Amphotericin B severely affects expression and activity of the endothelial constitutive nitric oxide synthase involving altered mRNA stability.

The therapeutic use of the antifungal drug amphotericin B (AmB) is limited due to severe side effects like glomerular vasoconstriction and risk of renal failure during AmB administration. As nitric oxide (NO) has substantial functions in renal autoregulation, we have determined the effects of AmB on endothelial constitutive NO synthase (ecNOS) expression and activity in human and rat endothelial cell cultures. AmB used at concentrations of 0.6 to 1.25 microg ml(-1) led to increases in ecNOS mRNA and protein expression as well as NO production. This was the result of an increased ecNOS mRNA half-life. In contrast, incubation of cells with higher albeit subtoxic concentrations of AmB (2.5 - 5.0 microg ml(-1)) resulted in a decrease or respectively in completely abolished ecNOS mRNA and protein expression with a strongly reduced or inhibited ecNOS activity, due to a decrease of ecNOS mRNA half-life. None of the AmB concentrations affected promoter activity as found with a reporter gene construct stably transfected into ECV304 cells. Thus, our experiments show a concentration-dependent biphasic effect of AmB on expression and activity of ecNOS, an effect best explained by AmB influencing ecNOS mRNA stability. In view of the known renal accumulation of this drug the results reported here could help to elucidate its renal toxicity.

Acute disseminated encephalomyelitis after parenteral therapy with herbal extracts: a report of two cases.

Two patients with acute disseminated encephalomyelitis after repeated injection of extracts from several different plants are described. There was no evidence of prior infection or vaccination. Both patients recovered rapidly after treatment with methylprednisolone. Acute disseminated encephalomyelitis should be considered a rare complication of parenteral therapy with herbal extracts.

A regulatory defect of constitutive no-synthase in islet endothelial cells correlates with probability of disease manifestation in BBdp rats.

AIMS/HYPOTHESIS: Type I (insulin-dependent) diabetes mellitus is characterised by leucocyte infiltration of pancreatic islets and a progressive destruction of insulin-producing beta cells. As endothelial nitric oxide production is known to regulate adhesion molecule expression and leucocyte permeation, we examined the activity and expression of the constitutive nitric oxide synthase (ecNOS) of islet endothelial cells from prediabetic BBdp rats. METHODS: Cultures of aortic endothelial cells and islet capillary endothelial cells were established from young normoglycaemic BBdp rats, Wistar rats and diabetes-resistant BBdr rats, all matched for age. Nitrite and citrulline production was measured in all culture supernatants as indicators for ecNOS activities. Expression of ecNOS mRNA was assessed by reverse transcription-polymerase chain reaction. RESULTS: In contrast to those of the aorta, the Wistar rat islet derived endothelial cells exhibited a strong positive correlation of ecNOS activity with the culture medium glucose concentration but none of the BB rat-derived islet endothelial cells showed a similar glucose-responsiveness. Furthermore, at physiological as well as at increased glucose concentrations islet endothelia from all BBdp rats exhibited a considerable decrease in ecNOS activity by a factor of 3 to 6, indicating a specific dysfunction which is also found for the inducible nitric oxide synthase activity after cytokine challenge but effects were less (2.5 to 3 times) dramatic. In contrast, aorta endothelia from all rats exhibited identical ecNOS activities and no glucose responsiveness. We also found a correlation between ecNOS activities and ecNOS-mRNA expression and can exclude the involvement of the inducible isoform. CONCLUSION/INTERPRETATION: A reproducible and highly significant dysfunction of islet ecNOS expression and activity in young normoglycaemic BBdp rats, which strongly correlates with the probability for disease manifestation is shown.

Heparin inhibits induction of nitric oxide synthase by cytokines in rat brain microvascular endothelial cells.

