Early-phase redistribution of the cation-independent mannose 6-phosphate receptor by U18666A treatment in HeLa cells

It has been shown that the treatment with 3-[2-(diethylamino)ethoxy] androst-5-en-17-one (U18666A) causes the accumulation of cholesterol and the cation-independent mannose 6-phosphate receptor (CIMPR) in late endosomal/lysosomal compartments in BHK cells. The present study reports on a study of the effect of U18666A on CIMPR distribution in more detail in HeLa cells. When cells were treated with U18666A for 20 h, the intense perinuclear signal for CIMPR corresponding to the trans-Golgi network (TGN) disappeared and lamp1-negative punctate signals, scattered in the perinuclear region were detected. CIMPR then began to accumulate in lamp1-positive compartments 48 h after addition of the drug. Double immunofluorescence microscopy showed that U18666A-induced mannose 6-phosphate receptor-containing compartments (U-MPRCs), which were formed in the early phase of the redistribution, contained no marker for the TGN, late endosomes or lysosomes. Approximately half of the structures contained transferrin that had been internalized for 20 min, and cathepsin D, the majority of which appeared to be its precursor form. Immunoelectron-microscopic analysis revealed that U-MPRCs are composed of multivesicular bodies, irregularly shaped structures, and vesicular structures adjacent to the multivesicular bodies. These results suggest that U18666A treatment primarily suppresses the CIMPR transport pathways to late endosomes and from transferrin-containing endosomes, both of which may be dependent on cholesterol function.This work was supported by grants from Japan Ministry of Education, Culture, Sports, Science, and Technology.     

Faster HIV-1 disease progression among Brazilian individuals recently infected with CXCR4-utilizing strains

Introduction

Primary HIV infection is usually caused by R5 viruses, and there is an association between the emergence of CCXR4-utilizing strains and faster disease progression. We characterized HIV-1 from a cohort of recently infected individuals in Brazil, predicted the virus''s co-receptor use based on the env genotype and attempted to correlate virus profiles with disease progression.

Methods

A total of 72 recently infected HIV patients were recruited based on the Serologic Testing Algorithm for Recent HIV Seroconversion and were followed every three to four months for up to 78 weeks. The HIV-1 V3 region was characterized by sequencing nine to twelve weeks after enrollment. Disease progression was characterized by CD4+ T-cell count decline to levels consistently below 350 cells/µL.

Results

Twelve out of 72 individuals (17%) were predicted to harbor CXCR4-utilizing strains; a baseline CD4<350 was more frequent among these individuals (p = 0.03). Fifty-seven individuals that were predicted to have CCR5-utilizing viruses and 10 individuals having CXCR4-utilizing strains presented with baseline CD4>350; after 78 weeks, 33 individuals with CCR5 strains and one individual with CXCR4 strains had CD4>350 (p = 0.001). There was no association between CD4 decline and demographic characteristics or HIV-1 subtype.

Conclusions

Our findings confirm the presence of strains with higher in vitro pathogenicity during early HIV infection, suggesting that even among recently infected individuals, rapid progression may be a consequence of the early emergence of CXCR4-utilizing strains. Characterizing the HIV-1 V3 region by sequencing may be useful in predicting disease progression and guiding treatment initiation decisions.     

Adaptive Iterative Dose Reduction Using Three Dimensional Processing (AIDR3D) Improves Chest CT Image Quality and Reduces Radiation Exposure

Objective

To assess the advantages of Adaptive Iterative Dose Reduction using Three Dimensional Processing (AIDR3D) for image quality improvement and dose reduction for chest computed tomography (CT).

Methods

Institutional Review Boards approved this study and informed consent was obtained. Eighty-eight subjects underwent chest CT at five institutions using identical scanners and protocols. During a single visit, each subject was scanned using different tube currents: 240, 120, and 60 mA. Scan data were converted to images using AIDR3D and a conventional reconstruction mode (without AIDR3D). Using a 5-point scale from 1 (non-diagnostic) to 5 (excellent), three blinded observers independently evaluated image quality for three lung zones, four patterns of lung disease (nodule/mass, emphysema, bronchiolitis, and diffuse lung disease), and three mediastinal measurements (small structure visibility, streak artifacts, and shoulder artifacts). Differences in these scores were assessed by Scheffe''s test.

