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G Biesecker

Publications and source records attributed to G Biesecker.

At least 19 recordsLinked to original sources
PubMed1999

Derivation of RNA aptamer inhibitors of human complement C5.

Specific aptamer inhibitors of the human complement C5 component were produced by the SELEX methodology of directed evolution of nucleic acid ligands. The SELEX procedure started with a pool of random-sequence, 2'F-pyrimidine-modified nuclease-stabilized RNA, and after twelve rounds of iterative C5 binding and nucleic acid amplification an evolved RNA pool was obtained which contained the highest affinity binders to the C5 protein. The evolved RNA pool was then cloned and sequenced, and individual clones were analyzed for binding and function. Twenty-eight clones (out of sixty) were identified which bound C5 (termed aptamers). Seven of these aptamers formed a closely related sequence homology family; these aptamers bound C5 with a Kd 20-40 nM and also inhibited human serum hemolytic activity. In addition, these aptamers inhibited zymosan-induced generation of C5a. Aptamer inhibition of both C5b and C5a suggests that aptamer binding inhibits cleavage of C5 by the C5 convertase of both pathways. One of the inhibitory aptamer sequences was truncated to yield a 38-mer 2'F RNA aptamer which retained C5 binding and inhibitory activity. The structure of this aptamer is predicted to be a stem-loop containing thirteen base pairs, and also containing two bulges. The affinity of this aptamer was improved by performing a second biased SELEX experiment, where the randomized starting RNA pool uses a template where the individual base compositions are biased toward a specific sequence. This second SELEX experiment produced an aptamer with a Kd of 2-5 nM which retained functional activity. Another SELEX to rat C5 produced an aptamer with binding and inhibitory properties virtually identical with the human aptamer. The human and rat aptamers are being evaluated for complement inhibition in vitro and in vivo as potential therapeutics for treatment of human disease.

Animals↗
PubMed1993

Role of late complement components in experimental autoimmune thyroiditis.

Availability of a line of rabbits deficient in the sixth complement component (C6-D) made it possible to evaluate the role of the terminal complement complex (TCC) in the development of experimental autoimmune thyroiditis (EAT) of the rabbit. Immunization with saline extract of homologous thyroid, known to be composed predominantly of thyroglobulin, led in normocomplementemic (NC) rabbits to severe thyroiditis, with cellular infiltrates occupying 50-95% of the thyroid, and to minimal or moderate thyroiditis, with 1-35% of thyroids infiltrated in C6-D rabbits. Cellular infiltrates consisted predominantly of mononuclear cells with appreciable numbers of granulocytes. Destruction of thyroid follicles was extensive and diffuse in NC rabbits, but it was only minimal and focal in C6-D rabbits. Immunohistology revealed in both groups of rabbits deposits of IgG and C3 along follicular basal laminae. In addition, NC rabbits showed deposition of C6 and MAC in thyroid follicles. These results suggested that TCC is necessary for the development of fully expressed, severe EAT; simultaneously, however, they showed that a significantly reduced EAT can develop without TCC. Administration of NC but not of C6-D rabbit serum to C6-D rabbits resulted in a significant increase in the severity of EAT. It was also shown that C6-D rabbits have "normal" T-cell activity, since they developed experimental autoallergic encephalomyelitis as readily as NC rabbits. Therefore, it is likely that development of EAT is indeed impaired by the C6 deficiency in rabbits. The requirement for TCC observed in this study may be relevant to the understanding of the pathogenesis of Hashimoto's thyroiditis, in which thyroid tissue was recently shown to contain TCC deposits.

Animals↗
PubMed1993

Structure of complement poly-C9 determined in projection by cryo-electron microscopy and single particle analysis.

