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Since the dawn of history, human beings have witnessed the appearance of epidemic or epizootic diseases. The suddenness and the prevalence of these plagues were generally considered to be connected with occult influences of the stars or planets upon human affairs, climatic changes or religious reasons. Slowly, the principle of the origins of contagious diseases has become better understood and the role of transmissible influences such as parasites, bacteria and viruses has been accepted. A landmark was discovery of the germ theory, which included small parasites, bacteria and viruses. This theory was mainly based on the studies of Koch, Lister, Pasteur and many others.

IgG(T) and IgGa isotypes were isolated from horse hyperimmune anti-bothropic and anti-crotalic sera using a combination of two affinity chromatographic processes. IgG(T) and IgGa isotypes were isolated from these sera by chromatography on protein A-Sepharose followed by separation of the two isotypes by chromatography on a column of anti-IgG(T)-Sepharose. LO-HoGT-1, a rat anti-horse IgG(T) monoclonal antibody, was used. A comparative study of the efficiency of these isotypes in neutralizing the main toxic activities of the homologous venoms was carried out. It was found that IgG(T) was about three-fold and seven-fold more protective than IgGa for neutralization of the lethal activity of B. jararaca and C. d. terrificus venoms, respectively. IgG(T) was also more effective than IgGa for the neutralization of the haemorrhagic activity induced by B. jararaca venom, while both isotypes neutralized equally well the blood incoagulability induced by this venom. The results suggest that IgG(T) is the most protective isotype present in both anti-bothropic and anti-crotalic sera, followed by IgGa. Owing to their very low concentration in the serum, other IgG isotypes are not likely to be important in neutralizing the venoms' toxic activities.

Attachment and ingestion of 51Cr-labelled TNP-SRBC sensitized by rat IgG1, IgG2a or IgG2b-type antibodies by homologous, elicited peritoneal macrophages were studied. IgG1 was found to be the most efficient isotype in mediating these functions. The antibody doses required for a significant attachment were found to differ with the isotype of Ab, while doses needed for a significant phagocytosis and antibody-dependent cellular cytotoxicity (ADCC) varied between 400-700 Ab/SRBC with all the isotypes studied. Both binding and phagocytosis were also influenced by the degree of hapten conjugation when target cells were sensitized by IgG1. Inhibition of these functions by soluble immune complexes and monomeric immunoglobulins suggests the involvement of two Fc gamma R in binding of the three isotypes. Based on the present work and on previous results we conclude that IgG2a interacts with a receptor binding complexed IgG only (Fc gamma RII), IgG2b binds to a different receptor which appears to bind monomeric ligand as well (Fc gamma RI), while IgG1 seems to interact with both types of receptor. We propose that phagocytosis can be mediated by both Fc gamma RI and Fc gamma RII.

The interaction between macrophages (M phi) and antibody sensitised target cells was studied by the use of rat peritoneal macrophages, TNP hapten conjugated sheep red blood cells (SRBC) and homologous antibodies of subclasses IgG1 and IgG2a. Under optimal conditions, the great majority of the M phi formed rosettes with IgG1-sensitised antibodies while a maximum of 50% was achieved when target cells were sensitised by IgG2a. Using a double rosette technique, the major part of rosette-forming cells was found to bind both of the isotypes. IgG1-mediated rosette formation was observed at very low degrees of sensitisation as opposed to IgG2a-mediated target cell binding. Not only the amount of bound antibody but also the degree of hapten conjugation (epitope density) appear to influence the ratio of rosette-forming cells. IgG1-mediated rosette formation was partially inhibited by monomeric IgG1 and more efficiently by soluble ovalbumin (OVA)-anti-OVA complexes involving IgG1-type antibodies, while IgG2a mediated rosette formation was inhibited by OVA-anti-OVA complexes containing IgG2a type antibodies, and less efficiently by complexes involving IgG1. No inhibition was found by monomeric IgG2a. Based on the present data, we propose that two types of receptors are involved in the interaction of M phi and target cells coated by IgG1 and/or IgG2a type antibodies. One requires a multiple antibody-receptor interaction, binding both subclasses at overlapping binding sites; the other is able to interact with IgG1 and does not depend on the multiplicity of interactions.

Lysis of 51Cr-labeled TNP-SRBC sensitised by rat IgG1 or IgG2a type antibodies by homologous, paraffin oil-elicited peritoneal macrophages (ADCC) or by homologous complement was studied. IgG2a was found to be markedly more efficient in mediating both ADCC and complement dependent lysis compared to IgG1. Inhibition of the ADCC pointed to the involvement of separate but partially overlapping interaction sites for the two isotypes. We suggest that FcRII type receptors play a favoured role in both IgG2a and IgG1 mediated ADCC. The threshold amount of bound antibody required for ADCC was lower than that sufficient for complement dependent lysis regardless on the subclass or on hapten density. The extent of lysis (both ways) was found to depend on hapten density using equal amounts of antibody. The results are interpreted as terms of the possible requirements for association of IgG molecules on the target cell surface.

