Functional and structural requirements for the internalization of distinct BCR-ligand complexes.

Antigen (Ag) binding to the BCR rapidly initiates two important events: a phosphorylation cascade that results in the production of secondary signaling intermediaries and the internalization of Ag-BCR complexes. Previous studies using anti-BCR antibodies (Ab) have suggested that BCR signaling is an essential requirement for BCR endocytosis and have further implicated lipid rafts as essential platforms for both BCR functions. However, published data from our laboratory indicate that lipid rafts and consequently raft-mediated signaling are dispensable for BCR-mediated internalization of Ag-specific BCR.

BCR ubiquitination controls BCR-mediated antigen processing and presentation.

BCR-mediated antigen processing occurs at immunologically relevant antigen concentrations and hinges on the trafficking of antigen-BCR (Ag-BCR) complexes to class II-containing multivesicular bodies (MVBs) termed MIICs. However, the molecular mechanism underlying the trafficking of Ag-BCR complexes to and within MIICs is not well understood. In contrast, the trafficking of the epidermal growth factor receptor (EGFR) to and within MVBs occurs via a well-characterized ubiquitin-dependent mechanism, which is blocked by acute inhibition of proteasome activity.

Superoxide dismutase B gene (sodB)-deficient mutants of Francisella tularensis demonstrate hypersensitivity to oxidative stress and attenuated virulence.

A Francisella tularensis live vaccine strain mutant (sodB(Ft)) with reduced Fe-superoxide dismutase gene expression was generated and found to exhibit decreased sodB activity and increased sensitivity to redox cycling compounds compared to wild-type bacteria. The sodB(Ft) mutant also was significantly attenuated for virulence in mice. Thus, this study has identified sodB as an important F.

Manganese superoxide dismutase protects from TNF-alpha-induced apoptosis by increasing the steady-state production of H2O2.

Manganese superoxide dismutase (SOD2) has been well established to be essential for protection from a variety of apoptotic stimuli. Here we demonstrate that the antiapoptotic effects of SOD2 are attributed to its ability to generate H(2)O(2) and that its efficient removal resensitizes cells to tumor necrosis factor (TNF)-alpha-induced apoptosis. SOD2 overexpression in HT-1080 cells leads to a decrease in the fluorescence of the superoxidesensitive fluorophore, dihydroethidium, and a concomitant increase in oxidation of the H2O2-sensitive dye, dichlorodihydrofluorescein diacetate (DCFDA).

T-bet deficiency facilitates airway colonization by Mycoplasma pulmonis in a murine model of asthma.

Epidemiological and clinical evidence suggest a correlation between asthma and infection with atypical bacterial respiratory pathogens. However, the cellular and molecular underpinnings of this correlation remain unclear. Using the T-bet-deficient (T-bet(-/-)) murine model of asthma and the natural murine pathogen Mycoplasma pulmonis, we provide a mechanistic explanation for this correlation.

Separate origins of hepatitis B virus surface antigen-negative foci and hepatocellular carcinomas in transgenic HBsAg (alb/psx) mice.

We have examined the development and transgene expression in liver lesions of transgenic mice bearing the hepatitis B surface antigen (HBsAg) gene of hepatitis B virus under the control of the albumin promoter (alb/psx) to study liver regeneration and hepatocellular carcinoma (HCC) associated with hepatitis B virus infection. Storage of the HBsAg in the endoplasmic reticulum precedes loss of liver cells and regenerative hyperplastic nodules that do not express HBsAg. Histological analysis indicated that HBsAg-negative foci and nodules arose from liver progenitor cells in the portal zone and lacked mRNA expression.

Inhibition of highly productive HIV-1 infection in T cells, primary human macrophages, microglia, and astrocytes by Sargassum fusiforme.

Background: The high rate of HIV-1 mutation and increasing resistance to currently available antiretroviral (ART) therapies highlight the need for new antiviral agents. Products derived from natural sources have been shown to inhibit HIV-1 replication during various stages of the virus life cycle, and therefore represent a potential source of novel therapeutic agents. To expand our arsenal of therapeutics against HIV-1 infection, we investigated aqueous extract from Sargassum fusiforme (S.

Toll-like receptor 2 is required for control of pulmonary infection with Francisella tularensis.

Toll-like receptor 2 (TLR2) deficiency enhances murine susceptibility to infection by Francisella tularensis as indicated by accelerated mortality, higher bacterial burden, and greater histopathology. Analysis of pulmonary cytokine levels revealed that TLR2 deficiency results in significantly lower levels of tumor necrosis factor alpha and interleukin-6 but increased amounts of gamma interferon and monocyte chemoattractant protein 1. This pattern of cytokine production may contribute to the exaggerated pathogenesis seen in TLR2-/- mice.

