Autoimmunity and inflammation due to a gain-of-function mutation in phospholipase C gamma 2 that specifically increases external Ca2+ entry

The identification of specific genetic loci that contribute to inflammatory and autoimmune diseases has proved difficult due to the contribution of multiple interacting genes, the inherent genetic heterogeneity present in human populations, and a lack of new mouse mutants. By using N-ethyl-N-nitrosourea (ENU) mutagenesis to discover new immune regulators, we identified a point mutation in the murine phospholipase Cg2 (Plcg2) gene that leads to severe spontaneous inflammation and autoimmunity. The disease is composed of an autoimmune component mediated by autoantibody immune complexes and B and T cell independent inflammation. The underlying mechanism is a gain-of-function mutation in Plcg2, which leads to hyperreactive external calcium entry in B cells and expansion of innate inflammatory cells. This mutant identifies Plcg2 as a key regulator in an autoimmune and inflammatory disease mediated by B cells and non-B, non-T haematopoietic cells and emphasizes that by distinct genetic modulation, a single point mutation can lead to a complex immunological phenotype.     

Gains of 13q are correlated with a poor prognosis in liposarcoma.

Liposarcomas are a phenotypical heterogeneous group of tumors divided into four main subtypes: well-differentiated, dedifferentiated, myxoid/round cell, and pleomorphic. The aim of this study was to compare DNA sequence copy number changes of these subtypes as investigated by comparative genomic hybridization in 36 patients. Comparative genomic hybridization revealed genomic imbalances in tumors of 27 patients (mean 5.6 imbalances per tumor). The most frequent gains were within single regions of 1q, 12q, and 13q. We found a significant correlation of poor overall survival and gain of 13q21 (P=0.0221), 13q22 (P=0.0341), 13q31 (P=0.0410), and 13q32 (P=0.0074). The univariate Cox regression analysis revealed an increased risk of tumor-related death for patients whose liposarcomas possess with gains of 13q21 and 13q32 simultaneously (P=0.010; RR=7.1; 95% CI 1.6-31.7). Furthermore, 12 high-level amplifications were found in tumors of seven patients. In four cases 12q14-q15 and in two cases 13q32-q33 were amplified. We identified in different liposarcoma subtypes characteristic genomic changes: Gains and high-level amplifications of 12q occurred in all 11 investigated well-differentiated liposarcomas, and these changes were often present simultaneously with gains of 1q (mean 5.5 changes). In the two dedifferentiated liposarcomas, gains of 1q in both liposarcomas, and a high-level amplification of 13q were striking. Only eight of the 17 patients with myxoid/round cell liposarcomas showed changes in DNA copy number (mean 3.4 imbalances). In four of these eight cases gains of 13q occurred. The six pleomorphic liposarcomas possessed the most frequent genomic imbalances (mean number 16.3) of all liposarcoma subtypes investigated. These imbalances were in almost all chromosomal regions detected predominantly as over-representations of chromosomes 1, 5p, 13q, and 22q. Summarizing, all subtypes but well-differentiated liposarcomas showed gains of 13q, which were associated with a poor prognosis.     

Chorioallantoic membrane angiogenesis model for tissue engineering: a new twist on a classic model

Tissue-engineering (TE) applications include the isolation, culture, and seeding of cells into a suitable matrix or scaffold before in vivo transplantation. After transplantation, vascularization of the scaffold is a principal limiting factor for cell viability for the first 6-8 days posttransplantation. A model for systematic analysis of this process has been developed. Fertilized White Leghorn eggs were incubated (at 37.8 degrees C in 60% relative humidity) and opened on day 3 of incubation. Preadipocyte-seeded fibrin constructs were implanted in a specially designed plastic cylinder and placed through the opening on the surface of the chorioallantoic membrane (CAM) on day 8 of incubation. Vascularization of the constructs by chorioallantoic blood vessels was assessed for up to 8 days posttransplantation. The survival rate for embryos receiving transplanted constructs was about 90%. Histology confirmed transplant cell viability at day 4 posttransplantation and vascularization of the constructs by avian endothelial cells began at this time. A new in vivo model to study the effect of angiogenesis in TE constructs, including assessments of viability, proliferation, and differentiation of transplanted cells and biomaterial properties, is presented. Advantages include easy access to the vascular network of the CAM, lack of immunocompetence, low costs, and avoidance of animal experiments.     

First external quality assurance of antibody diagnostic for SARS-new coronavirus.

