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A major challenge for the development of a highly effective AIDS vaccine is the identification of mechanisms of protective immunity. To address this question, we used a nonhuman primate challenge model with simian immunodeficiency virus (SIV). We show that antibodies to the SIV envelope are necessary and sufficient to prevent infection. Moreover, sequencing of viruses from breakthrough infections revealed selective pressure against neutralization-sensitive viruses; we identified a two-amino-acid signature that alters antigenicity and confers neutralization resistance. A similar signature confers resistance of human immunodeficiency virus (HIV)-1 to neutralization by monoclonal antibodies against variable regions 1 and 2 (V1V2), suggesting that SIV and HIV share a fundamental mechanism of immune escape from vaccine-elicited or naturally elicited antibodies. These analyses provide insight into the limited efficacy seen in HIV vaccine trials., Among the five human efficacy trials of HIV-1 vaccines, only one has shown some success in preventing HIV infection. In the RV144 trial, a combination viral vector and protein immunization [...]

Dooley JL, Abdel-Latif D, St. Laurent CD, Puttagunta L, Befus D, Lacy P. Regulation of inflammation by Rac2 in immune complex-mediated acute lung injury. Am J Physiol Lung Cell Mol Physiol 297: L1091-L1102, 2009. First published October 2, 2009; doi: 10.1152/ajplung.90471.2008.--Acute lung injury (ALI) is an inflammatory disorder associated with recruitment and activation of neutrophils in lungs. Rac2, a member of the Rho GTPase subfamily, is an essential regulator of neutrophil degranulation, superoxide release, and chemotaxis. Here, we hypothesized that Rac2 is important in mediating lung injury. Using a model of IgG immune complex-mediated ALI, we showed that injury was attenuated in rac[2.sup.-/-] mice compared with wild-type (WT) mice undergoing ALI, with significant decreases in alveolar leukocyte numbers, vascular leakage, and the inflammatory mediators, myeloperoxidase (MPO) and matrix metal-loproteinases (MMPs). Reduced injury in rac[2.sup.-/-] mice was not associated with diminished cytokine and chemokine production, since bronchoalveolar lavage (BAL) levels of IL-17, TNF, CCL3, CXCL1, and CXCL2 were similarly increased in WT and [rac.sup.2-/-] mice with ALI compared with sham-treated mice (no ALI). BAL levels of MMP-2 and MMP-9 were significantly decreased in the airways of rac[2.sup.-/-] mice with ALI. Immunohistochemical analysis revealed that MMP-2 and MMP-9 expression was evident in alveolar macrophages and interstitial neutrophils in WT ALI. In contrast, MMP-positive cells were less prominent in rac[2.sup.-/-] mice with ALI. Chimeric mice showed that Rac2-mediated lung injury was dependent on hematopoietic cells derived from bone marrow. We propose that lung injury in response to immune complex deposition is dependent on Rac2 in alveolar macrophages and neutrophils. neutrophils; macrophages; bronchoalveolar lavage; cytokines; bone marrow chimeras; matrix metalloproteinases doi: 10.1152/ajplung.90471.2008

Deposits of Ig and complement are abundant in affected joints of patients with rheumatoid arthritis (RA) and in animal models of RA in which antibodies are demonstrably pathogenic. To identify molecular targets of the Igs deposited in arthritic joints, which may activate local inflammation, we used a combination of mass spectrometry (MS) and protein microarrays. Immune complexes were affinity-purified from surgically removed joint tissues of 26 RA and osteoarthritis (OA) patients. Proteins complexed with IgG were identified by proteomic analysis using tandem MS. A striking diversity of components of the extracellular matrix, and some intracellular components, copurified specifically with IgG from RA and OA tissues. A smaller set of autoantigens was observed only in RA eluates. In complementary experiments, IgG fractions purified from joint immune complexes were tested on protein microarrays against a range of candidate autoantigens. These Igs bound a diverse subset of proteins and peptides from synovium and cartilage, different from that bound by normal serum Ig. One type of intracellular protein detected specifically in RA joints (histones H2A/B) was validated by immunohistology and found to be deposited on the cartilage surface of RA but not OA joints. Thus, autoantibodies to many determinants (whether deposited as 'neoantigens' or normal constituents of the extracellular matrix) have the potential to contribute to arthritic inflammation. autoantibodies | histones | mass spectrometry | proteomics

