Mechanisms of nonopsonic phagocytosis of Pseudomonas aeruginosa.

The interaction of the macrophage cell line P388D1 with Pseudomonas aeruginosa in the absence of stimulators or opsonins led to substantial association of bacteria, as judged by visual counting and FACScan assays. This association was observable within 5 min of addition of bacteria, could not be disturbed by exhaustive washing, and occurred with pilus- or flagellum-deficient mutants but not with rpoN mutants, which have been proposed to lack a secondary adhesin. In contrast, specific antibody was capable of causing similar enhancement of bacterial uptake regardless of the rpoN phenotype. Fibronectin stimulated uptake of bacteria with the pilus as an adhesin, and stimulation was observable within 5 min. Both fibronectin-enhanced and antibody-opsonized uptake were susceptible to inhibition by pertussis toxin but not by cholera toxin. The influence of fibronectin on P388D1 cells was distinguishable from that of lipopolysaccharide, which caused substantial morphological changes in cells. Although lipopolysaccharide stimulated bacterial uptake, it actually suppressed fibronectin-mediated enhancement of uptake at high concentrations.     

Quantitative determination of cell surface β-adrenoceptors in different rat skeletal muscles

In the present study, the density of cell surface beta-adrenergic receptors was determined in different skeletal muscles using the hydrophilic ligand [3H]CGP 12177. The density of beta-adrenergic receptors was highest in the slow-twitch soleus muscle (32.8+/-0.9 fmol mg dw(-1)) and lowest in the fast-twitch glycolytic white gastrocnemius (10.4+/-0.5 fmol mg dw(-1)) beta-Adrenoceptor density correlated closely with the percentage of type-I fibres (r=0.979; P<0.0001) and inversely with the percentage of type-IIB fibres (r=696; P<0.03). Incubation with isoprenaline (10 microM) for 30 min decreased the density of beta-adrenergic receptors in the cell surface from 32.9+/-0.8 to 19.3+/-0.7 fmol mg dw(-1) in the soleus and from 16.8+/-1.0 to 12.0+/-0.7 fmol mg dw(-1) in the epitrochlearis. Internalisation appeared rapid (half-time less than 5 min). To study externalisation of beta-adrenergic receptors, soleus strips were incubated 30 min with 10 microM isoprenaline and then transferred to buffer without agonist. The first incubation reduced the density to approximately 50%, the subsequent incubation without agonist increased cell surface receptor density to approximately 80% of the initial density after 1 h. No further increase was observed over the next 2 h, suggesting that some of the receptors had been degraded. Insulin or contractile activity did not influence rate of externalisation.     

Identification of a novel <Emphasis Type="Italic">PMS2</Emphasis> alteration c.505C&gt;G (R169G) <Emphasis Type="Italic">in trans</Emphasis> with a PMS2 pathogenic mutation in a patient with constitutional mismatch repair deficiency

Constitutional mismatch repair deficiency syndrome (CMMRD) is a rare autosomal recessive predisposition to colorectal polyposis and other malignancies, often childhood-onset, that is caused by biallelic inheritance of mutations in the same mismatch repair gene. Here, we describe a patient with a clinical diagnosis of CMMRD based on colorectal polyposis and young-onset endometrial cancer who was identified to have two alterations in trans in PMS2: one known pathogenic mutation (c.1831insA; p.Ile611Asnfs*2) and one novel variant of uncertain significance (c.505C>G; p.Arg169Glu), a missense alteration. We describe the clinical and molecular features in the patient harboring this novel alteration c.505C>G, who meets clinical criteria for CMMRD and exhibits molecular evidence supporting a diagnosis of CMMRD. Although experimental validation is needed to confirm its pathogenicity, PMS2 c.505C>G likely has functional consequences that contributes to our patient’s phenotype based on the patient’s clinical presentation, tumor studies, and bioinformatics analysis.     

