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Biomedical subjects

D T Fearon

Publications and source records attributed to D T Fearon.

At least 163 records · Page 9Linked to original sources
PubMed1977

Activation of the alternative complement pathway with rabbit erythrocytes by circumvention of the regulatory action of endogenous control proteins.

Cleavage of C3 by the alternative complement pathway occurs in at least two distinct phases: continuous low grade generation of C3b by the interaction of native C3, B, D, and P, and subsequent amplified cleavage of C3 by the interaction of C3b, B, D, and P which forms the amplification convertase, P,C3b,Bb. Transition to C3b-dependent amplification is necessary to achieve substantial C3 cleavage and is normally limited by the combined action of C3b inactivator (C3bINA) and betalH. An activator of the alternative pathway, such as rabbit erythrocytes (E(r)), provides sites that protect bound C3b and P,C3b,Bb from the action of these regulatory proteins and permits C3b deposited by the low grade fluid phase reaction to assemble a membrane-associated amplification convertase which can deposit additional protected C3b. Under conditions in which the control proteins, C3bINA and beta1H, almost completely inactivated C3b bound to sheep erythrocytes (E(s)), which does not activate the alternative pathway, the function of C3b bound to E(r) was diminished by less than one-fifth. Further, the P- stabilized amplification convertase on E(r) was 10-fold less sensitive to beta1H-mediated decay-dissociation than the convertase on E(s). The addition of E(r) to a regulated mixture of purified C3, B, D, P, C3bINA, and beta1H resulted in amplified inactivation of C3 and B by formation of the amplification convertase on E(r) as indicated by its lysis with subsequent exposure to C3-C9. In contrast, E(s) did not advance the low grade fluid phase inactivation of C3 and B to amplified inactivation and the cell was not converted to an intermediate susceptible to lysis by C3- C9. Since E(r) and E(s) did not differ in their inefficient fixation of C3b generated during an unregulated fluid phase reaction, the activating capacity of E(r) must reside in its protection of bound C3b and P, C3b,Bb from the regulatory proteins rather than in enhanced capacity to bind C3b from the fluid phase. When the reaction is limited to low grade fluid phase turnover, introduction of E(r) but not E(s) results in a 100-fold increase in the deposition of C3b, indicating that surface-dependent activation of the alternative pathway is characterized by efficient deposition of C3b on the initiating surface. Thus, the activating surfaces advance the interaction of the alternative pathway proteins to the amplification phase because of the selective inability of the regulatory proteins to deal with their substrates when deposited on these surfaces and results in a specificity that is not necessarily dependent on adaptive immunity.

Animals↗
PubMed1977

Activation of the alternative complement pathway due to resistance of zymosan-bound amplification convertase to endogenous regulatory mechanisms.

The surface of zymosan (Zy), by affording a protected microenvironment for C3b and the amplification convertase stabilized by properdin, P,C3b,Bb, shifts the alternative complement pathway from slow fluid phase turnover to the amplification phase of its expression. This mode of activation is in contradistinction to that of the classical pathway, which follows conversion of a proenzyme, Cl, to its active form, C1. Under conditions in which the control proteins, C3b inactivator (C3bINA) and beta1H, completely, inactivated C3b on the sheep erythrocyte intermediate, EAC4b,3b, the activity of C3b bound to Zy,ZyC3b, was diminished by only one-third. Further, when ZyC3b was converted to ZyC3b,Bb,P there was an additional point of deregulation in that the convertase was resistant to beta1H-mediated decay-dissociation while P,C3b,Bb on the sheep erythrocyte exhibited the usual susceptibility to beta1H. That Zy alone could indeed promote rapid C3 cleavage by the alternative pathway through assembly and protection of the amplification convertase on its surface was demonstrated with a mixture of alternative pathway proteins, C3,B,D,P, C3bINA, and beta1H, that had each been purified to homogeneity. Interaction of these proteins at one-tenth their relative serum concentrations with Zy permitted low-grade inactivation of C3 and B to advance to the level of amplification after a 15 min lag period. Because the reaction of the purified proteins proceeded spontaneously when either regulatory protein was deleted, the effect of Zy was attributed to deregulation rather than to conversion of one of the proteins to a specific initiating state. The alternative pathway, through the normal presence of D, interacts with a microbial surface, such as Zy, to amplify deposition of C3b by circumvention of endogenous regulatory mechanisms, thereby augmenting host defense.

Animals↗
PubMed1977

Methyltestosterone therapy in hereditary angioedema.

