Transfer of ovine experimental allergic glomerulonephritis (EAG) with serum.

Serum globulin from donor sheep made nephritic by immunization with glomerular basement membrane and subsequently nephrectomized contained specific kidney-fixing antibody and was capable of inducing an immediate, although transient, glomerulonephritis when injected into unilaterally nephrectomized lambs. This nephritis was characterized by immediate proteinuria, PMN infiltration into the glomerulus, and localization of gammaG- and beta1C-globulins in a linear fashion along the recipients' glomerular capillary walls. The nephritogenic property of the serum could be absorbed in vitro with isolated sheep glomerular basement membranes.

The role of anti-glomerular basement membrane antibody in the pathogenesis of human glomerulonephritis.

These observations established the presence of anti-GBM antibodies in the sera and/or kidneys of six humans with glomerulonephritis. Further, it seems clear that these antibodies do combine with the host's glomeruli in vivo and with GBM antigen of several species in vitro. Transfer of acute glomerulonephritis to normal recipient monkeys was possible with serum or renal eluate IGG from the three patients with anti-GBM antibodies in whom sufficient material was available. Based on this transfer of nephritis and on the presence of these antibodies at the site of injury in the nephritic kidneys of both the patients and the recipient monkeys, it seems likely that they are at least a contributing, if not primary, cause of the glomerular injury. The frequency of anti-GBM antibodies in human nephritis is not certain, but on the basis of preliminary observations it would appear that they are present in all cases of Goodpasture's nephritis and somewhat less than half of the cases of subacute and chronic glomerulonephritis of adults. The nature and source of immunogen stimulating the production of anti-GBM antibodies is not known, but the presence of potentially nephritogenic GBM antigens in normal urine raises the question of possible autoimmunization. From a practical point of view, it appears that patients forming anti-GBM antibodies may not be good candidates for renal transplantation since they are likely to produce in the transplants the nephritic changes already suffered by their own kidneys.

Synthesis of plasma membrane-associated and secretory immunoglobulin in diploid lymphocytes.

The half disappearance time for detectable plasma membrane-associated and cytoplasmic immunoglobulin after treatment of continuously growing diploid lymphocytes with inhibitors of protein and RNA synthesis was studied. Also, the amount of plasma membrane-associated and cytoplasmic immunoglobulin of synchronized cells in the G(1) phase of the cell cycle has been studied. Plasma membrane-associated immunoglobulin has a half disappearance time of 45 min after inhibition of protein synthesis. By contrast, after treatment of cells with actinomycin D for 24 hr, plasma membrane-associated immunoglobulin remains relatively unchanged whereas cytoplasmic immunoglobulin decreased by almost 90%. In the G(1) phase of the cell cycle, plasma membrane-associated immunoglobulin and cytoplasmic immunoglobulin were 70 and 10%, respectively, of that in logarithmically growing cells, and the half disappearance of M-Ig after treatment of cells with puromycin was again 45 min. In toto, these results suggest that perhaps secreted and plasma membrane-associated immunoglobulin may be separately controlled by the cells.

The oncornavirus glycoprotein gp69/71: a constituent of the surface of normal and malignant thymocytes.

The oncornavirus related proteins associated with the surface of normal and malignant thymocytes were studied. Three virion-associated proteins (gp69/71, p45, p30) were associated with lymphoma cells from about 70% of the tumors studied. Two virion-associated proteins (gp69/71 and p45 were associated with normal thymocytes form some but not all strains of mice. In gp69/71- mice, conversion to the gp69/71+ phenotype accompanied leukemogenesis. An interesting difference in the apparent molecular size of virus related antigens of the 70,000 dalton size class was detected in lymphoma cells present in involved spleens as compared to involved thymuses. Mice infected as neonates with Scripps leukemia virus make antibody to gp69/71 and some make antibodies to molecules associated with the surface of their own tumors. The significance of the restricted presence of antigens coded for by the viral genome to the surface of some differentiated cells is discussed in reference to (a) the relationship between virion, leukemia associated, and differentiation dependent markers, and (b) the possible consequence to the host of having similar antigenic determinants on three independent structures with replicative potential (virus, normal thymocytes, and tumor cells).