Expression of Ig isotypes other than IgD together with IgM on the membranes of single B cells has been reported in different experimental models. This paper describes the co-expression of IgG2b or IgE with IgM-IgD on the surface of single B cell subpopulations from normal rats. Their expression was demonstrated with anti-IgE or IgG2b monoclonal antibodies and their F(ab')2 fragments. After pronase digestion, the re-expression of these isotypes together with IgM-IgD was observed in vitro and was inhibited by cycloheximide. These observations imply that mechanisms other than class switching may participate in the expression of membrane isotypes in vivo. The role of these membrane isotypes is still to be established, but could be important as IgG2b molecules are found on a large B cell subpopulation.

Immune complexes, prepared with monoclonal rat IgA antibodies directed against DNP, activate the alternative pathway of the complement system in rat serum. In this study, the interaction of these monoclonal IgA antibodies with the classical pathway of complement was investigated. Monoclonal polymeric IgA (p-IgA) was shown to inhibit the IgG2b-mediated classical pathway-dependent lysis of TNP-coated sheep red blood cells. In addition, the binding of C3 to solid phase IgG2b immune complexes was inhibited by p-IgA. Monoclonal monomeric IgA (m-IgA) was much less efficient in this respect. To further analyse the effect of p-IgA on the activation of the classical pathway by IgG2b immune complexes, the interaction of p-IgA with C1 was studied. It was found that p-IgA antibodies bind C1q. No species-specificity was observed, since both rat and human C1q were bound. Whereas binding of C1q in C1 to IgG2b resulted in activation of C1, binding to p-IgA did not. The binding of C1q to both p-IgA and IgG2b could be inhibited by monoclonal antibodies directed against the globular heads of C1q, but not by monoclonal antibodies directed against the collagen tail. The formation of insoluble p-IgA immune complexes was inhibited in the presence of rat serum or C1. These studies indicate that C1q binds to p-IgA by its globular heads, and thereby may modulate classical pathway-mediated reactions such as the inhibition of immune precipitate formation.

The binding sites of rat IgE to mast cell receptor were investigated by the use of proteolytic fragments and a monoclonal antibody to epsilon chain (MARE-1). Three main fragments were characterized by short-time papain digestion of IgE: F(ab')2-E, a fragment related to the C, 4 domain, and an asymmetric fragment corresponding probably to an IgE molecule with one proteolyzed C, 3 domain. Neither F(ab')2-E nor C, 4 could interfere with the binding of IgE to rat mast cells. These two fragments did not show significant polymerization upon heating at 56 degrees C, while large amounts of polymers were produced from whole IgE, MARE-1 monoclonal antibody was found to react neither with F(ab')2 nor with C, 4, thereby suggesting its interaction with the C, 3 domain. MARE-1 was found to inhibit partially (about 55%) the binding of IgE to its receptor. Taken together the results indicate that the binding sites of IgE to rat mast cell receptor are located within the C, 3 domain. In addition, isolation of the C, 4 domain will be useful to evaluate its participation in the affinity of IgE to receptors of other cells such as lymphocytes or macrophages.

Detection of bone marrow metastases by indirect immunofluorescence methods was investigated using three monoclonal antibodies (MoAbs) raised against small cell lung cancer (SCLC). These antibodies, designated anti-LCA1, -LCA2 and -LCA3, recognize three different antigens on the surface of SCLC cells. Eighty-four bone marrow samples from 74 different patients were studied. Whereas tumor cells were found in 32 (38%) by MoAb staining, only 10 (12%) were positively identified using conventional morphological methods. Nine out of the morphologically positive specimens showed reactivity with at least two monoclonal antibodies. Among the 32 samples proven positive by immunofluorescence, an important antigenic variability was noted. Anti-LCA1 recognized tumor cells in 62%, anti-LCA2 and anti-LCA3 in 53%. Due to the recognition of bone marrow involvement by fluorescence methods in 26% of the 34 patients classified as limited disease, a new subgroup of limited disease patients was defined whose prognosis remains undetermined. Our results confirm the utility of immunodetection in the diagnosis of SCLC bone marrow metastases and emphasize the advantage of using a panel of MoAbs with different antigenic specificities. Further study is needed to determine the prognostic significance of bone marrow involvement established by immunodetection.

Rat basophilic leukemia (RBL) cells carry two surface glycoprotein molecules named R (or alpha) and H which, when detergent solubilized, bind to rat IgE-Sepharose. The same two molecules also bind to rat IgG-Sepharose but with a lower affinity. R is a component of the high affinity Fc receptor for IgE. In the present study the inhibition of the binding of R and H to rat IgG-Sepharose by various homologous and heterologous immunoglobulins was used to assess their relative affinities for the two receptor molecules. Ranking the rat immunoglobulins in order of their affinities for the R receptor yielded: IgE much greater than IgG2a greater than IgG1 greater than IgG2b; and for H: IgE greater than IgG2b greater than IgG1 greater than IgG2a. Rat IgG2c inhibited the binding of both R and H but a precise ranking could not be assigned. Conclusive evidence has been obtained for the Fc specificity of these interactions. The affinities of the mouse IgG subclass/R interactions can be ranked: IgG1 greater than IgG2a greater than IgG2b; and for the H receptor: IgG1 greater than IgG2b greater than IgG2a. All of the mouse proteins and other heterologous IgGs, such as those of sheep, goat, equine and rabbit origin, interacted considerably more strongly with H than with R. No interaction with mouse IgG3 could be detected under the conditions tested.

