Hematopoietic progenitor cells and cellular microenvironment: behavioral and molecular changes upon interaction

Cell-cell contact between stem cells and cellular determinants of the microenvironment plays an essential role in controlling cell division. Using human hematopoietic progenitor cells (CD34+/CD38-) and a stroma cell line (AFT024) as a model, we have studied the initial behavioral and molecular sequel of this interaction. Time-lapse microscopy showed that CD34+/CD38- cells actively migrated toward and sought contact with stroma cells and 30% of them adhered firmly to AFT024 stroma through the uropod. CD44 and CD34 are colocalized at the site of contact. Gene expression profiles of CD34+/CD38- cells upon cultivation with or without stroma for 16, 20, 48, or 72 hours were analyzed using our human genome cDNA microarray. Chk1, egr1, and cxcl2 were among the first genes upregulated within 16 hours. Genes with the highest upregulation throughout the time course included tubulin genes, ezrin, c1qr1, fos, pcna, mcm6, ung, and dnmt1, genes that play an essential role in reorganization of the cytoskeleton system, stabilization of DNA, and methylation patterns. Our results demonstrate directed migration of CD34+/CD38- cells toward AFT024 and adhesion through the uropod and that upon interaction with supportive stroma, reorganization of the cytoskeleton system, regulation of cell division, and maintenance of genetic stability represent the most essential steps.     

Location of immunodominant antigenic determinants on fragments of the tick-borne encephalitis virus glycoprotein: Evidence for two different mechanisms by which antibodies mediate neutralization and hemagglutination inhibition

A model showing the topological distribution, functions, and serological specifities of eight distinct, monoclonal antibody-defined epitopes on the tick-borne encephalitis (TBE) virus glycoprotein has been presented in a previous publication [Heinz et al., 1983]. Virology 126, 525–537.) In the present report the influence of conformational change, chemical modification, and fragmentation on the antigenic reactivity of each epitope has been analyzed by the use of blocking enzyme immunoassays and “Western blotting.” One of the two major antigenic domains (A), composed of three different epitopes, completely lost its antigenicity upon incubation at pH 5.0 or by treatment with guanidine-HCl/urea, SDS, reduction and carboxymethylation, as well as by proteolytic (trypsin, α-chymotrypsin, thermolysin) and chemical (CNBr) fragmentation. The second major antigenic domain (B), however, defined by four distinct monoclonal antibodies, three of which are hemagglutination (HA)-inhibiting, neutralizing, and protective, was shown to be resistant to low pH, guanidine-HCl/urea treatment, and proteolytic cleavage of the native protein. Also, polyclonal immune sera from mice and rabbits contained antibody populations reactive with antigenic determinants which are resistant and others which are sensitive to conformational change and fragmentation. Glycoprotein fragments with molecular weights of about 9000, generated by proteolysis of the native protein, were immunoreactive with neutralizing and protective monoclonal antibodies (defining domain B) as well as with a polyclonal mouse immune serum. Thus, these fragments appear to contain antigenic determinants which are immunodominant on the native protein and play an important role in the induction of a protective immune response against TBE virus. In addition, these results show that antibody binding to antigenic domains which are topologically and structurally completely unrelated may result in neutralization and/or HA inhibition. As the presence of two receptor-binding sites is unlikely, different effector mechanisms may account for the effects of these antibodies. The antigenic reactivity of domain A is sensitive to the same treatments which also inactivate HA activity of TBE virus, whereas domain B is resistant. These treatments include a change of domain A induced by incubation at slightly acidic pH which also results in inactivation of virus infectivity. Antibodies to domain A therefore presumably block viral activities by direct binding at or near the putative receptor-binding site whereas antibodies to domain B may cause loss of biological activities by inducing a conformational change of the receptor-binding site.     