Glial-conditional deletion of aquaporin-4 (Aqp4) reduces blood-brain water uptake and confers barrier function on perivascular astrocyte endfeet

Tissue- and cell-specific deletion of the Aqp4 gene is required to differentiate between the numerous pools of aquaporin-4 (AQP4) water channels. A glial-conditional Aqp4 knockout mouse line was generated to resolve whether astroglial AQP4 controls water exchange across the blood-brain interface. The conditional knockout was driven by the glial fibrillary acidic protein promoter. Brains from conditional Aqp4 knockouts were devoid of AQP4 as assessed by Western blots, ruling out the presence of a significant endothelial pool of AQP4. In agreement, immunofluorescence analysis of cryostate sections and quantitative immunogold analysis of ultrathin sections revealed no AQP4 signals in capillary endothelia. Compared with litter controls, glial-conditional Aqp4 knockout mice showed a 31% reduction in brain water uptake after systemic hypoosmotic stress and a delayed postnatal resorption of brain water. Deletion of astroglial Aqp4 did not affect the barrier function to macromolecules. Our data suggest that the blood-brain barrier (BBB) is more complex than anticipated. Notably, under certain conditions, the astrocyte covering of brain microvessels is rate limiting to water movement.     

Asthma in adolescent athletes

Athletes active in endurance sports are at an increased risk of acquiring asthma through their sports activities, especially so for cross-country skiers, biathlon skiers, swimmers and athletes of other endurance sports. Asthma may be present from early childhood or develop while in active sports. This article focuses on the physical activity and sports activities in children and adolescents. Exercise-induced asthma (EIA) is found in 8-10% of a normal child population of school age and in about 35% of children with current asthma. EIA is caused by the markedly increased ventilation during exercise, with increased heat and water loss through respiration, leading to bronchial constriction. The risk of developing asthma in the young athlete is related to the repeated daily training activity with increased epithelial damage of the airways, delayed repair due to the daily repetition of the training and increased airway mucosal inflammation. The increased environmental exposure through the sports activity to environmental agents, such as cold, dry air in skiers and chlorine compounds in swimmers, increases symptoms and signs of asthma and bronchial hyper-responsiveness, either worsening an existing asthma or leading to a novel disease in a previously healthy athlete. Several specific aspects of daily training life, environmental exposure, diagnostic procedures and aspects of treatment related to the regulations of medication use in sports need particular attention when addressing the adolescent athlete with respiratory symptoms.     

Evidence that compromised K+ spatial buffering contributes to the epileptogenic effect of mutations in the human Kir4.1 gene (KCNJ10)

Mutations in the human Kir4.1 potassium channel gene (KCNJ10) are associated with epilepsy. Using a mouse model with glia-specific deletion of Kcnj10, we have explored the mechanistic underpinning of the epilepsy phenotype. The gene deletion was shown to delay K(+) clearance after synaptic activation in stratum radiatum of hippocampal slices. The activity-dependent changes in extracellular space volume did not differ between Kcnj10 mutant and wild-type mice, indicating that the Kcnj10 gene product Kir4.1 mediates osmotically neutral K(+) clearance. Combined, our K(+) and extracellular volume recordings indicate that compromised K(+) spatial buffering in brain underlies the epilepsy phenotype associated with human KCNJ10 mutations.     

Select types of supporting cell in the inner ear express aquaporin-4 water channel protein

Aquaporins (AQPs) confer a high water permeability on cell membranes and play important parts in secretory and absorptive epithelia in kidney and other organs. Here we investigate whether AQPs are expressed in the sensory epithelia of the inner ear, where a precise volume regulation is crucial. By use of specific antibodies it was found that the inner ear contains AQP1 and 4 while being devoid of detectable levels of AQP2, 3 or 5. Immunofluorescence and postembedding immunogold labelling revealed a strictly non-epithelial distribution of AQP1, confirming previous data. In contrast, AQP4 protein and mRNA (visualized by in situ hybridization) were concentrated in select types of supporting cell, including Hensen's cells and inner sulcus cells. Immunogold particles signalling AQP4 were confined to the basolateral plasma membrane of Hensen's cells and to the basal plasma membrane of Claudius cells and inner sulcus cells. AQP4 was also found in supporting cells of the vestibular end organs, but was absent from transitional epithelial cells and dark cells. Strong labelling for AQP4 and AQP4-mRNA was associated with the central part of the cochlear and vestibular nerves. Hair cells were consistently unlabelled. Our findings indicate that AQP4 may facilitate osmotically driven water fluxes in the sensory epithelia of the inner ear and thus contribute to the volume and ion homeostasis at these sites.