Localization and Quantitative Autoradiography of Glutamatergic Ligand Binding Sites in Chick Brain

The anatomical localization of glutamate receptor subtype-selective ligand binding sites was investigated in 1-day-old chick brain using quantitative autoradiography. Under the conditions used, the regional distributions of [3H]glutamate, [3H]AMPA (a selective quisqualate receptor ligand) and [3H]kainate binding sites are manifestly different. [3H]l-glutamate binding is densely localized in the telencephalon, particularly in the neostriatum (2.8 pmol/mg protein). In addition, [3H]l-glutamate labels the thalamus, the nucleus mesencephalicus lateralis pars dorsalis, the superficial layers of the optic tectum and the molecular layer of the cerebellum. [3H]AMPA binding sites are most densely localized in the hippocampus (0.90 pmol/mg protein), with an otherwise relatively uniform distribution of binding within the telencephalon. [3H]AMPA also labels the striatum griseum et fibrosum superficiale of the optic tectum and the molecular layer of the cerebellum. [3H]Kainate binding sites are extremely densely packed in the molecular layer of the cerebellum (10 pmol/mg protein). Other regions of [3H]kainate binding include the hyperstriatum and the thalamus. The binding of the NMDA receptor channel blocker [3H]MK-801 is increased in the presence of 1 mM l-glutamate. [3H]MK-801 binding is generally widespread in the telencephalon but is notably absent from the ectostriatum. No evidence of [3H]MK-801 binding sites was detected in the cerebellum, even in the presence of 1 mM l-glutamate. The relatively high densities and the well-defined localizations of the glutamate receptor subtype binding sites suggest that chick brain provides a useful system for the further study of excitatory amino acid receptors.     

Portal hypertension in Williams syndrome: report of two patients

Williams or Williams-Beuren syndrome (WBS) is a developmental disorder with multisystemic manifestations characterized by distinctive facial features, mental disability with unique cognitive and personality profiles, vascular stenoses, growth retardation, and occasional infantile hypercalcemia, caused by haploinsufficiency for genes deleted in chromosome band 7q11.23. However, with the exception of arterial stenoses caused by haploinsufficiency for the elastin gene (ELN), no specific implication of any other gene in the phenotype has been established. We present two patients with portal hypertension leading to splenomegaly and pancytopenia carrying the common 1.5 Mb WBS deletion. We propose this is an additional severe vascular complication of ELN deficiency and discuss the specific characteristics of the portal venous tract that could explain the impact of ELN deficiency in that venous territory. This complication is potentially lethal and should thus be considered in any patient with WBS and splenomegaly.     

Adoptive immunotherapy of feline immunodeficiency virus with autologous ex vivo-stimulated lymphoid cells modulates virus and T-cell subsets in blood

The potential of immunotherapy with autologous virus-specific T cells to affect the course of feline immunodeficiency virus (FIV) infection was explored in a group of specific-pathogen-free cats infected with FIV a minimum of 10 months earlier. Popliteal lymph node cells were stimulated by cocultivation with UV-inactivated autologous fibroblasts infected with recombinant vaccinia viruses expressing either FIV gag or env gene products, followed by expansion in interleukin-2. One or two infusions of both Gag- and Env-stimulated cells resulted in a slow increase in FIV-specific gamma interferon-secreting T cells in the circulation of cats. In the same animals, viral set points fluctuated widely during the first 2 to 3 weeks after adoptive transfer and then returned to pretreatment levels. The preexisting viral quasispecies was also found to be modulated, whereas no novel viral variants were detected. Circulating CD4(+) counts underwent a dramatic decline early after treatment. CD4/CD8 ratios remained instead essentially unchanged and eventually improved in some animals. In contrast, a single infusion of Gag-stimulated cells alone produced no apparent modulations of infection.     

Mild Wolf-Hirschhorn phenotype and partial GH deficiency in a patient with a 4p terminal deletion

Wolf-Hirschhorn syndrome (WHS) is caused by a variably-sized deletion of chromosome 4 involving band 4p16 whose typical craniofacial features are "Greek warrior helmet appearance" of the nose, microcephaly, and prominent glabella. Almost all patients show mental retardation and pre- and post-natal growth delay. Patient was born at term, after a pregnancy characterized by intra-uterine growth retardation (IUGR). Delivery was uneventful. Developmental delay was evident since the first months of life. At 2 years, he developed generalized tonic-clonic seizures. Because of short stature, low growth velocity and delayed bone age, at 4 years he underwent growth hormone (GH) evaluation. Peak GH after two provocative tests revealed a partial GH deficiency. Clinical observation at 7 years disclosed a distinctive facial appearance, with microcephaly, prominent eyes, and beaked nose. Brain MRI showed left temporal mesial sclerosis. GTG banded karyotype was normal. Because of mental retardation, subtelomeric fluorescence in situ hybridization (FISH) analysis was performed, disclosing a relatively large deletion involving 4p16.2 --> pter (about 4.5 Mb), in the proband, not present in the parents. The smallest deletion detected in a WHS patient thus far includes two candidate genes, WHSC1 and WHSC2. Interestingly, that patient did not show shortness of stature, and that could be due to the haploinsufficiency of other genes localized in the flanking regions. Contribution of GH alterations and possible GH therapy should be further considered in WHS patients.     

