Inhibition of LDL oxidation by antioxidants.

Low density lipoprotein (LDL) consists of about 3000 fatty acids (50% polyunsaturated) and a single molecule apolipoprotein B (500 kDa). The endogenous antioxidants of LDL consist mainly of tocopherols and few carotenoids, which protect the PUFAS against oxidation. That native LDL contains traces of oxidation products has not been proved yet. Oxidatively modified LDL (oLDL) exhibits cytotoxic and chemotactic activities, furthermore it leads to foam cell formation, a critical step in atherogenesis. The oxidation of LDL is a free radical process and leads to various aldehydic products. The oxidation of LDL is initiated by cells as well as by transition metals like Cu2+. In both cases the oxidation goes through three consecutive phases. The lag-phase is characterized by minimal degradation of PUFAs but a loss of the antioxidants. Thereafter the PUFAs are oxidized to lipid hydroperoxides, which are only intermediates (propagation-phase). These intermediates will decompose to aldehydic products, accompanied by several additional changes in the LDL particle (decomposition-phase). For increased macrophage uptake oLDL must reach the late decomposition-phase; the presence of lipid hydroperoxides in LDL is not sufficient. It is suggested that binding of aldehydes to free amino groups of Apo B is the reason for macrophage uptake. This is supported by the finding that antibodies against aldehyde-modified LDL are able to recognize oxidized LDL in atherosclerotic lesions. Antioxidants like alpha-tocopherol are able to protect LDL against oxidation. The duration of the lag-phase shows a linear relationship with the content of alpha-tocopherol in LDL. Yet the efficiency of alpha-tocopherol to protect LDL shows strong individual variation.


Desialylated low density lipoproteins and atherosclerosis.

Oxidative modification of LDL is accompanied by a number of compositional and structural changes, now well known. In addition, other atherogenic modifications of LDL exist, such as desialylation. The present article summarizes the recent data related to desialylated LDL and to the presence of these LDL in blood plasma of patients with coronary atherosclerosis. In addition, this review examines the sensitivity of these LDL to peroxidative stress.


Molecular basis of alpha-tocopherol inhibition of smooth muscle cell proliferation in vitro.

The molecular events responsible for the inhibition of cell proliferation by alpha-tocopherol have been investigated. Smooth muscle cells in vitro have been shown to be specifically inhibited by alpha-tocopherol with a concomitant inhibition of protein kinase C activity. beta-Tocopherol was inactive, despite its similar radical scavenging activity. The point of inhibition of alpha-tocopherol relative to the cell cycle was localized in the late G1 phase. A second effect of alpha-tocopherol observed with smooth muscle cells was the stimulation of protein kinase C biosynthesis in both the S and G2 phases of the cell cycle. The implications of these findings for the onset of arteriosclerosis are discussed.


Oxidative damage in Alzheimer's dementia, and the potential etiopathogenic role of aluminosilicates, microglia and micronutrient interactions.

While evidence implicating free radical oxidative processes in the etiopathogenesis of Alzheimer's dementia is accumulating, the specific cellular and biochemical mechanisms involved remain to be identified. The potential pathogenic role of microglial cells in neurodegenerative processes is indicated by the finding that purified murine microglial cells exposed in vitro to various model aluminosilicate particles stimulate the generation of tissue-injurious free radical reactive oxygen metabolites. Analogous inorganic aluminosilicate deposits have been reported to occur in the core of the characteristic senile plaques found in the brains of Alzheimer disease subjects. The possible modulation of free radical oxidative activity by antioxidant micronutrients and pharmacological agents, provides a rational basis for further preventative and therapeutic clinical investigations.


The role of alterations in free radical metabolism in mediating cognitive impairments in Down's syndrome.

Down's syndrome (DS) is a genetic disorder involving an excess of chromosome 21 (trisomy 21) in approximately 96% of the cases and comprises approximately 15% of the population with mental retardation (Heller, 1969). In addition to the constitutional mental deficiencies associated with the syndrome many DS patients develop dementia associated with Alzheimer's disease (AD) in their later years of life (Thase et al., 1984). The genetic locus for Cu,Zn-superoxide dismutase (SOD1), a key enzyme in free radical metabolism, is located on chromosome 21, and the activity level of this enzyme is elevated by approximately 50% in a variety of cells of DS patients (see Kedziora and Bartosz, 1988; Sinet, 1982). Because alterations in free radical metabolism may be involved in neuronal death and may be associated with a number of pathological manifestations of DS, it is important to understand the role of free radical metabolism in cognitive impairments of DS, the topic discussed in this chapter.


