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1.  Dysregulated humoral immunity to nontyphoidal Salmonella in HIV-infected African adults 
Science (New York, N.Y.)  2010;328(5977):508-512.
Nontyphoidal Salmonellae are a major cause of life-threatening bacteremia among HIV-infected individuals. Although cell-mediated immunity controls intracellular infection, antibody protects against Salmonella bacteremia. We report that high titer antibodies specific for Salmonella lipopolysaccharide (LPS) associate with absent Salmonella-killing in HIV-infected African adults. Killing was restored by genetically shortening LPS from target Salmonella, or removing LPS-specific antibodies from serum. Complement-mediated killing of Salmonella by healthy serum is shown to be induced specifically by antibodies against outer membrane proteins. This killing is lost when excess antibody against Salmonella LPS is added. Thus our study indicates impaired immunity against nontyphoidal Salmonella bacteremia in HIV infection results from excess inhibitory antibodies against Salmonella LPS, whilst serum killing of Salmonella is induced by antibodies against outer membrane proteins.
PMCID: PMC3772309  PMID: 20413503
2.  Germinal center B cells govern their own fate via antibody feedback 
High-affinity antibodies reenter germinal centers (GCs) and limit antigen access, thus causing sustained directional evolution in GCs toward higher-affinity antibody production.
Affinity maturation of B cells in germinal centers (GCs) is a process of evolution, involving random mutation of immunoglobulin genes followed by natural selection by T cells. Only B cells that have acquired antigen are able to interact with T cells. Antigen acquisition is dependent on the interaction of B cells with immune complexes inside GCs. It is not clear how efficient selection of B cells is maintained while their affinity matures. Here we show that the B cells’ own secreted products, antibodies, regulate GC selection by limiting antigen access. By manipulating the GC response with monoclonal antibodies of defined affinities, we show that antibodies in GCs are in affinity-dependent equilibrium with antibodies produced outside and that restriction of antigen access influences B cell selection, seen as variations in apoptosis, plasma cell output, T cell interaction, and antibody affinity. Feedback through antibodies produced by GC-derived plasma cells can explain how GCs maintain an adequate directional selection pressure over a large range of affinities throughout the course of an immune response, accelerating the emergence of B cells of highest affinities. Furthermore, this mechanism may explain how spatially separated GCs communicate and how the GC reaction terminates.
PMCID: PMC3600904  PMID: 23420879
3.  Chimeric antibodies to proteinase 3 of IgG1 and IgG3 subclasses induce different magnitudes of functional responses in neutrophils 
Annals of the Rheumatic Diseases  2007;66(5):676-682.
Antineutrophil cytoplasmic antibodies (ANCA) are associated with small‐vessel vasculitis and have been implicated in its pathogenesis. The subclass distribution of ANCA IgG deviates from normal patterns, and it has been suggested that the IgG3 subclass may have pathogenic potential over the IgG1 subclass and may be more likely to be associated with active disease and renal involvement.
To deal with potential pathogenicity, chimeric antibodies were constructed of IgG1 and three subclasses with human IgG1 or three constant regions and a murine‐derived variable region that binds an epitope within the ANCA antigen proteinase 3 (PR3) that is recognised by human autoantibodies.
The antibodies were characterised for binding to PR3, including affinity and avidity, before being used as tools to explore their ability to activate human neutrophils for superoxide release, cytokine release, degranulation and ability to induce neutrophil adhesion under flow.
Both subclass antibodies elicited similar neutrophil responses for superoxide release, degranulation and interleukin (IL) 8 production, although quantitative responses showed that the IgG1 subclass favoured degranulation and the IgG3 subclass favoured IL8 production. Both antibodies were able to convert neutrophils from selectin‐dependent rolling adhesion to integrin‐dependent stationary adhesion in a flow assay.
These findings indicate that humanised antibodies directed against a single epitope of PR3 can recapitulate the effects of polyclonal human ANCA, which recognises multiple PR3 epitopes. Further, PR3‐ANCA of both IgG1 and IgG3 subclasses can activate neutrophils, although the more potent IL8 response by IgG3 PR3‐ANCA may encourage further neutrophil recruitment and amplify injury.
PMCID: PMC1954608  PMID: 17204568
4.  Claudin Association with CD81 Defines Hepatitis C Virus Entry 
The Journal of Biological Chemistry  2010;285(27):21092-21102.
Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multistep process involving tetraspanin CD81, scavenger receptor class B member I, and the tight junction proteins Claudin-1 and Occludin. CD81 and scavenger receptor class B member I interact with HCV-encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there are minimal data supporting Claudin-1 association with HCV particles, raising questions as to its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by fluorescence resonance energy transfer and stoichiometric imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor-inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction-associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.