Two monoclonal IgEs (IR 2 and IR 162) were studied in terms of their conformational features by circular dichroism and differential u.v. absorption. The studies were performed both on the native proteins and under different conditions known to affect the biological properties of IgE (heating at 56 degrees C, acid pH, and presence of reducing agents). Whereas IgE IR 162 appeared to undergo significant conformational changes upon heating and at acid pH, IgE IR 2 was found to be unaffected or less affected by these treatments. The two monoclonal IgEs were found to be equally effective in blocking passive sensitization by IgE antibodies. However, the blocking capacity of IgE IR 162 was affected much more by heating at 56 degrees C than that of IgE IR 2.

Monoclonal rat IgE was reduced over a range of dithiothreitol (DTT) concns. The number of disulfide bonds reduced and their location in the IgE molecule were studied. One millimolar DTT was found to split the two inter-heavy-chain disulfide bonds of the C epsilon 2 domain while increasing DTT concn to 10 mM split the two inter-heavy-light-chain disulfide bridges. Therefore, the sensitivities to reduction of disulfide bonds in rat IgE were found to be the opposite of those in human IgE. In addition, the results indicated the absence, in rat IgE, of the intra-epsilon-chain labile disulfide bond of the C epsilon 1 domain, which is reduced by 2 mM DTT in human IgE. Circular dichroism studies showed significant modifications, mainly of tertiary structure, for rat IgE reduced with 10 mM DTT, but not for IgE reduced with 1 mM DTT. The ability to block passive sensitization with reaginic antibody was not modified when IgE was reduced with 1 mM DTT (which split the two inter-heavy-chain disulfide bonds), but was lost when inter-heavy-light-chain bridges were reduced with 10 mM DTT. In addition, a non-covalent epsilon-chain dimer was found to have the same blocking activity as native IgE (or IgE reduced with 1 mM DTT). Therefore, the results suggest that reduction of most or all the inter-chain disulfide bonds, in rat as in human IgE, induces changes in quaternary structure, more especially in the relationship between the Fab and Fc parts of the molecule, leading to steric blockade, by Fab, of the binding sites for mast cells present on Fc.

The binding kinetics of radiolabelled rat IgE to Fc epsilon receptors (R) of rat B-cells have already been studied in IgE-stimulated animals. The receptors expressed after Nippostrongylus brasiliensis infection or 2 injections of 5 mg IgE/100 g body wt were class-specific: IgE binding was not hindered by rat IgM, IgD, IgA, IgG1, IgG2a, IgG2b and IgG2c in immunocompetitive-binding assays. The rat B-cell Fc epsilon R were not species-specific, since mouse IgE competes with rat IgE for binding to these receptors. The apparent number of Fc epsilon R on rat mesenteric lymph node B-cells varied with temp and was 1.1-2.4 X 10(5) at 4 degrees C and 5.9-7.7 X 10(5) at 37 degrees C. The experimental Ka was not influenced by temp and had an average value of 1.38 X 10(8) M-1. At 4 degrees C the IgE binding to B-cell Fc epsilon R had an association rate of 4.9 X 10(3) M-1 sec-1 and a dissociation rate of 4.65 X 10(-5) sec-1. After a very strong stimulation produced by injecting 5 mg IgE/100 g body wt every 24 hr for 5 days, the equilibrium binding curve became diphasic, indicating a probable heterogeneity of the B-cell Fc epsilon R.

Digestion with trypsin of monoclonal rat IgG of IgG1 and IgG2a subclasses produced two fragments, isolated only in dissociating media. The larger fragment (mol. wt. 120,000 Da) was comprised of the two light chains covalently bound to shortened gamma chains. Amino acid sequence of the shortened gamma chain indicated that the site of cleavage is located at the beginning of the C gamma 1 domain at a position homologous to residue 139 of mouse gamma heavy chain of IgG1 MOPC 21. The smaller fragment (mol. wt. 13,000 Da) was found to consist of the entire variable domain of the heavy chain and probably a short stretch of the C gamma 1 domain. The unique susceptibility of rat monoclonal IgG1 and IgG2a is likely to be the result of the presence of a lysine residue in a loop of C gamma 1 domain, which therefore is accessible to trypsin. Tryptic cleavage of rat monoclonal antibodies of IgG1 and IgG2a subclasses can be considered as a simple method to produce a fragment related to the VH domain.