The amyloid beta peptide (A beta P) is a small fragment of the much larger, broadly distributed amyloid precursor protein (APP). Abundant A beta P deposition in the brains of patients with Alzheimer's disease suggests that altered APP processing may represent a key pathogenic event. Direct protein structural analyses showed that constitutive processing in human embryonic kidney 293 cells cleaves APP in the interior of the A beta P, thus preventing A beta P deposition. A deficiency of this processing event may ultimately prove to be the etiological event in Alzheimer's disease that gives rise to senile plaque formation.

Alzheimer disease (AD) is characterized by excessive deposition of the (3-amyloid peptide (QAP) in the central nervous system. Although several lines of evidence suggest that 3-AP is neurotoxic, a mechanism for (-AP toxicity in AD brain remains unclear. In this paper we provide both direct in vito evidence that S-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.Alzheimer disease (AD) is characterized by excessive central nervous system (CNS) deposition of the f-amyloid peptide ((3-AP), a 40-to 42-amino acid peptide derived from a larger amyloid precursor protein (APP) (1-3). Although no specific mechanism of -3-AP deposition has yet been formally proven, there are several lines ofevidence (4-6) that, once generated, 3-AP causes direct or indirect toxicity to CNS neurons. Proposed mechanisms of AD neurotoxicity include membrane changes (7), alterations in Ca2+ homeostasis (6,8), excitotoxicity (5,6), and disruption of cytoskeletal or axon transport systems (9, 10). However, no single AD pathogenetic mechanism has yet achieved a wide consensus of acceptance.In addition to studies of 3-AP, over the last decade a number ofinvestigators have noted that the AD brain exhibits many of the classical markers of immune-mediated damage. These include elevated numbers of major histocompatibility complex class I-and II-immunoreactive microglia (cells believed to be an endogenous CNS form of the peripheral macrophage) (11-15) and astrocytes expressing interleukin 1 (16) and a1-antichymotrypsin (17) (both acute phase reactants). Of particular importance, complement proteins of the classical pathway have been immunohistochemically detected in the AD brain (12,13,(18)(19)(20), and we have noted that they most often appear associated with ,3-AP-containing pathological structures such as senile plaques. Proteins specific to the alternative pathway do not appear to be present (12,13,18). The first step in the classical complement pathway entails binding of the Clq component of C1, with subsequent activation of the Clr and Cls components. This is followed by a complex series of autocatalytic reactions, proceeding through C4, C2, and C3, and culminating in formation of the membrane attack complex (MAC), C5b-9. The MAC inserts a lytic plug in adjacent cell membranes, mediating cellular toxicity (21). Although Clq binding to the Fc region of immunoglobulins is the most common mechanism for initiating the classical pathway, several substratesincluding viruses, parasites, and mannan-binding proteinhave also been demonstrated to activate C1 and to do so in an antibody-independent fashion (22). In this paper we present six converging lines of evidence suggesting that S-AP activates the classical pathway complement cascade without mediation by immunoglobulin. This previously unrecognized mechanism may contribute significantly to the neurotoxicity of (3-AP as well as to the patho...

The A4 protein (or beta-protein) is a 42- or 43-amino-acid peptide present in the extracellular neuritic plaques in Alzheimer's disease and is derived from a membrane-bound amyloid protein precursor (APP). Three forms of APP have been described and are referred to as APP695, APP751 and APP770, reflecting the number of amino acids encoded for by their respective complementary DNAs. The two larger APPs contain a 57-amino-acid insert with striking homology to the Kunitz family of protease inhibitors. Here we report that the deduced amino-terminal sequence of APP is identical to the sequence of a cell-secreted protease inhibitor, protease nexin-II (PN-II). To confirm this finding, APP751 and APP695 cDNAs were over-expressed in the human 293 cell line, and the secreted N-terminal extracellular domains of these APPs were purified to near homogeneity from the tissue-culture medium. The relative molecular mass and high-affinity binding to dextran sulphate of secreted APP751 were consistent with that of PN-II. Functionally, secreted APP751 formed stable, non-covalent, inhibitory complexes with trypsin. Secreted APP695 did not form complexes with trypsin. We conclude that the secreted form of APP with the Kunitz protease inhibitor domain is PN-II.

The influence of valence and heavy chain on antibody activity was investigated using transfectoma-derived, class-switched IgG1 and IgM human monoclonal antibodies (MAbs) reactive with the bacterial pathogens Escherichia coli K1 and group B Streptococcus species. IgG-IgM pairs were compared in vitro for antigen binding and opsonic activities and in vivo for protective efficacy in neonatal rats. For the anti-E. coli pair, the IgM MAb was 1000-fold more potent in all assay formats. Importantly, the 50% protection dose (PD50) of the IgM MAb was 10-20 ng/rat, while 100 micrograms of the IgG MAb was only minimally protective. For the group B streptococcal MAbs, the IgM was 100- and 4500-fold more potent in binding and opsonization assays, respectively. However, while 20 micrograms of IgM protected neonatal rats, 100 micrograms of IgG MAb was partly protective. These experiments demonstrate the utility of recombinant DNA technology for creating a panel of antibodies that may aid in selecting potential immunotherapeutic candidates.

One of the major clinical findings in Alzheimer's disease (AD) is the formation of deposits of beta-amyloid protein in amyloid plaques, derived from the beta-amyloid precursor protein (beta-APP). To determine the possible use of beta-APP as a diagnostic marker for AD in CSF, a monoclonal antibody-based immunoassay specific for this protein was developed. The assay does not differentiate between beta-APP695 and beta-APP751 forms but does preferentially recognize beta-APP751 complexed with a protease. Of the two sets of CSF samples tested, one set, obtained from living patients, gave a slightly lower level of beta-APP in AD and Parkinson's disease patients relative to controls, whereas the other set, composed of postmortem samples, showed no significant differences between the AD and control groups.