Dissociation of experimental allergic encephalomyelitis protective effect and allergic side reactions in tolerization with neuroantigen

Administration of autoantigens under conditions that induce type 2 immunity frequently leads to protection from T cell-mediated autoimmune diseases. Such treatments, however, are inherently linked to the induction of IgG1 Abs and to the risk of triggering anaphylactic reactions. We studied the therapeutic benefit vs risk of immune deviation in experimental allergic encephalomyelitis of SJL mice induced by MP4, a myelin basic protein-proteolipid protein (PLP) fusion protein. MP4 administration in IFA induced type 2 T cell immunity, IgG1 Abs, and experimental allergic encephalomyelitis protection, and all three were enhanced by repeat injections. Despite high Ab titers, anaphylactic side reactions were not observed when MP4 was repeatedly injected in IFA or as soluble Ag s.c. In contrast, lethal anaphylaxis was seen after s.c. injection of soluble PLP:139-151 peptide, but not when the peptide was reinjected in IFA. Therefore, the Ab response accompanying the immune therapy constituted an anaphylactic risk factor only when the autoantigen was not retained in an adjuvant and when it was small enough to be readily disseminated within the body. Taken together, our data show that treatment regimens can be designed to boost the protective type 2 T cell response while avoiding the risk of Ab-mediated allergic side effects.     

Cross-Cultural and Site-Based Influences on Demographic, Well-being, and Social Network Predictors of Risk Perception in Hazard and Disaster Settings in Ecuador and Mexico

Although virtually all comparative research about risk perception focuses on which hazards are of concern to people in different culture groups, much can be gained by focusing on predictors of levels of risk perception in various countries and places. In this case, we examine standard and novel predictors of risk perception in seven sites among communities affected by a flood in Mexico (one site) and volcanic eruptions in Mexico (one site) and Ecuador (five sites). We conducted more than 450 interviews with questions about how people feel at the time (after the disaster) regarding what happened in the past, their current concerns, and their expectations for the future. We explore how aspects of the context in which people live have an effect on how strongly people perceive natural hazards in relationship with demographic, well-being, and social network factors. Generally, our research indicates that levels of risk perception for past, present, and future aspects of a specific hazard are similar across these two countries and seven sites. However, these contexts produced different predictors of risk perception—in other words, there was little overlap between sites in the variables that predicted the past, present, or future aspects of risk perception in each site. Generally, current stress was related to perception of past danger of an event in the Mexican sites, but not in Ecuador; network variables were mainly important for perception of past danger (rather than future or present danger), although specific network correlates varied from site to site across the countries.     

Endothelium-Dependent Relaxation and Angiotensin II Sensitivity in Experimental Preeclampsia

Objective

We investigated endothelial dysfunction and the role of angiotensin (Ang)-II type I (AT1-R) and type II (AT2-R) receptor in the changes in the Ang-II sensitivity in experimental preeclampsia in the rat.

Methods

Aortic rings were isolated from low dose lipopolysaccharide (LPS) infused pregnant rats (experimental preeclampsia; n=9), saline-infused pregnant rats (n=8), and saline (n=8) and LPS (n=8) infused non-pregnant rats. Endothelium-dependent acetylcholine--mediated relaxation was studied in phenylephrine-preconstricted aortic rings in the presence of vehicle, N^G-nitro-L-arginine methyl ester and/or indomethacin. To evaluate the role for AT1-R and AT2-R in Ang-II sensitivity, full concentration response curves were obtained for Ang-II in the presence of losartan or PD123319. mRNA expression of the AT1-R and AT2-R, eNOS and iNOS, COX1 and COX2 in aorta were evaluated using real-time RT-PCR.

Results

The role of vasodilator prostaglandins in the aorta was increased and the role of endothelium-derived hyperpolarizing factor and response of the AT1-R and AT2-R to Ang-II was decreased in pregnant saline infused rats as compared with non-pregnant rats. These changes were not observed during preeclampsia.

