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J E Volanakis

Publications and source records attributed to J E Volanakis.

At least 91 records · Page 5Linked to original sources
PubMed1989

Measurement of complement proteins C2 and B in systemic lupus erythematosus and septic shock.

We developed sensitive radioassays to quantitate the homologous complement proteins C2 and B, in intact form, in sera from patients with diseases associated with hypocomplementemia. In a study of systemic lupus erythematosus (SLE) patients, we found a subset of patients (6/40) with low C2 concentrations, though overall, C2 concentration was not related to disease activity. Protein B concentration was increased in 23/40 SLE patients, apparently not due to a generalized acute phase response. Among patients with both gram-negative and gram-positive septic shock, C2 and B concentrations were low in a subset of patients with pre-existing cirrhosis, suggesting hepatic hyposynthesis of these proteins may be important in their prognosis and predisposition to severe infections.

Biomarkers↗
PubMed1989

Complement activation by the alternative pathway is modified in renal failure: the role of factor D.

Factor D, an essential enzyme of the alternative pathway (AP) of complement, is eliminated by the kidney, and its plasma concentration increases 10-fold in end-stage renal disease (ESRD). The purpose of this study was to analyze the consequences of factor D accumulation. A number of in vitro assays were used to analyze AP activation in normal human serum (NHS), in normal serum supplemented with purified factor D to 10-fold its normal concentration (10 x D), and in sera of patients with ESRD. When compared with NHS, in 10 x D: 1) Spontaneous fluid-phase activation of complement at 37 degrees C was greatly increased as measured by C3 cleavage, 2) The lysis of rabbit erythrocytes, a function of the AP, was accelerated, 3) More C3 fragments bound to cuprophane membranes and to immune precipitates; both reactions were accompanied by the formation of more C5a, 4) Complement mediated solubilization of antigen-antibody precipitates was enhanced. Sera of patients with ESRD behaved similarly to 10 x D in all assays used, i.e., enhanced AP function, although complement activation measured in these assays varied widely from one individual to another. Thus, the elevated factor D concentration observed in renal failure might have important pathophysiological consequences, some of which could be detrimental (e.g., C5a produced during hemodialysis), while others might be beneficial, e.g., solubilization of immune precipitates.

Aged↗
PubMed1988

Evidence for a C4b binding site on the C2b domain of C2.

We raised murine mAb against human C protein C2. The representative mAb 3A3.3 (IgG1 kappa) recognized an epitope on the C2b domain of C2, as determined by binding and inhibition of binding radioassays. The hemolytic activity of purified human C2 and of C2 in normal human serum was inhibited by the mAb. The rate of decay of the C3-convertase at 30 degrees C was not affected by the mAb. C2 binding to EAC4b was inhibited by intact IgG and the Fab fragment of the mAb; 50% inhibition required 1 microgram/ml of either. The data suggest the presence of a C4b-binding site on the C2b domain of C2 and that the mAb recognizes an epitope at, or adjacent to, this site. The C2b portion of the C2 molecule may be important in assembly of the classical pathway C3-convertase.

Animals↗
PubMed1988

Radioassays for quantitation of intact complement proteins C2 and B in human serum.

Availability of polyclonal and monoclonal antibodies recognizing determinants on the major cleavage fragments of complement proteins C2 and B enabled development of sensitive radioassays which can be used to quantitate the intact proteins in human sera. Changes in C2 and B concentrations indicative of classical or alternative pathway activation, or both, were seen in normal serum after incubation with complement activators. We determined the normal range (mean +/- 2 SD) of C2 concentration to be 11-35 micrograms/ml in 32 healthy individuals, and that of protein B to be 74-286 micrograms/ml. Sera from patients with systemic lupus erythematosus (SLE), septic shock, infections, and following orthopedic surgery were then assayed. Mean protein B concentration was significantly higher in SLE sera (P = 0.002) and in the infected and post-operative (acute-phase) sera (P less than 0.001), and the mean C2 concentration in the septic shock group (P less than 0.001) was significantly lower than the mean of healthy individuals. Intact C2 was not detected in known C2-deficient individuals. These assays allow parallel quantitation of the structurally and functionally homologous proteins of the classical (C2) and alternative (B) pathways, which is of interest in patients with genetic and acquired hypocomplementemia.

