Early arrest in B cell development in transgenic mice that express the E41K Bruton's tyrosine kinase mutant under the control of the CD19 promoter region.

Bruton's tyrosine kinase (Btk) is a nonreceptor protein kinase that is defective in X-linked agammaglobulinemia in humans and in X-linked immunodeficiency in mice. To study the effect of Btk activation in early B cell development in vivo, we have created transgenic mouse strains expressing Btk under the control of the human CD19 promoter region. The transgenic expression of wild-type human Btk corrected all X-linked immunodeficiency features in mice carrying a targeted disruption of the Btk gene. In contrast, expression of an activated form of Btk, the E41K mutant, resulted in an almost complete arrest of B cell development in the immature IgM+IgD- B cell stage in the bone marrow, irrespective of the presence of the endogenous intact Btk gene. Immature B cells were arrested at the progression from IgMlow into IgMhigh cells, which reflects the first immune tolerance checkpoint at which autoreactive B cells become susceptible to apoptosis. As the constitutive activation of Btk is likely to mimic B cell receptor occupancy by autoantigens in the bone marrow, our findings are consistent with a role for Btk as a mediator of B cell receptor-induced apoptotic signals in the immature B cell stage. Whereas the peripheral mature B cell pool was reduced to <1% of the normal size, significant numbers of IgM-secreting plasma cells were present in the spleen. Serum IgM levels were substantial and increased with age, but specific Ab responses in vivo were lacking. We conclude that the residual peripheral B cells were efficiently driven into IgM+ plasma cell differentiation, apparently without functional selection.     

Numerical bifurcation analysis of distance-dependent on-center off-surround shunting neural networks

 On-center off-surround shunting neural networks are often applied as models for content-addressable memory (CAM), the equilibria being the stored memories. One important demand of biological plausible CAMs is that they function under a broad range of parameters, since several parameters vary due to postnatal maturation or learning. Ellias, Cohen and Grossberg have put much effort into showing the stability properties of several configurations of on-center off-surround shunting neural networks. In this article we present numerical bifurcation analysis of distance-dependent on-center off-surround shunting neural networks with fixed external input. We varied four parameters that may be subject to postnatal maturation: the range of both excitatory and inhibitory connections and the strength of both inhibitory and excitatory connections. These analyses show that fold bifurcations occur in the equilibrium behavior of the network by variation of all four parameters. The most important result is that the number of activation peaks in the equilibrium behavior varies from one to many if the range of inhibitory connections is decreased. Moreover, under a broad range of the parameters the stability of the network is maintained. The examined network is implemented in an ART network, Exact ART, where it functions as the classification layer F2. The stability of the ART network with the F2-field in different dynamic regimes is maintained and the behavior is functional in Exact ART. Through a bifurcation the learning behavior of Exact ART may even change from forming local representations to forming distributed representations. Received: 23 January 1996 / Accepted in revised form: 1 July 1996     

Relationships of Plasma Leptin Levels to Changes in Plasma Free Fatty Acids in Women Who Are Lean and Women Who Are Abdominally Obese

HENNES, MAGDA MI, ARNAVAZ DUA, DIANA L MAAS, GABRIELE E SONNENBERG, GLENN R KRAKOWER, AHMED H KISSEBAH. Relationships of plasma leptin levels to changes in plasma free fatty acids in women who are lean and women who are abdominally obese. Regulation of leptin production by the hormonal and metabolic milieu is poorly understood. Because abdominal obesity is commonly associated with elevated plasma free fatty acid (FFA) flux, we examined the effects of augmenting FFA on plasma leptin levels in women who were lean and of suppressing FFA in women with abdominal obesity. In study 1, nine subjects who were lean, after a 12-hour overnight fast, received either intravenous saline or Intralipid plus heparin to increase the plasma FFA concentration to approximately 1000 μmol/ L. After 3 hours of additional fasting, subjects underwent 3-hour hyperglycemic clamps. In study 2, seven subjects with abdominal obesity were evaluated by a similar protocol, but lipolysis and plasma FFA flux were instead maximally suppressed by acipimox. In the individuals who were lean, leptin levels were unchanged during clamping. Increasing plasma FFA reduced plasma leptin from 7.66 ± 0.66 to 7.05 ±0 0.66 (p=0.03), but 3 hours of hyperglycemia plus hyperinsulinemia had no additional effect on leptin levels (7.15 ± 0.71). Basal leptin levels, 4-fold higher in the subjects with obesity, were reduced from 34.6 ± 2.4 μg/L to 32.3 ± 1.1 μg/L (p=0.004) during the clamp period. When plasma FFA flux was suppressed, however, plasma leptin levels after clamped hyperglycemia/hyperinsulinemia were increased to 38.9 ± 1.2 μg/L (p=0.014 vs. time 0 and 0.001 vs. saline protocol). Changes in leptin concentrations are not correlated with changes in FFA. These results suggest that plasma FFA concentration does not regulate plasma leptin levels in basal, extended fasting, or hyperglycemic/hyperinsulinemic states.     

