Slight respiratory irritation but not inflammation in mice exposed to (1-->3)-beta-D-glucan aerosols

Airway irritation effects after single and repeated inhalation exposures to aerosols of beta-glucan (grifolan) were investigated in mice. In addition, the effects on serum total immunoglobulin E (IgE) production and histopathological inflammation in the respiratory tract were studied. The beta-glucan aerosols provoked slight sensory irritation in the airways, but the response was not concentration dependent at the levels studied. Slight pulmonary irritation was observed after repeated exposures. No effect was found on the serum total IgE levels, and no signs of inflammation were seen in the airways 6 h after the final exposure. The results suggest that, irrespective of previous fungal sensitization of the animals, inhaled beta-glucan may cause symptoms of respiratory tract irritation but without apparent inflammation. Respiratory tract irritation reported after inhalation of fungi may not be entirely attributed to beta-glucan.     

Epidermal ornithine decarboxylase and polyamines in mice exposed to 50 Hz magnetic fields and UV radiation

We studied the influence of magnetic fields (MFs) and simulated solar radiation (SSR) on ornithine decarboxylase (ODC) and polyamines in mouse epidermis. Chronic exposure to combined MF and SSR did not cause persistent effects on ODC activity or polyamines compared to the animals exposed only to UV, although the same MF treatment was previously found to accelerate skin tumor development. In an acute 24-h experiment, an elevation of putrescine and down-regulation of ODC activity was observed in the animals exposed to a 100-μT MF. No effect was seen 24 h after a single 2-MED (minimal erythemal dose) exposure to SSR. The results indicate that acute exposure to 50 Hz MF does exert distinctive biological effects on epidermal polyamine synthesis. Bioelectromagnetics 19:388–391, 1998. © 1998 Wiley-Liss, Inc.     

Characterization of high affinity monoclonal antibodies specific for chlamydial lipopolysaccharide

Pathogens belonging to the genus Chlamydia contain lipopolysaccharide with a 3-deoxy-D- manno- oct-2-ulosonic acid (Kdo) trisaccharide of the sequence alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo. This lipopolysaccharide is recognized in a genus-specific pattern by murine monoclonal antibodies (mAbs), S25-23 and S25-2 (both IgG1kappa), which bind as the minimal structures the trisaccharide and the terminal Kdo-disaccharide, respectively. The variable domains of these mAbs were reverse transcribed from mRNA which was isolated from hybridomas and cloned as single-chain variable fragments (scFvs) in E.coli TG1. The kinetics of binding of whole antibodies, Fab fragments and scFvs to natural and synthetically modified ligands were determined by surface plasmon resonance (SPR) using synthetic neoglycoconjugates. As examples of an antibody-carbohydrate interaction involving anionic carboxyl groups on the ligand, we report that the affinities of these antibodies are higher than usually observed in carbo-hydrate-protein interactions (K(D)of 10(-3)to 10(-5)M). SPR analy-ses of monovalent Fab and scFv binding to the natural trisaccharide epitope gave dissociation constants of 770 nM for S25-2 and 350 nM for S25-23, as determined by global fitting (simultaneous fitting of several measurements at different antibody concentrations) of sensorgram data to a one-to-one interaction model. Local fitting (separate fitting of individual sensorgram data at different antibody concentrations) and Scatchard analysis of the data gave kinetic and affinity constants that were in good agreement with those obtained by global fitting. The SPR data also showed that while S25-2 bound well to several Kdo disaccharides and carboxyl-reduced Kdo ligands, S25-23 did not. Identification of amino acids in the complementarity determining regions revealed the presence of a large number of positively charged amino acids which were located towards the center of the combining site, thus suggesting a different recognition mechanism than that observed for neutral ligands. The latter mainly involves aromatic amino acids for hydrophobic stacking inter-actions and hydrogen bonds.     

Biosynthesis of nucleotide-activated D-glycero-D-manno-heptose

The glycan chain repeats of the S-layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 contain d-glycero-d-manno-heptose, which has also been described as constituent of lipopolysaccharide cores of Gram-negative bacteria. The four genes required for biosynthesis of the nucleotide-activated form GDP-d-glycero-d-manno-heptose were cloned, sequenced, and overexpressed in Escherichia coli, and the corresponding enzymes GmhA, GmhB, GmhC, and GmhD were purified to homogeneity. The isomerase GmhA catalyzed the conversion of d-sedoheptulose 7-phosphate to d-glycero-d-manno-heptose 7-phosphate, and the phosphokinase GmhB added a phosphate group to form d-glycero-d-manno-heptose 1,7-bisphosphate. The phosphatase GmhC removed the phosphate in the C-7 position, and the intermediate d-glycero-alpha-d-manno-heptose 1-phosphate was eventually activated with GTP by the pyrophosphorylase GmhD to yield the final product GDP-d-glycero-alpha-d-manno-heptose. The intermediate and end products were analyzed by high performance liquid chromatography. Nuclear magnetic resonance spectroscopy was used to confirm the structure of these substances. This is the first report of the biosynthesis of GDP-d-glycero-alpha-d-manno-heptose in Gram-positive organisms. In addition, we propose a pathway for biosynthesis of the nucleotide-activated form of l-glycero-d-manno-heptose.     

