Activated T cells complicate the identification of regulatory T cells in rheumatoid arthritis

Most cell surface markers for CD4⁺CD25⁺ regulatory T cells (Tregs) are also expressed by activated non-regulatory T cells. Recently, CD127 down-regulation was found to identify functional Tregs in healthy individuals, but there are no data from patients with inflammatory conditions. We examined peripheral blood mononuclear cells (PBMC) from rheumatoid arthritis patients with active inflammation and from healthy controls, and found that CD4⁺ T cells contained an equal proportion of CD25⁺CD127⁻/low cells in both groups. In patients, not all these cells expressed intracellular FOXP3. Upon activation by anti-CD3/anti-CD28, PBMC rapidly down-regulated CD127, while FOXP3 up-regulation was transitory and occurred in fewer cells. The activated cells were not anergic to restimulation and had no suppressive effects. The distinct kinetics indicate that the FOXP3⁻CD127⁻/low cells in rheumatoid arthritis patients most likely represent activated non-regulatory T cells. This complicates the use of CD127 for identification of Tregs in inflammatory diseases.     

Titrating Theileria parva: single stocks against combination of stocks

Theileria parva is the causative agent of East Coast fever (ECF), an important cattle disease in East and Central Africa. One of the methods for control of ECF is 'infection and treatment', a procedure in which an animal is infected with the live parasite and at the same time treated with a long-acting oxytetracycline formulation, restraining the infection and allowing a protective cellular immune response to develop. Optimal immunizing doses were estimated using models of trichotomous response: dysimmunization (death or severe reaction during immunization), immunization failure (death or severe reaction during lethal challenge) and successful immunization (neither dysimmunization nor immunization failure). In this paper we present methods of interpreting immunization trials and apply these methods to previously unpublished data from two such trials: one with a mixture of three T. parva stocks and one with a single T. parva stock. We explain why titration trials conducted with a cocktail of antigens could predict a suboptimal immunization dose. Indeed it is possible for a combination of three individually efficient stocks to result in a mixture with which optimal immunization response might be difficult to achieve, because of averaging effects. The corresponding interpretation provides insights into why standard immunization trials for T. parva have not yielded the results that might be expected of them. The results of this work may also have implications for the use of antigen cocktails in cancer, HIV and malaria vaccine trials.     

Restoring natural seepage conditions on former agricultural grasslands does not lead to reduction of organic matter decomposition and soil nutrient dynamics

In the central part of the Netherlands, wetland restoration projects involve the rewetting of former agricultural land, where low water levels were artificially maintained (polders). Many of these projects do not result in the expected reduction of nitrogen and phosphorus availability and subsequent re-establishment of a diverse wetland vegetation. The aim of the present study was to investigate which mechanisms are responsible for this lack of success. Thereto, we studied the effect of rewetting of former agricultural grasslands on acidified peat soil (pH = 3.5) on organic matter decomposition, nitrogen cycling and phosphorus availability in the soil for three seasons. To provide an explanation for the observed effects, we simultaneously studied a set of potentially controlling abiotic soil conditions that were expected to change after rewetting. It was found that rewetting of these grasslands with natural, unpolluted seepage water did not affect nitrogen cycling, but raised decomposition rates and almost doubled phosphorus availability. The main cause of these effects is a raise of soil pH to about 7 due to the hydrochemical composition of the soil pore water after rewetting, which reflects groundwater with high amounts of buffering ions. This effect overruled any reduction in process rates by the lowered soil redox potential. The counterintuitive finding of eutrophication after rewetting with natural and unpolluted water is considered to represent a new form of internal eutrophication, triggered by the restoration of natural site conditions of former agricultural land on acid peat soil.     