Results

At each tube current, scans using AIDR3D had higher scores than those without AIDR3D, which were significant for lung zones (p<0.0001) and all mediastinal measurements (p<0.01). For lung diseases, significant improvements with AIDR3D were frequently observed at 120 and 60 mA. Scans with AIDR3D at 120 mA had significantly higher scores than those without AIDR3D at 240 mA for lung zones and mediastinal streak artifacts (p<0.0001), and slightly higher or equal scores for all other measurements. Scans with AIDR3D at 60 mA were also judged superior or equivalent to those without AIDR3D at 120 mA.

Conclusion

For chest CT, AIDR3D provides better image quality and can reduce radiation exposure by 50%.     

Nonstandard peptide binding revealed by crystal structures of HLA-B*5101 complexed with HIV immunodominant epitopes

The crystal structures of the human MHC class I allele HLA-B*5101 in complex with 8-mer, TAFTIPSI, and 9-mer, LPPVVAKEI, immunodominant peptide epitopes from HIV-1 have been determined by x-ray crystallography. In both complexes, the hydrogen-bonding network in the N-terminal anchor (P1) pocket is rearranged as a result of the replacement of the standard tyrosine with histidine at position 171. This results in a nonstandard positioning of the peptide N terminus, which is recognized by B*5101-restricted T cell clones. Unexpectedly, the P5 peptide residues appear to act as anchors, drawing the peptides unusually deeply into the peptide-binding groove of B51. The unique characteristics of P1 and P5 are likely to be responsible for the zig-zag conformation of the 9-mer peptide and the slow assembly of B*5101. A comparison of the surface characteristics in the alpha1-helix C-terminal region for B51 and other MHC class I alleles highlights mainly electrostatic differences that may be important in determining the specificity of human killer cell Ig-like receptor binding.     

Changes in the endurance shuttle walk test in COPD patients with chronic respiratory failure after pulmonary rehabilitation: the minimal important difference obtained with anchor- and distribution-based method

Background

Although the endurance shuttle walk test (ESWT) has proven to be responsive to change in exercise capacity after pulmonary rehabilitation (PR) for COPD, the minimally important difference (MID) has not yet been established. We aimed to establish the MID of the ESWT in patients with severe COPD and chronic hypercapnic respiratory failure following PR.

Methods

Data were derived from a randomized controlled trial, investigating the value of noninvasive positive pressure ventilation added to PR. Fifty-five patients with stable COPD, GOLD stage IV, with chronic respiratory failure were included (mean (SD) FEV₁ 31.1 (12.0) % pred, age 62 (9) y). MID estimates of the ESWT in seconds, percentage and meters change were calculated with anchor based and distribution based methods. Six minute walking distance (6MWD), peak work rate on bicycle ergometry (Wpeak) and Chronic Respiratory Questionnaire (CRQ) were used as anchors and Cohen’s effect size was used as distribution based method.

Results

The estimated MID of the ESWT with the different anchors ranged from 186–199 s, 76–82% and 154–164 m. Using the distribution based method the MID was 144 s, 61% and 137 m.

Conclusions

Estimates of the MID for the ESWT after PR showed only small differences using different anchors in patients with COPD and chronic respiratory failure. Therefore we recommend using a range of 186–199 s, 76–82% or 154–164 m as MID of the ESWT in COPD patients with chronic respiratory failure. Further research in larger populations should elucidate whether this cut-off value is also valid in other COPD populations and with other interventions.

Trial registration

ClinicalTrials.Gov (ID {"type":"clinical-trial","attrs":{"text":"NCT00135538","term_id":"NCT00135538"}}NCT00135538).

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0182-x) contains supplementary material, which is available to authorized users.     

