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Biomedical subjects

J P Leddy

Publications and source records attributed to J P Leddy.

At least 19 recordsLinked to original sources
PubMed1989

Secretion of the terminal complement proteins, C5-C9, by human platelets.

The terminal complement components, C8 and C9, and to a lesser extent C5, C6, and C7, but minimal amounts of C3, were shown to be associated with washed human platelets. In unactivated platelets, the complement components were detected in the platelet pellet by hemolytic assays after centrifugation and disruption of the platelets by freeze-thawing. However, after platelets had been activated by collagen, thrombin, or aggregated IgG to induce aggregation, the complement components were released into the supernatant. The rank order of hemolytic activity of C9, C8, C7, C6, and C5 detected in the supernatants of activated platelets was quite different from that found in serum from the same donors, in the same assays. In particular, the serum C7 hemolytic titer was more than twice the serum C9 hemolytic titer, whereas the activity of C9 detected from platelets was more than twice that of C7. This argues against a purely nonspecific uptake of these proteins by platelets from plasma. The functional role of terminal complement components released from platelets during activation is unknown, but it is tempting to speculate that these proteins may have a role in platelet-dependent immunological tissue injury. Because the C5b-9 membrane attack complex activates platelets, it is possible that release of terminal complement proteins serves to amplify platelet activation and may also play a role in diseases in which complement membrane attack complexes have been implicated.

Blood Platelets↗
PubMed1987

Human Fc gamma receptors: stable inter-donor variation in quantitative expression on platelets correlates with functional responses.

Evidence has recently been presented that a 40,000 dalton membrane sialoglycoprotein (p40) shared by monocytes and granulocytes serves as the human platelet receptor for aggregated IgG. We now report that the platelets of normal donors exhibit stable quantitative differences in the expression of this receptor molecule, as determined by flow cytometry using fluorescent staining with murine monoclonal antibody to p40 (mab IV.3). These inter-donor differences were reproducible on repeated testing over at least 4 mo. In concurrent assays, the binding of mab IV.3 to each donor's platelets was highly correlated with the binding of heat-aggregated human IgG, also assayed by flow cytometry. The biological relevance of this quantitative variation in IV.3 binding is suggested by its reproducible correlation with platelet responsiveness to aggregated IgG measured by aggregometry. Such stable quantitative variation in platelet Fc receptor expression among individual humans could contribute to differences in severity of certain pathologic processes initiated by IgG-containing immune complexes.

Antibodies, Monoclonal↗
PubMed1986

Enhanced reactive lysis of paroxysmal nocturnal hemoglobinuria erythrocytes. Studies on C9 binding and incorporation into high molecular weight complexes.

As part of a broader analysis of the mechanism(s) by which the most sensitive (type III) paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes are excessively sensitive to reactive lysis by isolated C5b6, C7, C8, and C9, we have compared type III PNH (PNH-III) and normal human E in respect to both total specific binding of 125I-C9 and the proportion of cell-bound C9 appearing in high molecular weight (HMW) complexes. In a previous report, we found that after exposure to purified C5b6 and 125I-C7, specific C7 binding and, by implication, EC5b-7 formation were equal for PNH-III E and normal E. In the present study, C8-dependent binding of 125I-C9 to PNH-III EC5b-7 and normal EC5b-7 was also similar, although lysis of the PNH-III E was up to five times greater; that is, PNH-III E required fewer bound C9 molecules to produce an effective lytic site than did normal E. To quantify radioactivity in monomeric and HMW forms of membrane-bound C9, lysed and unlysed E were subjected to low ionic strength buffers to convert all E to ghosts. These ghosts were solubilized in 0.1 or 2% SDS (without reduction) and electrophoresed on 2.4-11% polyacrylamide gradient gels followed by autoradiography and densitometric scanning. With 0.1% SDS, broad, heterodisperse zones of HMW C9 were recovered from both PNH and normal ghosts; the amounts of C9 incorporated into the HMW complexes were similar for PNH-III E and normal E. In selected experiments, 125I-C7 could be shown in these same HMW bands. When membranes were solubilized in 2% SDS, the overall proportion of HMW C9 complexes compared with dimer and monomer C9 was reduced on both PNH and normal membranes. In many, but not all experiments, more of the highest mol wt C9 complexes were detected from PNH-III E membranes solubilized in 2% SDS than from normal or PNH-II E membranes similarly treated. When antibody-sensitized E were lysed by purified C1-C9, PNH-III EA bound far more C9 than did normal EA, and both lysis and C9 incorporation into HMW complexes were markedly and proportionately increased over normal; however, lytic efficiency of 125I-C9 bound to PNH EA was equal to or less than that bound to normal EA.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies↗
PubMed1985

High-density lipoprotein and its apolipoproteins inhibit cytolytic activity of complement. Studies on the nature of inhibitory moiety.

