Pneumococcal trafficking across the blood-brain barrier. Molecular analysis of a novel bidirectional pathway

Although Streptococcus pneumoniae is a major cause of meningitis in humans, the mechanisms underlying its traversal from the circulation across the blood-brain barrier (BBB) into the subarachnoid space are poorly understood. One mechanism might involve transcytosis through microvascular endothelial cells. In this study we investigated the ability of pneumococci to invade and transmigrate through monolayers of rat and human brain microvascular endothelial cells (BMEC). Significant variability was found in the invasive capacity of clinical isolates. Phase variation to the transparent phenotype increased invasion as much as 6-fold and loss of capsule approximately 200-fold. Invasion of transparent pneumococci required choline in the pneumococcal cell wall, and invasion was partially inhibited by antagonists of the platelet-activating factor (PAF) receptor on the BMEC. Pneumococci that gained access to an intracellular vesicle from the apical side of the monolayer subsequently were subject to three fates. Most opaque variants were killed. In contrast, the transparent phase variants were able to transcytose to the basal surface of rat and human BMEC in a manner dependent on the PAF receptor and the presence of pneumococcal choline-binding protein A. The remaining transparent bacteria entering the cell underwent a previously unrecognized recycling to the apical surface. Transcytosis eventually becomes a dominating process accounting for up to 80% of intracellular bacteria. Our data suggest that interaction of pneumococci with the PAF receptor results in sorting so as to transcytose bacteria across the cell while non-PAF receptor entry shunts bacteria for exit and reentry on the apical surface in a novel recycling pathway.     

Contrast-enhanced gradient-echo MR imaging of the head and neck: experimental and clinical assessment

To clarify the enhancement pattern of gradient-echo (GE) MR imaging, phantom experiments and 24 clinical examinations of the head and neck region were performed. Both long repetition time (TR) GE and short TR GE pulse sequences demonstrated an enhancement pattern similar to T1-weighted spin-echo imaging in phantom experiments, although the signal intensity of water and gadopentetate dimeglumine (Gd) solution was higher and the signal intensity of fat was lower with the GE technique. In clinical examinations, contrast-enhanced GE imaging was beneficial for tumors within fatty tissue, and for tumors with calcification or other magnetic susceptibility difference.     

Systematic ultrasonography in asymptomatic dense breasts

Case report of a 64-year-old woman with increasing dyspnea and cough. She cared at home for 8 parakeets (Melopsittacus undulatus). Subsequent studies revealed a restrictive pulmonary defect and transbronchial biopsy, a histological bronchiolitis obliterans (BO). No significant elevated anti-avian IgG could be detected, probably because of a transient hypogammaglobulinaemia. The implication of antigenic exposure to avian antigens in the pathogenesis of BO is discussed.     

Immunological characterization of Asp f 2, a major allergen from Aspergillus fumigatus associated with allergic bronchopulmonary aspergillosis

The 37-kDa recombinant protein Asp f 2, encoding an allergen of Aspergillus fumigatus, was expressed in a prokaryotic expression system and immunologically evaluated for its functional and structural properties. The open reading frame for a 310-amino-acid-long protein was shown to encode a signal peptide of 31 amino acids. A native 37-kDa culture filtrate protein and a 55-kDa mycelial glycoprotein (gp55) exhibited complete N-terminal sequence homology to Asp f 2. A GenBank search for homologous proteins revealed 60 and 44% sequence homologies to the cytosolic protein ASPND1 from Aspergillus nidulans and fibrinogen binding protein from Candida albicans, respectively. The glycosylation sites and cysteine molecules are conserved in all the three proteins. The extracellular matrix protein laminin showed a dose-dependent interaction with Asp f 2. This protein, expressed as a major cell-associated protein within 24 h of in vitro fungal culture, comprises 20 to 40% of total fungal protein. Furthermore, both native and recombinant Asp f 2 exhibited specific immunoglobulin (IgE) binding with allergic bronchopulmonary aspergillosis (ABPA) and cystic fibrosis-ABPA patients, whereas A. fumigatus-sensitized allergic asthma and normal control subjects failed to show IgE binding with Asp f 2. These results indicate that Asp f 2 is a major allergen of A. fumigatus exhibiting IgE antibody binding with sera from patients with ABPA. The antigen should be explored further for its potential role in the differential diagnosis of A. fumigatus-associated allergic diseases.     