Adult↗
PubMed1988

Metabolism of complement factor D in renal failure.

Factor D is an essential enzyme of the alternative pathway of complement. Its plasma concentration increases approximately tenfold in end-stage renal failure (ESRF). To analyze its metabolism in humans, we injected purified radiolabelled factor D into 5 healthy individuals and 12 patients with various renal diseases or renal failure. Fractional metabolic rates (FMR) and extravascular/intravascular distributions (EV/IV) were calculated using a compartmental model. The FMR was very rapid in normal individuals (mean 59.6%/hr; range 74.1 to 50.5), significantly diminished in the five patients with ESRF (5.7%/hr; 7.0 to 2.8; P less than 0.004), and correlated well with the creatinine clearance (r = 0.89; P less than 0.001). The extrarenal catabolic rate was not modified in renal failure. Despite a significant inverse correlation between plasma levels of factor D and creatinine clearance [r = 0.68; P less than 0.002], factor D levels were not a sensitive indicator of renal function because the synthesis rate (SR) varied widely from one individual to another (mean SR: 62.9 micrograms/kg/hr; 14.9 to 136.5). Factor D synthesis was not significantly altered by renal function, and did not correlate with C-reactive protein, suggesting that factor D is not an acute phase protein. The proportion of intact factor D elimination in the urine was increased in patients with tubular dysfunction (up to 15% compared to less than 0.2% in normal individuals) confirming that under normal circumstances factor D is filtered through the glomerulus and catabolized by tubular cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement Activating Enzymes↗
PubMed1987

Cholera toxin and pertussis toxin regulate the Fc receptor-mediated phagocytic response of human neutrophils in a manner analogous to regulation by monoclonal antibody 1C2.

Data presented in this paper indicate that polymorphonuclear leukocyte (PMN) Fc receptor-mediated phagocytosis can be markedly augmented and that this augmentation is under regulatory control. Stimulation of PMN with either a low m.w., heat-labile cytokine(s) (the culture supernatant effluent from a YM-10 Centricon unit, YM-10E), phorbol esters (phorbol dibutyrate), or the polyene antibiotic, amphotericin B, enhances Fc-mediated ingestion in a dose-dependent manner. YM-10 effluent- and amphotericin B-stimulated ingestion is completely abrogated by treating the PMN with either pertussis toxin (PT), cholera toxin (CT), or a monoclonal antibody (mAb), 1C2. However, neither toxin nor mAb 1C2 affects nonstimulated ingestion or phagocytosis stimulated by phorbol esters or synthetic diacylglycerol. Increasing intracellular cyclic adenosine monophosphate levels by stimulation with prostaglandin E1 and the phosphodiesterase inhibitor, isobutylmethylxanthine, does not mimic the effect of either toxin or mAb 1C2. In addition, toxin-mediated inhibition is not due to loss of either the Fc receptor recognized by mAb 3G8 or the antigen recognized by mAb 1C2. These data indicate that both CT and PT regulate the phagocytic response of PMN, in a manner like mAb 1C2, probably by affecting a guanosine 5'-triphosphate-binding protein distinct from those that regulate adenylate cyclase. Since phorbol ester-stimulated ingestion is not inhibited by either PT, CT, or mAb 1C2 and phorbol esters activate protein kinase C directly, phagocytosis amplification regulated by PT, CT, and mAb 1C2 may involve protein kinase C activation.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Preliminary X-ray study of crystals of human C-reactive protein.

Two different crystal forms of human C-reactive protein have been grown from solutions of 2-methyl-2,4-pentanediol. Both crystal forms are tetragonal, the space group for form I is P4(1)22 (or P4(3)22), and that for form II is P4(2)22. The unit cell parameters for form I are a = b = 103.0(5) A, c = 308.5(7) A and for form II are a = b = 103.1(2) A, c = 312.7(6) A. The crystals of form II diffract to at least 3.0 A resolution, and are suitable for detailed structural studies.

C-Reactive Protein↗
PubMed1987

Isolation of complement protein D from urine of patients with Fanconi's syndrome.

