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R C Siegel

Publications and source records attributed to R C Siegel.

At least 19 recordsLinked to original sources
PubMed1994

Phase 1 clinical trial of chimeric monoclonal anti-CD4 antibody in multiple sclerosis.

We conducted an open trial of cM-T412, a chimeric monoclonal anti-CD4 antibody, in 29 patients with MS. This antibody caused a prompt and long-lasting depletion of circulating CD4 (helper/inducer) lymphocytes. The mean (+/- SE) CD4 count for the group decreased from 870 (+/- 66) cells/mm3 at baseline to 76 (+/- 11) 3 hours after treatment, and then increased to 425 (+/- 38) at 1 month after treatment and 475 (+/- 39) at 6 months after treatment. Numbers of CD8 (cytotoxic/suppressor) lymphocytes, B lymphocytes, granulocytes, and monocytes changed transiently but showed no significant long-term effects. The most common side effects were headache, nausea, myalgia, fever, and tachycardia occurring in the first few hours after treatment. No serious or unexpected infections or other significant adverse effects occurred. Kurtzke EDSS scores remained stable, and MRI scans showed less contrast enhancement 1 week after treatment. We conclude that treatment of MS patients with cM-T412 chimeric anti-CD4 antibody is well tolerated at the doses tested and produces a long-lasting, selective depletion of CD4 lymphocytes.

Adult↗
PubMed1993

Human monoclonal antibody HA-1A binds to endotoxin via an epitope in the lipid A domain of lipopolysaccharide.

HA-1A, a human IgM mAb, has been shown to significantly reduce mortality in septic patients with Gram-negative bacteremia, especially those with septic shock, in a controlled clinical trial. To confirm the reported specificity of this antibody for the lipid A domain of endotoxin, several assay systems were developed. These assay systems included an ELISA, which measured the binding of HA-1A to lipid A adsorbed to a solid phase; a rate nephelometry assay, which measured the ability of HA-1A to bind and aggregate lipid A in solution; and a dot-blot immunoassay, which measured the ability of HA-1A to interact with lipid A adsorbed to Immobilon-P. In all three assay systems, HA-1A bound in a dose-dependent manner to lipid A prepared from Salmonella minnesota R595 LPS, whereas negative control human IgM mAb or polyclonal antibodies did not. Several experimental approaches were employed to demonstrate the specificity of HA-1A in these assay systems. Both polymyxin B and murine IgG mAb (8A1) with a specificity for lipid A were able to competitively inhibit HA-1A reactivity with lipid A in a dose-dependent manner. Furthermore, a murine IgG anti-Id mAb (9B5.5) developed against HA-1A was also able to block the binding of HA-1A to lipid A in these assay formats. HA-1A reactivity with synthetic lipid A confirmed that HA-1A binding to the natural lipid A was not the result of contaminants in the latter. Finally, the reactivity of HA-1A against a variety of glucosamine-containing and fatty acid-containing compounds was assessed. Some weak interaction was seen with cardiolipin and chitin, but not with serum proteins, lipoteichoic acid, or DNA. Collectively, these results conclusively establish that HA-1A binds to the lipid A region of LPS by an interaction with the V region of the antibody.

Antibodies, Monoclonal↗
PubMed1985

Segmental flexibility of the C1q subcomponent of human complement and its possible role in the immune response.