Conclusion

Pregnancy induced adaptations in endothelial function, which were not observed in the rat model for preeclampsia. This role of lack of pregnancy induced endothelial adaptation in the pathophysiology of experimental preeclampsia needs further investigation.     

SNP array analysis in constitutional and cancer genome diagnostics--copy number variants, genotyping and quality control

Array-based comparative genomic hybridization analysis of genomic DNA was first applied in postnatal diagnosis for patients with intellectual disability (ID) and/or congenital anomalies (CA). Genome-wide single-nucleotide polymorphism (SNP) array analysis was subsequently implemented as the first line diagnostic test for ID/CA patients in our laboratory in 2009, because its diagnostic yield is significantly higher than that of routine cytogenetic analysis. In addition to the detection of copy number variations, the genotype information obtained with SNP array analysis enables the detection of stretches of homozygosity and thereby the possible identification of recessive disease genes, mosaic aneuploidy, or uniparental disomy. Patient-parent (trio) information analysis is used to screen for the presence of any form of uniparental disomy in the patient and can determine the parental origin of a de novo copy number variation. Moreover, the outcome of a genotype analysis is used as a final quality control by ruling out potential sample mismatches due to non-paternity or sample mix-up. SNP array analysis is now also used in our laboratory for patients with disorders for which locus heterogeneity is known (homozygosity pre-screening), in prenatal diagnosis in case of structural ultrasound anomalies, and for patients with leukemia. In this report, we summarize our array findings and experiences in the various diagnostic applications and demonstrate the power of a SNP-based array platform for molecular karyotyping, because it not only significantly improves the diagnostic yield in both constitutional and cancer genome diagnostics, but it also enhances the quality of the diagnostic laboratory workflow.     

Inducible costimulator regulates Th2-mediated inflammation, but not Th2 differentiation, in a model of allergic airway disease

A novel costimulatory molecule expressed on activated T cells, inducible costimulator (ICOS), and its ligand, B7-related protein-1 (B7RP-1), were recently identified. ICOS costimulation leads to the induction of Th2 cytokines without augmentation of IL-2 production, suggesting a role for ICOS in Th2 cell differentiation and expansion. In the present study, a soluble form of murine ICOS, ICOS-Ig, was used to block ICOS/B7RP-1 interactions in a Th2 model of allergic airway disease. In this model, mice are sensitized with inactivated Schistosoma mansoni eggs and are subsequently challenged with soluble S. mansoni egg Ag directly in the airways. Treatment of C57BL/6 mice with ICOS-Ig during sensitization and challenge attenuated airway inflammation, as demonstrated by a decrease in cellular infiltration into the lung tissue and airways, as well as by a decrease in local IL-5 production. These inhibitory effects were not due to a lack of T cell priming nor to a defect in Th2 differentiation. In addition, blockade of ICOS/B7RP-1 interactions during ex vivo restimulation of lung Th2 effector cells prevented cytokine production. Thus, blockade of ICOS signaling can significantly reduce airway inflammation without affecting Th2 differentiation in this model of allergic airway disease.     

RSCA genotyping of MHC for high-throughput evolutionary studies in the model organism three-spined stickleback Gasterosteus aculeatus

Background  

In all jawed vertebrates, highly polymorphic genes of the major histocompatibility complex (MHC) encode antigen presenting molecules that play a key role in the adaptive immune response. Their polymorphism is composed of multiple copies of recently duplicated genes, each possessing many alleles within populations, as well as high nucleotide divergence between alleles of the same species. Experimental evidence is accumulating that MHC polymorphism is a result of balancing selection by parasites and pathogens. In order to describe MHC diversity and analyse the underlying mechanisms that maintain it, a reliable genotyping technique is required that is suitable for such highly variable genes.     

