Michael Carroll - US grants

Covalent attachment of the fourth component of complement to the immune complex (IC) is a critical events in the classical pathway. This event not only focuses assembly of the later components at the site of inflammation but is though to be important in inhibiting the formation of insoluble complexes. Two isotypes of C4 protein are found in the serum of most individuals and through their structures are nearly identifical, they differ significantly in their efficiency of covalent binding to IC. While C4A selectively forms an amide linkage with protein, C4B more readily forms an ester bond with carbohydrate. It has been proposed that the selectively in binding evolved as a mechanism to insure attachment of C4 to a wide range of pathogens or structurally different forms of antibody. Genetic studies have shown that there is an increased relative risk to a number of autoimmune diseases, particularly those complicated by immune complex deposition such as SLE, with certain C4 allotypes. The association is greatest with partial and complete deficiencies of C4 protein. The objective of this proposal is to determine the chemical basis for the binding differences between the two isotypes of C4 protein. This will be accomplished by analysing hybrid C4 proteins constructed by the technique of site-directed mutagenesis and includes: (1) determining which amino acid residues are critical for the selectivity in transacylation of C4 to either protein or carbohydrate; (2) determining what role allotypic differences have in the interaction and covalent binding of C4 to antigen and antibody; (3) determining what role carbohydrate has in covalent binding; (4) determining which residues are critical for stability of the thiolester.

The long-term objective of this proposal is to increase our understanding of the role of complement in human disease and its interaction with other components of the immune system. The serum complement system in man is important in both the afferent and efferent immune response. The central component or C3 participates as both a cofactor in formation of convertase in the cascade and interacts directly with both humoral and cellular components of the immune system. A critical step in this intersection is covalent attachment of C3 to antibody-antigen complexes on activation of the alternative pathway. Following activation, split products of C3 serve as ligands for specific receptors on blood cells, e.g. CD35, CD21 & CD11b,c/CD18, which are important in such diverse roles as cellular adhesion and B-cell signaling. As a first step towards the long-term objective, this proposal will examine the biochemical interaction between C3 and the antibody molecule. The hypothesis that C3 binds to a specific site on the IgG heavy chain will be tested. The first specific aim is to identify the site/sites of covalent attachment of human C3 onto human IgG1 heavy chain. Preliminary results have shown that the major site of attachment is within the CH1 domain. This aim will use a biochemical approach (amino acid sequencing and mass spectrometry) to identify the site/sites of attachment. The second specific aim is to identify specific residues for C3 attachment within this region of the heavy chain for both human and murine isotypes by using a site-directed mutagenesis approach. The approach in general is to synthesize recombinant antibody bearing neutral substitutions (e.g., Ala) at potential acceptor sites and test their activity for activation of the alternative pathway. Understanding the role of complement in its interaction with the immune system in host protection is to significant importance. Genetic deficiencies in complement often lead to autoimmune disorders and increased susceptibility to disease. By increasing our understanding of its role, a therapeutic approach can be realized.

The long-term objective of this proposal is to increase our understanding of the role of complement in human disease and its interaction with other components of the immune system. The serum complement system in man is important in both the afferent and efferent immune response. The central component or C3 participates as both a cofactor in formation of convertase in the cascade and interacts directly with both humoral and cellular components of the immune system. A critical step in this intersection is covalent attachment of C3 to antibody-antigen complexes on activation of the alternative pathway. Following activation, split products of C3 serve as ligands for specific receptors on blood cells, e.g. CD35, CD21 & CD11b,c/CD18, which are important in such diverse roles as cellular adhesion and B-cell signaling. As a first step towards the long-term objective, this proposal will examine the biochemical interaction between C3 and the antibody molecule. The hypothesis that C3 binds to a specific site on the IgG heavy chain will be tested. The first specific aim is to identify the site/sites of covalent attachment of human C3 onto human IgG1 heavy chain. Preliminary results have shown that the major site of attachment is within the CH1 domain. This aim will use a biochemical approach (amino acid sequencing and mass spectrometry) to identify the site/sites of attachment. The second specific aim is to identify specific residues for C3 attachment within this region of the heavy chain for both human and murine isotypes by using a site-directed mutagenesis approach. The approach in general is to synthesize recombinant antibody bearing neutral substitutions (e.g., Ala) at potential acceptor sites and test their activity for activation of the alternative pathway. Understanding the role of complement in its interaction with the immune system in host protection is to significant importance. Genetic deficiencies in complement often lead to autoimmune disorders and increased susceptibility to disease. By increasing our understanding of its role, a therapeutic approach can be realized.

DESCRIPTION (provided by applicant): The goal of the Graduate Program in Immunology at Harvard Medical School is to recruit students of exceptional ability regardless of gender or ethnicity and provide these students with the best attainable training environment in which they may develop into independently thinking scientists, specializing in the field of immunology. In this context, the field of immunology is defined broadly and includes not only the strictly immunological methodologies, but also approaches such as genetics, structural and molecular biology, cell biology, tumor biology and computational biology. This goal will be accomplished through formal course-work, laboratory rotations, literature discussions, discussions with faculty and mentored research. Students may choose from 47 faculty for rotations and laboratory work in all major areas of immunology. Policies and guidelines for graduate work are set by the Graduate Committee, a body of senior and junior faculty active in the teaching immunology program, in accordance with the Executive Committee in Immunology and those of the Division of Medical Sciences. Students'progress is monitored through the administration of a comprehensive qualifying examination, comprised of an oral presentation and a written proposal (NIH style post-doctoral application). This phase of the student's training -including coursework and rotations- is usually completed by middle of the second year. Dissertation research is supervised by the dissertation advisor and its progress is monitored by a dissertation advisory committee. The experimental component usually requires 3-4 years to complete a body of work considered adequate for a written dissertation.