Fluorescence polarization techniques were used to study the rotational dynamics of the C1q subcomponent of human complement. C1q was covalently labeled with dansyl (DNS) chloride. Digestion of either C1q-DNS4.0 or C1q-DNS1.8 conjugates with pepsin showed that about 75% of the DNS probes were attached to the C1q globular heads and that the remainder were on the collagen-like stalk (peptic fragment). C1q-DNS conjugates readily agglutinated IgG-coated latex beads and combined with C1r2C1s2 to form hemolytically active 16 S C1-DNS. Both C1q-DNS and C1-DNS samples displayed steady-state rotational correlation time and fluorescence lifetime transitions near 48 degrees C. Hydrodynamic studies showed that C1q formed soluble aggregates near the transition temperature. In contrast, stalk samples with a DNS probe apparently attached to the large central fibril showed no thermal transitions or aggregation even when heated above 50 degrees C. Nanosecond fluorescence depolarization measurements detected restricted flexible motions of the C1q heads with an associated rotational correlation time, phi s, of about 25 ns. The C1q anisotropy decay was dominated, however, by a long component, phi L, of perhaps 1000 ns. Except for probe wiggle, the stalk-DNS anisotropy profile was essentially flat. The rapid rotations associated with phi s could represent restricted twisting motions of the arm-head segments or wobbling motions of the heads themselves. Such motions may facilitate binding of the C1q heads to immune complexes. Straightforward diffusion calculations indicated that phi L could represent either global tumbling of the entire C1q molecule or wagging motions of the individual arm-head segments, as suggested by electron micrographs. Upon binding of the C1q heads to an activator, some of the C1q segments may be held in a slightly more open or more closed conformation, which in turn may trigger activation of the C1 proenzymes. In conclusion, we suggest a plausible triggering mechanism for C1 activation that is compatible with the flexible properties of its subcomponents.

Complement Activating Enzymes↗
PubMed1984

Inhibition of collagen fibril formation in vitro and subsequent cross-linking by glucose.

Glucose inhibits collagen fibril formation in vitro. A linear dose response was observed, with half-maximum inhibition of fibril formation occurring at 50 mM glucose. Nonfibrillar collagen cannot be cross-linked by lysyl oxidase, an enzyme that catalyzes the initial cross-linking reaction. The degree of decreased fibril formation correlated with the loss of ability of the collagen to serve as a substrate for lysyl oxidase. Collagen that is not cross-linked is unstable and more susceptible to collagenolytic attack. Interference with collagen cross-linking and more rapid degradation may explain the decreased amounts of interstitial collagen and the poor healing of wounds associated with diabetes mellitus.

Animals↗
PubMed1983

Measurement of the association constants of the complexes formed between intact C1q or pepsin-treated C1q stalks and the unactivated or activated C1r2C1s2 tetramers.

The association constants between C1q and C1r2C1s2 and between C1q and C1r2C1s2 were measured in solution using a new technique which employs sucrose gradient ultracentrifugation to estimate thermodynamic association constants. In this technique, zones of dilute, radioiodine-labeled C1q were sedimented through uniform concentrations of either C1r2C1s2 or C1r2C1s2. The zones remained intact, indicating that the dynamic equilibrium was rapid compared with the time of centrifugation. The observed increases in the sedimentation coefficients of the C1q zones were assumed to be directly proportional to the fraction of C1q bound in the dynamic equilibrium. Binding curves were constructed by performing the measurements at many C1r2C1s2 and C1r2C1s2 concentrations. The association constants were estimated from the midpoints of the binding curves and found to be 6.7 X 10(7)M-1 for C1r2C1s2 binding to 125I-C1q. After activation of the C1r2C1s2 the association constant decreased 10-fold to 7.1 X 10(6)M-1. These association constants refer to solvent conditions of pH 7.35, 1 mM Tris, 5 mM Ca2+ and 150 mM NaC1, pH 7.35. Similar measurements were performed with the collagenous peptic fragment of C1q and both 125I-C1r2C1s2 and 125I-C1r2C1s2. The association constants were independent of the state of activation and both found to be about 2 X 10(7) M-1, suggesting that most if not all of the interactions between C1q and C1r2C1s2 were confined to the collagenous portion of C1q.

Binding Sites↗
PubMed1982

Ultrastructure of the first component of human complement: electron microscopy of the crosslinked complex.