The ring-like complement 'lesions' found on membranes of complement lysed cells comprise a complex of components C5b through C9 that coalesce to form hollow cylinders which penetrate the membrane bilayer and create lytic pores. Walls of these C5b-9 membrane attack complex cylinders may consist primarily of the C9 component, since samples of purified, isolated C9 can polymerize into cylindrical structures which appear identical with the fully assembled C5b-9 complex. The structure of these poly-C9 molecules has been investigated using the techniques of cryo-electron microscopy and single particle analysis. Sets of single poly-C9 particles viewed as rings were selected from cryo-EM images, then particles were aligned and treated by correspondence analysis to identify the principle interparticle similarities and variations. The highest ranking variation found was the presence or absence of a dense inner ring of protein density. Other important variations were interpreted as different types of particle tilt. These results were used in selecting a subgroup of untilted particles for averaging and symmetry analysis. The rotational power spectrum of the initial average suggested 13-fold symmetry. The 13-fold symmetry was used to select and group particles for further analysis. Individual particles were 13-fold rotational averaged and those with enhanced peripheral features were placed into either a right-handed subgroup or into a left-handed subgroup based on orientation of the peripheral features. Particles within each group were aligned and averaged, and a poly-C9 structure was produced which shows important structural details and from which the C9 monomer structure can be deduced. The poly-C9 structure contains a dense inner ring of diameter between 113-181 A and which is modulated into 13 discrete peaks with peak-to-peak separation of approx. 35 A. The dense inner ring is surrounded by a less dense, concentric outer rim extending to 254 A diameter. The outer rim contains projections that are contiguous with the inner peaks but are skewed relative to the ring radius to produce the appearance of a pin-wheel. These projections correspond with the peripheral features picked up in the rotationally averaged individual particles; the left- or right-handed orientation of projections may result from the up/down orientation of individual particles in ice. The C9 monomer structure within the cylinder is suggested by the density distribution. The monomer would be a rod with diameter of 35 A, oriented parallel to the cylinder axis and would be roughly perpendicular to a membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement C9↗
PubMed1993

Role of the membrane attack complex of complement in lung injury mediated by antibodies to endothelium.

The potential pathogenic role of the membrane attack complex (MAC) of the complement system was investigated in two models of lung injury mediated by antibodies to angiotensin-converting enzyme (ACE), an endothelial cell enzyme. In the first model, acute and fatal lung edema was induced in rabbits by intravenous administration of divalent anti-ACE antibodies. These animals died acutely. C6-deficient rabbits tolerated anti-ACE antibodies without apparent ill effects. On the other hand, C6-deficient rabbits reconstituted with C6 and then receiving anti-ACE antibodies developed acute pulmonary edema and died. These results indicate that the MAC is required for the pathogenesis of this lung injury. In the second model, intravenous administration of monovalent anti-ACE Fab fragments over 4 consecutive days induced fatal interstitial pneumonitis in normal rabbits. For C6-deficient rabbits there was a reduced inflammatory response, and no animals died, implicating a mediator function for the MAC in this model as well. These results demonstrate that MAC is an important mediator of acute pulmonary edema induced by divalent antibodies to an endothelial antigen. Moreover, the complement system was also, to some extent, involved in the recruitment of inflammatory cells leading to the development of interstitial pneumonitis in the experimental lung injury induced by monovalent anti-ACE Fab fragments that 'per se' do not activate complement.

Acute Disease↗
PubMed1990

The complement SC5b-9 complex mediates cell adhesion through a vitronectin receptor.

Adhesion of cells to the terminal complement complex of C5b through C9 containing the serum S-protein (SC5b-9) was investigated using a microtiter plate attachment assay with L8 myoblast indicator cells. The skeletal muscle-derived L8 myoblasts bound and spread on substratum coated with SC5b-9, and with the vitronectin/S-protein component of SC5b-9. The myoblasts did not adhere to substratum coated with collagen, laminin, or fibronectin. The cell attachment was blocked by antibody to vitronectin/S-protein, whereas antibody to the other components C5, C6, C7, C8, or C9 had minimal effect. The cells were not bound to free vitronectin because attachment activity was removed by adsorption with an anti-C6 antibody column. The L8 cell attachment was dependent on divalent cations, was blocked by synthetic peptides containing the amino acid sequence Arg-Gly-Asp, and was inhibited by antivitronectin receptor antibody. These results indicate that cells adhere to the SC5b-9 complex through interaction of the vitronectin component with an integrin vitronectin receptor. Cell attachment to terminal C complexes could be used for leukocyte adherence and migration during inflammation, and also for attachment of tissue cells during regeneration after disease or traumatic injury.