Streptococcus pneumoniae recruits complement factor H through the amino terminus of CbpA.

Streptococcus pneumoniae, a human pathogen, is naturally capable of colonizing the upper airway and sometimes disseminating to remote tissue sites. Previous studies have shown that S. pneumoniae is able to evade complement-mediated innate immunity by recruiting complement factor H (FH), a complement alternative pathway inhibitor.

Redox-dependent matrix metalloproteinase-1 expression is regulated by JNK through Ets and AP-1 promoter motifs.

Reactive oxygen species have been shown to play an important role in the regulation of distinct signaling cascades, many of which act upon the production of matrix metalloproteinases (MMP). Using a series of redox-engineered cell lines we have previously demonstrated that MMP-1 expression is sensitive to the alterations in the steady state production of H2O2 (Ranganathan, A. C.

Dynamics of MHC class II-activating signals in murine resting B cells.

MHC class II (MHC II) proteins are competent signaling molecules on APC. However, little is known about the mechanisms that control generation of their activating signals. Previous reports highlighted a number of factors that could affect the nature and outcome of MHC II signals, including the inability of MHC II ligation on resting vs activated murine B cells to induce mobilization of Ca2+.

Natural killer and CD8 T cells dominate the response by human peripheral blood mononuclear cells to inactivated Francisella tularensis live vaccine strain.

Francisella tularensis is a category A biothreat agent, and as a result, it has recently generated much research interest. F. tularensis live vaccine strain (LVS) is an attenuated form of the virulent F.

Enhanced antigen-specific antibody and cytokine responses when targeting antigen to human FcGAMMA receptor type I using an anti-human FcGAMMA receptor type I-streptavidin fusion protein in an adjuvant-free system.

There is a continuing need for alternatives to current human adjuvants. Recombinant protein vaccines, which target antigen to human Fc gamma receptor type I (hFcgammaRI) on hFcgammaRI-expressing antigen presenting cells, provide one potential alternative. Using a recombinant anti-hFcgammaRI-antigen fusion protein and adjuvant independent mouse model, we demonstrate enhanced antigen-specific antibody responses to low doses of antigen, when targeting antigen to hFcgammaRI in vivo.

Redox response of the endogenous calcineurin inhibitor Adapt 78.

Adapt 78 (DSCR 1/calcipressin/MCIP 1) is a potent natural inhibitor of calcineurin, an important intracellular phosphatase that mediates many cellular responses to calcium. We previously reported two major cytosolic isoforms (1 and 4) of Adapt 78, and that isoform 4 is an oxidative and calcium stress-response protein. Using a higher cell culture density and new antibody, we again observed that both major isoforms localized to the cytosol, but a significant level of isoform 4 (but not isoform 1) was also detected in the nucleus where it was present in the non-soluble region and not associated with RNA.

Mucosal B cell deficiency in IgA-/- mice abrogates the development of allergic lung inflammation.

We have investigated the consequence of lack of IgA on host immunity using a murine model of allergic lung inflammation. Mice with a targeted disruption of the alpha-switch region and 5' H chain gene (IgA(-/-) mice), which lack total IgA, developed significantly reduced pulmonary inflammation with fewer inflammatory cells in lung tissue and bronchoalveolar lavage fluids, as well as reduced levels of total and IgG1 OVA-specific Abs and decreased IL-4 and IL-5 in bronchoalveolar lavage fluids compared with IgA(+/+) controls, following allergen sensitization and challenge. This defect was attributable to fewer B cells in the lungs of IgA(-/-) mice.

Early activation of NK cells after lung infection with the intracellular bacterium, Francisella tularensis LVS.

Francisella tularensis is a gram-negative intracellular bacterium that has been classified as a Category A biothreat because of its ability to induce deadly pneumonic tularemia when inhaled. In the present study, an experimental model of F. tularensis LVS intranasal infection was used to study the immune cells involved in cytokine secretion in the lungs after infection.

Intranasal interleukin-12 treatment for protection against respiratory infection with the Francisella tularensis live vaccine strain.

Francisella tularensis is a gram-negative intracellular bacterium that can induce lethal respiratory infection in humans and rodents. However, little is known about the role of innate or adaptive immunity in protection from respiratory tularemia. In the present study, the role of interleukin-12 (IL-12) in inducing protective immunity in the lungs against intranasal infection of mice with the live vaccine strain (LVS) of F.

Low-level signaling generated by FcgammaRIIB-B cell receptor co-ligation establishes a state of global B cell receptor nonresponsiveness.