To confirm an infection with the new coronavirus (SARS-CoV) causing the severe acute respiratory syndrome (SARS) diagnostic assays for detection of SARS-CoV specific antibody are necessary. To evaluate the diagnostic performance of laboratories an external quality assurance (EQA) study was performed in 2004. Participating laboratories (9/20) correctly detected anti-SARS antibodies in serum samples without false positive results in an immunofluorescence assay. In contrast, only 4/13 laboratories detected most of the anti-SARS antibody positive samples without false positive results using enzyme immunoassays (EIA) and/or immunoblot. The overall results clearly demonstrate that serological diagnosis of SARS-CoV remains at an early stage of development, with further technical improvements required, particularly with respect to the use of SARS specific EIAs.     

Modulation of associative human motor cortical plasticity by attention

The role of attention in generating motor memories remains controversial principally because it is difficult to separate the effects of attention from changes in kinematics of motor performance. We attempted to disentangle attention from performance effects by varying attention while plasticity was induced in human primary motor cortex by external stimulation in the absence of voluntary movement. A paired associative stimulation (PAS) protocol was employed consisting of repetitive application of single afferent electric stimuli, delivered to the right median nerve, paired with single-pulse transcranial magnetic stimulation (TMS) over the optimal site for activation of the right abductor pollicis brevis muscle (APB) to generate near-synchronous events in the left primary motor cortex. In experiment 1, the spatial location of attention was varied. PAS failed to induce plasticity when the subject's attention was directed to their left hand, away from the right target hand the cortical representation of which was being stimulated by PAS. In experiment 2, the grade of attention to the target hand was manipulated. PAS-induced plasticity was maximal when the subject viewed their target hand, and its magnitude was slightly reduced when the subject could only feel their hand. Conversely, plasticity was completely blocked when the subject's attention was diverted from the target hand by a competing cognitive task. A similar modulation by attention was observed for PAS-induced changes in the duration of the silent period evoked by TMS in voluntarily contracted muscle. Associative plasticity in the human motor cortex depends decisively on attention.     

The N-linked glycan g15 within the V3 loop of the HIV-1 external glycoprotein gp120 affects coreceptor usage,cellular tropism,and neutralization

We have studied infectivity and neutralization of X4, R5, and R5X4 tropic HIV-1 mutants, which are lacking N-linked glycosylation sites for glycans g13, g14, g15, and g17 in the V3 loop region of gp120. X4-tropic NL4-3 mutants lacking combinations of g14/15 or g15/17 showed markedly higher infectivity in CXCR4-specific infection. The role of g15 in CCR5-specific infection was investigated using viruses with high (NL-918, R5-monotropic), medium (NL-991, R5-monotropic), and low (NL-952, R5X4-dualtropic) CCR5-specific infectivity. For NL-991, a reduction of infectivity on GHOST-CCR5 cells was observed for a mutant lacking g15. For NL-952 mutants all lacking g15, a complete loss of CCR5-specificity was observed and NL-952 was shifted from R5X4 to X4 tropism. For all mutants of NL4-3, NL-991, and NL-952, where the lack of g15 markedly influenced infectivity or coreceptor usage, neutralization was enhanced. In contrast, NL-918 mutants with or without g15 showed no difference in neutralization and no difference in GHOST-CCR5 infection rates. Thus, for viruses with a low or medium CCR5-specificity the role of g15 for changing CCR5-usage and sensitivity to neutralization was more significant than for viruses with high infection rates on GHOST-CCR5 cells. Our data demonstrate that V3 glycans play an important role in the usage of CXCR4 and CCR5. The lack of g15 was relevant for a more efficient use of CXCR4, whereas interaction with CCR5 was facilitated in the presence of g15. This study also demonstrates that glycan g15 is involved in blocking of neutralizing antibodies and shifting HIV tropism from R5X4 to X4.     

Comparison of three methods to measure absolute cerebral hemoglobin concentration in neonates by near-infrared spectrophotometry

