Direct binding of peptides to MHC class I molecules on living cells. Analysis at the single cell level.

To directly assess the binding of exogenous peptides to cell surface-associated MHC class I molecules at the single cell level, we examined the possibility of combining the use of biotinylated peptide derivatives with an immunofluorescence detection system based on flow cytometry. Various biotinylated derivatives of the adenovirus 5 early region 1A peptide 234-243, an antigenic peptide recognized by CTL in the context of H-2Db, were first screened in functional assays for their ability to bind efficiently to Db molecules on living cells. Suitable peptide derivatives were then tested for their ability to generate positive fluorescence signals upon addition of phycoerythrin-labeled streptavidin to peptide derivative-bearing cells. Strong fluorescent staining of Db-expressing cells was achieved after incubation with a peptide derivative containing a biotin group at the C-terminus. Competition experiments using the unmodified parental peptide as well as unrelated peptides known to bind to Kd, Kb, or Db, respectively, established that binding of the biotinylated peptide to living cells was Db-specific. By using Con A blasts derived from different H-2 congenic mouse strains, it could be shown that the biotinylated peptide bound only to Db among > 20 class I alleles tested. Moreover, binding of the biotinylated peptide to cells expressing the Dbm13 and Dbm14 mutant molecules was drastically reduced compared to Db. Binding of the biotinylated peptide to freshly isolated Db+ cells was readily detectable, allowing direct assessment of the relative amount of peptide bound to distinct lymphocyte subpopulations by three-color flow cytometry. While minor differences between peripheral T and B cells could be documented, thymocytes were found to differ widely in their peptide binding activity. In all cases, these differences correlated positively with the differential expression of Db at the cell surface. Finally, kinetic studies at different temperatures strongly suggested that the biotinylated peptide first associated with Db molecules available constitutively at the cell surface and then with newly arrived Db molecules.

Setting boundaries: brain dynamics of modal and amodal illusory shape completion in humans.

Normal visual perception requires differentiating foreground from background objects. Differences in physical attributes sometimes determine this relationship. Often such differences must instead be inferred, as when two objects or their parts have the same luminance. Modal completion refers to such perceptual "filling-in" of object borders that are accompanied by concurrent brightness enhancement, in turn termed illusory contours (ICs). Amodal completion is filling-in without concurrent brightness enhancement. Presently there are controversies regarding whether both completion processes use a common neural mechanism and whether perceptual filling-in is a bottom-up, feedforward process initiating at the lowest levels of the cortical visual pathway or commences at higher-tier regions. We previously examined modal completion (Murray et al., 2002) and provided evidence that the earliest modal IC sensitivity occurs within higher-tier object recognition areas of the lateral occipital complex (LOC). We further proposed that previous observations of IC sensitivity in lower-tier regions likely reflect feedback modulation from the LOC. The present study tested these proposals, examining the commonality between modal and amodal completion mechanisms with high-density electrical mapping, spatiotemporal topographic analyses, and the local autoregressive average distributed linear inverse source estimation. A common initial mechanism for both types of completion processes (140 msec) that manifested as a modulation in response strength within higher-tier visual areas, including the LOC and parietal structures, is demonstrated, whereas differential mechanisms were evident only at a subsequent time period (240 msec), with amodal completion relying on continued strong responses in these structures.

Differential peptide binding to CD40 evokes counteractive responses.

The antigen-presenting cell-expressed CD40 is implied in the regulation of counteractive immune responses such as induction of pro-inflammatory and anti-inflammatory cytokines interleukin (IL)-12 and IL-10, respectively. The mechanism of this duality in CD40 function remains unknown. Here, we investigated whether such duality depends on ligand binding. Based on CD40 binding, we identifed two dodecameric peptides, peptide-7 and peptide-19, from the phage peptide library. Peptide-7 induces IL-10 and increases Leishmania donovani infection in macrophages, whereas peptide-19 induces IL-12 and reduces L. donovani infection. CD40-peptide interaction analyses by surface plasmon resonance and atomic force microscopy suggest that the functional differences are not associated with the studied interaction parameters. The molecular dynamic simulation of the CD40-peptides interaction suggests that these two peptides bind to two different places on CD40. Thus, we suggest for the first time that differential binding of the ligands imparts functional duality to CD40.