State-dependent NMDA receptor antagonism by Ro 8-4304, a novel NR2B selective, non-competitive, voltage-independent antagonist

1. Subunit-selective blockade of N-methyl-D-aspartate (NMDA) receptors provides a potentially attractive strategy for neuroprotection in the absence of undesirable side effects. Here, we describe a novel NR2B-selective NMDA antagonist, 4-?3-[4-(4-fluoro-phenyl)-3,6-dihydro-2H-pyridin-1-yl]-2-hydroxy-propoxy ?-benzamide (Ro 8-4304), which exhibits >100 fold higher affinity for recombinant NR1(001)/NR2B than NR1(001)/NR2A receptors. 2. Ro 8-4304 is a voltage-independent, non-competitive antagonist of NMDA receptors in rat cultured cortical neurones and exhibits a state-dependent mode of action similar to that described for ifenprodil. 3. The apparent affinity of Ro 8-4304 for the NMDA receptor increased in an NMDA concentration-dependent manner so that Ro 8-4304 inhibited 10 and 100 microM NMDA responses with IC50s of 2.3 and 0.36 microM, respectively. Currents elicited by 1 microM NMDA were slightly potentiated in the presence of 10 microM Ro 8-4304, and Ro 8-4304 binding slowed the rate of glutamate dissociation from NMDA receptors. 4. These results were predicted by a reaction scheme in which Ro 8-4304 exhibits a 14 and 23 fold higher affinity for the activated and desensitized states of the NMDA receptor, respectively, relative to the agonist-unbound resting state. Additionally, Ro 8-4304 binding resulted in a 3 4 fold increase in receptor affinity for glutamate site agonists. 5. Surprisingly, whilst exhibiting a similar affinity for NR2B-containing NMDA receptors as ifenprodil, Ro 8-4304 exhibited markedly faster kinetics of binding and unbinding to the NMDA receptor. This spectrum of kinetic behaviour reveals a further important feature of this emerging class of NR2B-selective compounds.     

Vascular endothelial growth factor in premenopausal women--indicator of the best time for breast cancer surgery?

Timing of surgery in premenopausal patients with breast cancer remains controversial. Angiogenesis is essential for tumour growth and vascular endothelial growth factor (VEGF) is one of the most potent angiogenic cytokines. We aimed to determine whether the study of VEGF in relation to the menstrual cycle could help further the understanding of this issue of surgical intervention. Fourteen premenopausal women were recruited, along with three post-menopausal women, a woman on an oral contraceptive pill and a single male subject. Between eight and 11 samples were taken per person, over one menstrual cycle (over 1 month in the five controls) and analysed for sex hormones and VEGF165. Serum VEGF was significantly lower in the luteal phase and showed a significant negative correlation with progesterone in all 14 premenopausal women. No inter-sample variations of VEGF were noted in the controls. Serum from both phases of the cycle from one subject was added to MCF-7 breast cancer cells; VEGF expression in the supernatant was lower in the cells to which the luteal phase serum was added. The lowering of a potent angiogenic cytokine in the luteal phase suggests a possible decreased potential for micrometastasis establishment in that phase. This fall in VEGF may be an effect of progesterone and should be the focus of future studies.     

