Analysis of mast cell subpopulations (MCT, MCTC) in cutaneous inflammation using novel enzyme-histochemical staining techniques

Abstract In order to gain insights into the dynamics of mast cell subpopulations in normal and diseased skin, a novel enzyme-histochemical double and triple staining method was employed that allowed the detection of metachromasia (toluidine blue) and the mast cell proteases tryp-tase and chymase within the same cell. Cryostat sections were used of skin biopsies from the following specimens: normal skin (N=4), psoriasis (N=13), atopic eczema (N=7), lichen planus (N=6), interferon α2a injection sites (N=l) of a leukemic infiltrate and corresponding normal skin of the same patient before and after treatment. (i) Equal numbers of tryptase-and chymase-positive mast cells (MCTC) were obtained in all normal and diseased specimens in papillary and reticular dermis, with threefold increases around appendages, (ii) Tryptase-positive mast cells (MCT) were absent in normal skin, but were markedly increased in a disease-specific pattern within the papillary dermis, the inflammatory infiltrate and around appendages, (iii) Marked increases of MCT were also noted at interferon injection sites within the leukemic infiltrate, but not in the normal skin of the same patient. These data suggest that disease-dependent mast cell dynamics involve only MCT in cutaneous inflammation and that MCT numbers are controlled by distinct, disease-specific local tissue factors.     

Distribution and potential biologic function of the thrombin receptor PAR-1 on human keratinocytes

Thrombin has recently been shown not only to exert procoagulant activities, but also to induce mitogenic responses of different cell types involved in wound healing via binding to and cleavage of the thrombin receptor. In order to further explore these aspects of thrombin function, human keratinocytes (HaCaT cell line) were examined for their potential mitogenic responsiveness to thrombin and for the dependency of this process on the expression of the high-affinity thrombin receptor. Quiescent keratinocytes were stimulated in the mitogenic assay with alpha-thrombin and the thrombin receptor activating peptides TRAP42-55 (SFLLRNPNDKYEPY) and TRAP42-46 (SFLLR). A strong induction of cell proliferation was noted with alpha-thrombin, TRAP42-55 and TRAP42-46, but not with the "scrambled" peptide (FSLLR). These findings confirm that keratinocytes express the thrombin receptor and that the sequence of the first two amino acids of the generated neo-N-terminus are important for the activation of the receptor. Using cDNA fragments of the 5' coding sequence of the receptor, Northern blot analysis confirmed that HaCaT keratinocytes express the thrombin receptor. Expression of the receptor was also detected on normal human keratinocytes by immunohistochemistry and in situ hybridization. These data demonstrate the expression and biologic function of the human thrombin receptor on human keratinocytes, suggesting that thrombin, among other mediators, plays an important part in the orchestration of epidermal growth and repair processes.     

Transcranial harmonic power duplex sonography for the evaluation of cerebral arteries.

Harmonic power-based duplex sonography is a new ultrasound method that improves the signal-to-noise ratio of extracranial vascular imaging. The authors evaluated this new method for transtemporal imaging of the basal cerebral arteries. Fundamental power-based duplex sonography (p-TCCS) and harmonic power-based duplex sonography (HI-p-TCCS) in combination with a novel perfluoropropane-containing ultrasound contrast agent (Optison) were investigated for the evaluation of the basal cerebral arteries in 12 healthy volunteers. The number of identified vascular segments and the blood flow velocities in the middle and posterior cerebral arteries were determined for p-TCCS and for two doses of Optison (0.5 and 1.5 mL) using HI-p-TCCS. Furthermore, the authors determined the time course of signal enhancement after Optison bolus injections. The results were compared using Friedman two-way ANOVA test. Significantly more arterial segments were visualized using HI-p-TCCS with enhancement of either 0.5 mL or 1.5 mL Optison (p < 0.01, each) than using p-TCCS. The spatial resolution was markedly increased with HI-p-TCCS, resulting in a striking difference in the detection of distal arterial segments and cortical and parenchymal branches. Except for the diastolic blood flow velocities (BFVs) in the M1 segment, the BFVs did not differ significantly between p-TCCS and HI-p-TCCS. Comparing HI-p-TCCS with 0.5 mL and 1.5 mL Optison, the authors found a small but significant reduction of the latency period (18.2 vs. 15.9 seconds, respectively; p < 0.01), a significant increase of the blooming phase (62.7 vs. 99.8 seconds, respectively; p < 0.0006) and a significant prolongation of the diagnostically useful signal enhancement (233.7 vs. 427.6 seconds, respectively; p < 0.004).     