Animals↗
PubMed1990

Identification of an Arg-Gly-Asp (RGD) cell adhesion site in human immunodeficiency virus type 1 transactivation protein, tat.

Tat, the transactivation factor of human immunodeficiency virus type 1 (HIV-1), contains the highly conserved tripeptide sequence Arg-Gly-Asp (RGD) that characterizes sites for integrin-mediated cell adhesion. The tat protein was assayed for cell attachment activity by measuring the adhesion of monocytic, T lymphocytic, and skeletal muscle-derived cell lines to tat-coated substratum. All cell lines tested bound to tat in a dose-dependent manner and the tat cell adhesion required the RGD sequence because tat mutants constructed to contain an RGE or KGE tripeptide sequence did not mediate efficient cell adhesion. The tat-mediated cell attachment also required divalent cations and an intact cytoskeleton. In addition, cell adhesion to tat was inhibited in the presence of an RGD-containing peptide GRGDSPK or an anti-tat mAb that recognizes the RGD epitope. These results strongly suggest that cells are bound to tat through an integrin. Interestingly, myoblast cells bound to tat remained round, whereas the same cells attached through an integrin for a matrix protein typically flatten and spread. The role of this RGD-dependent cellular adhesion of tat in HIV-1 infection remains to be determined.

Amino Acid Sequence↗
PubMed1989

The release of C5a in complement-activated serum does not require C6.

The influence of terminal complement components on the generation and release of the complement C5a fragment was investigated by comparing the levels of C5a in complement-activated serum with the levels of C5a produced in serum depleted of complement C6. In order to investigate the release of C5a, a modified C5a assay was developed that utilizes an anti-C5b monoclonal antibody to remove C5, C5b, and C5b-C5a complexes from samples prior to C5a assay. The modified assay was developed because the standard methodology, which includes an acid-precipitation step designed to dissociate C5a and C5b, cannot distinguish free C5a from the C5a that is bound to C5b. Therefore, the standard methodology is not capable of monitoring the influence of terminal components on C5a/C5b dissociation. Levels of C5a were measured in complement-activated whole human serum, in serum depleted of C6, and in serum containing inhibitory levels of anti-C6 Fab using both the modified C5a assay and the standard methodology. Sera were complement-activated with either zymosan to activate the alternative complement pathway or with antibody-coated sheep erythrocytes to activate the classical pathway. The levels of free C5a in C6-depleted sera after activation were equivalent to the C5a levels in activated whole serum, indicating that C6 is not required for the release of C5a from C5b. In addition, the quantity of C5a detected in zymosan-activated sera using the standard acid-precipitation methodology was greater than C5a levels when assayed using the modified immunoadsorption technique, confirming that acid-treatment enhances the C5a dissociation and promotes C5a recovery. Since the other terminal components, C7, C8, and C9, bind to C5b only after C5b only after C6 is bound, these results indicate that none of the terminal components are required for the release of C5a. Although the terminal components could influence the rate of C5a release, the quantity of C5a released in serum was entirely independent of terminal components.

Animals↗
PubMed1989

Inhibition of acute passive transfer experimental autoimmune myasthenia gravis with Fab antibody to complement C6.

