Central role of complement in passive protection by human IgG1 and IgG2 anti-pneumococcal antibodies in mice

Streptococcus pneumoniae is an important cause of morbitity and mortality worldwide. Capsule-specific IgG1 and IgG2 Abs are induced upon vaccination with polysaccharide-based vaccines that mediate host protection. We compared the protective capacity of human recombinant serogroup 6-specific IgG1 and IgG2 Abs in mice deficient for either leukocyte FcR or complement factors. Human IgG1 was found to interact with mouse leukocyte FcR in vitro, whereas human IgG2 did not. Both subclasses induced complement activation, resulting in C3c deposition on pneumococcal surfaces. Passive immunization of C57BL/6 mice with either subclass before intranasal challenge with serotype 6A induced similar degrees of protection. FcgammaRI- and III-deficient mice, as well as the combined FcgammaRI, II, and III knockout mice, were protected by passive immunization, indicating FcR not to be essential for protection. C1q or C2/factor B knockout mice, however, were not protected by passive immunization. Passively immunized C2/factor B(-/-) mice displayed higher bacteremic load than C1q(-/-) mice, supporting an important protective role of the alternative complement pathway. Spleens from wild-type and C1q(-/-) mice showed hyperemia and thrombotic vessel occlusion, as a result of septicemic shock. Notably, thrombus formation was absent in spleens of C2/factor B(-/-) mice, suggesting that the alternative complement pathway contributes to shock-induced intravascular coagulation. These studies demonstrate complement to play a central role in Ab-mediated protection against pneumococcal infection in vivo, as well as in bacteremia-associated thrombotic complications.     

Ablation of tumor necrosis factor receptor type I (p55) alters oxygen-induced lung injury

Hyperoxic lung injury, believed to be mediated by reactive oxygen species, inflammatory cell activation, and release of cytotoxic cytokines, complicates the care of many critically ill patients. The cytokine tumor necrosis factor (TNF)-alpha is induced in lungs exposed to high concentrations of oxygen; however, its contribution to hyperoxia-induced lung injury remains unclear. Both TNF-alpha treatment and blockade with anti-TNF antibodies increased survival in mice exposed to hyperoxia. In the current study, to determine if pulmonary oxygen toxicity is dependent on either of the TNF receptors, type I (TNFR-I) or type II (TNFR-II), TNFR-I or TNFR-II gene-ablated [(-/-)] mice and wild-type control mice (WT; C57BL/6) were studied in >95% oxygen. There was no difference in average length of survival, although early survival was better for TNFR-I(-/-) mice than for either TNFR-II(-/-) or WT mice. At 48 h of hyperoxia, slightly more alveolar septal thickening and peribronchiolar and periarteriolar edema were detected in WT than in TNFR-I(-/-) lungs. By 84 h of oxygen exposure, TNFR-I(-/-) mice demonstrated greater alveolar debris, inflammation, and edema than WT mice. TNFR-I was necessary for induction of cytokine interleukin (IL)-1beta, IL-1 receptor antagonist, chemokine macrophage inflammatory protein (MIP)-1beta, MIP-2, interferon-gamma-induced protein-10 (IP-10), and monocyte chemoattractant protein (MCP)-1 mRNA in response to intratracheal administration of recombinant murine TNF-alpha. However, IL-1beta, IL-6, macrophage migration inhibitory factor, MIP-1alpha, MIP-2, and MCP-1 mRNAs were comparably induced by hyperoxia in TNFR-I(-/-) and WT lungs. In contrast, mRNA for manganese superoxide dismutase and intercellular adhesion molecule-1 were induced by hyperoxia only in WT mice. Differences in early survival and toxicity suggest that pulmonary oxygen toxicity is in part mediated by TNFR-I. However, induction of specific cytokine and chemokine mRNA and lethality in response to severe hyperoxia was independent of TNFR-I expression. The current study supports the prediction that therapeutic efforts to block TNF-alpha receptor function will not protect against pulmonary oxygen toxicity.     