IgA nephropathy (IgAN) is characterized by circulating immune complexes composed of galactose-deficient IgA1 and a glycan-specific IgG antibody. These immune complexes deposit in the glomerular mesangium and induce the mesangioproliferative glomerulonephritis characteristic of IgAN. To define the precise specificities and molecular properties of the IgG antibodies, we generated EBV-immortalized IgG-secreting lymphocytes from patients with IgAN and found that the secreted IgG formed complexes with galactose-deficient IgA1 in a glycan-dependent manner. We cloned and sequenced the heavy- and light-chain antigen-binding domains of IgG specific for galactose-deficient IgA1 and identified an A to S substitution in the complementarity-determining region 3 of the variable region of the gene encoding the IgG heavy chain in IgAN patients. Furthermore, site-directed mutagenesis that reverted the residue to alanine reduced the binding of recombinant IgG to galactose-deficient IgA1. Finally, we developed a dot-blot assay for the glycan-specific IgG antibody that differentiated patients with IgAN from healthy and disease controls with 88% specificity and 95% sensitivity and found that elevated levels of this antibody in the sera of patients with IgAN correlated with proteinuria. Collectively, these findings indicate that glycan-specific antibodies are associated with the development of IgAN and may represent a disease-specific marker and potential therapeutic target., Introduction IgA nephropathy (IgAN), also called Berger disease, was described in 1968 (1) based on the immunohistochemical finding of IgA- and IgG-containing immune complexes in the glomerular mesangium of the [...]

Antibodies are among the most highly selective tight-binding ligands for proteins. Because the human genome project has deciphered the proteome, there is an opportunity to use combinatorial antibody libraries to select high-affinity antibodies to every protein encoded by the genome. However, this is a large task because the selection formats used today for combinatorial antibody libraries are geared toward generating antibodies to one antigen at a time. Here, we describe a method that accelerates the identification of antibodies to a multitude of antigens simultaneously by matching combinatorial antibody libraries against eukaryotic antigen libraries so that replication-competent cognate antigen-antibody pairs can be directly selected. Phage and yeast display systems are used because they each link genotype to phenotype and can be replicated individually. When combined with cell sorting, the two libraries can be selected against each other for recovery of cognate antigen-antibody clones in a single experiment. antibody libraries | human genome | phage display | yeast display

The integration of immune and neuroendocrine responses that the body uses to deals with dynamic changes in the environment is discussed. This is explained using the bow-tie architecture wherein a number of stimuli are converted in broad range of fine responses after passing through an integrated activity of a core comprising of a limited number of elements interacting with each other and this helps in reducing energy expenditure for the immune response.

Molecular recognition by the adaptive immune system relies on specific high-affinity antibody receptors that are generated from a restricted set of starting sequences through homologous recombination and somatic mutation. The steroid binding antibody DB3 and the catalytic Diels--Alderase antibody 1E9 derive from the same germ line sequences but exhibit very distinct specificities and functions. However, mutation of only two of the 36 sequence differences in the variable domains, [Leu.sup.H47]Trp and [Arg.sup.H100]Trp, converts 1E9 into a high-affinity steroid receptor with a ligand recognition profile similar to DB3. To understand how these changes switch binding specificity and function, we determined the crystal structures of the 1E9 [Leu.sup.H47]Trp and [Arg.sup.H100]Trp double mutant (1E9dm) as an unliganded Fab at 2.05 [Angstrom] resolution and in complex with two configurationally distinct steroids at 2.40 and 2.85 [Angstrom]. Surprisingly, despite the functional mimicry of DB3, 1E9dm employs a distinct steroid binding mechanism. Extensive structural rearrangements occur in the combining site, where residue H47 acts as a specificity switch and H100 adapts to different ligands. Unlike DB3, 1E9dm does not use alternative binding pockets or different sets of hydrogen-bonding interactions to bind configurationally distinct steroids. Rather, the different steroids are inserted more deeply into the 1E9dm combining site, creating more hydrophobic contacts that energetically compensate for the lack of hydrogen bonds. These findings demonstrate how subtle mutations within an existing molecular scaffold can dramatically modulate the function of immune receptors by inducing unanticipated, but compensating, mechanisms of ligand interaction. antibody-antigen complex | modulation of receptor specificity | molecular recognition | protein engineering | x-ray crystallography

