Neil R. Cooper

Inflammation and Alzheimer’s disease

Inflammation clearly occurs in pathologically vulnerable regions of the Alzheimers 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. Because these stimuli are discrete, microlocalized, and present from early preclinical to terminal stages of AD, local upregulation of complement, cytokines, acute phase reactants, and other inflammatory mediators is also discrete, microlocalized, and chronic. Cumulated over many years, direct and bystander damage from AD inflammatory mechanisms is likely to significantly exacerbate the very pathogenic processes that gave rise to it. Thus, animal models and clinical studies, although still in their infancy, strongly suggest that AD inflammation significantly contributes to AD pathogenesis. By better understanding AD inflammatory and immunoregulatory processes, it should be possible to develop anti-inflammatory approaches that may not cure AD but will likely help slow the progression or delay the onset of this devastating disorder.

The Classical Complement Pathway: Activation and Regulation of the First Complement Component

Publisher Summary This chapter focuses on the activation and regulation of the first complement component. . The chapter outlines the history of the classical pathway of the complement system. Clq and C1 are bound and activated by immune complexes or aggregates containing IgG or IgM but not by those containing IgA, IgD, or IgE . Among IgG subclasses, IgG 3 is most reactive followed by IgG 1 , and IgG 2 ; intact IgG 4 is minimally reactive, although its Fc region binds C1. C1-In does not efficiently regulate C1 activation induced by immune complexes. It, thus, appears that immune complex dependent activation is not under positive host regulation. This is apparently largely true. However, C1 activation by small immune complexes or complexes formed with nonavid antibody or with ratios of antigen to antibody far from equivalence, all of which are poor C1 activators, may well be regulated by C1-In.

The reaction of monomeric and aggregated immunoglobulins with Cl

Abstract The relative capacity of different immunoglobulin classes and subclasses in different states of aggregation to bind the first component of complement (C l ) was studied. Both monomeric γG and 7S γM immunoglobulins were found to be able to bind C l . The binding capacity of γG myeloma proteins was dependent upon the γG subclass, the order of reactivity being γ G3 > γ Gl > γ G2 > γ G4. No differentiation into functional subclasses was possible on the examination of 12 different monomeric γM preparations. The relative C l binding capacity of γM polymers was: 7S γ M = 1, 19S γ M = 15, 27–35S γ M = 64, and > 35S γ M = 116. Comparable amounts of the 7S subunit of γM and γG bound C l to a similar extent, and aggregated γM and γG were also comparable in C l binding, suggesting that the differences in the relative C l binding capacity of these immunoglobulins is dependent upon the degree of polymerization that is present in the native state of these proteins. The Fc fragment of γCl was as efficient as the intact protein in fixing C l , when compared on a molar basis; human F(ab′) 2 and the cyanogen bromide treated Fc fragment were unable to bind C l .

Identification of an epitope in the major envelope protein of Epstein-Barr virus that mediates viral binding to the B lymphocyte EBV receptor (CR2)

The Epstein-Barr virus gp350/220 envelope protein mediates virus attachment to the EBV/C3dg receptor (CR2) of human B lymphocytes. Synthetic peptides corresponding to two regions in gp350/220, which have a similar amino acid sequence with the complement C3dg protein, were used to identify a receptor binding epitope. A peptide corresponding to the N terminus of gp350/220, EDPGFFNVE, bound to purified CR2 and to CR2 positive but not CR2 negative B and T lymphoblastoid cell lines. Soluble monomeric gp350/220 peptide blocked CR2 binding to immobilized EBV, while multimeric forms of the N-terminal gp350/220 peptide conjugated to albumin efficiently blocked recombinant gp350/220 and C3dg binding to B cells as well as EBV-induced B cell proliferation and transformation. These studies indicate that the N-terminal region of gp350/220 plays a crucial role in mediating the earliest stages of EBV infection of B cells and provides a molecular basis for the restricted host cell EBV tropism.

Neuronal Expression of a Functional Receptor for the C5a Complement Activation Fragment

The present studies were undertaken to determine whether neuronal subsets in normal brains constitutively express functionally competent C5a receptors. In situ hybridization studies coupled with immunohistochemical approaches revealed that most neurons in the hippocampal formation, many pyramidal cortical neurons, and cerebellar Purkinje neurons in normal human and murine brains constitutively express C5a receptors. Neuronal C5a receptors bound C5a-coated fluorescent microspheres, and primary rodent hippocampal neurons responded to C5a with increased calcium fluxes via a pertussis-sensitive, presumably Gi-coupled protein. Additional studies with human neuroblastoma cells conducted to address the functional role of C5a receptors revealed that C5a triggered rapid activation of protein kinase C and activation and nuclear translocation of the NF-κB transcription factor. In addition, C5a was found to be mitogenic for undifferentiated human neuroblastoma cells, a novel action for the C5aR. In contrast, C5a protected terminally differentiated human neuroblastoma cells from toxicity mediated by the amyloid Aβ peptide. Thus, normal rodent hippocampal neurons as well as undifferentiated and differentiated human neuroblastoma cells express functional C5a receptors. These results have implications for understanding the role of neuronal C5aR receptors in normal neuronal development, neuronal homeostasis, and neuroinflammatory conditions such as Alzheimer’s disease.