In a double-blind study of four patients with hereditary angioedema, the efficacy of methyltestosterone (taken daily in 10-mg linguet form) in preventing attacks was shown. There were 19 episodes during 11.8 months of placebo administration, compared with only four attacks during the 46 months of cumulative methyltestosterone treatment (P less than 0.001). The mean serum C4 protein level was twice as high in all patients when they were taking the drug (176 +/- 36 mug/ml) as compared with the placebo (84 +/- 21 mug/ml), and rose to normal range in three of four patients.

Adolescent↗
PubMed1977

Pathways of complement activation in membranoproliferative glomerulonephritis and allograft rejection.

The complement system is comprised of at least 18 plasma proteins and consists of four functional divisions: two pathways for activation (classical and alternative), a common amplification mechanism for the activating pathways, and a final common effector pathway to which the activating and amplifying sequences are directed. The classical pathway is activated by certain antigen-antibody complexes, while the alternative pathway may be initiated non-immunologically by various microbial polysaccharides. Indeed, mixtures of purified C3, B, D, and P regulated to low-grade interaction by the presence of C3bINA and beta1 H respond to zymosan with amplified C3 and B inactivation. Both pathways form enzymes termed C3 convertases that cleave C3 to generate its major fragment, C3b. C3b interacts with each C3 convertase to permit C5 cleavage in activation of the effector complement sequence, and it interacts with alternative-pathway factors B and D to generate additional C3 convertase, C3bBb, in the amplification pathway. As C3 cleavage represents the most critical step in the elaboration of the biologic effects of the complement system, modulation of this reaction by generation, stabilization, and inactivation of the amplification convertase C3bBb may well determine whether initial activation of the complement sequence eventuates in beneficial or detrimental effects for the host. Initial generation of C3bBb is dependent on prior cleavage of C3, which may occur by the classical pathway or the alternative pathway. Stabilization of C3bBb is achieved with either P or C3NeF after their binding to C3b and C3bBb, respectively. Control of this amplifying step occurs at three levels: intrinsic decay of the inherently labile C3bBb complex, extrinsic decay-dissociation of Bb from the complex by beta1H, and inactivation of C3b by C3bINA. In the presence of stabilizing factors the control proteins must function in sequence, since C3bINA cannot act on C3bBb; beta1H-mediated decay of protective Bb must precede C3b inactivation by C3bINA. C3NeF, which is found in the sera of some patients with MPGN and persistent depressions of serum C3, circumvents all three controls because of its capacity to create a stabilized convertase that is relatively resistant to decay-dissociation by beta1H. The effector complement sequence is activated by cleavage of C3 and C5, which releases vasoactive and chemotactic peptides, C3a and C5a, and generates the major fragments C3b and C5b. C3b, in addition to its function in the amplifying reaction and the C5 convertases, mediates immune adherence to cells possessing membrane-associated receptors for C3b; this in turn promotes the phagocytic and secretory functions unique to each cell type. Cell-bound C5b serves to assemble the cytolytic complex C5b6789, while fluid-phase C5d generates the hemolytically inactive chemotactic complex C567d...

Complement C3↗
PubMed1977

Purification of C3b inactivator and demonstration of its two polypeptide chain structure.

C3b inactivator (C3bINA) was isolated from plasma by sequential ammonium sulfate gradient solubilization, anion exchange chromatography, gel filtration, and repeat ammonium sulfate gradient solubilization. The final product was pure as assessed by alkaline disc gel electrophoresis, isoelectric focusing, and SDS polyacrylamide gel electrophoresis, and elicited a monospecific rabbit antiserum. The normal serum concentration of C3bINA was found to be 53 +/- 9 microgram/ml (mean +/- S.D.). Heterogeneity of purified C3bINA was apparent on alkaline disc gel electrophoresis and isoelectric focusing, but not with SDS polyacrylamide gel electrophoresis and thus is attributed to forms of C3bINA that differ in charge rather than in size. SDS polyacrylamide gel electrophoresis of unreduced, alkylated C3bINA yielded a single stained band with an apparent m.w. of 93,000, whereas the reduced protein demonstrated two bands of 55,000 and 42,000 m.w., thereby establishing a composition of two disulfide-linked polypeptide chains for C3bINA.

Chemical Phenomena↗
PubMed1976

Control of the amplification convertase of complement by the plasma protein beta1H.