The anti-LPS IgG mAb F22-4, raised against Shigella flexneri serotype 2a bacteria, protects against homologous, but not heterologous, challenge in an experimental animal model. We report the crystal structures of complexes formed between Fab F22-4 and two synthetic oligosaccharides, a decasaccharide and a pentadecasaccharide that were previously shown to be both immunogenic and antigenic mimics of the S. flexneri serotype 2a O-antigen. F22-4 binds to an epitope contained within two consecutive 2a serotype pentasaccharide repeat units (RU). Six sugar residues from a contiguous nine-residue segment make direct contacts with the antibody, including the nonreducing rhamnose and both branching glucosyl residues from the two RUs. The glucosyl residue, whose position of attachment to the tetrasaccharide backbone of the RU defines the serotype 2a O-antigen, is critical for recognition by F22-4. Although the complete decasaccharide is visible in the electron density maps, the last four pentadecasaccharide residues from the reducing end, which do not contact the antibody, could not be traced. Although considerable mobility in the free oligosaccharides can thus be expected, the conformational similarity between the individual RUs, both within and between the two complexes, suggests that short-range transient ordering to a helical conformation might occur in solution. Although the observed epitope includes the terminal nonreducing residue, binding to internal epitopes within the polysaccharide chain is not precluded. Our results have implications for vaccine development because they suggest that a minimum of two RUs of synthetic serotype 2a oligosaccharide is required for optimal mimicry of O-Ag epitopes. antibody complex | carbohydrate | crystal structure | polyliposaccharide | shigellosis

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) functions as a negative regulator of endogenous and vaccine-induced antitumor immunity. The administration of fully human anti-CTLA-4 blocking monoclonal antibodies to advanced-cancer patients increases immune-mediated tumor destruction in some subjects. Nonetheless, patients that respond also frequently manifest serious inflammatory pathologies, raising the possibility that the therapeutic and toxic effects of CTLA-4 blockade might be linked. Here we show that periodic infusions of anti-CTLA-4 antibodies after vaccination with irradiated, autologous tumor cells engineered to secrete GM-CSF (GVAX) generate clinically meaningful antitumor immunity without grade 3 or 4 toxicity in a majority of metastatic melanoma patients. The application of this sequential immunotherapy to advanced ovarian carcinoma patients also revealed that tumor destruction and severe inflammatory pathology could be dissociated, although further refinements are required to increase clinical responses and to minimize toxicity in this population. The extent of therapy-induced tumor necrosis was linearly related to the natural logarithm of the ratio of intratumoral CD[8.sup.+] effector T cells to FoxP[3.sup.+] regulatory T cells (Tregs) in posttreatment biopsies. Together, these findings help clarify the immunologic and clinical effects of CTLA-4 antibody blockade in previously vaccinated patients and raise the possibility that selective targeting of antitumor Tregs may constitute a complementary strategy for combination therapy. CTLA-4 | regulatory T cells (Tregs) | vaccine | GM-CSF

Relatively little is known about immune evasion during the productive phase of infection by the [[gamma].sub.1]-herpesvirus Epstein--Barr virus (EBV). The use of a unique system to isolate cells in lytic cycle allowed us to identify a host shutoff function operating in productively EBV-infected B cells. This impairment of protein synthesis results from mRNA degradation induced upon expression of the early lytic-cycle gene product BGLF5. Recently, a [[gamma].sub.2]-herpesvirus, Kaposi sarcoma herpesvirus, has also been shown to encode a host shutoff function, indicating that host shutoff appears to be a general feature of [gamma]-herpesviruses. One of the consequences of host shutoff is a block in the synthesis of HLA class I and II molecules, reflected by reduced levels of these antigen-presenting complexes at the surface of cells in EBV lytic cycle. This effect could lead to escape from T cell recognition and elimination of EBV-producing cells, thereby allowing generation of viral progeny in the face of memory T cell responses. herpesvirus | HLA | immune escape | RNA degradation