The Cdc42-selective GAP rich regulates postsynaptic development and retrograde BMP transsynaptic signaling

Retrograde bone morphogenetic protein signaling mediated by the Glass bottom boat (Gbb) ligand modulates structural and functional synaptogenesis at the Drosophila melanogaster neuromuscular junction. However, the molecular mechanisms regulating postsynaptic Gbb release are poorly understood. In this study, we show that Drosophila Rich (dRich), a conserved Cdc42-selective guanosine triphosphatase-activating protein (GAP), inhibits the Cdc42-Wsp pathway to stimulate postsynaptic Gbb release. Loss of dRich causes synaptic undergrowth and strongly impairs neurotransmitter release. These presynaptic defects are rescued by targeted postsynaptic expression of wild-type dRich but not a GAP-deficient mutant. dRich inhibits the postsynaptic localization of the Cdc42 effector Wsp (Drosophila orthologue of mammalian Wiskott-Aldrich syndrome protein, WASp), and manifestation of synaptogenesis defects in drich mutants requires Wsp signaling. In addition, dRich regulates postsynaptic organization independently of Cdc42. Importantly, dRich increases Gbb release and elevates presynaptic phosphorylated Mad levels. We propose that dRich coordinates the Gbb-dependent modulation of synaptic growth and function with postsynaptic development.     

Differential expression of T‐type calcium channels in P/Q‐type calcium channel mutant mice with ataxia and absence epilepsy

Mutations in P/Q‐type calcium channels generate common phenotypes in mice and humans, which are characterized by ataxia, paroxysmal dyskinesia, and absence seizures. Subsequent functional changes of T‐type calcium channels in thalamus are observed in P/Q‐type calcium channel mutant mice and these changes play important roles in generation of absence seizures. However, the changes in T‐type calcium channel function and/or expression in the cerebellum, which may be related to movement disorders, are still unknown. The leaner mouse exhibits severe ataxia, paroxysmal dyskinesia, and absence epilepsy due to a P/Q‐type calcium channel mutation. We investigated changes in T‐type calcium channel expression in the leaner mouse thalamus and cerebellum using quantitative real‐time polymerase chain reaction (qRT‐PCR) and quantitative in situ hybridization histochemistry (ISHH). qRT‐PCR analysis showed no change in T‐type calcium channel α1G subunit (Cav3.1) expression in the leaner thalamus, but a significant decrease in α1G expression in the whole leaner mouse cerebellum. Interestingly, quantitative ISHH revealed differential changes in α1G expression in the leaner cerebellum, where the granule cell layer showed decreased α1G expression while Purkinje cells showed increased α1G expression. To confirm these observations, the granule cell layer and the Purkinje cell layer were laser capture microdissected separately, then analyzed with qRT‐PCR. Similar to the observation obtained by ISHH, the leaner granule cell layer showed decreased α1G expression and the leaner Purkinje cell layer showed increased α1G expression. These results suggest that differential expression of T‐type calcium channels in the leaner cerebellum may be involved in the observed movement disorders. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

A new model of pneumococcal lipoteichoic acid structure resolves biochemical, biosynthetic, and serologic inconsistencies of the current model

Lipoteichoic acid (LTA) is an essential bacterial membrane polysaccharide (cell wall component) that is attached to the membrane via a lipid anchor. According to the currently accepted structure of pneumococcal LTA, the polysaccharide is comprised of several repeating units, each of which starts with glucose and ends with ribitol, with the lipid anchor predicted to be Glc(beta1-->3)AATGal(beta1-->3)Glc(alpha1-->3)-acyl(2)Gro, where AATGal is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose. However, this lipid anchor has not been detected in pneumococcal membranes. Furthermore, the currently accepted structure does not explain the Forssman antigen properties of LTA and predicts a molecular weight for LTA that is larger than its actual observed molecular weight. To resolve these problems, we used mass spectrometry to analyze the structure of LTA isolated from several pneumococcal strains. Our study found that the R36A pneumococcal strain produces LTA that is more representative of pneumococci than that previously characterized from the R6 strain. Analysis of LTA fragments obtained after hydrofluoric acid and nitrous treatments showed that the fragments were consistent with an LTA nonreducing terminus consisting of GalNAc(alpha1-->3)GalNAc(beta1-->, which is the minimal structure for the Forssman antigen. Based on these data, we propose a revised model of LTA structure: its polysaccharide repeating unit begins with GalNAc and ends with AATGal, and its lipid anchor is Glc(alpha1-->3)-acyl(2)Gro, a common lipid anchor found in pneumococcal membranes. This new model accurately predicts the observed molecular weights. The revised model should facilitate investigation of the relationship between LTA's structure and its function.     

