Rosuvastatin upregulates the antioxidant defense protein heme oxygenase-1.

Cholesterol-independent, pleiotropic actions of HMG-CoA reductase inhibitors (statins) lead to anti-inflammatory and antioxidant actions by as yet unidentified mechanisms. This study explores the role of heme oxygenase-1 (HO-1) as target and potential mediator of rosuvastatin. In cultured human endothelial cells (ECV 304), rosuvastatin increased HO-1 mRNA and protein levels in a concentration-dependent fashion. HO-1 induction by rosuvastatin remained unaffected by mevalonate and N-nitro-L-arginine-methylester, showing that isoprenoid- and NO-dependent pathways were not involved. Pretreatment of endothelial cells with rosuvastatin reduced NADPH-dependent production of oxygen radicals. The HO-1 metabolite bilirubin, when added exogenously to the cells, virtually abolished NADPH-dependent oxidative stress. Rosuvastatin-induced inhibition of free radical formation was rescued in the presence of the HO inhibitor, tin protoporphyrin-IX. Our results demonstrate that HO-1 is a target site and antioxidant mediator of rosuvastatin in endothelial cells. This novel pathway may contribute to and partially explain the pleiotropic antiatherogenic actions of rosuvastatin.

Aspirin induces nitric oxide release from vascular endothelium: a novel mechanism of action.

1. The study was designed to test the hypothesis that aspirin may stimulate nitric oxide (NO) release from vascular endothelium, a pivotal factor for maintenance of vascular homeostasis. 2. Clinical evidence suggests that low-dose aspirin may improve vascular endothelial function. Since other cyclooxygenase (COX) inhibitors showed no beneficial vascular effects, aspirin may exhibit a vasculoprotective, COX-independent mechanism. 3. Luminal NO release was monitored in real time on dissected porcine coronary arteries (PCA) by an amperometric, NO-selective sensor. Additionally, endothelial NO synthase (eNOS) activity was measured in EA.hy 926 cell homogenates by an l-[(3)H]citrulline/l-[(3)H]arginine conversion assay. Superoxide scavenging capacity was assessed by lucigenin-enhanced luminescence. 4. Aspirin induced an immediate concentration-dependent NO release from PCA with an EC(50) of 50 nm and potentiated the NO stimulation by the receptor-dependent agonist substance P. These effects were independent of an increase in intracellular calcium and could be mimicked by stimulation with acetylating aspirin derivatives. The aspirin metabolite salicylic acid or the reversible cyclooxygenase inhibitor indomethacin failed to modulate NO release. Incubation of soluble eNOS for 15 min with 100 microm aspirin or acetylating aspirin analogues increased the l-[(3)H]citrulline yield by 40-80%, while salicylic acid had no effect. Aspirin and salicylic acid showed a similar, but only modest, magnitude and velocity of superoxide scavenging. 5. Our findings demonstrate that therapeutically relevant concentrations of aspirin elicit NO release from vascular endothelium. This effect appears to be due to a direct acetylation of the eNOS protein, but is independent of COX inhibition or inhibition of superoxide-mediated NO degradation.

A common pathway for nitric oxide release from NO-aspirin and glyceryl trinitrate.

NO-Aspirin (NCX-4016) releases nitric oxide (NO) in biological systems through as yet unidentified mechanisms. In LLC-PK1 kidney epithelial cells, a 5-h pretreatment with glyceryl trinitrate (GTN, 0.1-1 microM) significantly attenuated the cyclic GMP response to a subsequent challenge with both NO-aspirin or GTN. Similarly, NO-aspirin (10-100 microM) was found to induce tolerance to its own cyclic GMP stimulatory action and to that of GTN. In contrast, cyclic GMP stimulation by the spontaneous NO donor SIN-1, which releases NO independently of enzymatic catalysis, remained unimpaired in cells pretreated with GTN or NO-aspirin. The observed cross-tolerance between NO-aspirin and GTN cells indicates that bioactivation pathways of organic nitrates, which have been shown to involve cytochrome P450, may also be responsible for NO release from NO-aspirin. Prolonged treatment with NO-aspirin causes down-regulation of the cellular cyclic GMP response, suggesting that tolerance may occur during therapy with NO-aspirin.

Learning to deal with drug-dependent patients: one physician's reflections.