Surfactant protein (SP)-A and SP-D, immunoglobulins, and complement all modulate inflammation within the lung by regulating pathogen clearance. For example, SP-A binds to and opsonizes a variety of bacteria and viruses, thereby enhancing their phagocytosis by innate immune cells such as alveolar macrophages. Immunoglobulins, which bind to antigen and facilitate Fc receptor-mediated phagocytosis, can also activate complement, a family of soluble proteins with multiple host defense functions. Previous studies showed that SP-A and complement protein Clq interact. Since complement protein Clq binds to IgG and IgM immune complexes, the hypothesis tested in this study was that SP-A, which is structurally homologous to Clq, also binds to IgG and affects its functions. SP-A binds to the Fc, rather than the Fab, region of IgG. Binding is calcium dependent but not inhibited by saccharides known to bind to SP-A's carbohydrate recognition domain. The binding of SP-A does not inhibit the formation of immune complexes or the binding of IgG to Clq. In contrast, SP-A enhances the uptake of IgG-coated erythrocytes, suggesting that SP-A might be influencing Fc receptor-mediated uptake. In summary, this study shows a novel interaction between SP-A and IgG and a functional consequence of the binding. collectin; immune complex; innate immunity; lung

Immune complexes (ICs) are potent inflammatory mediators in peripheral tissues. However, very few studies have examined the ability of ICs to induce inflammatory responses in the brain. Therefore, using preformed ICs or the reverse passive Arthus (RPA) model to localize ICs to the pial microvasculature of mice, we aimed to investigate the ability of ICs to induce an inflammatory response in the cerebral (pial) microvasculature. Application of preformed ICs immediately increased pial microvascular permeability, with a minimal change in leukocyte adhesion in pial postcapillary venules. In contrast, initiation of the RPA response in the pial microvasculature induced changes in cerebral microvascular permeability and increased leukocyte adhesion in pial postcapillary venules. The RPA response induced deposition of C3 in perivascular regions adjacent to sites of IC formation. Depletion of C3 abrogated RPA-induced microvascular permeability and leukocyte adhesion, indicating that the complement pathway was critical for this response. Inhibition of leukocyte adhesion via CD18 blockade also reduced IC-induced microvascular permeability. However, this did not require intercellular adhesion molecule-l, inasmuch as blockade of intercellular adhesion molecule-1 did not alter RPA-induced microvascular permeability and adhesion. These findings demonstrate that ICs are capable of rapidly inducing inflammatory responses in the cerebral microvasculature, with the complement pathway and leukocyte recruitment playing critical roles in microvascular dysfunction. inflammation; cell trafficking; adhesion molecules doi:10.1152/ajpheart.01271.2005

Antibody (human IgG, anti-E, coli O157:H7, and anti-Salmonella) complexes on the surface of IR-transparent Ge-containing chalcogenide glass films were formed via thiol chemistry on 20-nm-thick gold islands. As a first step, the protocol was validated by monitoring fluorescently tagged targets to validate binding. FT-IR spectroscopy confirmed that the coating of the films with 20-nm gold did not have a significant effect on the propagation and penetration of IR evanescent waves through the film. The films functionalized with anti-E. coli O157:H7 and anti-Salmonella antibodies were used to detect E. coli O157:H7 and S. enteriditis through label-free IR finger-printing. Highly selective detection of bacterial targets was achieved at both the species (E. coli vs. S. enteriditis) and strain level (E. coli O157:H7 vs E. coli K12). A mid-infrared approach could thus be used as a biosensor as well as a molecular fingerprinting tool.

Life history theory provides a predictive framework for identifying prenatal and early postnatal factors that are likely to shape immune function over the life course. A series of hypotheses derived from key life history issues are proposed and evaluated with data from ongoing research on immune function in the Philippines and Bolivia.