Genome sequence of tick-borne encephalitis virus (Western subtype) and comparative analysis of nonstructural proteins with other flaviviruses

The genome sequence of tick-borne encephalitis (TBE) virus (Western subtype vaccine strain Neudoerfl) was determined. This extends the previously published sequence of the structural proteins to the nonstructural protein region and noncoding sequences at the 5'- and 3'-termini. The amino-termini of the individual proteins were assigned by comparison with other flavivirus sequences. Amino acid homology calculations between TBE virus and mosquito-borne flaviviruses were performed for all nonstructural proteins. An evolutionary tree based on protein NS1 is presented that reveals the molecular basis of relationships among flaviviruses. Tick-borne and mosquito-borne flaviviruses share a common hydrophilicity profile and also other features of their primary sequences, such as the presumably functional Gly-Asp-Asp sequence element within protein NS5. Other characteristics, such as the potential N-glycosylation sites of protein NS1 and a potential proteolytic cleavage site within protein NS4B, are conserved within the mosquito-borne group, but differ in the TBE virus sequence.     

Are Compulsive Checkers Impaired in Memory? A Meta‐Analytic Review

Researchers have hypothesized that compulsive checkers suffer from impairment in explicit memory (e.g., Sher, Frost, & Otto, 1983 ), low confidence in explicit memory (e.g., McNally & Kohlbeck, 1993 ), or both. However, empirical findings have been equivocal, possibly due to variability in effect sizes produced by small samples. Combining data across studies may yield more meaningful conclusions than can be surmised from a narrative review. Following a brief review of the literature on checking and memory, we present meta-analytic results suggesting that checkers are impaired on many types of memory tasks (e.g., verbal free recall, verbal cued recall, and recall of actions) and are less confident in recognition than noncheckers. We discuss implications of these findings, suggestions for future research, and limitations of this analysis.     

C4d]FlowPRA screening--a specific assay for selective detection of complement-activating anti-HLA alloantibodies

On waiting lists, transplant candidates are routinely screened for potentially harmful complement-fixing alloantibodies using complement-dependent cytotoxicity (CDC) panel-reactive antibody (PRA) testing. We have recently developed a novel cell-independent assay for assessment of complement-activating panel reactivity ([C4d]FlowPRA), which is based on selective flow-cytometric detection of alloantibody-triggered C4 complement split product deposition to human leukocyte antigen (HLA)-coated FlowPRA beads. Serum specimens selected from 120 transplant candidates were evaluated by [C4d]FlowPRA (HLA class I vs II) in comparison with FlowPRA IgG alloantibody screening (HLA class I vs II), a method detecting both complement- and noncomplement-activating alloantibodies, and with CDC-PRA on separated T (T-CDC) or B cells (B-CDC, evaluation on platelet-absorbed sera). For each assay, >/=10% PRA reactivity was considered positive. Comparing complement-dependent PRA assays with standard FlowPRA, the specificity calculated for [C4d]FlowPRA (HLA class I: 92%; class II: 100%) was found to be superior to that of CDC testing (T-CDC-PRA: 79%; B-CDC-PRA: 86%). Because noncomplement-activating alloreactivities were not detected for both techniques, low sensitivities were calculated ([C4d]FlowPRA HLA class I: 61%; class II: 31%; T-CDC-PRA: 70%; B-CDC-PRA: 55%). Compared with CDC-PRA, [C4d]FlowPRA gave a high specificity (HLA class I compared with T-CDC: 89%, HLA class II compared with B-CDC: 95%) but, at least in part because of false-positive CDC results, a modest sensitivity (66% and 38%, respectively). For both HLA classes, we found a highly significant association between absolute [C4d]FlowPRA and CDC-PRA levels (p < 0.0001). Our results suggest that detection of C4 split product deposition to FlowPRA beads may represent an attractive HLA-specific and time-effective alternative to CDC-PRA screening.     

