High-performance liquid chromatography method with radiochemical detection for measurement of nitric oxide synthase, arginase, and arginine decarboxylase activities.

Nitric oxide has been shown to be involved in numerous biological processes, and many studies have aimed to measure nitric oxide synthase (NOS) activity. Recently, it has been demonstrated that arginase and arginine decarboxylase (ADC), two enzymes that also employ arginine as a substrate, may regulate NOS activity. We aimed to develop a HPLC-based method to measure simultaneously the products of these three enzymes. Traditionally, the separation of amino acids and related compounds with HPLC has been carried out with precolumn derivatization and reverse phase chromatography. We describe here a simple and fast HPLC method with radiochemical detection to separate radiolabeled L-arginine, L-citrulline, L-ornithine, and agmatine. 3H-labeled L-arginine, L-citrulline, agmatine, and 14C-labeled L-citrulline were used as standards. These compounds were separated in the normal phase column (Allure Acidix 250 x 4.6 mm i.d.) under isocratic conditions in less than 20 min with good sensitivity. Using the current method, we have shown the formation of L-citrulline and L-ornithine in vitro using brain tissue homogenate of rats and that of agmatine by Escherichia coli ADC.

Proliferation of T lymphocytes from atopic dermatitis skin is enhanced upon anti-CD3, reduced upon mitogen and superantigen, and negligible upon tuberculin stimulation.

Knowledge about the nature of lymphocytes infiltrating atopic dermatitis skin is restricted to allergen-specific T cells. We investigated the proliferative capacities of T lymphocytes cultured in an antigen-independent way from biopsies of atopic dermatitis skin. When compared with peripheral blood mononuclear cells (PBMC) from healthy donors or atopic dermatitis patients, the skin-homing lymphocytes proliferated more vigorously in response to stimulation with anti-CD3 antibodies (1 microglml), reflecting their high response capacity. When stimulated with phytohemagglutinin (10 microg/ml) or staphylococcal enterotoxin A (0.1 microg/ml) the skin-homing lymphocytes achieved significantly lower proliferation levels than PBMC. In contrast to normal and atopic PBMC the skin-homing lymphocytes did not respond to tuberculin purified protein derivative (10 microg/ml). In the mixed lymphocyte reaction the skin-homing lymphocytes did not stimulate autologous PBMC to proliferate. We conclude that skin-homing lymphocytes have more pronounced immune deviations than PBMC in patients with atopic dermatitis. They represent a valuable approach for further investigating the pathogenesis of the disease.

Telomerase activity is spontaneously increased in lymphocytes from patients with atopic dermatitis and correlates with cellular proliferation.

Telomerase is a ribonucleoprotein enzyme involved with cellular proliferation and cellular senescence. The aim of the present study was to investigate telomerase activity in lymphocytes from patients with atopic dermatitis (AD) and to observe its regulation of cellular proliferation. Peripheral blood mononuclear cells (PBMC) were isolated from 15 patients with AD and 13 healthy donors. Cells were stimulated with purified protein derivative (PPD) of tuberculin (10 microg/ml), interleukin 2 (IL-2) (100 U/ml), anti-CD3 monoclonal antibody (anti-CD3) (1 microg/ml), anti-CD3 plus IL-2, and staphylococcal enterotoxin A (SEA) (0.1 microg/ml). Telomerase activity was measured by the telomeric repeat amplification protocol-based telomerase polymerase chain reaction enzyme-linked immunosorbent assay at 0 and 72 h of incubation. In addition, DNA synthesis of the cells was assayed using 3H-thymidine incorporation. We found that telomerase activity in non-stimulated PBMC from patients with AD was significantly up-regulated without any stimulation during the 72 h of in vitro incubation. The most potent stimulator of telomerase activity was SEA, followed by anti-CD3 plus IL-2, anti-CD3 alone, and PPD. IL-2 did stimulate telomerase activity and DNA proliferation with increasing dosage of IL-2. The DNA proliferation was paralleled by increase in telomerase activity. There was no significant difference between telomerase activity in stimulated lymphocytes from AD patients and normal donors, but the relative increase in telomerase activity tended to be less in AD patients. A spontaneously higher telomerase activity in lymphocytes from AD patients could indicate that T lymphocytes are already stimulated in vivo or that a population of T cells in peripheral blood exhibits an increased telomerase activity compatible with cellular immaturity.

[The accuracy of angiography and CT angiography of the carotid bifurcation compared to macro-morphological correlation]. / Genauigkeit der Angiographie und CT-Angiographie der Karotisbifurkation im Vergleich mit dem makromorphologischen Korrelat.

PURPOSE: To compare the degree of carotid artery stenosis in angiography and CT angiography with the degree of stenosis measured in an intact eversion endarterectomy specimen. METHODS: Preoperative angiograms (intraarterial DSA, 512 x 512 matrix) and CT-angiograms (24 sec spiral scan, slice thickness 2 mm, pitch 1.5) were taken in 12 patients with symptomatic carotid stenosis. Evaluation of the degree of stenosis was performed according to the NASCET ("distal" degree) and ECST ("local" degree) methods. These data were compared with measurements of the surgical specimens. RESULTS: The median "local" degree of stenosis in angiograms was 81.5% (range: 70-99%), in CT angiograms 83% (59-94%) and in specimens 85.5% (65-96%). The "distal" degree of stenosis was 79% (50-99%) in angiograms, 85.5% (55-99%) in CT angiograms and 81% (52-95%) in specimens. CT angiography slightly overestimated the degree of stenosis compared with the specimen, whereas angiography slightly underestimated the true degree of stenosis. However, these differences were not statistically significant. CONCLUSION: CT angiography is able to predict the degree of internal carotid stenosis when compared with an intact surgical specimen. It is as accurate as the "gold standard" of invasive angiography.