Complement research experienced a renaissance with the discovery of a third activation route, the lectin pathway. We developed a unique model of total lectin pathway deficiency, a mouse strain lacking mannan-binding lectin-associated serine protease-2 (MASP-2), and analyzed the role of MASP-2 in two models of postischemic reperfusion injury (IRI). In a model of transient myocardial IRI, MASP-2-deficient mice had significantly smaller infarct volumes than their wild-type littermates. Mice deficient in the downstream complement component C4 were not protected, suggesting the existence of a previously undescribed lectin pathway-dependent C4-bypass. Lectin pathway-mediated activation of C3 in the absence of C4 was demonstrated in vitro and shown to require MASP-2, C2, and MASP-1/3. MASP-2 deficiency also protects mice from gastrointestinal IRI, as do mAb-based inhibitors of MASP-2. The therapeutic effects of MASP-2 inhibition in this experimental model suggest the utility of anti-MASP-2 antibody therapy in reperfusion injury and other lectin pathway-mediated disorders.

The intricate system of serum complement proteins provides resistance to infection. A pivotal step in the complement pathway is the assembly of a C3 convertase, which digests the C3 complement component to form microbial binding C3 fragments recognized by leukocytes. The spleen and C3 provide resistance against blood-borne S. pneumoniae infection. To better understand the mechanisms involved, we studied SIGN-R1, a lectin that captures microbial polysaccharides in spleen. Surprisingly, conditional SIGN-R1 knockout mice developed deficits in C3 catabolism when given S. pneumoniae or its capsular polysaccharide intravenously. There were marked reductions in proteolysis of serum C3, deposition of C3 on organisms within SIGN-R1(+) spleen macrophages, and formation of C3 ligands. We found that SIGN-R1 directly bound the complement C1 subcomponent, C1q, and assembled a C3 convertase, but without the traditional requirement for either antibody or factor B. The transmembrane lectin SIGN-R1 therefore contributes to innate resistance by an unusual C3 activation pathway.

The targeted delivery of Ags to dendritic cell (DCs) in vivo greatly improves the efficiency of Ag presentation to T cells and allows an analysis of receptor function. To evaluate the function of Langerin/CD207, a receptor expressed by subsets of DCs that frequently coexpress the DEC205/CD205 receptor, we genetically introduced OVA into the C terminus of anti-receptor Ab H chains. Taking advantage of the new L31 mAb to the extracellular domain of mouse Langerin, we find that the hybrid Ab targets appropriate DC subsets in draining lymph nodes and spleen. OVA is then presented efficiently to CD8+ and CD4+ T cells in vivo, which undergo 4–8 cycles of division in 3 days. Peptide MHC I and II complexes persist for days. Dose response studies indicate only modest differences between Langerin and DEC receptors in these functions. Thus, Langerin effectively mediates Ag presentation.

A new sulfide-selective chemosignaling system was devised based on a Cu(2+) complex of fluorescein derivative having a dipicolylamine (DPA) binding site; the fluorescein-DPA conjugate 1, in the presence of Cu(2+) ions, revealed a selective turn-on type signaling behavior toward sulfide ions with a detection limit of 420 nM in 100% aqueous solution.

The cis-replicating RNA elements in the 5 and 3 nontranslated regions (NTRs) of the hepatitis C virus (HCV) genome have been thoroughly studied before. However, no cis-replicating elements have been identified in the coding sequences of the HCV polyprotein until very recently. The existence of highly conserved and stable stem-loop structures in the RNA polymerase NS5B coding sequence, however, has been previously predicted (A. Tuplin, J. Wood, D. J. Evans, A. H. Patel, and P. Simmonds, RNA 8:824-841, 2002). We have selected for our studies a 249-nt-long RNA segment in the C-terminal NS5B coding region (NS5BCR), which is predicted to form four stable stem-loop structures (SL-IV to SL-VII). By deletion and mutational analyses of the RNA structures, we have determined that two of the stem-loops (SL-V and SL-VI) are essential for replication of the HCV subgenomic replicon in Huh-7 cells. Mutations in the loop and the top of the stem of these RNA elements abolished replicon RNA synthesis but had no effect on translation. In vitro gel shift and filter-binding assays revealed that purified NS5B specifically binds to SL-V. The NS5B-RNA complexes were specifically competed away by unlabeled homologous RNA, to a small extent by 3 NTR RNA, and only poorly by 5 NTR RNA. The other two stem-loops (SL-IV and SL-VII) of the NS5BCR domain were found to be important but not essential for colony formation by the subgenomic replicon. The precise function(s) of these cis-acting RNA elements is not known.Plus-strand RNA viruses belong to many different families, but they all have similar strategies for replicating their genomes. The nonstructural viral proteins in concert with cellular proteins form a complex with the viral RNA on membranous vesicles, where RNA replication takes place. The input RNA is first transcribed into a minus strand, which in turn serves as the template for production of progeny plus strands. For many years it was generally believed that the specificity of genome translation and replication resides in cis-acting RNA elements located in the 5Ј and 3Ј nontranslated regions (NTRs) of viral genomes. However, recently the importance of cis-replicating elements in the coding sequences of the viral polyprotein of a number of plus-strand RNA viruses has been demonstrated.Hepatitis C virus (HCV) is the major causative agent of transfusion-related and sporadic non-A, non-B hepatitis (9). HCV, a member of the Flaviviridae family, is an enveloped plus-stranded RNA virus with a genome length of about 9,600 nucleotides (nt) (reviewed in reference 52). The viral RNA contains a long 5Ј NTR, a single open reading frame, and a 3Ј NTR (Fig. 1A). The 5Ј NTR contains the IRES (internal ribosomal entry site), which promotes the translation of a large polyprotein. The virus-encoded polypeptide is processed by host and viral proteinases into at least 10 distinct proteins: NH 2 -C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH.Sequences of the HCV genome, the identities of viral proteins, and the basic steps in replication have been known ...

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Ruane et al. demonstrate a role for the microbiota in modulating protective immunity to intranasal vaccination via the ability of lung dendritic cells to induce B cell IgA class switching.

Langerin CD207 is a type II transmembrane protein. It is responsible for the formation of Birbeck granules, which are intracellular organelles within Langerhans cells, the dendritic cells of stratified squamous epithelia like the epidermis. Because current anti-CD207 antibodies have limitations, we prepared new monoclonals by immunizing rats with the extracellular region of mouse Langerin followed by a boost with enriched Langerhans cells (LCs). We secured a large panel of mAbs, most of which reacted with the carboxy terminal carbohydrate recognition domain. These mAbs could be used to immunoblot and immunoprecipitate mouse Langerin and to stain the cell surface and intracellular pools of CD207 by FACS analysis. Labeling of Birbeck granules was also achieved by immunoelectron microscopy. Anti-CD207 identified LCs in the epidermis and skin draining lymph nodes of BALB/c and C57BL/6 mice, but BALB/c mice had an additional Langerin + population in spleen, thymus and mesenteric lymph node. This additional subset had higher levels of CD8 and CD205 than epidermal LCs, and also had a less mature phenotype, i.e., lower MHC II, CD40 and CD86. Subcutaneous injection of IgG but not IgM forms of these new anti-CD207 mAbs led to rapid and selective labeling of the Langerin + cells in skin draining lymph nodes as well as spleen. The new IgG anti-CD207 mAbs should be useful for further research on LCs and dendritic cells including an evaluation of the consequences of antigen delivery within anti-CD207 mAbs in vivo.