Detection of mutant protein in complex biological samples: glucocerebrosidase mutations in Gaucher's disease

We report a sensitive method to detect point mutations in proteins from complex samples. The method is based on surface-enhanced laser desorption/ionization time-of-flight (SELDI-ToF) MS but can be extended to other MS platforms. The target protein in this study is the lysosomal enzyme glucocerebrosidase (GC), the key enzyme in Gaucher's disease. Deficiency of GC activity results in accumulation of glucosylceramide in macrophages. The relationship between GC genotypes and Gaucher's patient phenotypes is not strict. The possibility to measure protein levels of GC in clinical samples may provide deeper insight into the phenomenology of Gaucher's disease. For this purpose, GC was isolated in a single enrichment step through interaction with an immobilized monoclonal antibody, 8E4. After on-chip digestion of the antibody-antigen complex with trypsin, a total of 25 GC peptides were identified (sequence coverage approximately 60%), including several peptides containing mutated amino acid residues. The described methodology allows mutational analysis on the protein level, directly measured on complex biological samples without the necessity of elaborate purification procedures.     

Crystal structure of Sulfolobus acidocaldarius aspartate carbamoyltransferase in complex with its allosteric activator CTP

Aspartate carbamoyltransferase (ATCase) is a paradigm for allosteric regulation of enzyme activity. B-class ATCases display very similar homotropic allosteric behaviour, but differ extensively in their heterotropic patterns. The ATCase from the thermoacidophilic archaeon Sulfolobus acidocaldarius, for example, is strongly activated by its metabolic pathway′s end product CTP, in contrast with Escherichia coli ATCase which is inhibited by CTP. To investigate the structural basis of this property, we have solved the crystal structure of the S. acidocaldarius enzyme in complex with CTP. Structure comparison reveals that effector binding does not induce similar large-scale conformational changes as observed for the E. coli ATCase. However, shifts in sedimentation coefficients upon binding of the bi-substrate analogue PALA show the existence of structurally distinct allosteric states. This suggests that the so-called “Nucleotide-Perturbation model” for explaining heterotropic allosteric behaviour, which is based on global conformational strain, is not a general mechanism of B-class ATCases.     

Current measures of nutrient resorption efficiency lead to a substantial underestimation of real resorption efficiency: facts and solutions

Nutrient resorption is an important process during leaf senescence, which helps plants to minimize nutrient losses. To quantify nutrient resorption, the parameter resorption efficiency is commonly used. This parameter describes the percentage of the nutrient pool withdrawn before leaf abscission. The nutrient pool is generally expressed on the basis of leaf mass or leaf area, assuming that these bases do not change during senescence. In this paper we firstly present a mathematical formula describing the effect of change in measurement basis on the difference between the real resorption efficiency (RRE) value and the measured resorption efficiency (MRE). This formula shows that even moderate senescence-related changes in a measurement basis can lead to considerable underestimation of RRE. Secondly, to estimate the general change in measurement basis we quantified leaf mass loss and leaf shrinkage during senescence from literature data. These data shows that mass loss percentages can be as high as 40%, and leaf shrinkage can be up to 20%. This level of change in basis seriously compromises the MRE when not corrected for. Using our formula and the reported average literature values of changes in leaf mass (21%) and leaf shrinkage (11%) during senescence, we calculated that the average RRE for nitrogen and phosphorous of terrestrial plants is 6% (leaf area) to 10% (leaf mass) higher than the 50%, respectively 52% as reported by Aerts (1996) . This implies that nutrient resorption from senescing leaves is even more important for nutrient retention in terrestrial plants than thought so far. We advocate that preselecting leaves and monitoring the measurement basis throughout the duration of the experiment should minimize the difference between MRE and RRE.     

Modeling heterogeneity for count data: A study of maternal mortality in health facilities in Mozambique

Count data are very common in health services research, and very commonly the basic Poisson regression model has to be extended in several ways to accommodate several sources of heterogeneity: (i) an excess number of zeros relative to a Poisson distribution, (ii) hierarchical structures, and correlated data, (iii) remaining “unexplained” sources of overdispersion. In this paper, we propose hierarchical zero‐inflated and overdispersed models with independent, correlated, and shared random effects for both components of the mixture model. We show that all different extensions of the Poisson model can be based on the concept of mixture models, and that they can be combined to account for all different sources of heterogeneity. Expressions for the first two moments are derived and discussed. The models are applied to data on maternal deaths and related risk factors within health facilities in Mozambique. The final model shows that the maternal mortality rate mainly depends on the geographical location of the health facility, the percentage of women admitted with HIV and the percentage of referrals from the health facility.