Diffusion-Weighted Imaging in 3.0 Tesla Breast MRI: Diagnostic Performance and Tumor Characterization Using Small Subregions vs. Whole Tumor Regions of Interest

Introduction

Apparent diffusion coefficient (ADC) values are increasingly reported in breast MRI. As there is no standardized method for ADC measurements, we evaluated the effect of the size of region of interest (ROI) to diagnostic utility and correlation to prognostic markers of breast cancer.

Methods

This prospective study was approved by the Institutional Ethics Board; the need for written informed consent for the retrospective analyses of the breast MRIs was waived by the Chair of the Hospital District. We compared diagnostic accuracy of ADC measurements from whole-lesion ROIs (WL-ROIs) to small subregions (S-ROIs) showing the most restricted diffusion and evaluated correlations with prognostic factors in 112 consecutive patients (mean age 56.2±11.6 years, 137 lesions) who underwent 3.0-T breast MRI.

Results

Intra- and interobserver reproducibility were substantial (κ = 0.616–0.784; Intra-Class Correlation 0.589–0.831). In receiver operating characteristics analysis, differentiation between malignant and benign lesions was excellent (area under curve 0.957–0.962, cut-off ADC values for WL-ROIs: 0.87×10⁻³ mm²s^-1; S-ROIs: 0.69×10⁻³ mm²s^-1, P<0.001). WL-ROIs/S-ROIs achieved sensitivities of 95.7%/91.3%, specificities of 89.5%/94.7%, and overall accuracies of 89.8%/94.2%. In S-ROIs, lower ADC values correlated with presence of axillary metastases (P = 0.03), high histological grade (P = 0.006), and worsened Nottingham Prognostic Index Score (P<0.05). In both ROIs, ADC values correlated with progesterone receptors and advanced stage (P<0.01), but not with HER2, estrogen receptors, or Ki-67.

Conclusions

ADC values assist in breast tumor characterization. Small ROIs were more accurate than whole-lesion ROIs and more frequently associated with prognostic factors. Cut-off values differed significantly depending on measurement procedure, which should be recognized when comparing results from the literature. Instead of using a whole lesion covering ROI, a small ROI could be advocated in diffusion-weighted imaging.     

Transcription factor snail1 expression and poor survival in pharyngeal squamous cell carcinoma

Snail1, a key regulator of epithelial-mesenchymal transition (EMT), plays an important role in tumour progression. Previous studies of snail1 have mainly focused on the epithelial tumour cells. The objective of this study was to evaluate the expression of snail1 protein in endothelial cells, stromal myofibroblasts and malignant epithelial cells of pharyngeal squamous cell carcinomas (PSCC), as well as its relation to clinicopathological features and survival. One hundred and ten tissue microarray samples were analyzed for snail1 expression using immunohistochemistry. In endothelial cells snail1 expression was observed in 51 (48%) of 107 cases and it predicted reduced disease specific survival (DSS) (p=0.009). In 49 (46%) tumour samples snail1 immunostaining was detected in stromal myofibroblasts and there was a tendency to poorer DSS in that group (p=0.067). Snail1 expression in endothelial cells and stromal myofibroblasts is also associated with hypopharyngeal tumours (p=0.01 and p=0.038 respectively), increasing T category (T3-4) (p=0.005, p=0.037 respectively) and poorer general condition of the patient (Karnofsky performance status score <70; p=0.029, p=0.039 respectively). Moreover endothelial expression correlated with advanced stage (III-IV) (p=0.005) and poorer differentiation (grade 2-3; p=0.012). In malignant epithelial cells snail1 immunostaining was detected in 75 of 110 cases (68%). Expression of the protein was more common in hypopharyngeal tumours (p=0.044). Snail1 positive tumours associated with a lower Karnofsky performance status score (p=0.039) and regional failure (p=0.042). Our findings indicate that snail1 protein expression in endothelial cells and to some extent also in tumour stromal myofibroblasts seems to be a predictor of poor survival in PSCC. The presence of snail1 protein in tumour microenvironment rather than in malignant epithelial tumour cells may induce tissue remodelling and tumour progression.     

Synthesis of methyl 4'-O-methyl-beta-D-cellobioside-13C12 from D-glucose-13C6. Part 2: solid-state NMR studies

Double Quantum (DQ) NMR, which utilizes the magnetic dipole interaction between the (13)C atoms, was used for the complete assignment of the (13)C NMR resonances to the corresponding carbon ring positions for the monoclinic and triclinic allomorphs of methyl 4'-O-methyl-beta-D-cellobioside-(13)C(12)(1-(13)C(12)), a cellodextrin model compound of cellulose (13)C-perlabeled at the cellobiose core. The through-space interactions were used to identify the direct chemical bonds between adjacent carbon atoms in the rings. More importantly, the (13)C NMR signals of the carbon sites C1' and C4 involved in the glycosidic bond were identified. This allowed for the complete (13)C chemical shift assignment, that when combined with the X-ray crystallography data provides a complete characterization.