Electron micrographs are shown of the first component of human complement (C1) which has been crosslinked with a water-soluble carbodiimide to prevent dissociation into its C1q and C1r2C1s2 subunits. Two projections of the crosslinked molecule are seen in the electron micrographs, which are called "top" and "profile." In both views, the C1q heads are visible. From the top, the C1r2C1s2 tetrameric subunits appears to be located centrally on the C1q and folded to form a compact mass obscuring most of the arms and central bundle. In profile, the tetramer appears to be located in the region of the arms between the C1q heads and the central bundle. Both the heads and the rod-like central bundle appear to be free of C1r2C1s2 in these profile projections. Sometimes it is possible to count more than six domains in the region of the C1q heads, as though a portion of the tetramer had unfolded to protrude among the heads.

Complement C1↗
PubMed1981

Marfan syndrome: abnormal alpha 2 chain in type I collagen.

Cells in culture from a woman with a variety of the Marfan syndrome produce two species of the alpha 2 chains of type I collagen. One alpha 2 chain appears normal; the abnormal chain has a higher apparent molecular weight than normal and migrates more slowly during electrophoresis in sodium dodecyl sulfate/polyacrylamide gels. A similar change in electrophoretic behavior is seen in the prepro alpha 2 chain and the pN alpha 2 chain (which contains the amino-terminal extension). Asymmetric cleavage of the pepsin-treated procollagens with a fibroblast collagenase locates the abnormal segment amino terminal to the cleavage site, and analysis of cyanogen bromide peptides of collagenase cleavage peptides and of whole collagens indicates that the abnormal segment is in either the alpha 2CB3 peptide or the short segment of alpha 2CB5 amino terminal to the collagenase site of the altered alpha 2 chain. The higher apparent molecular weight is consistent with the insertion of a small peptide fragment of approximately 20 amino acids. This alteration in chain size has marked effects on crosslinking because collagen from the patient's skin was 5-10 times more extractable in nondenaturing solvents than that from control skins. Although the abnormal chain was not found in several other individuals with the Marfan syndrome, these findings suggest that the phenotype may be the expression of a variety of primary structure alterations in the chains of type I collagen that interfere with normal crosslink formation.

Adult↗
PubMed1981

Stoichiometry and sedimentation properties of the complex formed between the C1q and C1r2C1s2 subcomponents of the first component of complement.

We have examined the functional and hydrodynamic properties of the first component of human complement, C1, and the activated first component, C1-, reassembled in the presence of Ca++ from C1q and either the C1r2C1s2 or the C1r-2C1s-2 tetramer. Reconstituted C1 has hemolytic activity similar to C1 in serum. As long as either tetramer is in excess and the total concentration is low, we find that only a 1:1 complex is formed between C1q and either the unactivated or activated tetramer. This complex sediments at 15.9 +/- 0.2 Svedbergs and has a complex sediments at 15.9 +/- 0.2 Svedbergs and has a m.w. of 739,000 +/- 37,000. The boundary shape of the sedimenting C1- preparation was broader than that of C1 suggesting the association constant between C1r2C1s2 and C1q may have decreased upon activation. At elevated concentrations, with more than a molar excess of C1q, C1 aggregated to form both 16S and 23S species.

Animals↗
PubMed1980

X-linked cutis laxa: defective cross-link formation in collagen due to decreased lysyl oxidase activity.

We studied several members of a family with an X-linked form of cutis laxa; the affected males have mild skin laxity, a characteristic facies, skeletal abnormalities, structural abnormalities of the genitourinary tract, and low serum copper levels. The activity of lysyl oxidase, a copper-dependent enzyme involved in cross-link formation in collagen, was decreased in skin-biopsy specimens (13 to 26 per cent of normal) and in culture medium from cells to two affected males (15 to 20 per cent of normal). Immunoreactive lysyl oxidase from skin of both patients was virtually undetectable by immunodiffusion assay. The amounts of lysyl-derived aldehydes (the product formed in collagen and elastin by lysyl oxidase) and of cross-links formed from these products were decreased in dermal fibroblasts in culture. Collagen extractability from these cells was increased in culture. These findings suggest that lysyl oxidase deficiency provides the biochemical basis of the X-linked form of cutis laxa.