Human high-density lipoprotein (HDL) and its apolipoproteins A-I and A-II inhibit complement-mediated lysis of human and sheep erythrocytes. This inhibitory activity under study is exerted after C9 is bound to membrane-associated C5b-8 complexes but prior to completed assembly and insertion of the C5b-9 complex. In this paper, we define some structure-activity relationships of the inhibitory moiety. With the exception of weak lytic inhibitory activity found in LDL/VLDL pools and in some unconcentrated minor fractions of plasma obtained by hydrophobic chromatography, all inhibitor activity was found in fractions which contained either apolipoprotein A-I, apolipoprotein A-II, or both. Intact HDL has a high level of inhibitor activity but delipidation by chloroform-methanol extraction was associated with an increase in activity on a protein-weight basis. Purified apolipoprotein A-I and apolipoprotein A-II exhibited equal inhibitory activity, greater than that exhibited by intact HDL. Nevertheless, ultracentrifugal fractions in which no free apolipoproteins could be demonstrated still possessed inhibitory activity. These experiments suggest that delipidation of HDL is not necessary for expression of inhibitor activity, although we could not rule out the possibility that apolipoproteins in dynamic equilibrium with HDL are responsible for the inhibitor activity observed in whole serum and plasma and in HDL preparations. Limited proteinase digestion completely abolished the inhibitory activity of partially delipidated HDL. Phospholipase C had little or no effect on the inhibitory activity of delipidated HDL, apolipoprotein A-I or apolipoprotein A-II, but reduced the inhibitory activity of intact HDL. These data suggest that the phospholipid polar headgroups are not necessary for inhibitory activity. However, the loss of these headgroups is associated with decreased activity, possibly due to increased hydrophobicity of HDL, or increased association among HDL micelles, and subsequent decrease in effective molar concentration of the inhibitory moiety.

Apolipoprotein A-I↗
PubMed1985

Human platelet Fc receptor for immunoglobulin G. Identification as a 40,000-molecular-weight membrane protein shared by monocytes.

We have recently shown that human monocytes and U937 cells possess two molecular classes of Fc gamma receptor. One, a 72,000-mol-wt sialoglycoprotein, has high affinity for certain subclasses of human and murine monomeric IgG. The other is a 40,000-mol-wt protein (p40) with low affinity for monomeric IgG but with the capacity to bind IgG aggregates or IgG-coated particles. In the present study, a 40,000-mol-wt single chain protein, apparently identical to p40 from U937 cells, was isolated from surface-radioiodinated human platelets by affinity purification using a murine IgG2b monoclonal antibody to p40. This 40,000-mol-wt protein was not seen when control IgG2b or unrelated murine monoclonal antibodies were employed in place of anti-p40. The same 40,000-mol-wt protein was also recovered from an IgG-Sepharose affinity adsorbent, but not from ovalbumin-or myoglobin-Sepharose. The 72,000-mol-wt Fc gamma receptor of monocytes was not identified on platelets. Monoclonal anti-p40 and Fab fragments derived from this antibody blocked platelet aggregation by heat-aggregated human IgG, whereas a control murine IgG2b protein had little or no inhibitory effect at 500-1,000-fold higher concentrations. A murine IgG1 monoclonal antibody, reactive with an unrelated platelet-specific membrane antigen, did not inhibit platelet responses to aggregated IgG. Anti-p40 did not affect platelet aggregation by thrombin, collagen, or fibrinogen plus ADP. Although anti-p40 did not directly aggregate platelets in the concentrations employed, cross-linking with F(ab')2 goat anti-murine Ig induced apyrase-sensitive aggregation of anti-p40-treated platelets. This indicates that p40 possesses transmembrane linkage for platelet activation and secretion. These observations strongly suggest that this newly recognized 40,000-mol-wt platelet membrane protein serves as an Fc gamma receptor.

Antibodies, Monoclonal↗
PubMed1985

Enhanced reactive lysis of paroxysmal nocturnal hemoglobinuria erythrocytes by C5b-9 does not involve increased C7 binding or cell-bound C3b.