In addition to the stimulatory, antigen-specific B cell receptor (BCR), B lymphocytes also express multiple inhibitory receptors, including Fc gamma receptor type IIB (FcgammaRIIB). Moreover, many laboratories have demonstrated that co-ligation of BCR molecules to inhibitory FcgammaRIIB molecules with high concentrations (10-15 microg/ml) of ligand results in altered BCR signaling. However, there are no reports on the effect of low concentrations of ligand on BCR-FcgammaRIIB co-ligation and subsequent signaling.

Signaling through CD14 attenuates the inflammatory response to Borrelia burgdorferi, the agent of Lyme disease.

Lyme disease is a chronic inflammatory disorder caused by the spirochetal bacterium, Borrelia burgdorferi. In vitro evidence suggests that binding of spirochetal lipoproteins to CD14, a pattern recognition receptor expressed on monocytes/macrophages and polymorphonuclear cells, is a critical requirement for cellular activation and the subsequent release of proinflammatory cytokines that most likely contribute to symptomatology and clinical manifestations. To test the validity of this notion, we assessed the impact of CD14 deficiency on Lyme disease in C3H/HeN mice.

The pathway of antigen uptake and processing dictates MHC class II-mediated B cell survival and activation.

The influence of the pathway of Ag uptake and processing on MHC class II (CII)-mediated B cell function is unknown. In this study, we investigate in resting and activated (via the BCR or CD40) B cells the biological properties of CII-peptide complexes (CII-peptide) generated by either the BCR-mediated Ag processing (type I complex) or fluid phase Ag processing (type II complex). Compared with type I complex, ligation of type II complex by either specific Ab or the TCR in Ag-presenting assay results in significant decreases in B cell survival rate (50-100%) and expression levels of CII, CD86, and CD54.

Azithromycin modulates murine immune responses to pneumococcal conjugate vaccine and inhibits nasal clearance of bacteria.

Macrolide antibiotics, including azithromycin, have been implicated in the modulation of host immune responses, independently of their antimicrobial properties. The present work was designed to study the effect that azithromycin has on protective humoral immune responses induced by a 7-valent, polysaccharide, pneumococcal conjugate vaccine (PCV7). By use of a murine vaccination/challenge model, it was found that inoculation with azithromycin led to significantly lower primary antibody responses, decreased recall proliferative responses, and, in nasal cavities, impaired clearance of Streptococcus pneumoniae serotype 14 from the nasal cavities.

An important role for polymeric Ig receptor-mediated transport of IgA in protection against Streptococcus pneumoniae nasopharyngeal carriage.

The importance of IgA for protection at mucosal surfaces remains unclear, and in fact, it has been reported that IgA-deficient mice have fully functional vaccine-induced immunity against several bacterial and viral pathogens. The role of respiratory Ab in preventing colonization by Streptococcus pneumoniae has now been examined using polymeric IgR knockout (pIgR(-/-)) mice, which lack the ability to actively secrete IgA into the mucosal lumen. Intranasal vaccination with a protein conjugate vaccine elicited serotype-specific anti-capsular polysaccharide Ab locally and systemically, and pIgR(-/-) mice produced levels of total serum Ab after vaccination that were similar to wild-type mice.

Mitochondrial redox control of matrix metalloproteinases.

Reactive oxygen species (ROS) are constantly generated in aerobic organisms during normal metabolism and in response to both internal and external stimuli. Imbalances in the production and removal of ROS have been hypothesized to play a causative role in numerous disease pathologies such as cancer, ischemia/reperfusion injury, and degenerative diseases such as photoaging, atherosclerosis, arthritis, and neurodegeneration. A feature often associated with these diseases is a malfunctioning of the connective tissue remodeling process due to increased activity of extracellular matrix-degrading metalloproteinases (MMPs).

Multiple oxidative stress-response members of the Adapt78 family.

Adapt78 is an oxidative and calcium stress-response gene. Its protein product is a potent natural inhibitor of the intracellular calcium signaling protein calcineurin. Much of what is known about Adapt78 protein is based on cell-transfection studies.

The effect of B cell receptor signaling on antigen endocytosis and processing.

B cell receptor (BCR)-mediated antigen processing and presentation involves both the BCR-mediated internalization and processing of cognate antigen as well as the formation and expression of antigenic peptide-MHC class II complexes. While BCR signaling is known to result in changes in the biosynthesis and intracellular trafficking of class II molecules, the effect of BCR signaling on the cell biology of antigen endocytosis and processing is less clear. Therefore, the effect of BCR signaling on the cell biology of fluid phase antigen endocytosis, processing and presentation was analyzed in both B cell lines or in normal splenic B cells.