Use of rbcL and trnL-F as a two-locus DNA barcode for identification of NW-European ferns: an ecological perspective

Although consensus has now been reached on a general two-locus DNA barcode for land plants, the selected combination of markers (rbcL + matK) is not applicable for ferns at the moment. Yet especially for ferns, DNA barcoding is potentially of great value since fern gametophytes--while playing an essential role in fern colonization and reproduction--generally lack the morphological complexity for morphology-based identification and have therefore been underappreciated in ecological studies. We evaluated the potential of a combination of rbcL with a noncoding plastid marker, trnL-F, to obtain DNA-identifications for fern species. A regional approach was adopted, by creating a reference database of trusted rbcL and trnL-F sequences for the wild-occurring homosporous ferns of NW-Europe. A combination of parsimony analyses and distance-based analyses was performed to evaluate the discriminatory power of the two-region barcode. DNA was successfully extracted from 86 tiny fern gametophytes and was used as a test case for the performance of DNA-based identification. Primer universality proved high for both markers. Based on the combined rbcL + trnL-F dataset, all genera as well as all species with non-equal chloroplast genomes formed their own well supported monophyletic clade, indicating a high discriminatory power. Interspecific distances were larger than intraspecific distances for all tested taxa. Identification tests on gametophytes showed a comparable result. All test samples could be identified to genus level, species identification was well possible unless they belonged to a pair of Dryopteris species with completely identical chloroplast genomes. Our results suggest a high potential of the combined use of rbcL and trnL-F as a two-locus cpDNA barcode for identification of fern species. A regional approach may be preferred for ecological tests. We here offer such a ready-to-use barcoding approach for ferns, which opens the way for answering a whole range of questions previously unaddressed in fern gametophyte ecology.     

Forest harvesting reduces the soil metagenomic potential for biomass decomposition

Soil is the key resource that must be managed to ensure sustainable forest productivity. Soil microbial communities mediate numerous essential ecosystem functions, and recent studies show that forest harvesting alters soil community composition. From a long-term soil productivity study site in a temperate coniferous forest in British Columbia, 21 forest soil shotgun metagenomes were generated, totaling 187 Gb. A method to analyze unassembled metagenome reads from the complex community was optimized and validated. The subsequent metagenome analysis revealed that, 12 years after forest harvesting, there were 16% and 8% reductions in relative abundances of biomass decomposition genes in the organic and mineral soil layers, respectively. Organic and mineral soil layers differed markedly in genetic potential for biomass degradation, with the organic layer having greater potential and being more strongly affected by harvesting. Gene families were disproportionately affected, and we identified 41 gene families consistently affected by harvesting, including families involved in lignin, cellulose, hemicellulose and pectin degradation. The results strongly suggest that harvesting profoundly altered below-ground cycling of carbon and other nutrients at this site, with potentially important consequences for forest regeneration. Thus, it is important to determine whether these changes foreshadow long-term changes in forest productivity or resilience and whether these changes are broadly characteristic of harvested forests.     