The most complement (C)-sensitive type of erythrocytes (E) occurring in paroxysmal nocturnal hemoglobinuria (type III PNH E) have previously been found to exhibit approximately twofold to fourfold greater lysis than normal human E when exposed to isolated human C5b6, C7, C8, and C9 (reactive lysis), in the absence of a known source of C3- or C5-convertases or fluid-phase C3. In further studies on the mechanism of this phenomenon, we now report that C5b6-dependent binding of 125I-C7 to two samples of PNH E (greater than 95% type III) is equal to that found with normal human E at each of several C5b6 inputs tested. Lysis developed by excess C8 and C9, however, was consistently greater for the PNH E. Thus, the exaggerated sensitivity of type III PNH E to reactive lysis cannot be explained by abnormally high uptake of C5b6 or C7 from the fluid phase. Rather, the data indicate that cell-bound C5b67 sites are converted to effective hemolytic sites with greater efficiency on type III PNH E than on normal human E, assuming that the distribution of cell-bound C7 throughout both cell populations is similar. In related studies we have addressed the proposal by other investigators that C3b putatively bound to PNH E in vivo might account for their increased sensitivity to reactive lysis in vitro, by analogy to prior observations on C3b-potentiated reactive lysis of sheep E. The latter hypothesis was made more appealing by the recent discovery that type III PNH E lack an integral membrane protein, decay-accelerating factor (DAF), which in normal E accelerates the decay of membrane-bound C3 convertases. Against this hypothesis, however, is our present finding that preincubation of PNH E with four different goat or rabbit polyclonal antibodies to human C3 failed to inhibit the subsequent reactive lysis of these cells. Under these same conditions, the C3b-dependent increment in reactive lysis of sheep EAC4b3b was abrogated by pretreatment with similar dilutions of these anti-C3 antibodies, generally in association with agglutination. Furthermore, sheep EAC4b3b displayed increased 125I-C7 binding in proportion to augmented lysis, in contrast to the findings with PNH E. Therefore, deficiency of DAF in type III PNH E does not adequately explain their supranormal sensitivity to reactive lysis unless DAF can modulate the terminal lytic steps by a mechanism distinct from its effect on C3 convertase decay. Alternatively, type III PNH E could have a more general abnormality in which DAF deficiency is one manifestation and increased sensitivity to reactive lysis is another.

Animals↗
PubMed1985

Avulsions of the flexor digitorum profundus.

Many physicians and trainers are not aware of the entity of avulsion of the profundus tendon insertion. This is unfortunate, since early diagnosis and prompt surgical treatment generally produce the best result. Late, untreated patients who are asymptomatic are best left alone. For late instability of the DIP joint, fusion should be considered. A one- to two-stage flexor tendon graft can be performed in carefully selected, untreated patients, but the potential risks may outweigh the possible advantages in some individuals.

Arthrodesis↗
PubMed1979

Complement lysis of human erythrocytes. Differeing susceptibility of two types of paroxysmal nocturnal hemoglobinuria cells to C5b-9.

Although enhanced sensitivity of erythrocytes to complement-mediated lysis is a hallmark of paroxysmal nocturnal hemoglobinuria (PNH), subpopulations of erythrocytes in such patients vary significantly in this respect. One PNH erythrocyte subpopulation (termed type III) comprises exquisitely sensitive cells, whereas type II PNH erythrocytes are intermediate in complement sensitivity between PNH type III and normal human erythrocytes. Differences in the action of the terminal complement components that would account for the differing lytic behavior of types II and III PNH erythrocytes have been proposed but not directly demonstrated. The present studies, making use of carefully selected cases with pure populations of type II or type III erythrocytes, confirm a prior observation that antibody-coated PNH erythrocytes of both types II and III display comparably supranormal C3 binding in whole human serum. However, when lysis was induced by the isolated C5b-9 membrane attack mechanism, bypassing the requirement for C3 binding, only type III PNH cells exhibited greater than normal lysis. This finding suggests that type III PNH erythrocytes have an additional membrane abnormality not present in type II cells. Thus, the differing lytic behavior of these two cell types in whole serum may reflect the additive effects on type III cells of both exaggerated C3 binding and enhanced sensitivity to C5b-9, whereas the more moderate lysis of type II PNH cells may be determined mainly or entirely by the earlier-acting mechanism producing augmented C3 binding. The failure of guinea pig C8 and C9, as opposed to human C8 and C9, to reveal the true lytic sensitivity of PNH-III E in our earlier study is illustrated, and its implications briefly discussed.

Animals↗
PubMed1978

Responses of human platelets to immunologic stimuli: independent roles for complement and IgG in zymosan activation.

