Molecular properties of the Goodpasture epitope

Goodpasture disease fulfils all criteria for a classical autoimmune disease, where autoantibodies targeted against the non-collagenous domain of the alpha3-chain of collagen IV initiates an inflammatory destruction of the basement membrane in kidney glomeruli and lung alveoli. This leads to a rapidly progressive glomerulonephritis and severe pulmonary hemorrhage. Previous studies have indicated a limited epitope for the toxic antibodies in the N-terminal part of the non-collagenous domain. The epitope has been partially characterized by recreating the epitope in the non-reactive alpha1-chain by exchanging nine residues to the corresponding ones of alpha3. In this study we have investigated to what extent each of these amino acids contribute to the antibody binding in different patient sera. The results show that seven of the nine substitutions are enough to get an epitope that is recognized equally well as the native alpha3-chain by all sera from 20 clinically verified Goodpasture patients. Furthermore, the patient sera reactivity against the different recombinant chains used in the study are very similar, with some minor exceptions, strongly supporting a highly defined and restricted epitope. We are convinced that the restriction of the epitope is of significant importance for the understanding of the etiology of the disease. Thereby also making every step on the way to characterization of the epitope a crucial step on the way to specific therapy for the disease.     

Capillary electrophoresis of highly sulfated flavanoids and flavonoids

Flavanoids and flavonoids are natural products present in our diet and known to possess multiple biological activities. Sulfated species of these natural products represent highly charged water-soluble organic molecules that possess unique biochemical properties. We describe here the first studies on capillary electrophoresis of these highly charged molecules. Fully sulfated flavanoids and flavonoids can be electrophoresed and resolved under reverse polarity at pH 3.5 using 5-10 kV in less than 20 min. In contrast, at high pH under normal polarity these species can be electrophoresed only if a pressurized capillary is employed. (+/-)-Catechin sulfate, a racemic sulfated flavanoid, was resolved into its enantiomers using 15% beta-cyclodextrin, a chiral selector, but not with alpha- or gamma-cyclodextrins. Yet, the high charge density of these molecules challenges the resolving capability of capillary electrophoresis as diastereomers (-)-epicatechin sulfate and (+)-catechin sulfate do not resolve, even in the presence of cyclodextrins or chiral positively charged amino acids. Overall, capillary electrophoresis of highly sulfated flavanoids and flavonoids is expected to be useful in rapid structure analysis of sulfated flavonoids, either synthetic or natural.     

Experimental design as a tool when evaluating stationary phases for the capillary electrochromatographic separation of basic peptides

Two different capillary electrochromatography (CEC) stationary phases, Hypersil phenyl and Hypersil C(18), have been characterised with respect to their ability to separate the four basic peptides H-Tyr-(D)Ala-Phe-Phe-NH(2) (TAPP), H-Tyr-(D)Ala-Phe-NH(2) (TAP), H-Phe-Phe-NH(2) (PP) and H-Phe-NH(2) (P). Optimal separation conditions were first established separately for the two phases by applying experimental design in a stepwise procedure. The first step comprised a study to acquire basic knowledge about the variables, their influence on the response and their respective experimental domains for each of the two stationary phases. The second step was screening the significant variables and the third step was an optimisation with response surface modelling (RSM) to locate the optimum separation conditions for each stationary phase. The experimental procedure was identical for both stationary phases, but their respective experimental domains were different. The response functions were peak resolution and peak efficiency. This procedure enables specific optimal experimental conditions to be identified for each of the two stationary phases. The optimal conditions identified for the separation on the phenyl stationary phase were to use 50% ACN, 20% 50 mM Tris(hydroxymethyl)aminomethane (TRIS) pH 7.5, 30% H(2)O as BGE, operating at 20 degrees C and 20 kV high voltage. For the C(18) stationary phase optimal separation was achieved using a BGE with 80% ACN, 20% 30 mM TRIS pH 8.5, again operating at 20 degrees C and 20 kV high voltage. Results show that the phenyl stationary phase is better suited for the separation of basic, hydrophilic peptides.     

Analysis of Antigens in Mycobacterium Paratuberculosis

Analysis of antigens in Mycobacterium paratuberculosis. Acta vet. scand. 1979, 20, 200–215. — Using crossed immunoelectrophoresis (GIE) and crossed line immunoelectrophoresis (GLIE), antigens from different strains and variants of Mycobacterium paratuberculosis were compared, and cross-reactions between 1 of these strains and Mycobacterium avium and BGG studied. In each of 4 bovine laboratory strains of M. paratuberculosis examined, altogether 44 different antigens were demonstrated. This is the largest number of antigens in M. paratuberculosis which has been described so far. No important difference in the antigenic structure of the strains was found. The 4 laboratory strains are being used routinely in the production of vaccine against Johne’s disease in Norway and Iceland. One of the aims of the present work was to investigate the antigenic relationship between these strains and the goat-pathogenic Norwegian and the Icelandic variant of M. paratuberculosis. Out of 44 different antigens demonstrated in the laboratory strains, 39 and 31 gave cross-reactions against the Norwegian and the Icelandic variant, respectively. This is in accordance with practical experience, as the results of vaccination against Johne’s disease, performed in Norway for many years, are very good. Twenty-seven and 24 cross-reacting antigens between M. paratuberculosis and strains of M. avium and BGG, respectively, were observed. This finding agrees with clinical observations. Another aim of the investigation was to identify species-specific antigens as regards M. paratuberculosis. One antigen showed a marked cross-reaction between the strains of M. paratuberculosis examined, but did not react with antisera against M. avium and BGG. Some other antigens showed partial specificity. The results obtained stress the complicated antigenic situation in mycobacteria which is of decisive significance as regards the diagnosis and classification of mycobacterial infections.     