The inflammatory reaction following ischemic brain injury involves bioactive mediators released mainly from leukocytes. The aim of this in vitro-study was to evaluate possible modulatory actions of heparin on nitric oxide synthesis induced by proinflammatory cytokines. Rat microvascular brain endothelial cells were isolated from adult rat brains and treated with interferon-gamma (IFN-gamma) and interleukin-1beta (IL-1beta) with or without heparin. The expression of inducible nitric oxide synthase (iNOS) mRNA and the iNOS protein activity was analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Griess reagent, respectively. Heparin in a dose-dependent manner attenuated the increase in iNOS expression and NO release after cytokine activation. Thus, in addition to its anticoagulatory effect, heparin may also be effective in the prevention of inflammatory reaction after cerebral ischemia.

Manganese augments nitric oxide synthesis in murine astrocytes: a new pathogenetic mechanism in manganism?

Since manganese (Mn2+) is known to be sequestered in glial cells, we investigated possible neurotoxic mechanisms involving astrocytes in vitro. Low concentrations of Mn2+ were toxic only in astrocyte-neuronal cocultures but not in pure astrocyte or neuronal cultures. As a possible mediator of manganese-derived neurotoxicity, we measured the production of nitric oxide in astrocytes. Manganese, but not other transition metals, dose dependently increased iNOS mRNA and protein levels and the release of nitric oxide in activated astrocytes. This effect was specific for astrocytes, since we observed no stimulation in microglial cells. The observations suggest that besides the known inhibition of mitochondrial function the neurotoxic effect of manganese in low concentrations might be mediated by the increased production of nitric oxide in astrocytes.

The dominant role of exogenous or endogenous interleukin-1 beta on expression and activity of inducible nitric oxide synthase in rat microvascular brain endothelial cells.

In the brain large amounts of nitric oxide are produced in response to various pathological stimuli such as infectious agents, ischemia and trauma. Although it is known that endothelial cells can express the inducible isoform of nitric oxide synthase (iNOS) upon activation, the impact of different cytokines on iNOS expression in rat microvascular endothelial cells remains unclear. We now investigated iNOS mRNA expression and enzyme activity in primary cell cultures of rat microvascular brain endothelial cells after treatment with the proinflammatory cytokines interleukin-1beta (IL-1beta), Tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) alone or in combination. Cells were characterized by immunocytochemistry staining for von-Willebrand-factor and the rat brain endothelial antigen recognized by monoclonal antibody Ox2. iNOS-enzyme activity was determined by measurement of nitrite in the supernatants of cell culture using the Griess-reaction. In addition mRNA expression was analysed by RT-PCR with iNOS and IL-1beta specific primers. All cells in the endothelial cell culture were found to express the antigenic phenotype vWF+/Ox2+/Ox43-, thus identifying the cells as rat brain endothelial cells of microvascular origin. IL-1beta was the only cytokine that as a single stimulus induced iNOS mRNA expression and iNOS-enzyme activity in these endothelial cells. All combinations of two cytokines, including that of TNF-alpha and IFN-gamma--or the triple combination led to expression of iNOS-mRNA and active protein. Cell activation by the combination of TNF-alpha + IFN-gamma led to an early expression of IL-1beta by the endothelial cells suggesting iNOS induction as a consequence of endogenous IL-1beta production under this challenge. The experiments prove that rat brain microvascular endothelial cells express iNOS and produce large amounts of NO under inflammatory conditions. Furthermore, our results indicate a decisive role of IL-1beta in iNOS expression and NO generation.

Cryoglobulinemia: a complication of infectious disease.

We report on a 23-year-old male patient with general weakness, subfebrile temperatures, and arthralgia. The first symptoms were observed some months after a severe cytomegalovirus mononucleosis-gastroenteritis. High titers of cryoglobulins suggested an autoimmunological process. This case is interesting because of the association between cytomegalovirus infection and cryoglobulinemia. In conclusion, the differential diagnosis of autoimmune disease should be considered in the course of viral infections.