The ability of a fluorescence-based array biosensor to measure and quantify the binding of an antigen to an immobilized antibody has been demonstrated using the four different immunoassay formats: direct, competitive, displacement, and sandwich. A patterned array of antibodies specific for 2,4,6-trinitrotoluene (TNT) was immobilized onto the surface of a planar waveguide and used to measure signals from different antigen concentrations simultaneously. For direct, competitive, and displacement assays, which are one-step assays, measurements were obtained in real time. Dose-response curves were calculated for all four assay formats, demonstrating the array biosensor's ability to quantify the amount of antigen present in solution.

Byline: P. C. Singhal (1,2), S. Gupta (1,2), P. Sharma (1,2), H. Shah (1,2), N. Shah (1,2), Puja Patel (1,2) Abstract: Background: Accumulation of immune complexes in the mesangium is a common finding. Since migration of macrophages (MI) in the mesangium has been demonstrated to be an important event in the development of glomerular lesions, we studied the role of immune complexes and mesangial cell (MC) interaction in the transmigration (Tm) of MI. Methods: To determine the effect of MC and immune complexes (aggregated IgG, IgGAg) on transmigration of MI, MC were incubated with or without IgGAg in the lower compartment of a modified Boyden Chamber. To determine the effects of the secretory products (as a result of endocytosis of IgGAg by mesangial cells), MC-IgAg conditioned media was prepared and placed in the lower compartment of the Boyden chamber. We evaluated the effects of MC alone, MC + IgGAg, or MC-IgGAg conditioned media on the transmigration of macrophages across a filter. To determine the effect of free radicals on MC-IgAg endocytosis-induced MI migration we evaluated the effect of free radical scavengers such as dimethyl thiourea (DMTU) and tetramethylthiourea (TMTU) in MC-IgAg endocytosis-induced MI migration. To determine the role of chemokines in MC-IgAs endocytosis-induced MI migration we evaluated the effect of ani-MCP-1 antibodies on MC-IgAg endocytosis-induced MI migration, and also studied the effects of IgAg on MC mRNA expression of MCP-1 and RANTES. In addition, we evaluated the role of Fc receptors and actin cytoskeleton of MC in transmigration of MI. Results: Mesangial cell endocytosis of IgG aggregates (IgGAg) is associated with enhanced (P &lt 0.001) transmigration of MI (control, 11.2 +- 0.2 vs. MC + IgGAg, 22.1 +- 0.9 migrated MI/field). IgGAg also induced MC mRNA expression for RANTES and MCP-1 on MC. DMTU and TMTU attenuated (P &lt 0.001) the MC + IgGAg-induced migration of MI as well as IgGAg-induced mRNA expression for RANTES and MCP-1. MC and IgGAg interaction products (MC-IgGAg conditioned media) also increased (P &lt 0.01) transmigration of MI (control, 18.3 +- 1.7 vs. MC-IgGAg conditioned media, 30.7 +- 0.6 MI/field). This effect of MC-IgGAg conditioned media on the migration of macrophages was dose dependent. Anti-MCP-1 antibody partially inhibited MC-IgGAg-induced migration of macropahges. MC and monomeric IgG (MIgG) interaction (MC-MIgG conditioned media) showed a lower (P &lt 0.05) migration of MI when compared to the MC-IgGAg conditioned media. MC-IgGAg conditioned media prepared from cytochalasin B pretreated MCs also showed a lower (P &lt 0.001) migration of MI when compared with MC-IgGAg conditioned media-induced migration. Conclusions: These results indicate that MC-IgGAg conditioned media-induced transmigration of macrophages may be mediated through the generation of RANTES and MCP-1 by MC. Author Affiliation: (1) Departments of Medicine and Pathology, Long Island Jewish Medical Center, New Hyde Park, New York, 11040 (2) The Long Island Campus for Albert Einstein College of Medicine, Bronx, New York, 10461 Article History: Registration Date: 05/10/2004