Production of macrophage migration inhibition factor by continuous cell lines.

Macrophage migration inhibitory factor (MIF) was found in media of human and mouse lymphocyte and fibroblast cell lines that were continuously growing. Its release was dependent on activation of the cells to enter the mitotic cycle, particularly on cells in S phase. The greatest quantity of MIF was detected in supernatants of lymphocytes collected during S phase after the cells were synchronized in G(1) and in supernatants of growing fibroblasts. When the latter were contact inhibited little or no MIF was found in media. MIF was also released into media of cells proliferating in homologous serum in the absence of fetal calf serum and into media lacking any protein. The MIF produced by lymphocyte lines eluted from Sephadex G-100 in the same fashion as MIF produced by the interaction of sensitized guinea pig cells and antigen. The results indicated that MIF is not a specific mediator of delayed hypersensitivity and cellular immunity and that MIF released by sensitized lymphocytes incubated with antigen merely reflects that fraction of cells activated by antigen to enter the mitotic cycle.

Endogenous oncornaviral gene expression in adult and fetal mice: quantitative, histologic, and physiologic studies of the major viral glycorprotein, gp70.

Endogenous expression of the murine leukemia virus (MuLV) genome has been studied in a number of strains of mice. Expression of the major envelope glycoprotein, gp70, is restricted to certain anatomical sites and cell types, prominent among which are lymphoid and epithelial cells. On a quantitative basis, the major site of gp70 expression is the male genital tract. During development, gp70 first appears in the hematopoietic liver of 14-day-old embryos and by day 18, it is already expressed at anatomical sites similar to those of the adult. In toto, these results show that control of expression of the MuLV genome in adult and developing mice is linked to differentiation.

Immunopathogenicity and oncogenicity of murine leukemia viruses. I. Induction of immunologic disease and lymphoma in (BALB-c times NZB)F1 mice by Scripps leukemia virus.

This report clearly demonstrates that a systemic lupus erythematosus (SLE)-like syndrome and lymphoma can be induced in immunologically normal (BALB/c x NZB)F(1) mice by infection of neonates with a murine leukemia virus (MuLV) (Scripps leukemia virus [SLV] 60A) isolated from NZB lymphoblasts. SLV 60A was titered in vitro (XC test) and administered to newborn and adult (BALB/c x NZB)F(1) mice over six log(10) dilutions. Propagation of MuLV in the newborn recipients was indicated by greatly elevated serum Mu gs-1 levels which were proportional to the dose of virus given. The SLE-like syndrome was characterized by antinuclear antibodies (ANA) and immune complex-type glomerulonephritis. ANA production was related to the dose of virus and reached the highest levels at 8-16 wk. The incidence of glomerulonephritis was also correlated with the dose of virus and reached nearly 50% in the animals given the highest virus dose. Both titers of ANA and incidence of glomerulonephritis were greater in females than in males, although the amounts of Mu gs-1 in sera of both sexes were equal. The incidence of direct Coombs' positivity was not significantly affected by inoculation of this virus. The incidence and time of onset of thymocytic lymphoma were linearly related to the amount of virus inoculated. High serum Mu gs-1 levels predicted lymphoma development and reflected increases in the amount of infectious virus in the spleen. No induction of tumors, autoimmunity, or high serum Mu gs-1 levels followed administration of SLV 60A to 6-wk old (BALB/c x NZB)F(1) mice or inactivated 60A or active AKR virus to newborns.

The sleep-inducing lipid oleamide deconvolutes gap junction communication and calcium wave transmission in glial cells.

Oleamide is a sleep-inducing lipid originally isolated from the cerebrospinal fluid of sleep-deprived cats. Oleamide was found to potently and selectively inactivate gap junction-mediated communication between rat glial cells. In contrast, oleamide had no effect on mechanically stimulated calcium wave transmission in this same cell type. Other chemical compounds traditionally used as inhibitors of gap junctional communication, like heptanol and 18beta-glycyrrhetinic acid, blocked not only gap junctional communication but also intercellular calcium signaling. Given the central role for intercellular small molecule and electrical signaling in central nervous system function, oleamide- induced inactivation of glial cell gap junction channels may serve to regulate communication between brain cells, and in doing so, may influence higher order neuronal events like sleep induction.