Monoclonal antibodies to streptococcal group A carbohydrate. I. A dominant idiotypic determinant is located on Vk

In an effort to better define the antibody repertoire to streptococcal group A carbohydrate (GAC), somatic cell hybrids were prepared from A/J mice immunized with streptococcal vaccine. Most antibodies were IgG3K and IgMK, while 2 of 26 antibodies were lambda type. Each of the IgG3 antibodies had a distinct isoelectric point consistent with previous estimates of clonal repertoires of approximately 200. IEF analysis of the L chains, however, showed that about half of the antibodies produce a common L chain, called VK1GAC, previously identified in A/J anti-GAC serum antibodies. Additional support for the structural similarity of these L chains was gained by developing an idiotype antiserum to VK1GAC. All proteins with the common L chain spectrotype react strongly with anti-VK1GAC. Thus, it appears that anti-GAC antibodies are composed of H chains bearing a few VH regions pairing with a few L chains.     

Functional properties of human germinal center B cells.

Germinal centers (GCs) are histologically defined areas where B cells undergo extensive proliferation and maturation, or die of apoptosis. GC B cells isolated from human tonsils can be phenotypically identified by expression of peanut agglutinin (PNA)-binding sites and can be further divided into subpopulations based on their expression of CD77. To assess the functional potential of GC B cells, we studied CD77+ PNA+ B cells isolated from tonsils by examining their differentiation status and their ability to proliferate in vitro to various cytokines and costimulants. We found that CD77+ GC B cells are less differentiated than CD77- GC B cells; GC B cells less frequently express cytoplasmic IgG and IgM, and spontaneously secrete less Ig compared to CD77- GC B cells. To identify conditions capable of inducing GC B cell proliferation, we examined IL-4, IL-2, IFN-gamma, low molecular weight BCGF (LMW-BCGF), and an MLR supernatant along with costimulants such as anti-IgM antibody, Staphylococcus aureus Cowan I (SAC), PMA, and pokeweed mitogen (PWM). While non-GC B cells proliferate strongly in response to these stimuli, GC B cells did not proliferate. However, CD77+ as well as CD77- GC B cells mounted a rapid and strong proliferative response upon stimulation with IL-4, but only in the presence of anti-CD40 antibody. Moreover, although nine additional cytokines were examined, only IL-4 was capable of supporting CD77+ GC B cell proliferation in the presence of anti-CD40 antibody. When cells were stimulated with IL-4 and anti-CD40 antibody, we also found that IFN-gamma consistently decreased the proliferative response of CD77+ GC B cells without affecting the response of non-GC B cells. Taken together, these data indicate that GC B cells have characteristic growth requirements and that IL-4 may be important for GC B cell growth in vivo.     

Identification of anti-adipogenic proteins in adult bovine serum suppressing 3T3-L1 preadipocyte differentiation

Adipocyte differentiation is a complex developmental process forming adipocytes from various precursor cells. The murine 3T3-L1 preadipocyte cell line has been most frequently used in the studies of adipocyte differentiation. Differentiation of 3T3-L1 preadipocytes includes a medium containing fetal bovine serum (FBS) with hormonal induction. In this study, we observed that differentiation medium containing adult bovine serum (ABS) instead of FBS did not support differentiation of preadipocytes. Impaired adipocyte differentiation was due to the presence of a serum protein factor in ABS that suppresses differentiation of preadipocytes. Using a proteomic analysis, alpha-2-macroglobulin and paraoxonase/arylesterase 1, which were previously shown to suppress differentiation of preadipocytes, were identified as anti-adipogenic proteins. Although their functional mechanisms have not yet been elucidated, the anti-adipogenic effects of these proteins are discussed. [BMB Reports 2013; 46(12): 582-587]