In most vertebrates, a primary antibody repertoire is created through the recombination of a diverse set of Ig variable (V), diversity (D), and joining (J) gene segments. In contrast, an avian immune repertoire is generated by gene conversion of rearranged Ig genes during B cell development within the bursa of Fabricius, a lymphoid organ unique to birds. To investigate the properties of antigen-specific Igs created through the process of gene conversion, we have developed a system for the production of avian-derived mAbs. This system was used to produce multiple antibodies after a single immunization with a conserved peptide from the human cystic fibrosis transmembrane conductance regulator gene. Each antibody isolated was found to have arisen independently through a distinct series of gene conversion events. These primary antibodies displayed evidence of diversity in all of the complementarity determining regions of both heavy and light chains, and both the heavy and the light chains contributed to antigen specificity. In the light chains, diversity could be attributed to gene conversion events. The measured affinity constants of two of the antibodies were between [10.sup.8] and [10.sup.9] [M.sup.-1], and the antibodies were functional in quantitative ELISA as well as immunohistochemical studies of cystic fibrosis transmembrane conductance regulator expression. These data demonstrate that antigen-specific antibodies produced by Ig gene conversion display both high affinity and specificity. In addition, the methods developed here provide the description of a system for the production of mAbs derived from a nonmammalian species.

The interaction of histidine-rich glycoprotein (HRG) with human IgG and Clq as well as its role in the formation of insoluble immune complexes were investigated. Enzyme-linked immunosorbent assay (ELISA) and IAsys resonant mirror biosensor results have shown that physiological concentrations of HRG binds strongly to Clq and to IgG. Furthermore, HRG inhibits the formation of insoluble immune complexes (IIC).

The O-antigenic repeating units of lipopolysaccharides from Salmonella serogroups A, B, and D1 serve as receptors for the phage P22 tailspike protein, which also has receptor destroying endoglycosidase (endorhamnosidase) activity, integrating the functions of both hemagglutinin and neuraminidase in influenza virus. Crystal structures of the tailspike protein in complex with oligosaccharides, comprising two O-antigenic repeating units from Salmonella typhiumurium, Salmonella enteritidis, and Salmonella typhi 253Ty were determined at 1.8 [Angstrom] resolution. The active-site topology with Asp-392, Asp-395, and Glu-359 as catalytic residues was identified. Kinetics of binding and cleavage suggest a role of the receptor destroying endorhamnosidase activity primarily for detachment of newly assembled phages.

The formation of intrapulmonary immune complexes in mice generates a vigorous inflammatory response characterized by microvascular permeability and polymorphonuclear neutrophil influx. Gene-targeted disruption of the substance P receptor (NK- 1R) protected the lung from immune complex injury, as did disruption of the C5a anaphylatoxin receptor. Immunoreactive substance P was measurable in fluids lining the lung at time points before neutrophil influx and may thus be involved in an early step in the inflammatory response to immune complexes in the lung., Immune complexes underlie the inflammatory response seen in a variety of rheumatologic illnesses, including arthritis, vasculitides, and systemic lupus erythematosus (1). Antigen-antibody aggregates may be deposited locally and incite edema [...]

Secondary forces or the interactions between the antigen and regions immediately adjacent to the antibody active site were examined through the use of monofluorescein-derivatized synthetic peptides of various electrostatic characteristics. Unimolecular rate constants were correlated with transition-state theory to understand secondary effects. An acidic peptide caused a large temperature-dependent effect upon binding including a substantial enthalpic factor of 33.28 kcal/mol relative to the binding of fluorescein ligand.

Phosphocholine was linked to tyrosine or histidine as single amino acid carriers and to tripeptides and octapeptides containing a central tyrosine or histidine molecule to assess the effects of haptens and carriers on each other. The hapten phosphocholine retained its bent conformation regardless of being free or bound to a carrier. Hapten affinity, however was affected by the presence of carrier residues.

Weaknesses in the low-affinity immuno-globulin-G receptor Fc(gamma)RII or its signalling pathway in B cells upset the immune-complex-mediated feedback inhibition of the generation of antibodies. A study shows that the immunoglobulin levels of mice lacking in Fc(gamma)RII soar in reaction to antigens regardless of the latter's thymus-dependence. Findings indicate that Fc(gamma)RII negatively regulates immune-complex-triggered activation in vivo for the afferent as well as the efferent limbs of the immune reaction.

Immune complexes (IC) activate endothelial cells adhesiveness in the presence of complement-sufficient normal human serum and not with heat inactivated serum. The adhesiveness for leukocytes depends upon the presence of heat-labile serum factor, the C1q indicating that C1q is an important serum cofactor necessary for ICs to activate endothelial cells.

