Signaling lymphocyte activation molecule-associated protein is a negative regulator of the CD8 T cell response in mice

The primary manifestation of X-linked lymphoproliferative syndrome, caused by a dysfunctional adapter protein, signaling lymphocyte activation molecule-associated protein (SAP), is an excessive T cell response upon EBV infection. Using the SAP-/- mouse as a model system for the human disease, we compared the response of CD8+ T cells from wild-type (wt) and mutant mice to various stimuli. First, we observed that CD8+ T cells from SAP-/- mice proliferate more vigorously than those from wt mice upon CD3/CD28 cross-linking in vitro. Second, we analyzed the consequence of SAP deficiency on CTL effector function and homeostasis. For this purpose, SAP-/- and wt mice were infected with the murine gamma-herpesvirus 68 (MHV-68). At 2 wk postinfection, the level of viral-specific CTL was much higher in mutant than in wt mice, measured both ex vivo and in vivo. In addition, we established that throughout 45 days of MHV-68 infection the frequency of virus-specific CD8+ T cells producing IFN-gamma was significantly higher in SAP-/- mice. Consequently, the level of latent infection by MHV-68 was considerably lower in SAP-/- mice, which indicates that SAP-/- CTL control this infection more efficiently than wt CTL. Finally, we found that the Vbeta4-specific CD8+ T cell expansion triggered by MHV-68 infection is also enhanced and prolonged in SAP-/- mice. Taken together, our data indicate that SAP functions as a negative regulator of CD8+ T cell activation.     

In situ diversification of the antibody repertoire in chronic Lyme arthritis synovium

Lyme arthritis is initiated by the tick-borne spirochete, Borrelia burgdorferi. In a subset of patients, symptoms do not resolve in response to standard courses of antibiotics. Chronic joint inflammation may persist despite spirochetal killing, suggesting an autoimmune etiology. The pathogenic mechanisms that sustain chronic Lyme arthritis have not been fully elucidated, although T cells are believed to play a role. The synovial lesion contains elements of a peripheral lymph node, with lymphoid aggregates, plasma cells and follicular dendritic cells. An analysis of activated cells at the site of injury could yield clues regarding the nature of the response and the identity of potential autoantigens. Using laser-capture microdissection, we have isolated plasma cells from the joint tissue of chronic Lyme arthritis patients who underwent synovectomy. Expressed Ig V regions were amplified by RT-PCR. A majority of isolated cells expressed gamma H chains, which is indicative of a class-switched response. There were a large number of nucleotide substitutions from germline, with a higher fraction of replacement mutations in the CDRs, suggesting a process of Ag-driven selection. We have recovered clonal clusters of cells containing identical junctions and V(D)J rearrangements. Sequence analysis reveals a hierarchy of shared somatic mutations between members of a given clone. Intraclonal diversity among plasma cells of close physical proximity points toward an ongoing process of diversification and affinity maturation, possibly driven by the chronic presence of an autoantigen.     

Inhibition of dynamin-dependent endocytosis increases shedding of the amyloid precursor protein ectodomain and reduces generation of amyloid β protein

Background  

The amyloid precursor protein (APP) is transported via the secretory pathway to the cell surface, where it may be cleaved within its ectodomain by α-secretase, or internalized within clathrin-coated vesicles. An alternative proteolytic pathway occurs within the endocytic compartment, where the sequential action of β- and γ-secretases generates the amyloid β protein (Aβ). In this study, we investigated the effects of modulators of endocytosis on APP processing.     

Chemokines and Toll-like receptors in Lyme disease pathogenesis

Lyme disease is a tick-transmitted inflammatory disorder, caused by the spirochete Borrelia burgdorferi (Bb). Recent discoveries cast new light on Bb dissemination and the ensuing pathogenesis of inflammation. Although the strong proinflammatory Bb lipoproteins have been implicated in the induction of inflammation, they do not seem to act exclusively through Toll-like receptor (TLR) engagement. In fact, mice that are deficient for MyD88, a component of the TLR signaling pathway, manifest similar or increased recruitment of cells into Bb-infected tissues. By contrast, the absence of the chemokine receptor CXCR2 results in reduced inflammation. Overall, these findings highlight the complexity of Lyme disease pathogenesis and identify chemokine pathways as novel therapeutic targets for the control of Bb-induced inflammation.     