Efficient aldolase catalytic antibodies that use the enamine mechanism of natural enzymes.

Antibodies that catalyze the aldol reaction, a basic carbon-carbon bond-forming reaction, have been generated. The mechanism for antibody catalysis of this reaction mimics that used by natural class I aldolase enzymes. Immunization with a reactive compound covalently trapped a Lys residue in the binding pocket of the antibody by formation of a stable vinylogous amide. The reaction mechanism for the formation of the covalent antibody-hapten complex was recruited to catalyze the aldol reaction. The antibodies use the epsilon-amino group of Lys to form an enamine with ketone substrates and use this enamine as a nascent carbon nucleophile to attack the second substrate, an aldehyde, to form a new carbon-carbon bond. The antibodies control the diastereofacial selectivity of the reaction in both Cram-Felkin and anti-Cram-Felkin directions.

Catalytic antibodies.

Monoclonal antibodies elicited to haptens that are analogs of the transition state for hydrolysis of carboxylic esters behaved as enzymic catalysts with the appropriate substrates. These substrates are distinguished by the structural congruence of both hydrolysis products with haptenic fragments. The haptens were potent inhibitors of this esterolytic activity, in agreement with their classification as transition state analogs. Mechanisms are proposed to account for the different chemical behavior of these antibodies with two types of ester substrates. The generation of an artificial enzyme through transition state stabilization by antibodies was thus demonstrated. These studies indicate a potentially general approach to catalyst design.

From molecular diversity to catalysis: lessons from the immune system.

By combining the enormous molecular diversity of the immune system with basic mechanistic principles of chemistry, one can produce catalytic antibodies that allow control of reactions in ways heretofore not possible. Mechanistic and structural studies of these antibodies are also providing insights into important aspects of enzymatic catalysis and the evolution of catalytic function. Moreover, the ability to rationally direct the immune response to generate selective catalysts for reactions ranging from pericyclic and redox reactions to cationic rearrangement reactions underscores the chemical potential of this and other large combinatorial libraries.

Sequence-specific peptide cleavage catalyzed by an antibody.

Monoclonal antibodies have been induced that are capable of catalyzing specific hydrolysis of the Gly-Phe bond of peptide substrates at neutral pH with a metal complex cofactor. The antibodies were produced by immunizing with a Co(III) triethylenetetramine (trien)-peptide hapten. These antibodies as a group are capable of binding trien complexes of not only Co(III) but also of numerous other metals. Six peptides were examined as possible substrates with the antibodies and various metal complexes. Two of these peptides were cleaved by several of the antibodies. One antibody was studied in detail, and cleavage was observed for the substrates with the trien complexes of Zn(II), Ga(III), Fe(III), In(III), Cu(II), Ni(II), Lu(III), Mg(II), or Mn(II) as cofactors. A turnover number of 6 x 10(-4) per second was observed for these substrates. These results demonstrate the feasibility of the use of cofactor-assisted catalysis in an antibody binding site to accomplish difficult chemical transformations.

At the crossroads of chemistry and immunology: catalytic antibodies.

Immunochemistry has historically focused on the nature of antigenicity and antibody-antigen recognition. However, in the last 5 years, the field of immunochemistry has taken a new direction. With the aid of mechanistic and synthetic chemistry, the vast network of molecules and cells of the immune system has been tapped to produce antibodies with a new function--catalytic antibodies. Because antibodies can be generated that selectively bind almost any molecule of interest, this new technology offers the potential to tailor-make highly selective catalysts for applications in biology, chemistry, and medicine. In addition, catalytic antibodies provide fundamental insight into important aspects of biological catalysis, including the importance of transition-state stabilization, proximity effects, general acid and base catalysts, electrophilic and nucleophilic catalysis, and strain.

Antibody catalysis of a disfavored chemical transformation.