The role of the lytic complement C5b-9 membrane attack complex (MAC) in acute passive transfer experimental autoimmune myasthenia gravis (EAMG) produced in rats was investigated by in vivo inhibition of MAC formation with anti-C6 Fab. Anti-C6 Fab totally inhibited in vitro serum hemolytic activity, but did not consume or inhibit early complement pathways. Injection of rats with 0.12 mg/ml anti-C6 Fab reduced serum C6 to 8% and inhibited the muscle weakness, electrophysiologic abnormalities and loss of acetylcholine receptor (AChR) associated with acute EAMG. This level of C6 inhibition reduced the total serum complement hemolytic activity to 29% of normal but did not reduce the serum levels of complement components C3, C5, or C7. Treatment of rats with lower amounts of anti-C6 Fab (0.08 mg/ml) also inhibited clinical and electrophysiologic signs of EAMG, however, the lower amount of anti-C6 did not prevent the loss of muscle AChR. Both the higher and the lower amount of anti-C6 Fab inhibited the accumulation of macrophages at muscle motor end-plates. The inhibition by anti-C6 indicates that muscle weakness and electrophysiologic abnormalities associated with EAMG are dependent on the complement MAC, and that muscle weakness results from tissue injury in addition to loss of muscle membrane and AChR.

Animals↗
PubMed1988

Resistance to experimental autoimmune myasthenia gravis in genetically inbred rats. Association with decreased amounts of in situ acetylcholine receptor-antibody complexes.

Genetically related susceptibility for experimental autoimmune myasthenia gravis was investigated in nine inbred strains of rats immunized with heterologous acetylcholine (AChR) from Torpedo californica. Wistar Munich and Fischer strain animals consistently developed severe, fatal disease associated with impaired neuromuscular transmission and increased sensitivity to low doses of curare. A lower incidence of disease was induced in Wistar Kyoto, ACI, Brown Norway, Buffalo, and Lewis strain animals. In contrast, Wistar Furth and Copenhagen strain animals were resistant to experimental autoimmune myasthenia gravis, electrophysiologic responses were normal, and animals were insensitive to curare. All strains of animals manifested equivalent amounts of serum antibody to AChR and total muscle AChR was reduced to the same extent in both resistant and susceptible animals. In contrast, the amount of antibody-bound AChR was greater in susceptible Wistar Munich animals than the amount observed in resistant Wistar Furth animals. These data suggest that impaired neurotransmission is correlated with the extent of antibody binding to the AChR. The discordance in the amount of antibody bound to the AChR of resistant and susceptible animals may result from heritable differences in antibody properties. Cross-breeding experiments with Wistar Munich and Wistar Furth animals show that resistance for development of experimental autoimmune myasthenia gravis is recessive and indicate that disease susceptibility is linked to one or two genetic loci.

Animals↗
PubMed1987

Heymann antibodies induce complement-dependent injury of rat glomerular visceral epithelial cells.

The aim of this study was to investigate the in vitro role of the complement membrane attack complex (MAC) in the injury induced by nephritogenic anti-brush border vesicle (Fx1A) antibodies on rat glomerular visceral epithelial cells (GEC). Both sheep and rabbit anti-rat brush border vesicle IgG-induced complement-dependent lysis of cultured GEC. Fab fragments of sheep anti-rat brush border vesicles and polyclonal or monoclonal gp330 IgG were devoid of lytic activity. Shedding of cell-surface antigens induced by sheep or rabbit anti-rat brush border vesicle IgG protected GEC from subsequent exposure to lytic antibodies and complement, an effect that was not obtained with Fab fragments. When GEC were incubated with sheep or rabbit anti-rat brush border vesicle IgG in capping conditions, the C3 component was co-redistributed with Heymann immune complexes; in contrast, the MAC remained diffusely bound to the cell surface, indicating that it was not associated with the antigen-antibody complexes. The MAC was demonstrated on the surface of GEC by immunofluorescence staining with anti-MAC neoantigen and by electron microscopy of negatively stained membranes showing focal clusters of 110 A MAC lesions. When GEC were treated with sheep IgG or rabbit IgG plus C6-deficient sera, the cells were not lysed and MAC was not demonstrable on the surface; however, lytic activity was restored when C6-deficient sera were reconstituted with purified C6. The results are consistent with the interpretation that injury induced by Heymann antibodies on GEC is MAC-dependent.