Complement protein D is the least abundant of all complement proteins and, thus, one of the most difficult to purify. We report a new method for obtaining pure D from urine of patients with Fanconi's syndrome. The method is simple and allows the purification of milligram amounts of D within a few days. It involves three chromatographic steps using Bio-Rex 70, hydroxylapatite HPLC, and reverse-phase HPLC. Protein D purified by this method is suitable for both functional and structural studies.

Chromatography, High Pressure Liquid↗
PubMed1987

Blood clearance of Streptococcus pneumoniae by C-reactive protein.

C-reactive protein (CRP) has long been known to be an acute phase protein associated with infection and various forms of tissue damage. Recent studies have shown that human CRP can be used to passively protect mice from lethal infection with Streptococci pneumoniae. In this study we have undertaken a detailed examination of the ability of human CRP and rabbit CRP (CxRP) to mediate the blood clearance of pneumococci in mice. We have shown that the optimal activity of these acute-phase proteins requires a functioning complement system, and it can take place even in the xid mouse, which has virtually no naturally occurring anti-pneumococcal antibody in its serum. These studies provide additional evidence that CRP may play a protective role in pneumococcal infections, and it may help postpone the development of fatal levels of pneumococci in the blood, long enough for an effective anti-pneumococcal antibody response to be generated.

Animals↗
PubMed1987

Human complement proteins D, C2, and B. Active site mapping with peptide thioester substrates.

The specificity and reactivity of complement serine proteases D, B, Bb, C2, and C2a were determined using a series of peptide thioester substrates. The rates of thioester hydrolysis were measured using assay mixtures containing the thiol reagent 4,4'-dithiodipyridine at pH 7.5. Each substrate contained a P1 arginine residue, and the effect of various groups and amino acids in the P2, P3, P4, and P5 positions was determined using kcat/Km values to compare reactivities. Among peptide thioesters corresponding to the activation site sequence in B, dipeptide thioesters containing a P2 lysine residue were the best substrates for D. Extending the chain to include a P3 or P4 amino acid resulted in loss of activity, and neither the tripeptide nor the tetrapeptide containing the cleavage sequence of B was hydrolyzed. Overall, D cleaved fewer substrates and was 2-3 orders of magnitude less reactive than C1s against some thioester substrates. C2 and fragment C2a had comparable reactivities and hydrolyzed peptides containing Leu-Ala-Arg and Leu-Gly-Arg, which have the same sequence as the cleavage sites of C3 and C5, respectively. The best substrates for C2 and C2a were Z-Gly-Leu-Ala-Arg-SBzl and Z-Leu-Gly-Leu-Ala-Arg-SBzl, respectively, where Bzl is benzyl. B was the least reactive among these complement enzymes. The best substrate for B was Z-Lys-Arg-SBzl with a kcat/Km value of 1370 M-1 s-1. The catalytic fragment of B, Bb, had higher activity toward these peptide thioester substrates. The best substrate for Bb was Z-Gly-Leu-Ala-Arg-SBzl with a kcat/Km similar to C2a and 10 times higher than the value for B. Both C2a and Bb were considerably more reactive against C3-like than C5-like substrates. Bovine trypsin hydrolyzed thioester substrates with kcat/Km approximately 10(3) higher than the complement enzymes. These thioester substrates for D, B, and C2 should be quite useful in kinetic and active site studies of the purified enzymes.

Animals↗
PubMed1987

Probing functional sites on complement protein B with monoclonal antibodies. Evidence for C3b-binding sites on Ba.

We used four mouse monoclonal antibodies (Mab) as probes of functional sites of human complement protein B. Two Mab, HA4-1B (gamma 2a kappa) and HA4-15 (gamma 2a kappa), reacted with the same or adjacent epitopes on the Bb fragment of B, while the other two, HA4-1A (gamma 1 kappa) and FD3-20 (gamma 1 kappa), reacted with distinct epitopes on Ba. All reactive epitopes were expressed on native B and only one, recognized by the anti-Ba Mab HA4-1A was more reactive on isolated Ba than on B. These binding specificities were determined by direct binding radioassays and confirmed by inhibition studies. Immunoelectron microscopy of B and Bb in complex with anti-Ba and anti-Bb revealed that the recognized epitopes are on opposite sides of the molecule and are on discrete domains. All four Mab inhibited the hemolytic activity of B, although with different efficiencies and through different mechanisms. The main effect of the two anti-Bb Mab was an increased rate of loss of hemolytic sites from preformed EC3bBb C3 convertase presumably through accelerated dissociation of Bb. On the other hand, the main effect of the two anti-Ba Mab was inhibition of binding of B to C3b. HA4-1A was more efficient, inhibiting by 50% the binding of [125I]B to EC3b at 10 micrograms/ml as IgG and at 13 micrograms/ml as Fab. The data suggest that a binding site for C3b on intact B is located on the Ba portion of the molecule.