An inhibitory activity for an erythrocyte in termediate bearing the properdin (P)-stabilized amplification C3 convertase, PC3bBb, was recognized in whole normal human serum and separated from C3b inactivator by its distinct physicochemical and functional characteristics. The inhibitory activity was found to reside in a protein that was purified to homogeneity and elicited a monospecific antibody in a rabbit. This protein was identified as beta1H and found to have a serum concentration of 516 +/- 89 mug/ml (mean +/- 1 SD). beta1H produced a dose related, first-order loss of convertase function and release of 125I-Bb from the P-stabilized intermediate, indicating a mechanism of action by decay-dissociation of Bb from the complex, PC3bBb. beta1H exhibited only a limited capacity to accelerate decay of C3bBb sites stabilized with C3 nephritic factor or to release 125I-Bb from such sites. Amplification of C3 cleavage by C3bBb may well determine whether initial complement activation by the classical or alternative activating sequence is beneficial or detrimental to the host. Regulation of this amplifying function is now recognized to occur at at least three steps: intrinsic decay which reflects the inherent lability of the C3bBb convertase; extrinsic decay-dissociation of Bb which is mediated by the effect of beta1H; and inactivation of exposed C3b by C3b inactivator. The stabilization of C3bBb by activated properdin minimizes intrinsic decay and protects C3b in the bimolecular complex from C3b inactivator. beta1H restores control of the system by decay-dissociation of the bimolecular complex, therby exposing C3b to C3b inactivator whose irreversible action prevents regeneration of the convertase at that site.

Animals↗
PubMed1976

The natural modulation of the amplification phase of complement activation.

As C3 cleavage represents the most critical step in the elaboration of the biologic effects of the complement system, the modulation of this reaction by formation and function of the C3b-dependent C3 convertase may well determine whether the initial activation of the complement sequence eventuates in beneficial or detrimental effects to the host. Stabilization of the amplification C3b-dependent convertase, C3bBb, is achieved with P and C3NeF, respectively, after their binding which exhibits different molecular and temperature requirements. Control of this amplifying step occurs at three levels: intrinsic decay of the inherently labile C3bBb convertase; extrinsic decay by displacement of Bb from the convertase with beta1H; and inactivation by C3bINA of C3b after its generation from native C3 or removal of protective Bb by intrinsic or extrinsic decay. In the presence of the stabilizing factors the control proteins must function in sequence with beta1H-mediated decay preceding C3b inactivation.

Complement C3↗
PubMed1976

C3 nephritic factor (C3NeF): stabilization of fluid phase and cell-bound alternative pathway convertase.

C3 nephritic factor (C3NeF) defined by the capacity of nephritic serum and its fractions to initiate loss of the B antigen of C3 in normal serum was purified from the serum of three different donors and shown to function by stabilization of membrane-bound and fluid phase alternative pathway C3 convertase. C3NeF converts cell-bound C3B sites in a dose-related manner to CEB(NeF) sites, which exhibit an approximate 10-fold increase in half-life. The linear relationship between the C3NeF input and the residual hemolytic sites on EAC43B present after incubation for 20 min at 30 degrees C, during which labile C3B sites have decayed, indicates that the number of residual C3B sites is directly related to the dose of C3NeF. The capacity of C3NeF to stabilize the C3B convertase in a temperature- and dose-dependent manner, which is independent of binding or consumption of C3NeF, in a fluid phase reaction mixture of 125I-B, 131I-C3 and D permits isolation of a 10S complex containing radiolabeled C3 and B and exhibiting C3, convertase activity on an exogenous C3 source. Thus, the stabilizing effect of C3NeF is not limited to membrane-bound C3B but is also sufficient to permit recovery of a fluid phase C3 convertase formed during the interaction of C3, B, and D.

Binding Sites↗
PubMed1976

C3 requirements for formation of alternative pathway C5 convertase.

Although alternative pathway C3 and C5 convertases both have active proteolytic sites dependent on the same protein, Bb, the quantitative requirements for the expression of these activities are sufficiently different to permit their delineation in terms of B input an cell-bound C3b. That the labile component of each active site is Bb was established by their parallel decay rates, regeneration of the original specificities with B in the presence of D, and stabilization of each convertase by C3NeF. The evidence that the spatial relationships of Bb and C3b on the cell surface for C3 and C5 convertase activities are distinct is based not only upon the decay and regeneration of each original convertase but more so upon their interconversion. C3 convertase is converted to C5 convertase by interaction with additional C3 whereas C5 convertase reverts to a C3 convertase by treatment with C3 INA. The capacity of C3 INA treatment to abolish C5 convertase sites without affecting C3 convertase sites indicates the existence of two functional species of C3b, one of which is protected in the C3bBb complex whereas the other is exposed.

