulinaemia, one bone marrow sample showed no immunoglobulin synthesis, the other sample synthesized a trace of IgG. Immunofluorescent staining has demonstrated that in the spleen and lymph nodes plasma cells and large and medium-sized lymphocytes were positive for IgG, IgA or IgM; small lymphocytes were only positive for IgM. In bone-marrow samples, however, only plasma cells were positive for immunoglobulins. It is discussed whether in the bone marrow the cells that synthesize immunoglobulins do not originate in this organ, but derive from other lymphopoietic organs.

The normal thymus showed a different pattern because it synthesized only IgG and IgA. The plasma cells and medium-sized lymphocytes, which synthesize immunoglobulins, were localized predominantly in the interstitial connective tissue and ocasionally in the medulla, both near blood vessels. In all likelihood these cells did not originate in the thymus, but were trapped in this organ from the circulation. In autoimmune diseases, however, the thymus showed IgM-positive germinal centres and plasma cells and synthesized IgG, IgA and also IgM.

     

A simple method for the simultaneous detection of human immunoglobulins of both light chain types

In the method presented, an immuno-reagent is used which consists of a mixture of specific anti-light chain sera. When this mixture is reacted in double immunodiffusion or in immunoelectrophoresis with appropriate dilutions of normal human serum, two parallel precipitin lines appear: in the one, the precipitins are complexed with κ immunoglobulins and in the other with λ immunoglobulins. The immunoreagent is thus a κ/λ double-line (DL) antiserum. In fluids with disturbed κ/λ immunoglobulin distribution, shifting of one or other line and, consequently, a convergence or divergence of precipitin lines occur. The sensitivity of the method is demonstrated by analysis of normal serum to which critical amounts of isolated monoclonal IgC, IgA and IgM protein are added. The light chain typing of monoclonal proteins of all classes in myeloma sera can be done in a single experiment. In addition, the technique demonstrates a disbalance of κ/λ immunoglobulin distribution in chromatographically isolated ‘normal’ human IgG.     

Immunoglobulin synthesis by human peripheral lymphocytes and thymocytes in vitro. Specificity of immunochemical methods and stimulation by phytohaemagglutinin

Antibody–antigen precipitates were formed in solutions of radioactive products of human lymphocytes. The amount of radioactive material co-precipitating with non-specific, apparently unrelated, antibody–antigen precipitates was of the same magnitude as that co-precipitating with related, specific precipitates of IgG–anti-IgG or Tg–anti-Tg. Treatment of the solutions with an unrelated EA–anti-EA precipitate readily removed material capable of reacting with IgG–anti-IgG or Tg–anti-Tg precipitates.

By contrast the evidence obtained with the technique of autoradiography of immunoelectrophoresis patterns indicated that the labelling of immunoglobulin arcs, and of a specific antibody–antigen precipitate, was specific. Only products of the lymphocyte from a subject with Hashimoto's disease labelled a Tg–anti-Tg line, although the control preparations from normal lymphocytes labelled the immunoglobulin arcs on the other side of the pattern. When mouse serum was used as carrier, and the immunoelectrophoresis was developed with rabbit anti-mouse serum, the immunoglobulin lines were not labelled. Products of human lymphocytes did not label an EA–anti-EA line in the immunoelectrophoresis.

The anodal ends of gels were eluted after electrophoresis of radioactive products synthesized by lymphocytes. The amount of material co-precipitating in these eluates with specific precipitates was 1·8 times greater than that adhering to non-specific precipitates. These data raise the possibility that immunoglobulin molecules may be non-specifically adsorbed to unrelated antigen–antibody precipitates.

The data indicated that phytohaemagglutinin stimulated synthesis of immunoglobulin was less than the overall increase of protein synthesis. Phytohaemagglutinin sometimes stimulated the total protein synthesis while inhibiting IgG synthesis.