Mutations in genes encoding complement inhibitors CD46 and CFH affect the age at nephritis onset in patients with systemic lupus erythematosus

Introduction

Inherited deficiencies of several complement components strongly predispose to systemic lupus erythematosus (SLE) while deficiencies of complement inhibitors are found in kidney diseases such as atypical hemolytic uremic syndrome (aHUS).

Methods

The exons of complement inhibitor genes CD46 and CFH (factor H) were fully sequenced using the Sanger method in SLE patients with nephritis originating from two cohorts from southern and mid Sweden (n = 196). All identified mutations and polymorphisms were then analyzed in SLE patients without nephritis (n = 326) and in healthy controls (n = 523).

Results

We found nonsynonymous, heterozygous mutations in CFH in 6.1% patients with nephritis, in comparison with 4.0% and 5.4% in patients without nephritis and controls, respectively. No associations of SLE or nephritis with common variants in CFH (V62I/Y402H/E936D) were found. Furthermore, we found two nonsynonymous heterozygous mutations in CD46 in SLE patients but not in controls. The A353V polymorphism, known to affect function of CD46, was found in 6.6% of nephritis patients versus 4.9% and 6.1% of the non-nephritis SLE patients and controls. The presence of mutations in CD46 and CFH did not predispose to SLE or nephritis but was associated with earlier onset of nephritis. Furthermore, we found weak indications that there is one protective and one risk haplotype predisposing to nephritis composed of several polymorphisms in noncoding regions of CD46, which were previously implicated in aHUS.

Conclusions

SLE nephritis is not associated with frequent mutations in CFH and CD46 as found in aHUS but these may be modifying factors causing earlier onset of nephritis.     

Individual Variation in Influenza A Virus Infection Histories and Long-Term Immune Responses in Mallards

Wild dabbling ducks (genus Anas) are the main reservoir for influenza A virus (IAV) in the Northern Hemisphere. Current understanding of disease dynamics and epidemiology in this virus-host system has primarily been based on population-level surveillance studies and infection experiments conducted in laboratory settings. Using a combined experimental-natural approach with wild-strain captive mallards (Anas platyrhynchos), we monitored individual IAV infection histories and immunological responses of 10 birds over the course of 15 months. This is the first detailed study to track natural IAV infection histories over several seasons amongst the same individuals growing from juvenile to adults. The general trends in the infection histories of the monitored birds reflected seasonal variation in prevalence at the population level. However, within the study group there were significant differences between individuals in infection frequency as well as in short and long term anti-IAV antibody response. Further observations included individual variation in the number of infecting virus subtypes, and a strong tendency for long-lasting hemagglutinin-related homosubtypic immunity. Specifically, all infections in the second autumn, except one, were of different subtypes compared to the first autumn. The variation among birds concerning these epidemiologically important traits illustrates the necessity for IAV studies to move from the level of populations to examine individuals in order to further our understanding of IAV disease and epidemiology.     

Climatic modifiers of the response to nitrogen deposition in peat-forming Sphagnum mosses: a meta-analysis

Peatlands in the northern hemisphere have accumulated more atmospheric carbon (C) during the Holocene than any other terrestrial ecosystem, making peatlands long-term C sinks of global importance. Projected increases in nitrogen (N) deposition and temperature make future accumulation rates uncertain. Here, we assessed the impact of N deposition on peatland C sequestration potential by investigating the effects of experimental N addition on Sphagnum moss. We employed meta-regressions to the results of 107 field experiments, accounting for sampling dependence in the data. We found that high N loading (comprising N application rate, experiment duration, background N deposition) depressed Sphagnum production relative to untreated controls. The interactive effects of presence of competitive vascular plants and high tissue N concentrations indicated intensified biotic interactions and altered nutrient stochiometry as mechanisms underlying the detrimental N effects. Importantly, a higher summer temperature (mean for July) and increased annual precipitation intensified the negative effects of N. The temperature effect was comparable to an experimental application of almost 4 g?N m(-2) yr(-1) for each 1°C increase. Our results indicate that current rates of N deposition in a warmer environment will strongly inhibit C sequestration by Sphagnum-dominated vegetation.