Stereoselective S-nitrosocysteine recognition sites in rat brain

We examined the effects of intracisternal (i.c.) injections (10-250 nmol) of the L- and D-isomers of S-nitrosocysteine (L- and D-S-nitrosocysteine) on the mean arterial blood pressure and heart rate of conscious rats, and the decomposition of L- and D-S-nitrosocysteine to nitric oxide (NO) upon addition to brain homogenates. The i.c. injection of L-S-nitrosocysteine produced initial falls in mean arterial blood pressure and heart rate which were followed by increases in these parameters. The i.c. injection of D-S-nitrosocysteine did not produce initial falls in mean arterial blood pressure or heart rate but produced the subsequent increases in these parameters. L- and D-S-nitrosocysteine decomposed equally to NO. These results suggest that the initial effects of L-S-nitrosocysteine may be due to the activation of stereoselective recognition sites on brain neurons.     

Ro 25-6981, a highly potent and selective blocker of N-methyl-D-aspartate receptors containing the NR2B subunit. Characterization in vitro

The interaction of Ro 25-6981 with N-methyl-D-aspartate (NMDA) receptors was characterized by a variety of different tests in vitro. Ro 25-6981 inhibited 3H-MK-801 binding to rat forebrain membranes in a biphasic manner with IC50 values of 0.003 microM and 149 microM for high- (about 60%) and low-affinity sites, respectively. NMDA receptor subtypes expressed in Xenopus oocytes were blocked with IC50 values of 0.009 microM and 52 microM for the subunit combinations NR1C & NR2B and NR1C & NR2A, respectively, which indicated a >5000-fold selectivity. Like ifenprodil, Ro 25-6981 blocked NMDA receptor subtypes in an activity-dependent manner. Ro 25-6981 protected cultured cortical neurons against glutamate toxicity (16 h exposure to 300 microM glutamate) and combined oxygen and glucose deprivation (60 min followed by 20 h recovery) with IC50 values of 0.4 microM and 0.04 microM, respectively. Ro 25-6981 was more potent than ifenprodil in all of these tests. It showed no protection against kainate toxicity (exposure to 500 microM for 20 h) and only weak activity in blocking Na+ and Ca++ channels, activated by exposure of cortical neurons to veratridine (10 microM) and potassium (50 mM), respectively. These findings demonstrate that Ro 25-6981 is a highly selective, activity-dependent blocker of NMDA receptors that contain the NR2B subunit.     

The effect of asphyxia on the pharmacokinetics of ceftazidime in the term newborn

The multiple-dose pharmacokinetics of ceftazidime (CAZ) (administered twice daily in a 50 mg/kg of body weight i.v. dose) were studied in 10 severely asphyxiated term infants with suspected septicemia on d 3 of life. Nine term infants with suspected septicemia but without asphyxia served as controls. Blood samples were collected from an arterial catheter at 0, 0.5, 1, 2, 4, 8, and 12 h after an i.v. bolus injection. A high performance liquid chromatography method was used to determine CAZ concentrations from serum. CAZ pharmacokinetics followed a one-compartment open model. The GFRs of all infants were simultaneously studied by means of the 24-h continuous inulin infusion technique. Elimination serum half-life (5.86 +/- 1.13 h versus 3.85 +/- 0.40 h) and serum trough concentrations (46 +/- 14 mg/L versus 23 +/- 7 mg/L) of CAZ were significantly (p < 0.001) increased in the asphyxiated newborn, whereas total body clearance of CAZ (128.4 +/- 25.1 mL/h versus 205.7 +/- 55.4 mL/h), CAZ clearance per kg (40.9 +/- 6.1 mL/h/kg versus 60.8 +/- 8.3 mL/h/kg), and the GFR expressed in mL/min (3.14 +/- 0.43 versus 4.73 +/- 0.89) were significantly (p < 0.001) decreased in the asphyxiated newborn. We conclude that twice daily administration of 50 mg/kg of body weight CAZ given to asphyxiated term newborns in the first days of life results in significantly higher serum trough levels in comparison with control infants. The impaired CAZ clearance is a result of a significantly decreased GFR.