Organic reactions are often limited by stereoelectronic constrains that appear along the reaction coordinate. An antibody has been generated that overcomes these constraints and catalyzes a highly disfavored chemical transformation. The antibody facilitates the difficult 6-endo-tet ring closure of an epoxy-alcohol to form a tetrahydropyran. The catalyzed process is in formal violation of what has become known as Baldwin's rules for ring-closure reactions. In addition to controlling the regiochemistry of the disfavored cyclization reaction, these catalytic antibodies resolve enantiomeric substrates to afford a stereochemically pure product. The principles demonstrated in this study may be applicable to other disfavored chemical processes.

Catalytic antibodies with lipase activity and R or S substrate selectivity.

The specific hydrolysis of unactivated esters bearing an R or S enantiomeric alcohol has been achieved by two separate classes of catalytic antibodies induced to bind either the R or S substrates. The antibodies exhibit rate accelerations (10(3) to 10(5] above background hydrolysis that, coupled with their antipodal specificity, provide a novel set of reagents for use in synthesis.

Quantitative aspects of plasma membrane-associated immunoglobulin in clones of diploid human lymphocytes.

The amount of kappa and lambda chains and Fc fragment associated with the plasma membrane and in the entire cell has been measured for eight lines of human lymphocytes and 21 clones derived from two diploid lines. There was considerable variation in the nature and amount of membrane-associated immunoglobulin and total immunoglobulin among different cell lines and clones. Cells with different phenotypes for membrane-associated immunoglobulin were utilized to show that it is not simply absorbed from the culture medium onto the plasma membrane. The methods are useful for selecting variants which lack or have altered membrane-associated immunoglobulin.

Reactive immunization.

For almost 200 years inert antigens have been used for initiating the process of immunization. A procedure is now described in which the antigen used is so highly reactive that a chemical reaction occurs in the antibody combining site during immunization. An organophosphorus diester hapten was used to illustrate this concept coined "reactive immunization." The organophosphonate recruited chemical potential from the immune response that resembled the way these compounds recruit the catalytic power of the serine hydrolases. During this recruitment, a large proportion of the isolated antibodies catalyzed the formation and cleavage of phosphonylated intermediates and subsequent ester hydrolysis. Reactive immunization can augment traditional immunization and enhance the scope of catalytic antibody chemistry. Among the compounds anticipated to be effective are those that contain appropriate reactive functionalities or those that are latently reactive, as in the mechanism-based inhibitors of enzymes.

An unexpectedly efficient catalytic antibody operating by ping-pong and induced fit mechanisms.

A transition state analogue was used to produce a mouse antibody that catalyzes transesterification in water. The antibody behaves as a highly efficient catalyst with a covalent intermediate and the characteristic of induced fit. While some features of the catalytic pathway were programmed when the hapten was designed and reflect favorable substrate-antibody interactions, other features are a manifestation of the chemical potential of antibody diversity. The fact that antibodies recapitulate mechanisms and pathways previously thought to be a characteristic of highly evolved enzymes suggests that once an appropriate binding cavity is achieved, reaction pathways commensurate with the intrinsic chemical potential of proteins ensue.

A stereospecific cyclization catalyzed by an antibody.

A monoclonal antibody elicited by a transition-state analog that is representative of an intramolecular six-membered ring cyclization reaction acted as a stereospecific, enzyme-like catalyst for the appropriate substrate. Formation of a single enantiomer of a delta-lactone from the corresponding racemic delta-hydroxyester was accelerated by the antibody by about a factor of 170, which permitted isolation of the lactone in an enantiomeric excess of about 94 percent. This finding demonstrates the feasibility of catalytic-antibody generation for chemical transformations that require stereochemical control.

Induction of an antibody that catalyzes the hydrolysis of an amide bond.

Catalysis of amide bond hydrolysis is of singular importance in enzymology. An antibody was induced to an analog of a high-energy intermediate anticipated along the reaction coordinate of amide hydrolysis. This antibody is an amidase with high specificity and a large rate enhancement (250,000) relative to the uncatalyzed reaction. This reaction represents the kinetically most difficult hydrolysis reaction yet catalyzed by an antibody.