Three methods by which to determine absolute total cerebral hemoglobin concentration (tHb in micromol/L) by near-infrared spectrophotometry (NIRS) have evolved: (1) tHbo, requiring oxygenation changes and arterial oxygen saturation measurements as a reference using a relative NIRS algorithm, (2) tHbg, using a geometrical multidistance principle and (3) tHbgo, a combination of both. The aim of this study was to compare the three methods quantitatively. Sixteen clinically stable preterm infants with a mean gestational age of 29.6 (range of 25.1-36.4) weeks, birthweight of 1386 (680-2820) g and a postnatal age of 2.5 (0.5-6) days, who needed supplemental oxygen, were enrolled. The mean+/-standard deviation tHbg was 150.2+/-41.8 micromol/L (range of 61.6-228.9 micromol/L), the tHbo was 62.1+/-27.2 micromol/L (26.0-110.8 micromol/L) and the tHbgo was 89.3+/-45.6 micromol/L (26.5-195.9 micromol/L). The correlation coefficient among the three methods were tHbg and tHbgo r=0.736; tHbo and tHbgo r=0.938; tHbg and tHbo r=0.598. A multiple regression with variable selection by Mellow's C(p) showed, that tHbg was correlated to the birthweight, the postnatal age, the heart rate and the pCO2 (r(2)=0.588), tHbo and tHbgo were associated with the hemoglobin concentration in the blood, the mean arterial blood pressure and the pCO2 (r(2)=0.493 and 0.406, respectively). The three methods (tHbg, tHbo, and tHbgo) give systematically different tHb readings and large intersubject variability.     

Cytoskeletal modulation of the response to mechanical stimulation in human vascular endothelial cells

Possible interactions of cytoskeletal elements with mechanically induced membrane currents and Ca²⁺ signals were studied in human endothelial cells by using a combined patch-clamp and Fura II technique. For mechanical stimulation, cells were exposed to hypotonic solution (HTS). The concomitant cell swelling activates a Cl^– current, releases Ca²⁺ from intracellular stores and activates Ca²⁺ influx. To interfere with the cytoskeleton, cells were loaded either with the F-actin-stabilizing agent phalloidin (10 mol/l), or the F-actin-depolymerizing substance cytochalasin B (50 mol/l). These were administered either in the bath or the pipette solutions. The tubulin structure of the endothelial cells was modulated by taxol (50 mol/l), which supports polymerization of tubulin, or by the depolymerizing agent colcemid (10 mol/l) both applied to the bath. Immunofluorescence experiments show that under the chosen experimental conditions the cytoskeletal modifiers employed disintegrate the F-actin and microtubuli cytoskeleton. Neither of these cytoskeletal modifiers influenced the HTS-induced Cl^– current. Ca²⁺ release was not affected by cytochalasin B, taxol or colcemid, but was suppressed if the cells were loaded with phalloidin. Depletion of intracellular Ca²⁺ stores by thapsigargin renders the intracellular [Ca²⁺] sensitive to the extracellular [Ca²⁺], which is indicative of a Ca²⁺ entry pathway activated by store depletion. Neither cytochalasin B nor phalloidin affected this Ca²⁺ entry. We conclude that F-actin turnover or depolymerization is necessary for Ca²⁺ release by mechanical activation. The tubulin network is not involved. The Ca²⁺ release-activated Ca²⁺ entry is not modulated by the F-actin cytoskeleton.     

Peptide analysis,stability studies,and structural modeling explain contradictory peptide motifs and unique properties of the NOD mouse MHC class II molecule H2-A(g7)

The MHC class II molecule H2-A(g7) is the chief genetic determinant in insulin-dependent diabetes mellitus of the non-obese diabetic (NOD) mice. Poor peptide binding ability, as well as presentation of a unique subset of peptides by this molecule was suggested to promote autoimmunity in this strain. However, several laboratories have presented results in favor of an H2-A(g7) molecule that can avidly bind many different peptides. The crystal structures of H2-A(g7) in complex with two different peptides did not completely resolve this issue. To analyze the peptide binding capacity and the motif requirements of H2-A(g7), we eluted natural ligands from purified H2-A(g7) molecules isolated from the H2-A(g7)-transfected M12-C3 cells. A low peptide yield dominated by a few peptide ligands was found. Pool sequencing and alignment of individual ligands on the basis of molecular modeling revealed a peptide-binding motif with basic/aliphatic/small hydrophilic amino acids at relative position 1 (p1), aliphatic amino acids at p4, Ala at p6, and acidic amino acids and Ser/Gly at p9, as well as acidic residues at p10/11. Though weak, the binding of individual ligands, as well as the importance of an acidic C-terminal residue was confirmed by peptide binding studies to isolated H2-A(g7) molecules. Furthermore, the H2-A(g7) molecule incompletely dissociated into its constituent chains in SDS-electrophoresis under nonreducing conditions. This provides additional evidence of its weak affinity for peptides, which probably arises from the combination of beta56His/beta57Ser/beta78Ala and other unique H2-A(g7) residues in contact with the antigenic peptide. These results allow a better understanding of the role of this molecule in the development of autoimmunity and the identification of epitopes relevant to diabetes.