     

The formation of immunoglobulins by human tissues in vitro: IV. Circulating lymphocytes in normal and pathological conditions

The formation of immunoglobulins by circulating lymphocytes was studied by three techniques: (1) Autoradiographic analysis of the immunoglobulins synthesized during the incubation of cell suspensions in a medium with radioactive amino acids; (2) direct immunofluorescent staining; and (3) examination of the cellular morphology.

Lymphocytes of the normal peripheral blood were found to synthesize a distinct amount of IgG and smaller amounts of IgA and IgM. Cells of the thoracic-duct lymph synthesized distinct amounts of all three immunoglobulins. A similar pattern was found in infectious mononucleosis and rubella. In infectious mononucleosis the significantly increased synthesis of IgM during the first 10 days of illness led to the supposition that this result may be due to primary antigenic stimulation. The pattern in chronic lymphatic leukaemia is characterized by the consistent absence of IgA and the labelling of IgG, mainly the medium to high mobility part, and of IgM. In agammaglobulinaemia a trace of IgG and IgA was found in one case; the other was entirely negative.

The immunofluorescent staining showed that in all samples some of the medium-sized lymphocytes contain IgG, IgA or IgM. Peripheral blood samples taken during an infectious mononucleosis or rubella infection and thoracic duct lymph revealed also positive large lymphocytes and plasma cells.

A remarkable observation was the weak fluorescence of small lymphocytes which were exclusively positive for IgM. It is postulated that these small lymphocytes indicate their initial synthesis of IgM antibodies when engaged in primary response.

     

Production and secretion of Leishmania braziliensis proteins

The kinetics of secretion of proteins by Leishmania braziliensis was followed by incorporation of [³H]leucine into macromolecules produced by the cells which are released into the growth medium. About 10% of the total protein synthesized by actively growing cells is secreted. Cycloheximide (100 μg/ml) and puromycin (0.5 mM) inhibited the incorporation of labelled leucine by 85 and 99%, respectively. The secreted proteins do not seem to results from cell lysis since, first, the kinetics of production are linear and, secondly, less than 1% the thymidine or uridine incorporated by the cells is found in the medium. Cells grownh with [³H]leucine and then transferred to fresh medium show two phases of secretion. During the first six hours, it is slow and reaches a plateau. The release increases about ten-fold during the next six hours. An analysis of the secreted material showed that following precipitation with methanol and sodium acetate, three isotopically labelled peaks were eluted from Sephadex G-120–150. The first of these, containing 50% of the radioactivity, did not react with anti-leishmanial serum, while the last two did. Since the last two fractions could be labelled with [³H]glucosamine as well as [³H]leucine it is suggested that they are glycoprotein in nature and are similar to the products released by other species of Leishmania.     

Established immunoglobulin producing myeloma (IgE) and lymphoblastoid (IgG) cell lines from an IgE myeloma patient

Two IgEL and one IgGK producing cell lines have been established in vitro from peripheral blood and bone marrow of an E myeloma patient. Morphologic and immunologic studies indicate that cells of the IgE producing lines are derived from the same clone of myeloma cells which grew in vivo. They have remained essentially unaltered with a stable near-diploid karyotype and continuous production of intact IgE molecules during 18 months in culture. The IgGK producing line has all the characteristics ascribed to permanent lymphoblastoid lines and is considered to be of non-malignant lymphoid origin. The process of establishment is described and it is suggested that lymphoblastoid cells have a selective advantage over myeloma cells. Permanent myeloma cell lines can therefore probably be obtained only if cells capable of forming established lymphoblastoid lines are very few or absent in the original biopsy. The establishment of a permanent cell line continuously producing intact molecules of IgE should permit further studies on the biological role of this class of immunoglobulins.     

Initial studies on the properties of a bovine lymphoid cell culture line infected with theileria parva

Observations were made on a bovine lymphoblast cell culture line, C2, permanently infected with the protozoan parasite Theileria parva. No specific parasite antigen was detected on the C2 cell surface, either by a fluorescent technique or by antibody-dependent cell-mediated cytotoxicity. There was no detectable surface immunoglobulin or secretion of immunoglobulins into the tissue culture medium.