Cell Membrane↗
PubMed1976

Formation in the presence of C3 nephritic factor (C3NeF) of an alternative pathway C3 convertase containing uncleaved B.

C3 nephritic factor (C3NeF) interacts with native C3 and B in the absence of D to generate a C3 convertase containing an uncleaved form of B. Dose response studies with C3NeF and B, respectively, revealed incremental C3 inactivation without loss of B. These findings are in agreement with the previous isolation from such reaction mixtures of a 10S complex containing haemolytically inactive C3 and active B and manifesting C3 convertase activity. Functional contamination of C3 with C3b was negated by demonstrating that pretreatment of C3 with C3bINA had no effect on its subsequent interaction with B and C3NeF to generate C3 convertase activity, while pretreatment of C3b eliminated its effective interaction with B and C3NeF. Relatively higher concentrations of C3bINA present during interaction of C3, B and C3NeF suppressed C3 inactivation, indicating its dependence on amplification by utilization of the initial C3b generated. Trace quantities of D were not found by functional analyses of C3, B and C3NeF and pretreatment of these proteins with a concentration of DFP sufficient to suppress D activity had no effect on their effective interaction. The introduction of D to mixtures of C3NeF, B, and C3 resulted in B clevage and more efficient expression of C3 convertase function as defined by a reduced requirement for C3NeF.

Complement C3↗
PubMed1975

Properdin: binding to C3b and stabilization of the C3b-dependent C3 convertase.

A function of P in the alternative complement pathway is to prolong the first order decay of the hemolytic sites on EAC43B in a dose-dependent manner. As the number of initial convertase sites is not changed, even when activated properdin (P) increases the t1/2 10-fold or more, P acts to stabilize rather than to uncover additional sites. P binds to EAC43 to generate EAC43P in a reaction that proceeds slightly more rapidly at 15 degrees C than at 0 degrees C, but reaches the same plateau and does not require divalent cations. The presence of P on EAC43P not only stabilizes the convertase subsequently formed on that cell, but, alternatively, permits transfer to convertase sites on other cells with the stability of the recipient intermediate being dependent on the P available for transfer. The capacity of P to bind to C3b and stabilize C3B contrasts with the inhibitory effect of the C3b inactivator on formation of this amplification convertase.

Animals↗
PubMed1975

Activation of the properdin pathway of complement in patients with gram-negative bacteremia.

To determine the pathway used for activation of complement component C3, serum levels of components C1, C4, C2, C3, C5, C6, and C9 and two properdin factors, properdin and factor B, were measured in 42 patients with gram-negative bacteremia, in 19 of whom shock subsequently developed. Mean levels of the classical components C1, C4, and C2 in bacteremic patients in whom shock subsequently developed did not differ significantly (p greater than 0.05) from those of patients with uncomplicated bacteremia. Levels of properdin, factor B and C3, C5, C6, and C9 were significantly (p less than 0.05) decreased in patients with shock in comparison with those with uncomplicated bacteremia. Taken together, these findings are consistent with activation of C3 and the terminal complement sequence, C5-C9, occurring primarily by the properdin pathway, in patients with gram-negative bacteremia eventuating in shock. Biologically active products released during activation of C3-C9 may contribute to the development of shock.

Bacteria↗
PubMed1975

Properdin: initiation of alternative complement pathway.

Activation of the classical complement (C) system involves conversion of C1 to its active state with subsequent cleavage of C4 and -d C2 so as to form the classical C3 convertase, C42 (a bar indicates the activated form of a protein), which sequentially cleaves C3 and C5 to initiate the cytolytic event associated with the complete reaction. An alternative, pr properdin-dependent, pathway to complement activation generates a C3 convertase, C3B, that is formed by cleavage of B with D in the presence of a C3b, the major cleavage fragment of C3. C3b is capable of binding activated properidin (P) with resultant stabilization of C3B, which otherwise rapidly decays by loss of B activity. Initial cleavage of C3, a prerequisite for formation of C3B, is demonstrated to occur through the interaction of native C3 and B in the presence of either D or P alone, or together. The effect of P on the interaction of D, B, and C3 is attributed to stabilization of C3B as has been shown for C3B. Larger amounts of P and B with C3 in the absence of D form a C3 convertase that is designated (P)C3B to indicate that demonstrable cleavage of B does not occur although the active site is available. The generation of this initial convertase, as assessed by C3 inactivation, is dose-related to P and B inputs. The presence of both P and D greatly augments initial cleavage of C3 with D fully uncovering the active site of B and P stabilizing that site.

Complement C3↗