Differential subcellular localization of phosphorylated neurofilament and tau proteins in degenerating neurons of the human entorhinal cortex.

A panel of novel monoclonal antibodies was tested on the human entorhinal cortex for the recognition of age- and disease-related changes of neurofilament proteins (NF). Several antibodies identified phosphorylated NF-H subunit, which occurred preferentially in those aged between 60 and 80 years and were localized in degenerating neurons. Such neurons also contained neurofibrillary tangles, but neurofilament aggregates did not co-localize with tangles, nor did the quantity nor the number of NF-positive neurons correlate with the severity of Alzheimer's disease. This points to a susceptibility of NF in a subset of neurons for phosphorylation- and metabolically related morphological changes during neurodegeneration.

Cured by tonsillectomy: was it really a PFAPA syndrome?

We show how an ultrafast pump-pump excitation induces strong fluorescence depletion in biological samples, such as bacteria-containing droplets, in contrast with fluorescent interferents, such as polycyclic aromatic compounds, despite similar spectroscopic properties. Application to the optical remote discrimination of biotic versus non-biotic particles is proposed. Further improvement is required to allow the discrimination of one pathogenic among other non-pathogenic micro-organisms. This improved selectivity may be reached with optimal coherent control experiments, as discussed in the paper.

Aging and motor programming: differential effects according to the selection of action

There are controversial reports about the effect of aging on movement preparation, and it is unclear to which extent cognitive and/or motor related cerebral processes may be affected. This study examines the age effects on electro-cortical oscillatory patterns during various motor programming tasks, in order to assess potential differences according to the mode of action selection. Twenty elderly (EP, 60-84 years) and 20 young (YP, 20-29 years) participants with normal cognition underwent 3 pre-cued response tasks (S1-S2 paradigm). S1 carried either complete information on response side (Full; stimulus-driven motor preparation), no information (None; general motor alertness), or required free response side selection (Free; internally-driven motor preparation). Electroencephalogram (EEG) was recorded using 64 surface electrodes. Alpha (8-12 Hz) desynchronization (ERD)/synchronization (ERS) and motor-related amplitude asymmetries (MRAA) were analyzed during the S1-S2 interval. Reaction times (RTs) to S2 were slower in EP than YP, and in None than in the other 2 tasks. There was an Age x Task interaction due to increased RTs in Free compared to Full in EP only. Central bilateral and midline activation (alpha ERD) was smaller in EP than YP in None. In Full just before S2, readiness to move was reflected by posterior midline inhibition (alpha ERS) in both groups. In Free, such inhibition was present only in YP. Moreover, MRAA showed motor activity lateralization in both groups in Full, but only in YP in Free. The results indicate reduced recruitment of motor regions for motor alertness in the elderly. They further show less efficient cerebral processes subtending free selection of movement in elders, suggesting reduced capacity for internally-driven action with age.

Differential contribution of mitochondria, NADPH oxidases, and glycolysis to region-specific oxidant stress in the anoxic-reoxygenated embryonic heart.