Using immunofluorescence and calf thymus and bone marrow antisera, C2 cells were found to share a membrane antigen with normal calf thymus cells, and also to possess a strong transplantation antigen. Bovine antisera against the latter antigen can initiate killing of C2 cells by normal bovine mononuclear leucocytes in antibody-dependent cell-mediated cytotoxicity. C2 cells can also act as weak stimulators in a mixed leucocyte reaction.

The possible role of such transformed parasite-infected lymphoblasts in the strong functional immunity that follows recovery from Theileria parva infection is discussed.

     

Serum immunoglobulin levels in various skin diseases

Serum immunoglobulin (IgG, IgA, IgM) determinations by the single radial immunodiffusion technique were done in patients with various skin diseases. The geometric means of the IgG, IgA and IgM concentrations of each of the patient groups were compared with those of a control group comprising 156 individuals.

A significantly raised IgG level was found only in patients with pemphigus; in patients with dermatitis herpetiformis, other eczemas, and psoriasis vulgaris the IgG level was significantly lowered. The IgA level was significantly elevated in patients with discoid lupus erythematosus and (bullous) pemphigoid, and the IgM level was significantly depressed in patients with atopic dermatitis.

IgE determinations, done with the Ouchterlony technique in a selected group of patients, showed raised levels in some patients with malignant reticulosis and other eczemas but not in those with atopic dermatitis.

Comparison of the results of the serum immunoglobulin determinations with the immunoglobulin synthesis of lesional skin showed no correlation between the local synthesis of immunoglobulins in the skin and changes in the serum levels.

     

Experimental allergic encephalomyelitis: immunological properties of encephalitogenic polypeptides and proteins

Two fractions, a protein and a polypeptide, from extracts of acetone dried powders of central nervous tissue produced experimental allergic encephalomyelitis (EAE) when injected with Freund's complete adjuvant, in doses as minute as 50 ng for the bovine encephalitogenic polypeptide (BEP).

All bovine and guinea-pig encephalitogens produced EAE in guinea-pigs but only guinea-pig and not bovine encephalitogens produced EAE in the Wistar rat.

The bovine and guinea-pig protein encephalitogens, in the guinea-pig particularly, induced antibody and delayed hypersensitivity against these proteins and EAE. The polypeptide encephalitogen, BEP, induced no humoral antibody reacting with BEP itself, but did induce delayed hypersensitivity to BEP and EAE; however, BEP induced antibody which cross-reacted with the protein encephalitogens from both species. Precipitin reactions with these basic proteins in immunodiffusion were optimal at a slightly acid rather than neutral pH.

Observations on cross-reactions among the neural fractions by immunodiffusion and delayed hypersensitivity suggested that a similar encephalitogenic determinant could be present in the various encephalitogens from the two species. Further work with enzymatic digests of our encephalitogenic polypeptides may bring us nearer our goal which is the identification of the molecular grouping in myelin essential for the induction of EAE.

     

Immunoglobulin production in the European pond tortoise, Emys orbicularis, immunized with serum protein antigens

The immunological responses of the European pond tortoise, Emys orbicularis, to BGG and sheep serum proteins indicate that in this tortoise there is no serum component corresponding with mammalian albumin and that both γG (7S) and γM (19S) immunoglobulins are involved when antibodies are produced. A prolonged period of γM antibody production occurs after primary immunization. The separation of the γM and γG immunoglobulins was performed using starch block electrophoresis and Sephadex G-200 gel filtration. The separated immunoglobulins were characterized by immunodiffusion and by starch gel, agar gel and immunoelectrophoresis.     

Concomitant synthesis, in separate cells, of non-reactive immunoglobulins and specific antibodies after immunization with tobacco mosaic virus

Injection of tobacco mosaic virus into rabbits induces the concomitant synthesis of specific antibodies and of non-reactive immunoglobulins. Labelling experiments show that the amount of non-specific immunoglobulins produced for a given amount of antibodies is much greater after a first stimulation than after a booster injection.