The Impact of Lactobacillus plantarum WCFS1 Teichoic Acid D-Alanylation on the Generation of Effector and Regulatory T-cells in Healthy Mice

To date it remains unclear how probiotics affect the immune system. Bacterial envelope components may play an essential role, as these are the first to establish bacterial-host cell interactions. Teichoic acids (TAs), and especially lipoteichoic acids, are the most pro-inflammatory components of the gram-positive bacterial envelope. This effect is dependent on D-alanyl substitution of the TA backbone and interactions with TLR2 on host cells. Although the pro-inflammatory properties of TAs have been established in vitro, it remains unclear how TAs affect immunomodulation in vivo. In this study, we investigated the role of TA D-alanylation on L. plantarum–induced intestinal and systemic immunomodulation in vivo. For this, we compared the effect of L. plantarum WCFS1 and its TA D-Alanylation negative derivative (dltX-D) on the distribution of dendritic cell and T cell populations and responses in healthy mice. We demonstrated that the majority of the L. plantarum-induced in vivo immunomodulatory effects were dependent on D-alanylation (D-Ala), as some L. plantarum WCFS1-induced immune changes were not observed in the dltX-D-treated group and some were only observed after treatment with dltX-D. Strikingly, not only pro-inflammatory immune responses were abolished in the absence of D-Ala substitution, but also anti-inflammatory responses, such as the L. plantarum-induced generation of regulatory T cells in the spleen. With this study we provide insight in host-microbe interactions, by demonstrating the involvement of D-alanylation of TAs on the bacterial membrane in intestinal and systemic immunomodulation in healthy mice.     

Platelet phosphoinositide turnover in streptozotocin-induced diabetes

Increased platelet aggregation and secretion in response to various agonists has been described in both diabetic humans and animals. Alterations in the platelet membrane fatty acid composition of phospholipids and changes in the prostacyclin and thromboxane formation could only partly explain the altered platelet function in diabetes. In the present study, we have examined the role of phosphoinositide turnover in the diabetic platelet function. We report alterations in 2-[³H] myo-inositol uptake, phosphoinositide turnover, inositol phosphate and diacylglycerol (DAG) formation, phosphoinositide mass, and phospholipase C activity in platelets obtained from streptozotocin (STZ)-induced diabetic rats. There was a significant increase in the 2-[³H) myo-inositol uptake in washed platelets from diabetic rats. Basal incorporation of 2-[³H] myo-inositol into phosphatidylinositol 4,5-bisphosphate (PIP₂), phosphatidylinositol 4-phosphate (PIP) or phosphatidylinositol (PI) in platelets obtained from diabetic rats was, however, not affected. Thrombin stimulation of platelets from diabetic rats induced an increase in the hydrolysis of [³²P]PIP₂ but indicated no change in the hydrolysis of [³²P]PIP and [³²P]PI as compared to their basal levels. Thrombin-induced formation of [³H]inositol phosphates was significantly increased in both diabetic as well as in control platelets as compared to their basal levels. This formation of [³H]inositol phosphates in diabetic platelets was greater than controls at all time intervals studied. Similarly, there was an increase in the release of DAG after thrombin stimulation in the diabetic platelets. Based on these results, we conclude that there is an increase in the transport of myoinositol across the diabetic platelet membrane and this feature, along with alterations in the hydrolysis of PIP₂, inositol phosphates and DAG in the diabetic platelets, may play a role in increased phosphoinositide turnover which could explain the altered platelet function in STZ-induced diabetes.     

Virtual nanoscopy: Generation of ultra-large high resolution electron microscopy maps

A key obstacle in uncovering the orchestration between molecular and cellular events is the vastly different length scales on which they occur. We describe here a methodology for ultrastructurally mapping regions of cells and tissue as large as 1 mm(2) at nanometer resolution. Our approach employs standard transmission electron microscopy, rapid automated data collection, and stitching to create large virtual slides. It greatly facilitates correlative light-electron microscopy studies to relate structure and function and provides a genuine representation of ultrastructural events. The method is scalable as illustrated by slides up to 281 gigapixels in size. Here, we applied virtual nanoscopy in a correlative light-electron microscopy study to address the role of the endothelial glycocalyx in protein leakage over the glomerular filtration barrier, in an immunogold labeling study of internalization of oncolytic reovirus in human dendritic cells, in a cryo-electron microscopy study of intact vitrified mouse embryonic cells, and in an ultrastructural mapping of a complete zebrafish embryo slice.