The ability of the developing myocardium to tolerate oxidative stress during early gestation is an important issue with regard to possible detrimental consequences for the fetus. In the embryonic heart, antioxidant defences are low, whereas glycolytic flux is high. The pro- and antioxidant mechanisms and their dependency on glucose metabolism remain to be explored. Isolated hearts of 4-day-old chick embryos were exposed to normoxia (30 min), anoxia (30 min), and hyperoxic reoxygenation (60 min). The time course of ROS production in the whole heart and in the atria, ventricle, and outflow tract was established using lucigenin-enhanced chemiluminescence. Cardiac rhythm, conduction, and arrhythmias were determined. The activity of superoxide dismutase, catalase, gutathione reductase, and glutathione peroxidase as well as the content of reduced and oxidized glutathione were measured. The relative contribution of the ROS-generating systems was assessed by inhibition of mitochondrial complexes I and III (rotenone and myxothiazol), NADPH oxidases (diphenylene iodonium and apocynine), and nitric oxide synthases (N-monomethyl-l-arginine and N-iminoethyl-l-ornithine). The effects of glycolysis inhibition (iodoacetate), glucose deprivation, glycogen depletion, and lactate accumulation were also investigated. In untreated hearts, ROS production peaked at 10.8 ± 3.3, 9 ± 0.8, and 4.8 ± 0.4 min (means ± SD; n = 4) of reoxygenation in the atria, ventricle, and outflow tract, respectively, and was associated with arrhythmias. Functional recovery was complete after 30-40 min. At reoxygenation, 1) the respiratory chain and NADPH oxidases were the main sources of ROS in the atria and outflow tract, respectively; 2) glucose deprivation decreased, whereas glycogen depletion increased, oxidative stress; 3) lactate worsened oxidant stress via NADPH oxidase activation; 4) glycolysis blockade enhanced ROS production; 5) no nitrosative stress was detectable; and 6) the glutathione redox cycle appeared to be a major antioxidant system. Thus, the glycolytic pathway plays a predominant role in reoxygenation-induced oxidative stress during early cardiogenesis. The relative contribution of mitochondria and extramitochondrial systems to ROS generation varies from one region to another and throughout reoxygenation.

Distinct expression pattern of microtubule-associated protein-2 in human oligodendrogliomas and glial precursor cells.

Microtubule-associated protein 2 (MAP2), a protein linked to the neuronal cytoskeleton in the mature central nervous system (CNS), has recently been identified in glial precursors indicating a potential role during glial development. In the present study, we systematically analyzed the expression of MAP2 in a series of 237 human neuroepithelial tumors including paraffin-embedded specimens and tumor tissue microarrays from oligodendrogliomas, mixed gliomas, astrocytomas, glioblastomas, ependymomas, as well as dysembryoplastic neuroepithelial tumors (DNT), and central neurocytomas. In addition, MAP2-immunoreactive precursor cells were studied in the developing human brain. Three monoclonal antibodies generated against MAP2A-B or MAP2A-D isoforms were used. Variable immunoreactivity for MAP2 could be observed in all gliomas with the exception of ependymomas. Oligodendrogliomas exhibited a consistently strong and distinct pattern of expression characterized by perinuclear cytoplasmic staining without significant process labeling. Tumor cells with immunoreactive bi- or multi-polar processes were mostly encountered in astroglial neoplasms, whereas the small cell component in neurocytomas and DNT was not labeled. These features render MAP2 immunoreactivity a helpful diagnostic tool for the distinction of oligodendrogliomas and other neuroepithelial neoplasms. RT-PCR, Western blot analysis, and in situ hybridization confirmed the expression of MAP2A-C (including the novel MAP2+ 13 transcript) in both oligodendrogliomas and astrocytomas. Double fluorescent laser scanning microscopy showed that GFAP and MAP2 labeled different tumor cell populations. In embryonic human brains, MAP2-immunoreactive glial precursor cells were identified within the subventricular or intermediate zones. These precursors exhibit morphology closely resembling the immunolabeled neoplastic cells observed in glial tumors. Our findings demonstrate MAP2 expression in astrocytic and oligodendroglial neoplasms. The distinct pattern of immunoreactivity in oligodendrogliomas may be useful as a diagnostic tool. Since MAP2 expression occurs transiently in migrating immature glial cells, our findings are in line with an assumed origin of diffuse gliomas from glial precursors.

Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that can be activated by various xenobiotics and natural fatty acids. These transcription factors primarily regulate genes involved in lipid metabolism and also play a role in adipocyte differentiation. We present the expression patterns of the PPAR subtypes in the adult rat, determined by in situ hybridization using specific probes for PPAR-alpha, -beta and -gamma, and by immunohistochemistry using a polyclonal antibody that recognizes the three rat PPAR subtypes. In numerous cell types from either ectodermal, mesodermal, or endodermal origin, PPARs are coexpressed, with relative levels varying between them from one cell type to the other. PPAR-alpha is highly expressed in hepatocytes, cardiomyocytes, enterocytes, and the proximal tubule cells of kidney. PPAR-beta is expressed ubiquitously and often at higher levels than PPAR-alpha and -gamma. PPAR-gamma is expressed predominantly in adipose tissue and the immune system. Our results suggest new potential directions to investigate the functions of the different PPAR subtypes.

IL28B polymorphism is significantly correlated with ifn anti-viral effectiveness already on first day of pegylated interferon-alpha and ribavirin therapy of chronic HCV infection

Background and Aims: Recently, single nucleotide polymorphisms (SNPs) in IL28B were shown to correlate with response to pegylated interferon-a (IFN) and ribavirin therapy of chronic HCV infection. However, the cause for the SNPs effect on therapy response and its application for direct anti-viral (DAV) treatment are not clear. Here, we analyze early HCV kinetics as function of IL28B SNPs to determine its specific effect on viral dynamics. Methods: IL28B SNPs rs8099917, rs12979860 and rs12980275 were genotyped in 252 chronically HCV infected Caucasian naïve patients (67% HCV genotype 1, 28% genotype 2-3) receiving peginterferonalfa- 2a (180 mg/qw) plus ribavirin (1000-1200 mg/qd) in the DITTO study. HCV-RNA was measured (LD = 50 IU/ml) frequently during first 28 days. Results: RVR was achieved in 33% of genotype 1 patients with genotype CC at rs12979860 versus 12-16% for genotypes TT and CT (P < 0.03). Significant (P < 0.001) difference in viral decline was observed already at day 1 (see Figure). First phase decline was significantly (P < 0.001) larger in patients with genotype CC (2.0 log) than for TT and CT genotypes (0.6 and 0.8), indicating IFN anti-viral effectiveness in blocking virion production of 99% versus 75-84%. There was no significant association between second phase slope and rs12979860 genotype in patients with a first phase decline larger than 1 log. HCV kinetics as function of IL28b SNP. The same trend (not shown) was observed for HCV genotype 2-3 patients with different SNP genotype distribution that may indicate differential selection pressure as function of HCV genotype. Similar results were observed for SNPs rs8099917 and rs12980275, with a strong linkage disequilibrium among the 3 loci allowing to define the composite haplotype best associated with IFN effectiveness. Conclusions: IFN effectiveness in blocking virion production/ release is strongly affected by IL28B SNPs, but not other viral dynamic properties such as infected cell loss rate. Thus, IFN based therapy, as standard-of-care or in combination with DAV, should consider IL28B SNPs for prediction and personalized treatment, while response to pure DAV treatment may be less affected by IL28B SNPs. Additional analyses are undergoing to pinpoint the SNP effect on IFN anti-viral effectiveness.

Differential distribution of tau proteins in developing cat cerebellum.