Early events in the infection of human B lymphocytes by Epstein-Barr virus: The internalization process

The early events in the infection of normal B lymphocytes and B lymphoblastoid cells by Epstein-Barr virus (EBV) were examined by electron and immunoelectron microscopy and by infectivity and inhibition studies. Purified EBV remained on the cell surface at 4 degrees and appeared as 250-nm ovoid particles in contact with the cell membrane through 50-nm envelope projections. Internalization of EBV in normal B lymphocytes into large (300-500 nm) uncoated vacuoles was initiated within 2 to 5 min at 37 degrees. At this stage approximately 1/3 of cell-associated virus was located in cellular invaginations while another 1/3 was in cell vacuoles. Direct fusion of EBV with the outer cell membrane was not observed. Instead, viral deenvelopment and nucleocapsid transit into the cytoplasm occurred from the large endocytic vesicles within 15 to 30 min at 37 degrees and did not involve lysosomal enzymes. During this time, the viral envelope became amorphous and its separation from the nucleocapsid was evident. After 60 to 90 min at 37 degrees, viral nucleocapsids were visualized in close proximity to the cell nucleus. Weak bases such as chloroquine, methylamine, and ammonium chloride retarded viral deenvelopment and fusion inside the endocytic vacuoles, resulting in abrogation of viral infectivity and accumulation of intact virions within cell vacuoles. These studies indicate that EBV enters normal B lymphocytes by a different endocytic pathway than the clathrin-receptosome-lysosome pathway utilized by many other ligands, including a number of viruses, to enter cells. In contrast to the pathway of entry into normal B lymphocytes, EBV entered B lymphoblastoid cells by direct fusion with the outer cell membrane within 2 to 5 min at 37 degrees.

Immune Adherence by the Fourth Component of Complement

An immune complex becomes reactive in immune adherence after the specific binding of the fourth component of complement (C4). Immune adherence produced by the fourth component of complement is indistinguishable from classical immune adherence in terms of all parameters tested, except that it is entirely independent of the participation of the second and third components of complement.

Inactivation and lysis of oncornaviruses by human serum

Abstract The phenomenon of oncornavirus inactivation by human serum was examined. As judged by 50% inhibition of plaque or focus formation, every fresh human serum tested inactivated eight different oncornaviruses at serum dilution endpoints of 1:16. Fresh human serum released RNA-dependent DNA polymerase (RDDP) activity from each of 10 tested oncornaviruses. Thus, at least one mechanism of the human serum-induced virus inactivation was by viral lysis. The RDDP assay provided us with a simple method to screen the virolytic properties of many sera against a number of viruses. Oncornaviruses were lysed by sera from monkeys and cats, but not by sera from guinea pigs, rabbits, chickens, rats, goats, and mice ( BALB c , CBA, NIH Swiss). Heated normal human sera did not inactivate or lyse oncornaviruses, nor did they sensitize virions to guinea pig serum-mediated lysis. However, serum from a human vaccinated against Rauscher leukemia virus did sensitize the virus to guinea pig serum-mediated lysis. This ability of human sera to lyse oncornaviruses may be a cause of the difficulties in finding oncornaviruses in humans.

A comparison of methods for the molecular quantitation of the fourth component of human complement

Abstract Quantitation of C′4 by the effective molecule titration procedure yielded values which were approximately 300 times lower than values based on nitrogen determination or on immunochemical analysis. To explore the reason for the disparate results, a quantitative analysis of the reaction mechanism of C′4 in immune hemolysis was performed employing highly purified, radioactively labeled C′4. It was found that the majority of the C′4 molecules escape detection by effective molecule titrations due to (a) inefficient binding of C′4 to the cell surface, and (b) hemolytic inefficiency of specifically cell bound C′4. Two factors were experimentally derived which permit conversion of the number of effective C′4 molecules to the absolute number of C′4 molecules. The number of effective molecules per μg of C′4 was similar for isolated C′4 and for C′4 in fresh human serum.

Consumption of human complement components by complexes of IgG with protein A of Staphylococcus aureus

Abstract Complexes between IgG or the Fc fragment of IgG and protein A from S. aureus activate the serum complement system in a manner which is entirely analogous to complement activation by antigen-antibody complexes. Thus C1,C4,C2,C3,C5,C6,C8 and C9 were found to be depleted on addition of protein A to serum. The alternate complement pathway was also activated on addition of protein A to serum. Consumption of the components and the C3 proactivator varied with the dose of protein A and the same dose response curve was obtained for activation of both pathways in serum. Maximal activation occurred with the same dose of protein A. Protein A was found to be able to inhibit binding of C 1 by IgG1 or the Fc fragment of IgG1, indicating that the complement activating site and protein A binding site are sterically close. Finally, it has been found that erythrocytes passively coated with protein A lyse in fresh normal human or guinea pig serum.