A subset of high-grade pulmonary neuroendocrine carcinomas shows up-regulation of matrix metalloproteinase-7 associated with nuclear β-catenin immunoreactivity,independent of EGFR and HER-2 gene amplification or expression

Nuclear translocation of β-catenin has been correlated with epidermal growth factor receptor (EGFR) overexpression/activation in nonsmall cell lung cancer. Less is known on β-catenin transactivation in high-grade pulmonary neuroendocrine tumors and on the status of β-catenin activating EGFR and human epidermal growth factor receptor 2 (HER-2) or β-catenin target genes cyclin D1 and matrix metalloproteinase-7 (MMP-7). β-catenin immunoreactivity was evaluated in 51 large-cell neuroendocrine carcinomas (LCNEC) and 45 small-cell lung carcinomas (SCLC). Nineteen cases were assessed for β-catenin gene exon 3 mutations, expression of MMP-7, and expression/gene amplification of EGFR, HER-2, and cyclin D1. β-catenin was expressed in all 96 high-grade neuroendocrine tumors, the vast majority (94%) showing >50% immunopositive cells. A disarrayed immunoreactivity, however, was commonly encountered consisting in variably altered membrane-associated patterns of staining along with progressive accumulation of cytoplasmic immunoreactivity. In LCNEC, but not in SCLC, the disarrayed patterns correlated with EGFR and HER-2 protein expression. β-catenin nuclear accumulation was found in nine tumors, including seven LCNEC and two SCLC, and was always associated with disarrayed immunoreactivity and increased MMP-7, but not cyclin D1 expression. These cases, however, did not show β-catenin gene mutations or EGFR and HER-2 gene amplification or expression. No association was found between nuclear β-catenin and any clinicopathological variable including patients' survival. The subcellular compartmentalization of β-catenin is profoundly altered in high-grade pulmonary neuroendocrine tumors. A minor subset of these tumors shows β-catenin nuclear accumulation in association with increased expression of MMP-7, but not of cyclin D1, independent of EGFR and HER-2 gene amplification or expression. The authors have no significant financial or other relationship with the manufacturers of any commercial products or commercial services presented in this paper     

Ionotropic glutamate receptor subunits are differentially regulated in the motoneuronal pools of the rat hypoglossal nucleus in response to axotomy

Unilateral hypoglossal nerve axotomy was used as a model to analyse immunohistochemically the expression of the GluR1, GluR2, GluR3, and GluR4 glutamate receptor subunits of the -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype and the NR1 subunit of the N-methyl-D-aspartate (NMDA) subtype in the different morphofunctional hypoglossal pools from 1 to 45 days postaxotomy. Following hypoglossal nerve axotomy, the percentage of motoneurons that were GluR1-immunopositive and the labeling intensity for this subunit was increased in some hypoglossal pools. Immunolabeling for the GluR2 subunit was undetectable. These results contrast with the unchanged pattern for these two subunits after sciatic nerve axotomy previously described. Image analysis showed a significant decrease in the intensity of immunohistochemical labeling for the GluR2/3 and GluR4 subunits in motoneurons, although most motoneurons were still immunopositive for these 2 subunits after axotomy. The intensity of immunolabeling for the NR1 subunit was slightly decreased postlesion, whereas the percentage of NR1-immunopositive motoneurons increased. Immunoreactivity returned to basal levels 45 days postlesion. These findings show that in axotomized hypoglossal motoneurons, i) AMPA and NMDA receptor subunits are still expressed, ii) the composition of the ionotropic glutamate receptor subunit pool is subjected to continuous changes during the regeneration process, iii) AMPA receptors, if functional, would have physiological properties different to those in intact motoneurons, and iv) the various AMPA receptor subunits are differentially regulated. The present results also suggest a faster recovery of basal levels of immunoreactivity for caudally localised groups of motoneurons which could reflect a caudo-rostral sequential functional revovery in the hypoglossal nucleus.     