The differential distribution and phosphorylation of tau proteins in cat cerebellum was studied with two well characterized antibodies, TAU-1 and TAU-2. TAU-1 detects tau proteins in axons, and the epitope in perikarya and dendrites is masked by phosphorylation. TAU-2 detects a phosphorylation-independent epitope on tau proteins. The molecular composition of tau proteins in the range of 45 kD to 64 kD at birth changed after the first postnatal month to a set of several adult variants of higher molecular weights in the range of 59 kD to 95 kD. The appearance of tau proteins in subsets of axons corresponds to the axonal maturation of cerebellar local-circuit neurons in granular and molecular layers and confirms previous studies. Tau proteins were also identified in synapses by immunofluorescent double-staining with synapsin I, located in the pinceau around the Purkinje cells, and in glomeruli. Dephosphorylation of juvenile cerebellar tissue by alkaline phosphatase indicated indirectly the presence of differentially phosphorylated tau forms mainly in juvenile ages. Additional TAU-1 immunoreactivity was unmasked in numerous perikarya and dendrites of stellate cells, and in cell bodies of granule cells. Purkinje cell bodies were stained transiently at juvenile ages. During postnatal development, the intensity of the phosphate-dependent staining decreased, suggesting that phosphorylation of tau proteins in perikarya and dendrites may be essential for early steps in neuronal morphogenesis during cat cerebellum development.

Differential species-specific ectoparasitic mite intensities in two intimately coexisting sibling bat species: resource-mediated host attractiveness or parasite specialization?

1. The mechanisms underlying host choice strategies by parasites remain poorly understood. We address two main questions: (i) do parasites prefer vulnerable or well-fed hosts, and (ii) to what extent is a parasite species specialized towards a given host species? 2. To answer these questions, we investigated, both in the field and in the lab, a host-parasite system comprising one ectoparasitic mite (Spinturnix myoti) and its major hosts, two sibling species of bats (Myotis myotis and M blythii), which coexist intimately in colonial nursery roosts. We exploited the close physical associations between host species in colonial roosts as well as naturally occurring annual variation in food abundance to investigate the relationships between parasite intensities and (i) host species and (ii) individual nutritional status. 3. Although horizontal transmission of parasites was facilitated by the intimate aggregation of bats within their colonial clusters, we found significant interspecific differences in degree of infestation throughout the 6 years of the study, with M. myotis always more heavily parasitized than M. blythii. This pattern was replicated in a laboratory experiment in which any species-specific resistance induced by exploitation of different trophic niches in nature was removed. 4. Within both host species, S. myoti showed a clear preference for individuals with higher nutritional status. In years with high resource abundance, both bat hosts harboured more parasites than in low-resource years, although the relative difference in parasite burden across species was maintained. This pattern of host choice was also replicated in the laboratory. When offered a choice, parasites always colonized better-fed individuals. 5. These results show first that host specialization in our study system occurred. Second, immediate parasite choice clearly operated towards the selection of hosts in good nutritional state.

Differential allocation and deployment of direct and indirect defences by Vicia sepium along elevation gradients

Dissecting drivers of plant defence investment remains central for understanding the assemblage of communities across different habitats. There is increasing evidence that direct defence strategies against herbivores, including secondary metabolites production, differ along ecological gradients in response to variation in biotic and abiotic conditions. In contrast, intraspecific variation in indirect defences remains unexplored. Here, we investigated variation in herbivory rate, resistance to herbivores, and indirect defences in ant-attracting Vicia species along the elevation gradient of the Alps. Specifically, we compared volatile organic compounds (VOCs) and ant attraction in high and low elevation ecotypes. Consistent with adaptation to the lower herbivory conditions that we detected at higher elevations in the field, high elevation plants were visited by fewer ants and were more susceptible to herbivore attack. In parallel, constitutive volatile organic compound production and subsequent ant attraction were lower in the high elevation ecotypes. We observed an elevation-driven trade-off between constitutive and inducible production of VOCs and ant attraction along the environmental cline. At higher elevations, inducible defences increased, while constitutive defence decreased, suggesting that the high elevation ecotypes compensate for lower indirect constitutive defences only after herbivore attack. Synthesis. Overall, direct and indirect defences of plants vary along elevation gradients. Our findings show that plant allocation to defences are subject to trade-offs depending on local conditions, and point to a feedback mechanism linking local herbivore pressure, predator abundance and the defence investment of plants.