Apical and basolateral expression of Aquaporin-1 in transfected MDCK and LLC-PK cells and functional evaluation of their transcellular osmotic water permeabilities

 Aquaporin-1 is present in the apical and basolateral membranes in proximal tubules and descending limbs of Henlé’s loop. In order to be able to study the routing of Aquaporin-1 and the regulation of Aquaporin-1-mediated transcellular water flow, we stably transfected LLC-PK₁ and MDCK-HRS cell lines with an Aquaporin-1 expression construct. LLC-PK₁ clone 7 and MDCK clone K integrated two and one copies, respectively, which was reflected in the amount of Aquaporin-1 mRNA expressed in both clones. The Aquaporin-1 protein levels, however, were similar. In both clones, immuno-electronmicroscopy showed extensive labelling of Aquaporin-1 on the basolateral plasma membrane, endosomal vesicles and the apical plasma membrane, including the microvilli. To measure transcellular water permeation, a simple method was applied using phenol-red as a cell-impermeant marker of concentration. In contrast to the native cell lines, both clones revealed a high transcellular osmotic water permeability, which could not be influenced by forskolin add/3-isobutyl-1-methylxanthine (IBMX) or the phorbol ester 12-O-tetradecanoyl 13-acetate (TPA). After glutaraldehyde fixation, it was inhibitable by HgCl₂. These results indicate that targeting of Aquaporin-1 to the apical and basolateral plasma membrane is independent of cell type and show for the first time that water flow through a cultured epithelium can be blocked by mercurial compounds. Received: 9 October 1996 / Received after revision: 3 January 1997 / Accepted: 8 January 1997     

Circulating immune complexes in ulcerative colitis.--II. Correlation with serum protein concentrations and complement conversion products

Several serum proteins were quantified in twenty-two patients with active ulcerative colitis, and the findings were related to disease activity and occurrence of circulating immune complexes (IC). Conversion of C3 was significantly more frequent in the IC-positive group (eight patients) as compared to the IC-negative group (fourteen patients). Factor B was demonstrable in fifteen out of the twenty-two patients and seven out of the eight IC-positive patients had detectable levels of factor B. There was no difference between the IC-positive and the IC-negative group as regards serum concentrations of the complement factors C3, C4 and factor B, or serum orosomucoid, albumin, IgM and IgG. In contrast, the serum IgA levels tended to be reduced in the IC-positive group. C3 and factor B were significantly elevated in four patients with severe disease activity. In addition, C3, factor B and C4 concentrations showed a positive correlation to the serum orosomucoid levels. The serum concentrations of orosomucoid and albumin were inversely correlated to each other.     

Adaptation of wild-type measles virus to CD46 receptor usage

Summary.  Vaccine strains of measles virus (MV) use CD46 as receptor and downregulate CD46 from the surface of infected cells. MVs isolated and passaged on B-lymphoid cells (wild-type MVs) seem to use another receptor and do not downregulate CD46. In the present study, we found that isolation of MV on human or marmoset B-lymphoid cells did not alter the MV haemagglutinin (H) protein relative to that in the patient. The wild-type isolates were adapted to the human epithelial HEp-2 cell line or the monkey fibroblast Vero cell line. All HEp-2 cell adapted viruses and 1 out of 4 Vero cell adapted viruses acquired the capacity to use CD46 as receptor, as measured by their ability to infect murine cells expressing human CD46. Adaptation to CD46 receptor usage was coupled to substitution of amino acid 481 of the MV H protein from asparagine to tyrosine but not to CD46 downregulation. The present study demonstrates that CD46 receptor usage can be induced by adaptation of wild-type MV to cells that do not express a wild-type receptor and suggests that a similar mechanism acted on the progenitor viruses of the present MV vaccine strains during their isolation and attenuation. Received June 5, 2000 Accepted October 5, 2000     

High-dose inhaled steroids in the management of asthma A comparison of the effects of budesonide and beclomethasone dipropionate on pulmonary function, symptoms, bronchial responsiveness and the adrenal function

Svendsen UG, Frølund L, Heinig JH, Madsen F, Nielsen NH, Weeke B. High-dose inhaled steroids in the management of asthma. A comparison of the effects of budesonide and beclomethasone dipropionate on pulmonary function, symptoms, bronchial responsiveness and the adrenal function.
The efficacy of budesonide (800 μg b.d.) and beclomethasone dipropionate (750 μg b.d.) in controlling the symptoms of asthma, pulmonary function, bronchial responsiveness to histamine, and adrenal function, was assessed in a double-blind, double-dummy cross-over study of 36 adult chronic asthmatic patients. The patients, the majority of whom were assessed to be affected to a severe degree, were insufficiently controlled in their current regimen of inhaled steroids and/or inhaled and oral bronchodilators. A 2 weeks baseline period preceded 6 weeks of treatment with each of the study drugs. Both treatment groups showed improvements from baseline in clinical assessment of lung function carried out after the first 6 weeks of treatment. No significant differences were seen throughout the entire 12 weeks study, when comparing the effects of the treatments on FEV₁ FVC, PEF or the histamine PC₂₀. Asthma severity, symptom score and inhaled bronchodilator use showed the same results after both treatments. It is concluded that inhalations of budesonide and beclomethasone dipropionate in high doses are equally potent in the treatment of severe asthma. There is no significant influence on the adrenal function and no significant side effects during a period equal to that of the present study.     

Ethylenediaminetetraacetic acid (disodium salt)-labile bovine immunoglobulin M Fc binding to Brucella abortus: a cause of nonspecific agglutination.