CaCO3 biomineralization: acidic 8-kDa proteins isolated from aragonitic abalone shell nacre can specifically modify calcite crystal morphology

Acidic proteins from many biogenic minerals are implicated in directing the formation of crystal polymorphs and morphologies. We characterize the first extremely acidic proteins purified from biomineralized aragonite. These abalone nacre proteins are two variants of 8.7 and 7.8 kDa designated AP8 (for aragonite proteins of approximately 8 kDa). The AP8 proteins have compositions dominated by Asx ( approximately 35 mol %) and Gly ( approximately 40 mol %) residues, suggesting that their structures have high Ca(2+)-binding capacity and backbone flexibility. The growth of asymmetrically rounded CaCO(3) crystals in the presence of AP8 reveals that both proteins preferentially interact with specific locations on the crystal surface. In contrast, CaCO(3) crystals grown with nacre proteins depleted of AP8 retain the morphology of unmodified calcite rhombohedra. Our observations thus identify sites of protein-mineral interaction and provide evidence to support the long-standing theory that acidic proteins are more effective crystal-modulators than other proteins from the same biomineralized material.     

Metallothionein concentration in sponges (Spongia officinalis) as a biomarker of metal contamination

The synthesis of metallothioneins (MTs) is often induced when organisms are exposed to heavy metals in the field. They are among the major "specific" biomarkers identified to date. With a view to include MTs in biomonitoring programs, the organisms most commonly studied are bivalves. Sponges present most of the characteristics researched in bioindicators of pollution and consequently have been proposed to constitute a "Sponge Watch Program". The detection of large quantities of metals in sponges suggests the existence of detoxification systems and indeed, the presence of metallothionein-like proteins (MTLPs) has been reported in two different species of sponges. In Spongia officinalis, the present study has demonstrated the presence of compounds exhibiting most of the characteristics of MTs: cytosolic, heat-stable, with apparent molecular mass of 4 to 15 kDa and binding (at least) Ag, Cu and Zn. Specimens have been collected along the French Mediterranean coast from three sites differing by their degree of contamination. Relationships between MTLP and metal concentrations have been established. For copper, mercury and zinc, the correlations were significantly positive.     

Engineered endophytic bacteria improve phytoremediation of water-soluble, volatile, organic pollutants

Phytoremediation of highly water soluble and volatile organic xenobiotics is often inefficient because plants do not completely degrade these compounds through their rhizospheres. This results in phytotoxicity and/or volatilization of chemicals through the leaves, which can cause additional environmental problems. We demonstrate that endophytic bacteria equipped with the appropriate degradation pathway improve the in planta degradation of toluene. We introduced the pTOM toluene-degradation plasmid of Burkholderia cepacia G4 into B. cepacia L.S.2.4, a natural endophyte of yellow lupine. After surface-sterilized lupine seeds were successfully inoculated with the recombinant strain, the engineered endophytic bacteria strongly degraded toluene, resulting in a marked decrease in its phytotoxicity, and a 50-70% reduction of its evapotranspiration through the leaves. This strategy promises to improve the efficiency of phytoremediating volatile organic contaminants.     

A functionally improved locked nucleic acid antisense oligonucleotide inhibits Bcl-2 and Bcl-xL expression and facilitates tumor cell apoptosis

We previously reported the Bcl-2/Bcl-xL-bispecific activity of the 2'-O-(2-methoxy)ethyl (2'-MOE)-modified gapmer antisense oligonucleotide 4625. This oligonucleotide has 100% complementarity to Bcl-2 and three mismatches to Bcl-xL. In the present study, the isosequential locked nucleic acid (LNA)-modified oligonucleotide 5005 was generated, and its ability to further improve the downregulation of the two antiapoptotic targets in tumor cells was examined. We demonstrate that compared with 4625, 5005 more effectively decreased the expression of the mismatching Bcl-xL target gene in MDA-MB-231 breast and H125 lung cancer cells. In both cell lines, antisense activity caused decreased cell viability by induction of apoptosis. Moreover, in combination with various anticancer agents, 5005 reduced tumor cell viability more effectively than 4625. We describe for the first time the functional comparison of isosequential Bcl-2/Bcl-xL-bispecific 2'-MOE and LNA-modified antisense oligonucleotides and report that the LNA analog more effectively downregulated the two apoptosis inhibitors overexpressed in human tumors. Our data underscore the ability of LNA modifications to enhance the efficacy and favorably modulate the target specificity of antisense oligonucleotides.