Animals↗
PubMed1984

Immunopathogenesis of Heymann's nephritis.

Renal localization of the C5b-9 membrane attack complex (MAC) in relation to other complement components and immunoglobulins was studied at different stages of Heymann's nephritis and following reimmunization of Lew rats with stage IV disease. Trace amounts of the MAC were found during stages I and IV of disease, whereas during stages II to III, a period of active glomerular and tubular injury, moderate deposits of MAC were observed in glomeruli, periluminal cytoplasm of proximal tubular epithelial cells, and desquamated intraluminal brush border material. Following reimmunization of stage IV animals, a striking increase in the amount of MAC was observed. Moderate to marked deposition of IgG and C3 was also found during stages II and III at sites containing the MAC. Although a slight decrease in detectable IgG was noted during stage IV, a bright, confluent ribbon-like staining pattern was present in reimmunized animals. These data suggest that the MAC is a mediator for acute in situ immune-complex-induced glomerular and tubular cytoplasmic injury, although subsequent proteinuria may persist without continued complement pathway activation and assembly of the MAC.

Animals↗
PubMed1983

Localization of the membrane attack complex (MAC) in experimental immune complex glomerulonephritis.

The role of the membrane attack complex (MAC) as a mediator of renal tissue injury was evaluated in rats affected by bovine serum albumin (BSA)-induced immune complex glomerulonephritis. Immunofluorescence studies revealed concurrent deposits of IgG, BSA, C3, and the MAC along glomerular capillary walls, although the MAC manifested a more restricted distribution than that observed for immune complexes. Immunoelectron microscopic techniques were utilized to demonstrate immune complexes, C3, and the MAC within dense deposits in the subepithelial aspect of the basement membrane. Visceral epithelial foot processes were fused in areas overlying large dense deposits and exhibited intense staining for the MAC, lesser reactivity for C3 but IgG was absent from the foot process membranes. Smaller granular deposits of immune complexes, C3, and the MAC were observed in the subendothelial region of the lamina rara interna and the lamina densa. Immune complexes may activate the classical complement pathway causing diffuse injury to the glomerular basement membrane (GBM), allowing subepithelial accumulation of complexes. These observations implicate the MAC as a mediator of GBM and juxtaposed podocyte membrane injury, thereby contributing to disruption of the glomerular filtration barrier. IgG and C3 were demonstrated within tubulointerstitial regions on the surface of collagen fibers in close proximity to the tubular basement membrane (TBM) of proximal convoluted tubules. Within the TBM, C3 localization was prominent with diminished reactivity for the MAC, but IgG was not detectable. The demonstration of C3 and scant MAC deposits in the TBM of nonimmunized control rats without evidence of interstitial IgG and C3 deposits suggests that both nonimmune and immune processes play a role in the pathogenesis of extraglomerular lesions. Evidence derived from these morphologic studies indicates that the MAC is associated with injury to the GBM, foot process membranes of visceral epithelium, and the TBM. Further experiments designed to selectively enhance or inhibit the deposition of MAC and assess consequent renal dysfunction are required to substantiate hypotheses concerning the in vivo membranolytic potential of the MAC in experimental immune complex glomerulonephritis.

Animals↗
PubMed1983

Immunology of myasthenia gravis.

Anti-acetylcholine-receptor antibody is demonstrable in more than 90 per cent of patients with myasthenia gravis. Serum antibody titers do not show a direct correlation with disease severity, although in certain patients antibody levels increase in association with disease activity. Impairment of neuromuscular transmission results from the loss of junctional receptors, either as a result of receptor internalization or destruction of junctional folds containing the acetylcholine receptor. Myasthenia gravis manifests immunologic, genetic, and clinical similarities to rheumatic syndromes, suggesting a generic immune dysfunction common to these disorders.

Acetylcholine↗