Peripheral complement interactions with amyloid β peptide in Alzheimer's disease: 2. Relationship to amyloid β immunotherapy.

Introduction: Our previous studies have shown that amyloid β peptide (Aβ) is subject to complement-mediated clearance from the peripheral circulation, and that this mechanism is deficient in Alzheimer's disease. The mechanism should be enhanced by Aβ antibodies that form immune complexes (ICs) with Aβ, and therefore may be relevant to current Aβ immunotherapy approaches.

Methods: Multidisciplinary methods were employed to demonstrate enhanced complement-mediated capture of Aβ antibody immune complexes compared with Aβ alone in both erythrocytes and THP1-derived macrophages.

Peripheral complement interactions with amyloid β peptide: Erythrocyte clearance mechanisms.

Introduction: Although amyloid β peptide (Aβ) is cleared from the brain to cerebrospinal fluid and the peripheral circulation, mechanisms for its removal from blood remain unresolved. Primates have uniquely evolved a highly effective peripheral clearance mechanism for pathogens, immune adherence, in which erythrocyte complement receptor 1 (CR1) plays a major role.

Methods: Multidisciplinary methods were used to demonstrate immune adherence capture of Aβ by erythrocytes and its deficiency in Alzheimer's disease (AD).

Survival benefit associated with human anti-mouse antibody (HAMA) in patients with B-cell malignancies.

Background: About one-third of patients with relapsed B-cell malignancies develop human anti-mouse antibody (HAMA) following mouse antibody treatment. The purpose of this study was to assess the relationship between HAMA and survival in patients given a mouse anti-lymphoma monoclonal antibody (mAb), Lym-1, directed against a unique epitope of HLA-DR antigen that is up-regulated on malignant B-cells.

Methods: ELISA was used to quantify HAMA in 51 patients with B-cell malignancies treated with iodine-131 (131I) labeled Lym-1.

Direct antilymphoma effects on human lymphoma cells of monotherapy and combination therapy with CD20 and HLA-DR antibodies and 90Y-labeled HLA-DR antibodies.

Purpose: Monoclonal antibodies (mAb) in combination and mAbs combined with a radionuclide (radioimmunotherapy) have both been more effective in patients than mAb monotherapy.

Experimental Design: Using assays of cell growth and viability, the dose response and temporal characteristics of CD20 (rituximab) and HLA-DR (Lym-1) mAbs, singly and in combination, and of 90Y-conjugated Lym-1 mAb have been characterized in five human lymphoma cell lines (B35M, Raji, SU-DHL-4, SU-DHL-6, and Ramos) spanning Burkitt's to diffuse large cell lymphoma. Although Ramos had a lower HLA-DR density, these cell lines were otherwise selected because of high cell surface CD20 and HLA-DR abundance.

A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words.

The United States Food and Drug Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ((90)yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ((131)I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) anti-CD20 MAbs for use in radioimmunotherapy (RIT) of non-Hodgkin's lymphoma (NHL), thus redefining the standard care of cancer patients.

Characterization of human IgG antimouse antibody in patients with B-cell malignancies.

Purpose: Immunotherapeutic approaches to cancer offer an attractive adjunct to conventional modalities, although human antiglobulin responses can be an obstacle to repeated treatment. This study of a large number of patients with B-cell malignancies, over an extended period of time, characterized their human antimouse antibody (HAMA) seroconversion.

Experimental Design: A total of 617 samples from 112 subjects were analyzed for HAMA titers.

Documentation of idiotypic cascade after Lym-1 radioimmunotherapy in a patient with non-Hodgkin's lymphoma: basis for extended survival?

Purpose: The purpose of this study was to examine idiotypic cascade mechanisms in the plasma of a prolonged survivor patient with aggressive non-Hodgkin's lymphoma (NHL). It is a follow-up to previously published seminal studies by this laboratory showing survival benefit associated with radioimmunotherapy in NHL patients. Immunoglobulin from the patient's plasma was purified, characterized, and shown to possess the activities expected of idiotypic antibodies.

Human antiglobulin response to foreign antibodies: therapeutic benefit?

Immunotherapies for cancer offer attractive alternatives to conventional therapies although human anti-globulin antibody (HAGA) against the antibody (Ab) administered to the patient can be an obstacle to repeated treatment. Monoclonal antibodies (mAb), whether foreign or human in origin, have been used safely in patients for two decades. Adverse events have not proven to be significant clinical obstacles, although alterations of pharmacokinetic behavior of subsequently administered Ab can lead to less effective therapy.

Constitutive expression of proinflammatory complement components by subsets of neurons in the central nervous system.