Antibodies, Monoclonal↗
PubMed1987

Peripheral blood mononuclear leukocytes release a mediator(s) that induces phagocytosis of C-reactive protein-coated cells by polymorphonuclear leukocytes.

We studied the phagocytosis by human polymorphonuclear leukocytes (PMN) of sheep erythrocytes (E) passively sensitized with pneumococcal C-polysaccharide (E-PnC), E-PnC coated with C-reactive protein (E-PnC-CRP), and E coated with rabbit antisheep E IgG (E-IgG). PMN isolated from the blood of normal individuals failed to ingest either E-PnC or E-PnC-CRP; however, after incubation with supernatants from stimulated peripheral blood mononuclear leukocytes, glass-adherent PMN ingested E-PnC-CRP with a mean phagocytic index of 47.8 +/- 14.9 (means +/- SD, n = 9) and E-PnC to a lesser extent with a phagocytic index of 9.6 +/- 2.9 (mean +/- SD, n = 4). We also observed a statistically significant increase in the ingestion of E-IgG by lymphokine-stimulated PMN with phagocytic indices of 85.2 +/- 21.2 (mean +/- SD, n = 12) for unstimulated PMN and 158 +/- 37.1 (mean +/- SD, n = 9) for stimulated PMN. The best conditions for stimulating release of this phagocytosis-promoting mediator included exposure to phytohemagglutinin (PHA) in the presence of monocytes that had ingested IgG-coated sheep erythrocytes. The induction of phagocytosis of E-PnC-CRP was rapid, reaching a maximal level after stimulation of the adherent PMN with the conditioned media for 30 min. The factor(s) responsible for the induction of the ingestion of E-PnC-CRP was less than 10,000 daltons and was heat stable (56 degrees C for 45 min). These data are similar to earlier results obtained with PMN activated by 12-0-tetradecanoyl-phorbol-13-acetate (PMA), an important contrast being that in the current studies PMN were activated by a soluble factor released from stimulated mononuclear cells under conditions simulating those in vivo.

Biological Products↗
PubMed1986

Stimulation of human neutrophil Fc receptor-mediated phagocytosis by a low molecular weight cytokine.

Human neutrophil Fc receptor-mediated phagocytosis can be markedly enhanced by a low m.w. (less than 10,000) heat-labile cytokine(s) derived from specifically stimulated human mononuclear cells and from a human T cell line, MO(t). PMN incubated with supernatant from control mononuclear leukocyte (MNL) culture bound EIgG (percentage of rosettes = 73.7% +/- 7.1) but did not ingest the attached targets (phagocytic index, PI = 40.7 +/- 9.5) as efficiently as PMN incubated with supernatant from adherent MNL, which had ingested EIgG and were then cocultured with nonadherent MNL (PI = 264.3 +/- 46.3). Cytokine-containing supernatants were fractionated on YM-10 Centricon microconcentrators, and the effluent (YM-10E) was found to contain the phagocytosis-enhancing activity. Optimal Fc receptor-mediated ingestion by YM-10E-stimulated PMN required a critical level of target-bound IgG; stimulation was dose dependent and detectable after 5 min at 37 degrees C with a maximal response by 15 min. Monoclonal antibody 3G8 (anti-PMN Fc receptor) inhibited in a dose-dependent fashion both Fc receptor-mediated rosette formation and ingestion by nonstimulated and YM-10E-stimulated PMN. Solid-phase 3G8 Fab had the same effect. A previously undescribed monoclonal antibody, 1C2, exhibited a different pattern of inhibition. It had no effect on rosetting or ingestion of EIgG by nonstimulated PMN; however, it inhibited EIgG phagocytosis by YM-10E-stimulated PMN down to the level of nonstimulated ingestion without affecting rosette formation. Solid-phase 1C2 had the same effect. These data indicate that phagocytosis mediated by 3G8-positive Fc receptors may be enhanced by cytokine(s) stimulation in a manner requiring the molecule recognized by 1C2. Monoclonal antibodies to the alpha-chain of CR3 had only minimal effects on YM-10E-stimulated ingestion. Fluorescence flow cytometry of YM-10E-stimulated PMN, indirectly stained with 3G8 or 1C2, indicated that cytokine enhancement of EIgG ingestion occurred without an increase in either 3G8 or 1C2 binding sites. These data show that the low avidity Fc receptor, which binds immune complexes, may be functionally modulated at sites of inflammation where PMN and macrophages mediate clearance and destruction of immune complexes and opsonized particles.