Decay-accelerating factor in the cardiomyocytes of normal individuals and patients with myocardial infarction

Summary The presence of decay-accelerating factor (DAF) was clearly demonstrated on the surface of normal cardiomyocytes. In patients who had died of myocardial infarction (MI) cardiomyocytes displayed different appearances: outside the ischaemically damaged region the myocytes showed no significant variations in DAF expression when compared with controls without MI. Within myocardial zones damaged by ischaemia, however, apparently normal myocytes showed large gaps in surface staining of DAF or formed clusters which were entirely devoid of reactivity with anti-DAF antibodies. The number of DAF-deficient myocytes increased with the extent of necrosis and also with the number of days between onset of MI and death. Even though injury to myocytes is to a large extent related to anoxia and to the presence of free oxygen radicals, the complement system also appears to be involved; DAF may have protective functions against complement-mediated injury. We speculate that phospholipase may be involved in the removal of DAF from the cardiomyocyte surface.This work was supported in part by grant no. 3.157.88 from the Swiss National Foundation for Scientific Research and a contribution from Sandoz Ltd. Pharma Division, Basel     

Induction of human airway smooth muscle apoptosis by neutrophils and neutrophil elastase

Neutrophils are an important component of airway inflammation and may interact with human airway smooth muscle cells (HASMC). We investigated the effect of neutrophils and of neutrophil-derived proteases on HASMC survival. When co-incubated with neutrophils (0.1-1 x 10(6) cells/ml), attachment of human ASMC was reduced to 12.3 +/- 4.3% compared with untreated controls after 72 h. HASMC showed nuclear condensation and fragmentation (41.6 +/- 8.1% compared with baseline of 3.1 +/- 0.4%), and the biochemical markers of apoptosis, annexin V binding (9.7 +/- 0.7%; baseline 1.1 +/- 0.3%) and cleaved caspase-3 expression, were observed. The proteolytic activity released by neutrophils was essential for the proapoptotic effect because inhibition of elastase activity by alpha(1)-antitrypsin and MeOSuc-Ala-Ala-Pro-Ala-CMK (MSACK) reduced HASMC apoptosis. Human neutrophil elastase (0.1-3 microg/ml) induced apoptosis of HASMC, as well as other neutrophil serine proteases, cathepsin G, and proteinase 3. Fibronectin degradation products were present in HASMC supernatants exposed to neutrophil-conditioned media and to neutrophil elastase. The local release of proteases from neutrophils present in airway smooth muscle cells may lead to HASMC apoptosis as a result of matrix degradation and loss of cell attachment. This may limit pathologic changes such as ASMC hyperplasia and extracellular matrix deposition seen in airway remodeling.     

Transgenic rat model of Huntington's disease

Huntington's disease (HD) is a late manifesting neurodegenerative disorder in humans caused by an expansion of a CAG trinucleotide repeat of more than 39 units in a gene of unknown function. Several mouse models have been reported which show rapid progression of a phenotype leading to death within 3-5 months (transgenic models) resembling the rare juvenile course of HD (Westphal variant) or which do not present with any symptoms (knock-in mice). Owing to the small size of the brain, mice are not suitable for repetitive in vivo imaging studies. Also, rapid progression of the disease in the transgenic models limits their usefulness for neurotransplantation. We therefore generated a rat model transgenic of HD, which carries a truncated huntingtin cDNA fragment with 51 CAG repeats under control of the native rat huntingtin promoter. This is the first transgenic rat model of a neurodegenerative disorder of the brain. These rats exhibit adult-onset neurological phenotypes with reduced anxiety, cognitive impairments, and slowly progressive motor dysfunction as well as typical histopathological alterations in the form of neuronal nuclear inclusions in the brain. As in HD patients, in vivo imaging demonstrates striatal shrinkage in magnetic resonance images and a reduced brain glucose metabolism in high-resolution fluor-deoxy-glucose positron emission tomography studies. This model allows longitudinal in vivo imaging studies and is therefore ideally suited for the evaluation of novel therapeutic approaches such as neurotransplantation.