Demonstration of high-affinity interleukin-2 receptors on B-chronic lymphocytic leukemia cells: functional and structural characterization

Functional and structural characteristics of interleukin 2 (IL-2) receptors on B-cell chronic lymphocytic leukemia (B-CLL) cells were analyzed by a proliferation assay, IL-2 binding assay and cross-linking study. In the 3H-thymidine incorporation assay, purified B-CLL cells from four out of sixteen cases, in which the percentage of Tac antigen (Tac Ag) positive cells in peripheral blood lymphocytes ranged from 0 to 48.8%, responded to IL-2 (100 U/ml) after both 3- and 6-day incubation. No relationship was found between the responsiveness to IL-2 and the percentage of Tac Ag positive cells. In the radiolabeled IL-2 binding assay, however, B-CLL cells from all seven cases examined, including three cases with mitogenic response to IL-2 and four cases without mitogenic response, were shown to have both high- and low-affinity receptors. The number of high- and low-affinity receptors per cell ranged from 29-186 and from 420 to 1,800, respectively. Furthermore, with the affinity cross-linking method p55 (Tac Ag) and p70/75 were found even in cases without mitogenic response in their B-CLL cells. In conclusion, the B-CLL cells so far examined possessed high-affinity IL-2 receptors consisting of p55 and p70/75; nevertheless, this was not sufficient to respond to the mitogenic signal of IL-2.     

Investigation of the protective effect of ellagic acid for preventing kidney injury in rats exposed to nicotine during the fetal period

We investigated the possible protective effects of ellagic acid on rat kidneys exposed to nicotine during the fetal period. Twenty pregnant female rats were divided randomly into four groups: control (C), nicotine (N), ellagic acid (EA) and nicotine + ellagic acid (N + EA). Nicotine and ellagic acid treatments were continued throughout the pregnancies and for 15 days after delivery. On day 15, all neonatal pups were sacrificed and their kidneys were removed for biochemical and histopathological examination. The nicotine treatment significantly decreased body weight, total glutathione (GSH), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities, and increased malondialdehyde (MDA) and nitric oxide (NO) levels in the N group compared to controls. EA treatment ameliorated decreased body weight, GSH, GSH-Px and SOD activities, and increased MDA and NO levels in group N + EA compared to group N (p < 0.05). Nicotine caused kidney damage as shown by incomplete development of glomeruli and Bowman's capsules. Nicotine also caused greater apoptosis in group N compared to group C. Ellagic acid treatment produced histological kidney structure that was closer to normal and it exerted an anti-apoptotic effect in the N + EA group compared to the N group. EA played a protective role against nicotine-induced nephrotoxicity and oxidative stress in rats owing to its antioxidant, radical scavenging and anti-apoptotic effects.     

Activation of 5-HT(1A) but not 5-HT(1B) receptors attenuates an increase in extracellular dopamine derived from exogenously administered L-DOPA in the striatum with nigrostriatal denervation

In order to determine whether L-DOPA-derived extracellular dopamine (DA) in the striatum with dopaminergic denervation is affected by activation of serotonin autoreceptors (5-HT(1A) and 5-HT(1B) receptors), we applied in vivo brain microdialysis technique to 6-hydroxydopamine-lesioned rats and examined the effects of the selective 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and the selective 5-HT(1B) receptor agonist CGS-12066 A on L-DOPA-derived extracellular DA levels. Single L-DOPA injection (50 mg/kg i.p.) caused a rapid increase and a following decrease of extracellular DA, with a peak value at 100 min after L-DOPA injection. Pretreatment with both 0.3 mg/kg and 1 mg/kg 8-OH-DPAT (i.p.) significantly attenuated an increase in L-DOPA-derived extracellular DA and the times of peak DA levels were prolonged to 150 min and 225 min after L-DOPA injection, respectively. These 8-OH-DPAT-induced changes in L-DOPA-derived extracellular DA were antagonized by further pretreatment with WAY-100635, a selective 5-HT(1A) antagonist. In contrast, intrastriatal perfusion with the 5-HT(1B) agonist CGS-12066 A (10 nM and 100 nM) did not induce any changes in L-DOPA-derived extracellular DA. Thus, stimulation of 5-HT(1A) but not 5-HT(1B) receptors attenuated an increase in extracellular DA derived from exogenous L-DOPA. These results support the hypothesis that serotonergic neurons are primarily responsible for the storage and release of DA derived from exogenous L-DOPA in the absence of dopaminergic neurons.