It was demonstrated by a radioimmunoassay procedure that Brucella abortus agglutinins from noninfected cattle sera, absorbed to B. abortus antigen and eluted with ethylenediaminetetraacetic acid (EDTA), was immunoglobulin M that bound to that bacterium by its Fc portion. The EDTA-eluted immunoglobulin M agglutinated intact B. abortus cells but not erythrocytes treated with B. abortus lipopolysaccharide. The specificity of the EDTA-eluted immunoglobulin was for B. abortus, although a small titer to Yersinia enterocolitica serotype O:9 was observed. In contrast, immunoglobulin M purified from the serum of a cow injected 7 days previously with heat-killed B. abortus bound to the antigen by its Fab portion, was not labile to EDTA treatment, cross-reacted extensively with Y. enterocolitica serotype O:9, and agglutinated various other bacterial antigens and normal erythrocytes.     

Anaerobic and aerobic skin bacteria before and after skin-disinfection with chlorhexidine: an experimental study in volunteers.

The amount, composition, and localization of anaerobic and aerobic bacteria in the normal skin before and after disinfection were the subject of a volunteer study. The superficial bacterial flora were sampled by velvet pad imprints, and the deep flora were determined from whole skin biopsies. Only one anaerobic species, Propionebacterium acnes, was encountered even though other and more strict anaerobic bacteria could have been grown with the anaerobic technique employed. Staphylococcus albus dominated among the aerobic superficial bacteria, while diphtheroids, Micrococcus spp., and lactobacilli occurred sporadically. The deep aerobic bacteria were present in a significantly greater amount than the anaerobic. A two-step cleansing/disinfection procedure was evaluated in vivo in volunteers as well as in surgical patients, and aqueous cetrimide/chlorhexidine (Savlon) followed by chlorhexidine in alcohol (Hibitane) almost eradicated both the superficial and deep anaerobic and aerobic skin flora.     

Expression of interleukin-15 in human skeletal muscle – effect of exercise and muscle fibre type composition

The cytokine interleukin-15 (IL-15) has been demonstrated to have anabolic effects in cell culture systems. We tested the hypothesis that IL-15 is predominantly expressed by type 2 skeletal muscle fibres, and that resistance exercise regulates IL-15 expression in muscle. Triceps brachii, vastus lateralis quadriceps and soleus muscle biopsies were obtained from normally physically active, healthy, young male volunteers ( n = 14), because these muscles are characterized by having different fibre-type compositions. In addition, healthy, normally physically active male subjects ( n = 8) not involved in any kind of resistance exercise underwent a heavy resistance exercise protocol that stimulated the vastus lateralis muscle and biopsies were obtained from this muscle pre-exercise as well as 6, 24 and 48 h post-exercise. IL-15 mRNA levels were twofold higher in the triceps (type 2 fibre dominance) compared with the soleus muscle (type 1 fibre dominance), but Western blotting and immunohistochemistry revealed that muscle IL-15 protein content did not differ between triceps brachii, quadriceps and soleus muscles. Following resistance exercise, IL-15 mRNA levels were up-regulated twofold at 24 h of recovery without any changes in muscle IL-15 protein content or plasma IL-15 at any of the investigated time points. In conclusion, IL-15 mRNA level is enhanced in skeletal muscles dominated by type 2 fibres and resistance exercise induces increased muscular IL-15 mRNA levels. IL-15 mRNA levels in skeletal muscle were not paralleled by similar changes in muscular IL-15 protein expression suggesting that muscle IL-15 may exist in a translationally inactive pool.     

Near-infrared spectrophotometry determined brain oxygenation during fainting

During orthostatic hypotension we evaluated whether presyncopal symptoms relate to a reduced brain oxygenation. Nine subjects performed 50° head-up tilt for 1 h and eight subjects were followed during 2 h of supine rest and during 1 h of 10° head-down tilt. Cerebral perfusion was assessed by transcranial Doppler determined middle cerebral artery blood velocity (MCA v_mean), while brain blood oxygenation was assessed by near-infrared spectrophotometry determined concentration changes for oxygenated (ΔHbO₂) and deoxygenated haemoglobin and brain cell oxygenation by the oxidized cytochrome c concentration (ΔCytO₂). During head-up tilt, six volunteers developed presyncopal symptoms and mean arterial pressure (88 (78–103) to 68 (57–79) mmHg; median and range), heart rate (96 (72–111) to 65 (50–107) beats min^?1), MCA v_mean (59 (51–82) to 41 (29–56) cm s^?1), ΔHbO₂ (by ?5.3 (?3.0 to ?14.8) μmol l^?1) and ΔCytO₂ were reduced (by ?0.2 (?0.1 to ?0.4) μmol l^?1; P < 0.05). During tilt down the cardiovascular variables recovered immediately and ΔHbO₂ increased to 2.2 (?0.9–12.0) mmol L^?1 above the resting value and also ΔCytO₂ recovered. In the nonsyncopal head-up tilted subjects as in the controls, blood pressure, heart rate, MCA v_mean and brain oxygenation indices remained stable. The results suggest that during orthostasis, presyncopal symptoms relate not only to cerebral hypoperfusion but also to reduced brain oxygenation.     

In situ hybridization for the detection of chicken anaemia virus in experimentally-induced infection and field outbreaks

A double stranded DNA probe, biotinylated by random primingj was used for in situ hybridization of chicken anaemia virus (CAV) in thymus tissue. Hybridization was visualized with an alkaline phosphatase conjugated streptavidin biotin complex and the substrate New Fuchsin. The test was performed on both uninfected and CAV-, adenovirus- or infectious bursal disease virus-infected specified pathogen free White Leghorn chickens. In addition, both healthy commercial broilers and broilers with blue wing disease or with colisepticemia, were tested. Genomic CAV was detected in all CAV-infected and blue wing-diseased chickens. With the exception of one chicken with colisepticemia, which showed a unexpectedly positive reaction, all control chickens tested negative in the test developed.