Amino Acid Oxidoreductases↗
PubMed1980

Conformation of Immunoglobulin M. III. Structural requirements of antigen for complement fixation by equine IgM.

Complexes of IgM equine anti-dansyl antibodies and different dansyl substituted carriers were tested for their ability to fix complement (C). Only dansyl92-Ficoll and dansyl12-poly-L-lysine were found to be effective. Dansyl13-bovine serum albumin, dansyl127-keyhole limpet hemocyanin, and reduced and alkylated dansyl10-ribonuclease were all ineffective. Lack of C fixation by the dansyl-ribonuclease was not due to lack of antibody-antigen complex formation, since binding at the concentrations employed for C fixation was established. However, in contrast, polymerized dansyl-ribonuclease (polydisperse, with m.w. = 74,000 to 230,000) was very effective in inducing C fixation. These results suggest that large antigen size is necessary for IgM to bind in a multivalent fashion to provide the correct conformation for C fixation. A similar conclusion had been made in earlier studies on rabbit IgM by Cunniff and Stollar. Since optimal C fixation occurred at lower antigen concentrations than maximal precipitation, it would appear that complexes in which several combining sites within a given IgM molecule may be bound to the same antigenic surface may be the most effective. The observation that the amount of C1q bound to antibody was the same in the presence and absence of antigen suggests that enhanced C fixation by antibody-antigen complexes is due to additional C component interactions such as C1r or C1s.

Animals↗
PubMed1979

Native cross-links in collagen fibrils induce resistance to human synovial collagenase.

A model system consisting of highly purified lysyl oxidase and reconstituted lathyritic chick bone collagen fibrils was used to study the effect of collagen cross-linking on collagen degradation by mammalian collagenase. The results indicate that synthesis of approx. 0.1 Schiff-base cross-link per collagen molecule results in a 2--3-fold resistance to human synovial collagenase when compared with un-cross-linked controls or samples incubated in the presence of beta-aminopropionitrile to inhibit cross-linking. These results confirm previous studies utilizing artificially cross-linked collagens, or collagens isolated as insoluble material after cross-linking in vivo, and suggest that increased resistance to collagenase may be one of the earliest effects of cross-linking in vivo. The extent of intermolecular cross-linking among collagen fibrils may provide a mechanism for regulating the rate of collagen catabolism relative to synthesis in normal and pathological conditions.

Aminopropionitrile↗
PubMed1979

Oxidative deamination of epsilon-aminolysine residues and formation of Schiff base cross-linkages in cell envelopes of Escherichia coli.

Oxidative deamination of the epsilon-amino group of lysyl residues to form allysine is the initial reaction in the cross-linking of collagen and elastin in vertebrates. The allysyl residues, generated by lysyl oxidase in this reaction, condense with either other allysyl residues or epsilon-amino groups of lysyl or hydroxylysyl to form aldol or Schiff base cross-links. This paper presents evidence that similar allysyl residues and Schiff base cross-links are synthesized in cell envelopes of Escherichia coli. Acid hydrolysis followed by amino acid analysis of envelopes either reduced with NaB[3H]4 or labeled with [14C]lysine and reduced with NaBH4 yielded allysine and two labeled fragments with elution profiles and molecular weights (250 and 330) consistent with Schiff base products derived at least in part from allysine. When [6-3H]lysine-labeled cell envelopes were incubated at 37 degrees C, gradual release of tritiated water occurred. This suggests that an enzymatic reaction catalyzes the deamination of lysine in E. coli membranes and that the higher molecular weight proteins detected in stationary phase or in log phase cell envelopes after NaBH4 reduction occur as a result of formation of Schiff base cross-links.

Amines↗
PubMed1979

Lysyl oxidase.

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Amino Acid Oxidoreductases↗