Antibodies, Monoclonal↗
PubMed1986

Quantitative levels of C-reactive protein in cerebrospinal fluid in patients with bacterial meningitis and other conditions.

We measured levels of C-reactive protein (CRP) in the cerebrospinal fluid in 145 children, using a solid-phase radioimmunoassay. The CRP levels in 49 patients with culture-proved bacterial meningitis ranged from 0 to 51,000 ng/ml (median 1460 ng/ml). In 33 patients with aseptic meningitis, values were much lower range 0 to 438 ng/ml; (median 17 ng/ml). In patients with CSF pleocytosis (greater than 10 WBC/microliter), CRP greater than 100 ng/ml was 95% accurate in identifying those with bacterial meningitis. However, a few patients with bacterial meningitis and little or no CSF pleocytosis had low levels of CRP at admission. Among the 63 patients with nonmeningitic conditions, those with bacterial infections frequently (10 of 13 had CRP greater than 100 ng/ml, whereas CRP elevations were infrequent (seven (18%) of 40) in patients with viral infections and other conditions. CRP diffuses into the CSF as readily as other proteins, but in bacterial meningitis the CSF/serum ratio of CRP was lower than that of albumin and IgG. The measurement of CRP in CSF is potentially a very useful diagnostic tool, but certain inherent limitations must be recognized, because some patients may fail to mount a prompt inflammatory response.

Adolescent↗
PubMed1986

Human complement protein D catabolism by the rat kidney.

Factor D (D) is an essential component of the alternative complement pathway. To determine whether D is catabolized by the kidney and, if so, at what site, we studied the renal handling of human D by in vivo nephron microperfusion and in vitro perfusion of rat kidneys. Human D was purified and labeled with 125I. Individual nephrons were perfused in vivo at varying rates with perfusate that contained 125I-D and [14C]inulin. When nephrons were perfused from proximal sites with perfusate 125I-D in a concentration of 3.0 micrograms/ml, urinary recovery of 125I-D increased (P less than 0.05) from 57.7 +/- 5.0 to 74.4 +/- 2.5% as tubule fluid flow rate was increased from 10 to 40 nl/min; recovery of 125I-D was less than (P less than 0.001) [14C]inulin recovery at all perfusion rates. At 20 nl/min, an increase in perfusate 125I-D concentration from 1.5 to 3.0 micrograms/ml was associated with an increase (P less than 0.001) in urinary 125I-D recovery (42.1 +/- 4.0 vs. 65.8 +/- 2.6%). Similarly, the addition of unlabeled D, 30 micrograms/ml, to 125I-D, 3.0 micrograms/ml, increased urinary 125I-D recovery (95.3 +/- 2.1%) at 20 nl/min. When nephrons were perfused from early distal segments at 10 nl/min, 125I-D recovery (91.2 +/- 4.3%) did not differ from [14C]inulin recovery (95.8 +/- 1.3%). In the isolated perfused filtering kidney, the concentration of intact 125I-D in the perfusate declined 60.3 +/- 14.6% over 1 h. 83.4 +/- 6.3% of the decrement in 125I-D was catabolized by the kidney; the remainder was excreted in the urine as intact D. When glomerular filtration was prevented by increasing perfusate albumin concentration to 16 g/dl, perfusate intact (125I-D) remained unchanged over 1 h. These data show that human D is catabolized by the kidney via glomerular filtration and reabsorption by the proximal nephron. Reabsorption of D appears to be a saturable process.

Animals↗