4-hydroxy-3-nitrophenyl acetyl; 4-hydroxy-5-nitrophenyl acetic acid; ATGN; Acylneuraminyl hydrolase; Adjuvant; Affect; Animal Model; Animal Models and Related Studies; Antibodies; Antigen Variation; Antigenic Determinants; Antigenic Variability; Antigenic Variation; Antigens; B blood cells; B-Cells; B-Lymphocytes; Binding; Binding (Molecular Function); Binding Determinants; Biological Models; Bursa-Dependent Lymphocytes; Bursa-Equivalent Lymphocyte; C3 d; C3d; CD 21 Antigens; CD19; CD19 gene; CD21; CD21 Antigens; CR2; CR3; CR3 Receptor; Capsid Proteins; Cells; Cleaved cell; Coat Proteins; Collaborations; Complement; Complement 3d; Complement 3d Receptors; Complement C3d; Complement Proteins; Complement Receptor; Complement Receptors 2; Coupled; Coupling; Dendritic Cells; Dendritic Cells, Follicular; Development; Egypt 101 virus; Epitopes; Epstein-Barr Virus Receptors; Follicular Dendritic Cells; Genetic Alteration; Genetic Change; Genetic defect; Germinal Center; Grippe; HHV-2; HSV-2; HSV2; Health; Hemagglutinin; Herpes Simplex Virus 2; Herpes Simplex Virus Type 2; Herpesvirus 2 (alpha), Human; Herpesvirus 2, Human; Herpesvirus progenitalis; Human; Human (alpha) herpes virus 2; Human Herpesvirus 2; Human herpes simplex virus type 2; Human, General; Humoral Immunities; Immune response; Immunities, Humoral; Immunity; Influenza; Influenza Virus; Integrin alpha-M beta-2; Integrin alphaMbeta2; Knock-in; Knock-in Mouse; Label; Ligands; Lymph; Lymph Node Subcapsular Sinus; Lymph node proper; Mac 1; Mac-1 Adhesive Receptor; Mac-1 Antigen; Mac-1 Receptor; Macrophage-1 Antigen; Maintenance; Maintenances; Mammalia; Mammals; Mammals, General; Mammals, Mice; Man (Taxonomy); Man, Modern; Mediating; Memory; Memory B Cell; Memory B-Lymphocyte; Mice; Microscopy; Model System; Models, Biologic; Molecular; Molecular Interaction; Murine; Mus; Mutation; N-Acylneuraminate Glycohydrolases; NP-hapten; Neuraminidase; Oligosaccharide Sialidase; Pathway interactions; Peripheral; Pneumonia; Pneumonitis; Predisposition; Proteins; Pulmonary Inflammation; Range; Receptor Protein; Receptor Signaling; Receptor, Complement 3; Receptor, Mo1 Antigen; Receptor, Mo1 Glycoprotein; Receptors, C3d; Receptors, CR2; Receptors, Complement 3d; Recombinants; Regulation; Reticuloendothelial System, Lymph; Reticuloendothelial System, Lymph Node; Role; Sialidase; Structure of germinal center of lymph node; Subcapsular Sinus; Susceptibility; System; System, LOINC Axis 4; Testing; Vaccination; Vaccines; Veiled Cells; Viral Coat Proteins; Viral Gene Products; Viral Gene Proteins; Viral Outer Coat Protein; Viral Proteins; WNV; West Nile; West Nile virus; abstracting; antibody-based immunity; base; cleaved; design; designing; exo alpha sialidase; experiment; experimental research; experimental study; flu; flu infection; gene product; genome mutation; herpes simplex ii; host response; human alphaherpesvirus 2; immunogen; immunoresponse; influenza infection; influenzavirus; influenzavirus (unspecified); insight; lymph gland; lymph nodes; lymphatic fluid; macrophage; model organism; neutralizing antibody; new vaccines; next generation vaccines; novel; novel vaccines; pathogen; pathway; receptor; research study; response; social role; trafficking; uptake; viral antigen variation; virus antigenic variation; virus protein

A research or resource commons requires agreement among providers or participants about its legal structure, the technical requirements for common resources, and a shared understanding about how to sustain the commons. Legal issues, usually involving intellectual property or contract law, often arise as researchers, or research funders, seek to build commons or commons-based tools, such as Creative Commons licenses. There are three major objectives for the workshop to be held at American University. First, the attendees will review lessons learned from those who have worked to build or to promote the use of commons structures to support scientific research from within the federal government and from the private sector, including the non-profit sector. Next, the members will identify the legal, technical, and cultural requirements for a successful commons, with a particular focus on scientific data. The key themes will be the respective roles of standardization and interoperability at the legal and technical levels necessary for resources to be shared in a commons, whether those resources are literature, data, physical inputs, or others. Third, they will discuss how the federal government, the university and non-profit sector, and industry can best work together to support existing successful resource commons in science and to create new commons or commons-based tools to improve the speed and efficiency of publicly funded scientific research. Attention will be given to how existing commons standards, such as legal and technical tools supplied by Creative Commons, are currently being used in the sciences and how these might be made more useful with respect to emergent forms of scientific communication. The report of the workshop and all case studies developed will be made publicly available and published on the Internet.