Ionotropic glutamate receptor subunit distribution on hypoglossal motoneuronal pools in the rat

The expression of ionotropic glutamate receptor subunits in the motoneuronal pools of the hypoglossal nucleus was studied using specific antibodies against subunits of the -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) subtypes. The highest numbers of intensely immunolabelled motoneurons were found in the dorsal tier and caudoventromedial part of the hypoglossal nucleus with all antibodies except that against the GluR1 AMPA subunit. Labelling for the GluR1 subunit was weak except for caudally located groups of motoneurons which innervate tongue muscles related to respiratory activity. By contrast, most motoneurons were intensely immunostained with antibodies against GluR2/3 and GluR4 subunits of the AMPA subtype. The low staining observed using an antibody specific for the GluR2 subunit (which prevents Ca²⁺-entry through AMPA channels) strongly suggests that AMPA receptors in hypoglossal motoneurons are Ca²⁺-permeable. Immunolabelling for the GluR5/6/7 kainate receptor subunits was found in many motoneuronal somata as well as in thin axon-like profiles and puncta that resembled synaptic boutons. Most motoneurons were intensely immunostained for the NMDA receptor subunit NR1. These results show that the hypoglossal nucleus contains five heterogeneous pools of motoneurons which innervate functionally defined groups of tongue muscles. The uneven expression of the different receptor subunits analysed here could reflect diverse phenotypic properties of hypoglossal motoneurons which might be expected to generate different patterns of motor responses under different physiological or pathological conditions.     

Circulating inflammatory mediators during start of fever in differential diagnosis of gram-negative and gram-positive infections in leukopenic rats

Gram-negative and gram-positive infections have been considered the most important causes of morbidity and mortality in patients with leukopenia following chemotherapy. However, discrimination between bacterial infections and harmless fever episodes is difficult. Because classical inflammatory signs of infection are often absent and fever is frequently the only sign of infection, the aim of this study was to assess the significance of serum interleukin-6 (IL-6), IL-10, macrophage inflammatory protein-2 (MIP-2), procalcitonin (PCT), and C-reactive protein (CRP) patterns in identifying bacterial infections during start of fever in normal and cyclophosphamide-treated (leukopenic) rats following an injection of lipopolysaccharide (LPS) or muramyl dipeptide (MDP) as a model for gram-negative and gram-positive bacterial infections. We found that, compared to normal rats, immunosuppressed animals exhibited significantly higher fevers and lesser production of all mediators, except IL-6, after toxin challenge. Moreover, compared to rats that received MDP, both groups of animals that received an equivalent dose of LPS showed significantly higher fevers and greater increase in serum cytokine levels. Furthermore, in contrast to those in immunocompetent rats, serum levels of IL-6 and MIP-2 were not significantly changed in leukopenic animals after MDP injection. Other serum markers such as PCT and CRP failed to discriminate between bacterial stimuli in both groups of animals. These results suggest that the use of the analyzed serum markers at an early stage of fever could give useful information for the clinician for excluding gram-negative from gram-positive infections.     

Clonal spread of a vancomycin-resistant Enterococcus faecium strain among bloodstream-infecting isolates in Italy

Recent data indicated that the rate of vancomycin resistance in bloodstream-infecting enterococcal isolates in Italy is one of the highest in Europe. The aims of this study were to characterize bloodstream-infecting vancomycin-resistant enterococci (VRE) obtained from various Italian hospitals and to establish whether the isolates were clonally related. During the years 2001 to 2003, a total of 39 VRE isolates were obtained from 19 hospital laboratories in various areas of Italy. Species identification and resistance genotypes of the isolates were obtained by multiplex PCR. Further characterization included antibiotic susceptibility testing, pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA, detection of virulence genes (esp and hyl), and multilocus sequence typing (MLST) of selected isolates. VRE were identified as 31 Enterococcus faecium (VREfm) isolates and 8 E. faecalis isolates. All but one isolate carried the vanA gene; one VREfm isolate carried the vanB gene. Analysis of the PFGE profiles showed that 28 VREfm isolates shared a similar electrophoretic profile, designed type 1, and were clonally related. All type 1 isolates were resistant to ampicillin, streptomycin, gentamicin, and rifampin and were positive for the esp gene. MLST identified an allelic profile (ST78) comprising purK allele 1, belonging to the C1 clonal lineage, characteristic of human infection and hospital outbreak isolates. The vanB-carrying VREfm isolate, of PFGE type 2, was shown to be a single-locus variant of ST78. Our data indicate that the recent increase in the number of bloodstream infections caused by VRE in Italy is due to the spread of a hospital-adapted, multidrug-resistant VREfm clone belonging to an internationally disseminated lineage.     

N19 polyepitope as a carrier for enhanced immunogenicity and protective efficacy of meningococcal conjugate vaccines

N19, a string of human universal CD4 T-cell epitopes from various pathogen-derived antigens, was shown to exert a stronger carrier effect than CRM197 for the induction of anti-group C Neisseria meningitidis capsular polysaccharide (MenC), after immunization of mice with various dosages of N19-MenC or CRM-MenC conjugate vaccines. After two immunizations, the N19-based construct induced anti-MenC antibody and protective bactericidal antibody titers higher than those induced by three doses of the CRM-MenC conjugate and required lower amounts of conjugate. N19-based conjugates are superior to CRM-based conjugates to induce protective immune responses to MenC conjugates.