A study was conducted to analyze the predictions of computer simulations of the molecular dynamics of the anti-fluorescein 4-4-20 antigen-binding fragment. Site-specific conjugation chemistry was used to attach a fluorescent probe to the 4-4-20 Fab at its C-terminus. Two rotational components were observed: a longer correlation time corresponding to global Fab rotation and a shorter correlation time attributed to local or segmental motion of the constant domain dimer.

A study was conducted to model the molecular dynamics of the anti-fluorescein 4-4-20 antigen-binding fragment with and without fluorescein using computer simulations. Time-resolved fluorescence experiments showed that the Fab minus fluorescein possessed more segmental flexibility than the Fab without fluorescein. The three-dimensional structure of the unliganded Fab was predicted using the simulations.

Results of an X-ray crystallization, thermodynamic characterization and nuclear magnetic resonance (NMR) agree with the prediction of a binding site that accommodates at least eight sialic acid residues. The earlier prediction of a helical nature for the antigenic form of the oligosaccharide is also affirmed by crystallographic evidence. Results of modeling studies on the nature of antibody-antigen complex conducted using the structure of the binding site described and the structure of the antigen reported in previous NMR experiments are also presented.

Various synthetic strategies help prepare antitumor prodrugs for use in antibody-directed catalysis (ADC) approaches to cancer chemotherapy. Examination of the chemistry of prodrugs, classified on the basis of enzyme/cleavage reaction type, reveals their capacity to mediate antigen-dependent cytotoxicity both in vitro and in vivo. Use of various enzymes leads to the synthesis of many prodrugs and three of them using carboxy peptidase G2, alkaline phosphatase and beta-lactamase show excellent antitumor activity. ADC based on cephem/beta-lactamase is more effective with various types of cytotoxic agents than other systems.

The three-dimensional structures of the antigen-binding fragment of the anti-digoxin monoclonal antibody do not alter in the process of forming the complex. Digoxin responds to the antibody heavy chain, carbohydrate groups are open to solvent and the lactonic ring is located deep in the combining site. The 26-10 digoxin complex is special with specificity and high affinity arising from shape complementarity.

The anti-hen egg lysozyme (HEL) monoclonal antibody (mAb) D11.15, which cross-reacts with various avian lysozymes, in certain instances with a greater affinity than for HEL, was investigated to explore the molecular foundations of the cross-reactions of antibodies. The crystal structure of the Fv protein of D11.15 complexed with pheasant lysozyme (PHL) was established. A stereochemically permissive setting for the variant antigen residues at the antibody-antigen interface is needed for cross-reactivity.

Heymann nephritis (HN) is the rat model of human glomerulonephritis, which is initiated by the binding of circulating antibodies to the HN antigenic complex (HNAC). A rat yolk sac carcinoma cell line capable of synthesizing a heterodimeric complex similar to HNAC was identified and characterized. The cell line, termed L2, expresses HNAC on the cell surface and culture medium, suggesting that it produces a soluble form of gp330, one of the components of HNAC. This cell line should be a useful model system for the study of the normal functions of HNAC and its role in HN.

The HyHEL series of antibodies are used to study the epitopic features of hen egg-white lysozyme (HEL). An investigation was conducted to determine thermodynamic contributions of specific antigen residues within a defined epitope of HEL recognized by HyHEL-10 by site-directed mutagenesis. The dissociation constants and free energies of dissociation attributed to mutations in specific residues are presented. The results demonstrate the applicability of site-directed mutagenesis in elucidating the detailed chemistry of immunocomplex formation.

Nuclear Overhauser effect spectroscopy examined the intramolecular interactions in bound cholera toxin peptide (CTP3) in three antibody complexes. Two beta-turns were found in the bound peptide that were not expressed in the free peptide. The beta-turn formations gave CTP3 a compact structure and greater flexibility in its conformation than in other secondary structures. Results showed that Tyr 32L, Tyr 32H and Trp 50H aromatic residues functioned in nonspecific hydrophobic interactions with the antigen.