Immunofluorescence studies on the spleen of animals before and after immunization clearly indicate that the increase in concentration of non-specific immunoglobulins observed in the serum is correlated with a significant increase in the number of cells secreting non-reactive immunoglobulins simultaneously with the appearance of antibody producing cells.

Finally cell counts and in vivo and in vitro synthesis by spleen fragments of specific antibodies and non-reactive immunoglobulins provide a strong argument in favour of a synthesis of non-reactive immunoglobulins and antibodies in different plasma cells.

The possible significance of the induced synthesis of non-specific immunoglobulins after antigenic stimulation are discussed.

     

The effect of phytohaemagglutinin and other lymphocyte mitogens on immunoglobulin synthesis by human peripheral blood lymphocytes in vitro

Amino acid incorporation techniques and immunofluorescence have been used to investigate the effect of mitogenic substances on immunoglobulin synthesis by human peripheral blood lymphocytes in vitro. Radioelectrophoresis, radioimmunoelectrophoresis and controlled immunological precipitation methods suggest that only a small amount of immunoglobulin is synthesized in the culture system used. Immunofluorescent staining of fixed cell preparations showed that during the first 24 hr in culture only a small percentage of cells reacted positively for immunoglobulin; after 24 hr these cells were no longer demonstrable. This suggests that the small amount of immunoglobulin detected was synthesized during the first few hours in culture by these cells, having the morphological appearance of medium lymphocytes. The slight enhancement of immunoglobulin synthesis obtained in one experiment with phytohaemagglutinin (PHA) probably occurred within this same cell type since after 24 hr in vitro no cells in the transformed cultures could be stained by the fluorescent anti-immunoglobulin. Fixed preparations of blast cells obtained by stimulation with anti-lymphocytic serum and staphylococcal filtrate also gave negative reactions. However, using a staining technique with suspensions of viable cells, it was possible to demonstrate positive staining for immunoglobulins with PHA stimulated cells as previously described by Ripps & Hirschhorn (1967). A number of controls suggest that this reaction depends upon the presence of exposed immunoglobulin groups or markers on the cell surface and that intracytoplasmic staining is the result of endocytosis of conjugate.     

In vitro synthesis of immunoglobulins, secretory component and complement in normal and pathological skin and the adjacent mucous membranes

A study on the synthesis of immunoglobulins (IgG, IgA, IgM, IgD, and IgE), secretory component and complement in normal and pathological skin and in the adjacent mucous membranes (i.e. conjunctiva, nasal, oral and vaginal mucosa) is reported. The results are based on the culture of tissue samples in a medium with two radioactive amino acids and the detection of synthesized proteins by autoradiography of the immunoelectrophoretic pattern of the culture fluid, except in the case of IgE for which the Ouchterlony technique was used.

The results indicate that the normal skin does not synthesize immunoglobulins, whereas normal mucous membranes produce IgG and IgA. In the lesions of various skin diseases immunoglobulins are synthesized, mainly IgG but sometimes also IgA and IgE. The cells responsible for the production of immunoglobulins are plasma cells and lymphoid cells present in the skin lesions and mucous membranes.

Synthesis of the free secretory component could be demonstrated only in certain mucous membranes (i.e. conjunctiva, nasal mucosa, and oral mucosa).

Complement (C3) synthesis was found in normal skin, mucous membranes (i.e. conjunctiva, nasal and oral mucosa), and in the lesions of such skin diseases as discoid lupus erythematosus, (bullous) pemphigoid, dermatitis herpetiformis, malignant reticulosis, eczema and lichen planus. Complement production was also demonstrated in allergic skin reactions (i.e. tissue from allergic-positive patch tests, positive Mantoux tests and drug eruptions), but no immunoglobulin synthesis was detected in these lesions.