The brain is largely protected from damage due to infection, trauma, and aberrant processes by the innate immune system. These studies were undertaken to determine whether neurons in normal brains constitutively express complement components. In situ hybridization and immunohistochemical studies with specific riboprobes and antibodies, respectively, revealed that most hippocampal neurons, many pyramidal cortical neurons and cerebellar Purkinje neurons in normal murine brains constitutively express C3, C5 and C6.

Complement activation by neurofibrillary tangles in Alzheimer's disease.

Brain inflammation is widely documented to occur in Alzheimer's disease (AD), but its sources are still incompletely understood. Here, we present in vitro and in situ evidence that, like amyloid beta peptide (Abeta), tau, the major protein constituent of the neurofibrillary tangle, is a potent, antibody-independent activator of the classical complement pathway. Complement activation, in turn, is known to drive numerous inflammatory responses, including scavenger cell activation and cytokine production.

Molecular cloning of the C6A form cDNA of the mouse sixth complement component: functional integrity despite the absence of factor I modules.

The sixth complement component (C6) is an essential component of the biologically active C5b-9 membrane attack complex of the complement system. The multimolecular C5b-9 complex is an important mediator of the biological effects of the activated complement system through its prominent cell signaling and cytolytic functions. To begin to provide essential information and reagents needed to analyze the functions of the complement system in mouse models of human diseases, the cDNA of the A form of mouse C6, which is present in all mouse strains, was cloned and characterized structurally and functionally.

Inflammation and Alzheimer's disease.

Inflammation clearly occurs in pathologically vulnerable regions of the Alzheimer's disease (AD) brain, and it does so with the full complexity of local peripheral inflammatory responses. In the periphery, degenerating tissue and the deposition of highly insoluble abnormal materials are classical stimulants of inflammation. Likewise, in the AD brain damaged neurons and neurites and highly insoluble amyloid beta peptide deposits and neurofibrillary tangles provide obvious stimuli for inflammation.

Focal inflammation in the brain: role in Alzheimer's disease.

We hypothesize that amyloid (Abeta) peptide-containing neuritic plaques in the brains of patients with Alzheimer's disease represent chronic inflammatory foci mediated by the actions of the complement system and proinflammatory cytokines. In support of this, in vitro studies show that the (Abeta) peptide is a potent complement activator and that such complement activation leads to the formation of covalent (Abeta)-C3 activation fragment complexes, the generation of the chemokine-like C5a complement activation peptide, and the formation of the proinflammatory C5b-9 complex in functionally active form able to insert into neuronal cell membranes. Other studies show that C5a, together with (Abeta), synergistically augments the release of proinflammatory cytokines from human monocytes.

Complement-dependent proinflammatory properties of the Alzheimer's disease beta-peptide.

Large numbers of neuritic plaques (NP), largely composed of a fibrillar insoluble form of the beta-amyloid peptide (Abeta), are found in the hippocampus and neocortex of Alzheimer's disease (AD) patients in association with damaged neuronal processes, increased numbers of activated astrocytes and microglia, and several proteins including the components of the proinflammatory complement system. These studies address the hypothesis that the activated complement system mediates the cellular changes that surround fibrillar Abeta deposits in NP. We report that Abeta peptides directly and independently activate the alternative complement pathway as well as the classical complement pathway; trigger the formation of covalent, ester-linked complexes of Abeta with activation products of the third complement component (C3); generate the cytokine-like C5a complement-activation fragment; and mediate formation of the proinflammatory C5b-9 membrane attack complex, in functionally active form able to insert into and permeabilize the membrane of neuronal precursor cells.

Molecular and cellular characterization of the membrane attack complex, C5b-9, in Alzheimer's disease.

The membrane attack complex, C5b-9, is of considerable importance in many inflammatory reactions. It is the terminal, cytolytic component of both classical and alternative pathway activation, and its presence presupposes other potentially destructive complement constituents, including anaphylotoxins and opsonins. We have characterized C5b-9 and its C9 constituent in the Alzheimer's disease (AD) and nondemented elderly (ND) brain using immunohistochemistry at the light and electron microscopic levels, Western blot analysis, and the reverse transcriptase polymerase chain reaction.

Aggregation state-dependent activation of the classical complement pathway by the amyloid beta peptide.

Activation of the classical complement pathway has been widely investigated in recent years as a potential mechanism for the neuronal loss and neuritic dystrophy characteristic of Alzheimer's disease (AD) pathogenesis. We have previously shown that amyloid beta peptide (A beta) is a potent activator of complement, and recent evidence suggesting that the assembly state of A beta is crucial to the progress of the disease prompted efforts to determine whether the ability of A beta to activate the classical complement pathway is a function of the aggregation state of the peptide. In this report, we show that the fibrillar aggregation state of A beta, as determined by thioflavin T fluorometry, electron microscopy, and staining with Congo red and thioflavine S, is precisely correlated with the ability of the peptide to induce the formation of activated fragments of the complement proteins C4 and C3.