Isotope-edited nuclear magnetic resonance techniques were used to investigate the dynamics and conformation of the peptide antigen bound to the Fab' fragment of the monoclonal antipeptide antibody B13A2. The peptide MHKDFLEKIGGL adopts a helical backbone conformation between residues F5 and G11 in the combining site of the Fab' fragment. It appears that the conformational ensemble which includes the nascent helix is driven toward the helical state upon binding to the antibody.

The binding of a heavy chain major histocompatibility (MHC) class I moleculeto an antigenic peptide was studied in the presence of various levels of the MHC class I light chain beta-2-microglobin (beta-2-m). The binding was conducted in an aqueous solution at 37 degrees celsius, using the H-2L(super d)class I heavy chain molecule and an antigenic peptide. The observed interactionwas controlled by the light chain beta-2-m subunit. Binding kinetics suggests that the class I MHC-peptide interaction follows the ternary-complex model.

Protein-containing dependent antigens elicit a response that is focused against immunogenic epitopes in the protein. Administration of adequate doses of fluoresceinated dextran can significantly lower T-cell dependent immune responses in mice with IgM, IgG and IgE anti-fluorescein antibodies. This suppression is not momentary and plasma cell secretion is eradicated. Masking of serum antibody by the inhibitory polymer is not the sole cause of lowering in antibody titer. These findings offer encouraging therapeutic applications and downgrade the need for toxic nonspecific treatments.

OLIGOSACCHARIDE EPITOPES OF bacterial and tumor cell-surface carbohydrates are important disease markers [1] and targets for therapeutic antibodies [2], but to date a crystal structure for an antibody-carbohydrate complex has [...]

IgM monoclonal gammopathy, a disorder characterized by abnormal multiplication of white blood cells producing a single (monoclonal) antibody of the IgM class, is frequently associated with polyneuropathy, nerve damage affecting several nerves and leading to weakness, tremor, and loss of sensation. The IgM antibody (M protein) of some patients actually reacts with components of the myelin sheath, the material that covers nerves and insulates them, allowing normal electrical transmission. It is possible that the demyelination, or loss of myelin, associated with this condition is the result of attack by M proteins. The mechanisms by which this might happen were studied. One way antibodies can cause cell damage is by means of the complement (C) proteins, a group of proteins activated in a chain-reaction manner (cascade) by antibody-antigen complexes. The antibodies involved in these complexes are called complement-fixing antibodies, and when they bind to the antigen they recognize, the molecular unit so formed can activate the first complement molecule to begin the cascade. The molecule formed as the last step in the cascade is called the terminal-complement complex; it is capable of causing the death of the cell to which the antibody has bound by introducing pores in its membrane. This is clearly of use if the antigen is dangerous to the body, but dysfunctional if the antigen is a component of the body's own myelin. The IgM molecules of seven IgM monoclonal gammopathy patients were studied. Six were found to have IgMs that reacted with molecules known to be associated with myelin, while the seventh had an M protein that reacted with an unidentified myelin component. Other methods demonstrated that these M proteins were preferentially deposited on the majority of myelinated fibers of nerves, and that some of the nerve fibers also had complement molecules on their surfaces. The presence of complement proteins from both early (C1q) and late (C9 neoantigen) stages of the cascade suggests that the demyelination in these patients may, indeed, be the result of antibody deposition and mediated by complement. None of these compounds were found on nerves from seven normal subjects, seven patients with hereditary sensorimotor neuropathy, or seven patients with diabetic neuropathy. Additional details regarding the role of complement in the pathogenesis of this type of polyneuropathy are provided. (Consumer Summary produced by Reliance Medical Information, Inc.)

Proteolytic pathways rely on the conjugation of ubiquitin with the protein to be degraded. Some antigenic peptides are also produced in the cytosol which bind with class I complexes and have the same labelling function for recognition by the degradative particle called the proteasome. Experiments with ovalbumin introduced into the cytosol show that ovalbumin must be conjugated with ubiquitin to generate antigenic peptides. This study will be helpful in recognition of certain virus peptides and the pathway for antigen action.

Table of Contents Abstract Introduction Materials and methods Results Discussion References Abstract Rabbit hyperimmune serum against intact whole immune complexes from a mammary tumor affected dog gave positive reactivity in [...]