Physicians with a contemporary education may not be adequately trained to deal effectively with drug-dependent patients. This paper details the problems that one physician encountered with such individuals in his practice. A retraining program was set up in which he received basic education in drug dependence and became involved in individual counselling with drug abusers and in research studies on alcoholism and drug abuse. Physicians must exercise caution when prescribing medications that are potentially addictive. They must have a responsible attitude in their care of drug-dependent patients. The assessment and treatment of such patients should be carried out only by a multidisciplinary team of health care professionals. These principles are best inculcated by the proper exposure of medical students to substance-abuse problems and by the availability of appropriate courses and studies in this area to practising physicians.

Isolation of human tumour-specific antigens associated with beta2 microglobulin.

In the present study the tube LAI assay was used to monitor the isolation of the TSA of 4 different types of human cancers. Each tumour antigen was found to be specific for tumours arising in the organ from which the TSA was initially derived and which were histopathologically similar. Immunochemical studies revealed that these molecules co-isolate with normal human HLA antigens and are associated with beta2m. On Sephadex G-150, the majority of the papain-solubilized tumour antigen eluted in the mol. wt range 70,000-150,000. Analysis of this material by SDS-PAGE and 6M guanidine-HC1 column chromatography indicated that the material is composed of smaller subunits with prominent peaks at approximately 40,000, 25,000 and 12,000 mol. wt. Immunoadsorbent affinity chromatography of the solubilized tumour-membrane constituents on AH-Sepharose-linked horse anti-human-beta2m indicated that the tumour antigens, like HLA molecules, contain a beta2m subunit. The specificity of binding of TSA to the immunoadsorbent columns and the immunologically specific abrogation of LAI reactivity were clearly shown. The present study, therefore, indicates that by the isolation of beta2m, human tumour antigens can also be isolated, since human tumour antigens are associated with beta2m. Whether human TSAs may perhaps be modified histocompatibility antigens remains to be answered. Although the change upon malignant transformation in the pattern of the cell-surface proteins expressing the TSA determinant remains obscure, it would appear that for tumours arising within a given organ, a consistent alteration of cell-surface proteins occurs.

An overview: antitumor immunity in breast cancer assayed by tube leukocyte adherence inhibition.

The adherence to glass of human peripheral blood leukocytes (PBL) incubated with tumor antigen in vitro, is specifically inhibited if the PBL are sensitized to the antigen. The presence of leukocyte adherence inhibition (LAI) to tumor extracts indicates the presence of systemic antitumor immunity. By the tube leukocyte adherence inhibition assay (tube LAI), it was shown that 85% (191 of 223) Stage I and II, 45% (15 of 34) Stage III and 29% (30 of 103) Stage IV breast cancer patients had LAI reactivity. LAI responsiveness diminished with an increased tumor burden and most patients with advanced cancer exhibited no LAI reactivity. When LAI reactivity was monitored for 1 to 6 months after surgery, 13 of 25 Stage I and II breast cancer patients were negative on the first repeat assay. In general, 7 months after mastectomy most patients clinically free of cancer showed no LAI reactivity. Of thirty-five patients tested between 7 and 18 months after mastectomy, 6 were positive and 4 of the positives had local recurrence. The phenomenon of tube LAI appears to be mediated by monocytes armed with cytophilic antitumor antibody. The serum of patients whose leukocytes responded in the tube LAI assay had free cytophilic antitumor antibody that "armed" or sensitized normal leukocytes to respond in the LAI assay. Serum arming paralleled leukocyte reactivity before and after surgery. Patients with advanced cancer whose leukocytes failed to react in the LAI assay had serum blocking factors (excess tumor antigen) that abrogated the LAI reactivity of leukocytes from reactive patients.

Tube leukocyte adherence inhibition assay for the detection of anti-tumor immunity. I. Monocyte is the reactive cell.

Peripheral blood leukocytes (PBL) from breast cancer patients with early or localized cancer fail to adhere to glass in the presence of breast cancer extract. The same leukocytes do not react to unrelated tumour extracts. Enrichment and depletion of certain PBL populations from patients with apparently localized breast cancer indicated that the indicator and/or reactive cell manifesting non-adherence in the presence of appropriate tumour antigen was phagocytic, glass adherent in the absence of tumour antigen and had cell surface Fc-receptors. The cell involved, therefore, appears to be the circulating monocyte. These results show that in the tube LAI assay, the peripheral blood monocyte appears to react directly with the tumour antigen resulting in a loss of its property of adherence to glass.