     

Isolation of t cell membrane proteins (IgT) with antisera to non-isotypic determinants of immunoglobulins: Evidence thaT IgT ‘Light’ chains are not identical to B cell kappa chains

Radiolabelled cell membrane proteins obtained by detergent lysis of murine T cells labelled by lactoperoxidase-catalysed radioiodination were specifically immunoprecipitated with an antiserum to immunoglobulin Fab determinants. Absorption of the antiserum by myeloma proteins or normal immunoglobulins coupled to Sepharose and the use of antibody proteins eluted from these absorbents indicated that the antibody activity in the serum responsible for binding detergent-soluble T cell membrane proteins was directed towards variable region and not to the constant region determinants of kappa light chains. Immunoglobulin light chain sized polypeptides isolated from T cells with this antiserum were found to possess serological characteristics which differed from serum or B cell membrane immunoglobulin kappa chains. Evidence is presented which suggests that a portion of the light chain sized polypeptides isolated from T cells may be proteolytic fragments of a larger polypeptide chain. The results suggest that immunoglobulin-like detergent-soluble T cell membrane proteins isolated with antisera to immunoglobulins (IgT) possess neither immunoglobulin heavy nor light chain constant regions determinants but do carry determinants associated with immunoglobulin variable regions.     

Mitogenic effect of HIV-infected human T cell lines on mouse B cells mediated by surface immunoglobulin

Following HIV-1 infection, a number of disorders are induced in both normal T and B cells by virus products derived from infected CD4⁺ T cells. In the present study, we found that HIV-infected, but not uninfected, human T cell lines generated vigorous blastogenesis and proliferation of freshly isolated mouse B cells in a short-term culture. Neither human B cells nor rat B cells showed significant responses to the HIV-infected T cell lines in the present condition. The mitogenic effect of HIV-infected human T cell line requires direct cell–cell interaction between mouse B cells and HIV-infected T cell lines. Since either mitomycin c treatment or paraformaldehyde fixation of HIV-infected T cell lines resulted in complete loss of the mitogenic effect, it seems that de novo synthesized viral products are responsible for this effect. Furthermore, anti-mouse immunoglobulin antibody inhibited completely the B cell stimulation by the HIV-infected human T cell lines. Thus, surface immunoglobulin (sIg) on mouse B cells appears to be an essential molecule which transduces activation signals from HIV-infected human T cells into cytoplasm of the B cells.     

Reactivity of radioiodinated serum antibody from Burkitt's lymphoma and nasopharyngeal carcinoma patients against culture lines derived from Burkitt's lymphoma

The IgG serum immunoglobulin fraction of two Burkitt's lymphoma (Mutua and Kiliopa) and one African nasopharyngeal carcinoma patient (Kipkoech) was conjugated to iodine-131 (¹³¹I). It is known from previous studies with fluorescein labelled conjugates that all three sera contain antibody against the Epstein–Barr virus (EBV)-associated membrane antigen complex, present on the surface of lymphoblastoid cells in EBV-carrier cultures. All three radioiodinated conjugates attached to live cells of an EBV-carrying Burkitt line (Maku), but not to EBV-free Raji cells. A Swedish control serum (Berith) did not block the binding of any of the three conjugates, whereas unconjugated sera of Mutua, Kiliopa and Kipkoech showed various degrees of blocking and cross-blocking. The blocking patterns were in good agreement with previous tests, performed with the same sera against their fluorescein conjugated derivatives.

Antibody release tests, involving preincubation of live cells with one of the three conjugates, followed by incubation with unlabelled serum revealed a certain `hierarchy' between the three sera with regard to their ability to displace radioiodinated surface-coupled immunoglobulin. This ability could be related to the competitive behaviour of the same sera in the cross blocking tests. The results are believed to reflect differences in the affinity of the three antibodies, due either to differences in fit in relation to the surface antigen(s) carried by the Maku target cell, or to differences in the duration of immunization in the three patients.