A Single Mutation at the Sheet Switch Region Results in Conformational Changes Favoring lambda 6 Light-Chain Fibrillogenesis

Systemic amyloid light-chain (LC) amyloidosis is a disease process characterized by the pathological deposition of monoclonal LCs in tissue. All LC subtypes are capable of fibril formation although lambda chains, particularly those belonging to the lambda 6 type, are overrepresented. Here, we report the thermodynamic and in vitro fibrillogenic properties of several mutants of the lambda 6 protein 6aJL2 in which Pro7 and/or His8 was substituted by Ser or Pro. The H8P and H8S mutants were almost as stable as the wildtype protein and were poorly fibrillogenic. In contrast, the P7S mutation decreased the thermodynamic stability of 6aJL2 and greatly enhanced its capacity to form amyloid-like fibrils in vitro. The crystal structure of the P7S mutant showed that the substitution induced both local and long-distance effects, such as the rearrangement of the V(L) (variable region of the light chain)-V(L) interface. This mutant crystallized in two orthorhombic polymorphs, P2(1)2(1)2(1) and C222(1). In the latter, a monomer that was not arranged in the typical Bence-Jones dimer was observed for the first time. Crystal-packing analysis of the C222(1) lattice showed the establishment of intermolecular beta-beta interactions that involved the N-terminus and beta-strand B and that these could be relevant in the mechanism of LC fibril formation. Our results strongly suggest that Pro7 is a key residue in the conformation of the N-terminal sheet switch motif and, through long-distance interactions, is also critically involved in the contacts that stabilized the V(L) interface in lambda 6 LCs. (C) 2009 Elsevier Ltd. All rights reserved.

Short-chain fatty acids stimulate the migration of neutrophils to inflammatory sites

SCFAs (short-chain fatty acids) are produced by anaerobic bacterial fermentation. Increased concentrations of these fatty acids are observed in inflammatory conditions, such as periodontal disease, and at sites of anaerobic infection. In the present study, the effect of the SCFAs acetate, propionate and butyrate on neutrophil chemotaxis and migration was investigated. Experiments were carried out in rats and in vitro. The following parameters were measured: rolling, adherence, expression of adhesion molecules in neutrophils (L-selectin and beta 2 integrin), transmigration, air pouch influx of neutrophils and production of cytokines [CINC-2 alpha beta (cytokine-induced neutrophil chemoattractant-2 alpha beta), IL-1 beta (interleukin-1 beta), MIP-1 alpha (macrophage inflammatory protein-1 alpha) and TNF-alpha (tumour necrosis factor-alpha)]. SCFAs induced in vivo neutrophil migration and increased the release of CINC-2 alpha beta into the air pouch. These fatty acids increased the number of rolling and adhered cells as evaluated by intravital microscopy. SCFA treatment increased L-selectin expression on the neutrophil surface and L-selectin mRNA levels, but had no effect on the expression of beta 2 integrin. Propionate and butyrate also increased in vitro transmigration of neutrophils. These results indicate that SCFAs produced by anaerobic bacteria raise neutrophil migration through increased L-selectin expression on neutrophils and CINC-2 alpha beta release.

Hematologically important mutations: X-linked chronic granulomatous disease (third update)

Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by a lack of superoxide production by the leukocyte enzyme NADPH oxidase. Superoxide is used to kill phagocytosed micro-organisms in neutrophils, eosinophils, monocytes and macrophages. The leukocyte NADPH oxidase is composed of five subunits, of which the enzymatic component is gp91-phox, also called Nox2. This protein is encoded by the CYBB gene on the X chromosome. Mutations in this gene are found in about 70% of all CGD patients. This article lists all mutations identified in CYBB in the X-linked form of CGD. Moreover, apparently benign polymorphisms in CYBB are also given, which should facilitate the recognition of future disease-causing mutations. (C) 2010 Elsevier Inc. All rights reserved.

Inconclusive results in conventional serological screening for Chagas` disease in blood banks: evaluation of cellular and humoral response

To find the most reliable screening method for Trypanosoma cruzi infection in blood banks. Epidemiological data, lymphoproliferation assay, parasitological, conventional serological tests: immunofluorescence, haemagglutination, ELISA with epimastigote and trypomastigote antigens and reference serological tests: trypomastigote excreted-secreted antigens (TESA) blot and chemiluminescent ELISA assay with mucine from trypomastigote forms were applied to individuals with inconclusive serology, non-chagasic individuals and chronic chagasic patients. TESA blot had the best performance when used as a single test in all the groups. In the inconclusive group 20.5% of individuals were positive for TESA blot, 23.3% for either lymphoproliferation or TESA blot, and 17.8% for lymphoproliferation only. Positive lymphoproliferation without detectable antibodies was observed in 5.47% of all inconclusive serology cases. Analysis of six parameters (three serological assays, at least one parasitological test, one lymphoproliferation assay and epidemiological data) in the inconclusive group showed that diagnosis of Chagas` disease was probable in 15 patients who were positive by two or more serological tests or for whom three of those six parameters were positive. TESA blot is a good confirmatory test for Chagas` disease in the inconclusive group. Although lymphoproliferation suggests the diagnosis of Chagas` disease in the absence of antibodies when associated with a high epidemiological risk of acquiring Chagas` disease, the data from this study and the characteristics of the lymphoproliferation assay (which is both laborious and time-consuming) do not support its use as a confirmatory test in blood-bank screening. However, our findings underscore the need to develop alternative methods that are not based on antibody detection to improve the diagnosis when serological tests are inconclusive.