Harmonic imaging of the brain parenchyma using a perfluorobutane-containing ultrasound contrast agent

We evaluated the signal-enhancing effect of the novel perfluorobutane-based ultrasound contrast agent BR 14 (Bracco Research, Switzerland) in grey-scale harmonic imaging of the brain parenchyma. Six sedated male beagle dogs were investigated with transcranial grey-scale harmonic imaging (SONOS 5500, 1.8/3.6 MHz). After bolus injection of two different doses of BR 14, acoustic densitometry was performed to quantify changes in regional contrast intensity. In the dogs' brain parenchyma, the mean relative peak increase in acoustic intensity was +61% after administration of 0.05 ml/kg BW of BR 14 and +24% after 0.2 ml/kg BW. In the masticatory muscle, application of the higher dose resulted in a stronger increase in contrast intensity compared to the lower dose. Evaluation of the contralateral base of the skull showed a dose-dependent decrease in acoustic intensity. Bolus injection of BR 14 produces an increase in acoustic intensity, which can be used for the visualization of contrast agent in the brain parenchyma. Using high dosages, a strong signal-enhancing effect in the regions near the ultrasound probe leads to a consecutive attenuation of signals from structures being located beyond ("shadowing-effect"). This is the explanation for the paradoxical result that the higher dose leads to a lower peak signal increase in the brain parenchyma.     

Quantification of flow rates using harmonic grey-scale imaging and an ultrasound contrast agent: an in vitro and in vivo study

It is unclear if the dye-dilution theory and its corresponding parameters are capable of measuring brain perfusion using harmonic grey-scale imaging. We performed a study on a flow phantom using a SONOS 5500 (1.8--3.6-MHz harmonic imaging) and Levovist as the ultrasound (US) contrast agent (UCA). We applied the UCA in six different doses (0.1 to 3.0 mL) and used eight different flow-rates (180 to 540 mL/min). Additionally, we performed a study on dog brain using Levovist boluses of 1.5 mL and 3 mL. We evaluated the influence of dose and flow-rate on the parameters of the time-intensity curve: peak signal intensity (PSI), area under the curve (AUC) and mean transit time (MTT). Along with an increase of the Levovist dose, the AUC and the PSI increased only in the dose range between 0.1 and 0.5 mL Levovist; further increase led to no change of parameters. Flow-rate showed no influence on AUC, MTT or PSI. The dye-dilution theory is not a useful theoretical model for the analysis of perfusion using harmonic grey-scale imaging. A possible explanation for this effect is the bubble saturation.     

Seltene Erkrankungen und Digitalisierung im Kontext des Nationalen Aktionsbündnisses für Menschen mit Seltenen Erkrankungen (NAMSE)

Bundesgesundheitsblatt - Gesundheitsforschung - Gesundheitsschutz - Menschen mit Seltenen Erkrankungen stehen im Gesundheitssystem vor besonderen Herausforderungen. Die Seltenheit der einzelnen...     

Recurrent NF1 gene mutation in a patient with oligosymptomatic neurofibromatosis type 1 (NF1)

We report a 21-year-old male with symptomatic optic glioma who does not fulfill the diagnosis of neurofibromatosis 1 (NF1) according to standard NIH criteria. Analysis of the NF1 gene revealed a recurrent mutation in exon 37 (C6792A or Y2264X). This nonsense mutation causes skipping of exon 37 during the splicing process and is predicted to result in a protein shortened by 34 amino acid residues. The mutation was detected in all tissues examined (blood lymphocytes, oral mucosa, and dermal fibroblasts). The same mutation was previously found in 3 patients with clinically confirmed NF1. To our knowledge, this is the first report of an adult patient carrying a putative (non-mosaic) NF1 gene mutation in multiple tissues but not fulfilling the NIH criteria for the clinical diagnosis of NF1. Am. J. Med. Genet. 86:328–330, 1999.     

Changes in the concentrations of hydroxyproline, glycine and serine in the plasma of haemodialysis patients undergoing erythropoietin therapy

The concentrations of proline, hydroxyproline, glycine and serine were determined in the plasma of 39 haemodialysis patients and 18 healthy subjects, using liquid chromatography with fluorescence detection. Plasma concentrations of the N-terminal immunoreactive parathyrin were also measured. In haemodialysis patients, the plasma concentrations of glycine (p less than 0.01), hydroxyproline (p less than 0.05) and proline (p less than 0.10) were significantly increased, whereas the serine concentrations (p less than 0.01) were decreased, compared with those of the healthy controls. Haemodialysis patients showed greatly elevated plasma N-terminal immunoreactive parathyrin values (greater than 30 pmol/l), which showed a significant correlation with the hydroxyproline values (r = 0.79). Fourteen haemodialysis patients received erythropoietin therapy. In these patients, changes in the concentrations of plasma amino acids were observed up to one year after the beginning of therapy. In the course of the erythropoietin therapy, the plasma concentrations of glycine (p less than 0.05) and hydroxyproline (p less than 0.10) of the haemodialysis patients decreased, whereas the concentration of serine increased (p less than 0.05) to approximately normal values. The results indicate that erythropoietin therapy leads to a normalization of amino acid metabolism.