Analysis of the zymosan (Z)-induced human platelet response with washed platelets, fresh agammaglobulinemic plasma, and purified human IgG has identified a previously unrecognized role of IgG in this reaction, in addition to the previously reported requirements for C and fibrinogen. The rate-limiting step involves the assembly of C components on the Z particles and is independent of immunoglobulin. After the heat-labile components (supplied by agammaglobulinemic plasma) are Z bound, Z-specific IgG (apparently antibody) can bind to Z very rapidly, conferring on the previously inactive Z-complement-fibrinogen intermediate the capacity to induce platelet aggregation and serotonin release. The critical function of this Z-bound IgG appears to be in the interaction of the active Z complex with the platelet through Fc receptors, since soluble Fc fragments of each IgG subclass can totally block platelet activation. It is proposed that the human platelet requires a dual, or multi-site, stimulation to become activated during some forms of immunologic injury. These findings may provide some insight into clinical syndromes involving thrombocytopenia and intravascular coagulation during sepsis.

Agammaglobulinemia↗
PubMed1977

Avulsion of the profundus tendon insertion in athletes.

A review of 36 avulsions of the flexor profundus tendon insertion in athletes seen during the past 5 years showed the injury to be most common in the ring finger. The injury was classified into three types depending upon (1) the presence or absence of a bony fragment on roentgenograms, (2) the level to which the tendon retracted, and (3) the status of the blood supply of the avulsed tendon. If the tendon retracts into the palm at the time of injury, it should be repaired within 7 days. If the tendon retracts only to the proximal interphalangeal joint, it often can be repaired a few months after the injury. Late untreated patients who were relatively asymptomatic were left alone. Those with unstable distal interphalangeal joints were treated by fusion or tenodesis of the distal joint. A flexor tendon graft through an intact functioning superficialis tendon in the ring finger seldom was indicated. Prompt diagnosis and surgical repair within the first week gave the best results.

Athletic Injuries↗
PubMed1977

Effect of selective complement deficiency on the rate of neutralization of enveloped viruses by human sera.

The capacity of human sera genetically deficient in selective complement (C) components to enhance neutralization of enveloped viruses was examined by kinetic plaque reduction assays. Vaccinia virus, a DNA virus, and vesicular stomatitis virus (VSV), an RNA virus, were studied. Exogenous rabbit: or human antibody to vaccinia virus, and guinea pig or human antibody to VSV were provided in limiting, C-dependent concentrations. IgG antibodies predominated in most of the antisera employed. C5-deficient and C6-deficient human sera consistently supported normal rates of neutralization of either virus; this effect was heat-labile. C4-deficient human serum did hot exceed heat-inactivated serum in any neutralization assay. C1r-deficient serum displayed slight heat-labile neutralizing capacity against vaccinia but none against VSV. C2- and C3-deficient sera consistently exhibited measurable but clearly subnormal rates of neutralization. Two fresh agammaglobulinemic sera failed to inactivate either virus in the absence of added antibody. These results confirm and extend earlier evidence, based on neutralization of herpes simplex and Newcastle disease viruses in the presence of early (IgM) antibody and functionally pure guinea pig C components or C-deficient animal sera, that the late-acting components C5-C9 are not required for C-dependent neutralization. Data on four enveloped viruses now agree that this function is mediated by C1-C3, although C1 plus C4 appear to have some neutralizing capacity. This requirement for C1-C3 is overcome, however, in the presence of higher antibody cohcentrations, suggesting that the contribution of the C system to viral neutralization in vivo may be chiefly in the early phase of infection when antibody is limited.

Antibodies, Viral↗
PubMed1977

Hereditary C5 deficiency in man. III. Studies of hemostasis and platelet responses to zymosan.

Platelet-rich-plasma from two hemostatically normal individuals, genetically lacking the fifth component of complement (C5), failed to exhibit normal platelet aggregation, or serotonin release, in the presence of zymosan. This abnormality was found to reside in the C5D plasma rather than in the platelets as demonstrated by the inability of the deficient plasma to activate zymosan for the aggregation of washed normal platelets. The defect could be corrected by the addition of normal plasma, normal serlm, or highly purified human C5. A plasma abnormality similar to that found in the C5D individuals was also noted in plasmas deficient in C3, C6, and C7; whereas C8 plasma D behaved normally. These data suggest that this platelet reaction requires late acting C components, perhaps as the C567 complex, bound to the zymosan particles.

Blood Platelets↗