PMCID: PMC2898367  PMID: 20375010
Fluorescence Resonance Energy Transfer (FRET); Receptor Structure-Function; Receptors; Tight Junction; Virus Entry
5.  The neglected role of antibody in protection against bacteremia caused by nontyphoidal strains of Salmonella in African children  
The Journal of Clinical Investigation  2008;118(4):1553-1562.
Nontyphoidal strains of Salmonella (NTS) are a common cause of bacteremia among African children. Cell-mediated immune responses control intracellular infection, but they do not protect against extracellular growth of NTS in the blood. We investigated whether antibody protects against NTS bacteremia in Malawian children, because we found this condition mainly occurs before 2 years of age, with relative sparing of infants younger than 4 months old. Sera from all healthy Malawian children tested aged more than 16 months contained anti-Salmonella antibody and successfully killed NTS. Killing was mediated by complement membrane attack complex and not augmented in the presence of blood leukocytes. Sera from most healthy children less than 16 months old lacked NTS-specific antibody, and sera lacking antibody did not kill NTS despite normal complement function. Addition of Salmonella-specific antibody, but not mannose-binding lectin, enabled NTS killing. All NTS strains tested had long-chain lipopolysaccharide and the rck gene, features that resist direct complement-mediated killing. Disruption of lipopolysaccharide biosynthesis enabled killing of NTS by serum lacking Salmonella-specific antibody. We conclude that Salmonella-specific antibody that overcomes the complement resistance of NTS develops by 2 years of life in Malawian children. This finding and the age-incidence of NTS bacteremia suggest that antibody protects against NTS bacteremia and support the development of vaccines against NTS that induce protective antibody.
PMCID: PMC2268878  PMID: 18357343
6.  Compromised Ox40 Function in Cd28-Deficient Mice Is Linked with Failure to Develop Cxc Chemokine Receptor 5–Positive Cd4 Cells and Germinal Centers 
The Journal of Experimental Medicine  1999;190(8):1115-1122.
Mice rendered deficient in CD28 signaling by the soluble competitor, cytotoxic T lymphocyte–associated molecule 4–immunoglobulin G1 fusion protein (CTLA4-Ig), fail to upregulate OX40 expression in vivo or form germinal centers after immunization. This is associated with impaired interleukin 4 production and a lack of CXC chemokine receptor (CXCR)5 on CD4 T cells, a chemokine receptor linked with migration into B follicles. Germinal center formation is restored in CTLA4-Ig transgenic mice by coinjection of an agonistic monoclonal antibody to CD28, but this is substantially inhibited if OX40 interactions are interrupted by simultaneous injection of an OX40-Ig fusion protein. These data suggest that CD28-dependent OX40 ligation of CD4 T cells at the time of priming is linked with upregulation of CXCR5 expression, and migration of T cells into B cell areas to support germinal center formation.
PMCID: PMC2195670  PMID: 10523609
CD28; OX40/OX40 ligand; germinal center; chemokine; T cell migration
7.  CD4 T Cell Cytokine Differentiation: The B Cell Activation Molecule, OX40 Ligand, Instructs CD4 T Cells to Express Interleukin 4 and Upregulates Expression of the Chemokine Receptor, Blr-1  
This report investigates the role of OX40 ligand (OX40L) and its receptor, OX40, expressed on activated B and T cells, respectively, in promoting the differentiation of T helper type 2 (Th2) CD4 T cells. These molecules are expressed in vivo by day 2 after priming with T cell– dependent antigens. Their expression coincides with the appearance of immunoglobulin (Ig)G switch transcripts and mRNA for interleukin (IL)-4 and interferon (IFN)-γ, suggesting that this molecular interaction plays a role in early cognate interactions between B and T cells. In vitro, we report that costimulation of naive, CD62Lhigh CD4 T cells through OX40 promotes IL-4 expression and upregulates mRNA for the chemokine receptor, blr-1, whose ligand is expressed in B follicles and attracts lymphocytes to this location. Furthermore, T cell stimulation through OX40 inhibits IFN-γ expression in both CD8 T cells and IL-12–stimulated CD4 T cells. Although this signal initiates IL-4 expression, IL-4 itself is strongly synergistic. Our data suggest that OX40L on antigen-activated B cells instructs naive T cells to differentiate into Th2 cells and migrate into B follicles, where T cell–dependent germinal centers develop.
PMCID: PMC2212448  PMID: 9670042
Th1–Th2 differentiation; OX40/OX40 ligand; cytokine; T cell priming; cognate B cell–T cell interaction

Results 1-7 (7)