Non-symbiotic Bradyrhizobium ecotypes dominate North American forest soils

The genus Bradyrhizobium has served as a model system for studying host–microbe symbiotic interactions and nitrogen fixation due to its importance in agricultural productivity and global nitrogen cycling. In this study, we identify a bacterial group affiliated with this genus that dominates the microbial communities of coniferous forest soils from six distinct ecozones across North America. Representative isolates from this group were obtained and characterized. Using quantitative population genomics, we show that forest soil populations of Bradyrhizobium represent ecotypes incapable of nodulating legume root hairs or fixing atmospheric nitrogen. Instead, these populations appear to be free living and have a greater potential for metabolizing aromatic carbon sources than their close symbiotic relatives. In addition, we identify fine-scaled differentiation between populations inhabiting neighboring soil layers that illustrate how diversity within Bradyrhizobium is structured by habitat similarity. These findings reconcile incongruent observations about this widely studied and important group of bacteria and highlight the value of ecological context to interpretations of microbial diversity and taxonomy. These results further suggest that the influence of this genus likely extends well beyond facilitating agriculture, especially as forest ecosystems are large and integral components of the biosphere. In addition, this study demonstrates how focusing research on economically important microorganisms can bias our understanding of the natural world.     

Bovine Immunoglobulin/Protein Isolate Binds Pro-Inflammatory Bacterial Compounds and Prevents Immune Activation in an Intestinal Co-Culture Model

Intestinal barrier dysfunction is associated with chronic gastrointestinal tract inflammation and diseases such as IBD and IBS. Serum-derived bovine immunoglobulin/protein isolate (SBI) is a specially formulated protein preparation (>90%) for oral administration. The composition of SBI is greater than 60% immunoglobulin including contributions from IgG, IgA, and IgM. Immunoglobulin within the lumen of the gut has been recognized to have anti-inflammatory properties and is involved in maintaining gut homeostasis. The binding of common intestinal antigens (LPS and Lipid A) and the ligand Pam3CSK4, by IgG, IgA, and IgM in SBI was shown using a modified ELISA technique. Each of these antigens stimulated IL-8 and TNF-α cytokine production by THP-1 monocytes. Immune exclusion occurred as SBI (≤50 mg/mL) bound free antigen in a dose dependent manner that inhibited cytokine production by THP-1 monocytes in response to 10 ng/mL LPS or 200 ng/mL Lipid A. Conversely, Pam3CSK4 stimulation of THP-1 monocytes was unaffected by SBI/antigen binding. A co-culture model of the intestinal epithelium consisted of a C2BBe1 monolayer separating an apical compartment from a basal compartment containing THP-1 monocytes. The C2BBe1 monolayer was permeabilized with dimethyl palmitoyl ammonio propanesulfonate (PPS) to simulate a damaged epithelial barrier. Results indicate that Pam3CSK4 was able to translocate across the PPS-damaged C2BBe1 monolayer. However, binding of Pam3CSK4 by immunoglobulins in SBI prevented Pam3CSK4 translocation across the damaged C2BBe1 barrier. These results demonstrated steric exclusion of antigen by SBI which prevented apical to basal translocation of antigen due to changes in the physical properties of Pam3CSK4, most likely as a result of immunoglobulin binding. This study demonstrates that immunoglobulins in SBI can reduce antigen-associated inflammation through immune and steric exclusion mechanisms and furthers the mechanistic understanding of how SBI might improve immune status and reduce inflammation in various intestinal disease states.     

Yearly hypertension screening in women with a history of pre-eclampsia: a cost-effectiveness analysis

Background

Women with a history of preeclampsia are at increased risk for future hypertension and cardiovascular disease (CVD); until now it is not clear whether preventive measures are needed.

Methods

A decision-analytic Markov model was constructed to evaluate healthcare costs and effects of screening and treatment (100 % compliance) for hypertension post preeclampsia based on the available literature. Cardiovascular events and CVD mortality were defined as health states. Outcomes were measured in absolute costs, events, life-years and quality-adjusted life-years (QALYs). Sensitivity and threshold analyses were performed to address uncertainty.

Results

Over a 20-year time horizon, events occurred in 7.2 % of the population after screening, and in 8.5 % of the population without screening. QALYs increased from 16.37 (no screening strategy) to 16.40 (screening strategy), an increment of 0.03 (95 % CI 0.01;0.05) QALYs. Total expected costs were € 8016 in the screening strategy, and € 9087 in the none screening strategy (expected saving of € 1071 (95 % CI − 3146;-87) per person).

Conclusion

Annual hypertension screening and treatment in women with a history of preeclampsia may save costs, for at least a similar quality of life and survival due to prevented CVD compared with standard care.

Electronic supplementary material

The online version of this article (doi:10.1007/s12471-015-0760-z) contains supplementary material, which is available to authorized users.