Enhanced cytotoxicity of amyloid beta-peptide by a complement dependent mechanism.

Amyloid beta-peptide (A beta) has been shown to activate the classical complement pathway in vitro. Here, we demonstrate that this interaction is fully capable of killing cells and damaging cellular processes in mixed hippocampal cultures from embryonic day 18 rat fetuses. Lactic acid dehydrogenase (LDH) release and morphologic changes were used to evaluate toxicity.

Substrate specificities of murine C1s.

An early step in the initiation of the classical C pathway is the proteolytic activation of component C4 by subcomponent C1-s. We have examined the substrate specificity of murine C1-s (mC1-s) by measuring its proteolytic activity on human and murine C4, and on the murine C4 isotype designated sex-limited protein (Slp). The latter substrate was examined because previous studies have demonstrated that Slp is not cleaved by C1-s, and hence Slp has been assumed to be nonfunctional in the C pathways.

Murine C4b-binding protein. Mapping of the ligand binding site and the N-terminus of the pre-protein.

We have constructed cell surface-bound forms of murine C4b-binding protein (mC4BP) that allowed us to monitor the binding of mC4BP to C4b with relatively simple erythrocyte rosette assays. We used two types of surface-bound mC4BP: one in which segments of mC4BP were fused directly to a peptide containing the transmembrane and cytoplasmic domains of human complement receptor CR2 (BPR1-type); and a second in which the same segments were fused to a longer peptide containing the five membrane-proximal short consensus repeats (SCR) of CR2 as well as the transmembrane and cytoplasmic domains (BPR2-type). COS cells transfected with either construct carrying all six mC4BP SCR rosetted with C4b-bearing EAC14 cells but not with C4b-lacking EAC1 cells; and rosetting was inhibited by excess inactivated C4 but not inactivated C3.

Complement activation by beta-amyloid in Alzheimer disease.

Alzheimer disease (AD) is characterized by excessive deposition of the beta-amyloid peptide (beta-AP) in the central nervous system. Although several lines of evidence suggest that beta-AP is neurotoxic, a mechanism for beta-AP toxicity in AD brain remains unclear. In this paper we provide both direct in vitro evidence that beta-AP can bind and activate the classical complement cytolytic pathway in the absence of antibody and indirect in situ evidence that such actions occur in the AD brain in association with areas of AD pathology.

Characterization of a Trypanosoma cruzi C3 binding protein with functional and genetic similarities to the human complement regulatory protein, decay-accelerating factor.

Evasion of the complement system by microorganisms is an essential event in the establishment of infection. In the case of Trypanosoma cruzi, the causative agent of Chagas disease, resistance to complement-mediated lysis is a developmentally regulated characteristic. Infectious trypomastigotes are resistant to complement-mediated lysis in the absence of immune antibodies, whereas the insect forms (epimastigotes) are sensitive to lysis via the alternative complement pathway.

CR2 complement receptor.

CR2, a membrane glycoprotein, is one of a number of cell-surface proteins which bind activation and processing fragments of the complement system. CR2, which is found on normal B lymphocytes, follicular dendritic cells in lymphoid organs, and epithelial cells, interacts preferentially with C3dg, the terminal activation/processing fragment of the third complement component. Attachment of C3dg to CR2 brings complement activators, bearing covalently bound C3dg, into direct membrane contact with CR2-bearing cells.

Murine complement component C4 and sex-limited protein: identification of amino acid residues essential for C4 function.

Murine sex-limited protein (Slp) is an isotype of murine complement component C4 that shares 95% sequence identity with C4 as well as the intramolecular thioester necessary for C4 function but has no complement activity. Slp is nonfunctional at least in part because it is not cleaved by the activated form of complement protease C1s (C1s), which proteolytically activates C4 in the classical complement pathway. Slp is also distinct from C4 in that its expression in some mouse strains is under testosterone control.

Transfected cDNA directs expression of hemolytically active murine C4 in cultured mouse and monkey cells.

Previously cloned and sequenced full-length cDNAs for murine C4 and the closely related sex-limited protein (Slp) have been placed into an eucaryotic expression vector. Transfer of these DNA constructs transiently into monkey COS cells or stably into mouse L cells results in the expression and secretion of hemolytically active mouse C4 and mature Slp. We estimate from hemolytic activities that COS and L cells secrete 0.