Tube leukocyte (monocyte) adherence inhibition assay for the detection of anti-tumour immunity. III. "Blockade" of monocyte reactivity by excess free antigen and immune complexes in advanced cancer patients.

Leukocytes from patients with limited cancer display LAI reactivity whereas leukocytes from patients with metastatic cancer frequently demonstrate no reactivity in the tube LAI assay. The leukocytes (monocytes) of reactive patients react with tumour antigen through specific cytophilic anti-tumour IgG antibody bound to the monocyte's Fc cell surface receptors. The non-reactive monocytes from patients with advanced cancer lacked the ability to bind free cytophilic anti-tumour antibody. Moreover, the serum of the non-reactive patient contained no free cytophilic anti-tumour antibody capable of "arming" normal leukocytes. The serum of patients with large tumour burdens contained free tumour antigenic determinants capable of absorbing free cytophilic anti-tumour antibody from the serum of reactive patients or when preincubated with reactive leukocytes abrogating their LAI responsiveness immunologically specifically. Blocking was immunologically specific; therefore, the specificity must reside in the tumour antigenic determinant since immune complexes are bound nonspecifically. The tumour antigen coat was removed by gentle trypsinization of the monocyte's surface. This restored the monocyte's capacity to react with the sensitizing tumour antigen and to bind free cytophilic antibody from the microenvironment. Nonreactivity in the tube LAI assay of patients with metastatic cancer was not the result of a numerical deficit of circulating monocytes but was mediated by an excess of tumour antigen in the microenvironment of the sensitized monocyte.

Tube leukocyte adherence inhibition assay for the detection of anti-tumour immunity. II. Monocyte reacts with tumour antigen via cytophilic anti-tumour antibody.

The peripheral blood monocyte is the reactive cell in the tube LAI assay. The monocyte loses its properties of adherence to glass upon exposure to specific antigen. Two different experiments to determine if lymphocytes, when they reacted with tumour, released mediators that were responsible for inhibiting monocyte glass adherence, gave negative results. The mechanism wherby the specific tumour antigen appeared to be recognized was the binding of cytophilic IgG antitumour antibody to receptors on the cell surface of the monocyte. The results of the experiments indicate that normal peripheral blood monocytes could be made specifically reactive ("armed") to the tumour extract by incubating normal peripheral blood leukocytes with serum from a reactive cancer patient. IgG isolated from "arming" sera was shown to have the capacity to sensitize normal leukocytes. Patients with breast cancer or malignant melanoma with limited tumour burdens had free cytophilic anti-tumour antibody in their serum, whereas the serum of patients with large tumour burdens (metastatic cancer), whose leukocytes did not react in the tube LAI assay, did not "arm".

Cell-mediated antitumor immunity in breast cancer patients evaluated by antigen-induced leukocyte adherence inhibition in test tubes.

Tumor antigen-induced inhibition of leukocyte adherence was adapted and modified for use in glass test tubes for the study of cell-mediated antitumor immunity to human adenocarcinoma of the breast. Peripheral blood leukocytes from 40 to 47 patients with proven breast cancer responded to an antigenic extract of breast cancer with significant leukocyte adherence inhibition, whereas only 2 of 32 controls showed a response. Further, 7 patients with histologically proven benign breast disease did not react to the breast adenocarcinoma extract, indicating that only breast cancer patients have leukocytes sensitized to the breast cancer antigen. The cell-mediated antitumor response of the breast cancer patient was dependent on the stage of the cancer, and patients with disseminated cancer had decreased responsiveness. In fact, 4 of 7 breast cancer patients who had no response in the assay had disseminated breast cancer. Also, surgery and irradiation depressed leukocyte adherence inhibition responsiveness. Chromatographic fractionation on Sepharose 4B of the breast cancer extract showed that the antigenic component was greater than 10(6) daltons. The responsive cell in the assay interacts directly with the tumor antigen, and as a result subsequent adherence to glass is inhibited. The assay described is a comparatively simple and sensitive technique for demonstrating cell-mediated antitumor immunity and appears to be immunologically specific.