Evidence for drug metabolism as a source of reactive species in the brain.

Several pathways for reactive species formation involving xenobiotic metabolism exist in the brain. They include oxidative activation by different enzymatic systems like cytochrome P-450 and monoamine oxidases, and superoxide radical production issued from reductive xenobiotic metabolism. They may contribute to cellular impairment observed in various physiopathological situations.


Anticarcinogenic activities of carotenoids in animals and cellular systems.

A large number of studies have indicated that carotenoid pigments act as anticarcinogenic agents in animals treated with either ultraviolet light, ultraviolet light with chemicals, or with chemical carcinogens alone. Although pharmacological doses of cartenoids were used in the early experiments, more recent evidence indicates that relatively small doses can be effective. These studies have been complemented by investigations in bacteria and mammalian tissue, either in cell culture or in organ culture, where it has been demonstrated that various carotenoid pigments can prevent mutagenesis, genotoxic effects, or malignant transformation. It would appear that these effects are intrinsic to the carotenoid molecule, and not necessarily due to the metabolic conversion to retinoids. Partially based on these observations, it has been suggested that carotenoid pigments may function as chemopreventive agents for reducing the risk of cancer in humans. Numerous studies are underway to test this hypothesis.


Aging and cancer: plasma antioxidants and lipid peroxidation in young and aged breast cancer patients.

The relationship between aging and cancer is complex because the intrication takes place at the cell, the organism and the environment levels. On the other hand, carcinogenesis is a multi-step process, and different mechanisms may be involved in each step. For example, oxidants and antioxidants may play a different role depending upon the phase considered. Tumors in older patients are generally described as slow growing. The difference in tumor aggressiveness between young and older patients is especially obvious in breast cancer patients. The age specificity of some breast risk factors suggests that breast cancer which has been diagnosed in an aged woman was induced late in her life. We address the question whether the characteristics of a senescent organism with regards to oxidant-antioxidant status could be causally related to the slow evolution of tumors in old patients.


DNA damage in mammalian cell lines with different antioxidant levels and DNA repair capacities.

A wide range of DNA damage is known to be caused by reactive oxygen species (ROS). Defence against the effects of such damage include damage prevention (e.g. antioxidant activity) and the removal of damaged moieties from DNA (DNA repair). Radiation (X-ray) sensitive murine lymphoma (LY) cells were seen to be more susceptible to ROS-induced damage than were radiation resistant cells. This difference was unlikely to be due to the marginally decreased DNA excision repair capacity of the sensitive cells. Radiation sensitive cells did, however, have lower endogenous antioxidant enzyme levels. Thus, the importance of assessing all levels of a cell's response to ROS, in determining the major factors leading to increased mutagen sensitivity, is emphasised.


Effect of photooxidation on the eye lens and role of nutrients in delaying cataract.

The function of the eye lens is to collect and focus light on the retina. To do so, it must remain clear during the decades of life. Upon aging, lens constituents are damaged and precipitate in opacities called senile cataracts. Laboratory and epidemiologic data indicate that the damage is due in part to light and active forms of oxygen. Antioxidant nutrients - ascorbate, carotenoids, and tocopherol - appear to offer protection against cataract. Fifty million persons worldwide are blind due to cataract, and, in the U.S., there are 1.2 million cataract surgeries performed at an annual cost (including physician visits) of over $3.2 billion. It has been estimated that a 10-year delay in the development of cataract would eliminate the need for half the surgeries. Since it will not be possible to replace most of the damaged lenses, it is essential to determine the efficacy of supplying adequate levels of antioxidant nutrients early in life to preserve lens function.


Carotenoids in the retina--a review of their possible role in preventing or limiting damage caused by light and oxygen.

Two of the circa 600 naturally occurring carotenoids, zeaxanthin and lutein, the major carotenoids of maize and melon respectively, are the constituents of the macula lutea, the yellow spot in the macula, the central part of the retina in primates and humans. Of the circa ten carotenoids found in the blood these two are specifically concentrated in this area, which is responsible for sharp and detailed vision. This paper reviews the ideas that this concentration of dietary carotenoids in the macula is not accidental, but that their presence may prevent or limit damage due to their physicochemical properties and their capability to quench oxygen free radicals and singlet oxygen, which are generated in the retina as a consequence of the simultaneous presence of light and oxygen. Additionally, in vitro and in vivo animal experiments are reviewed as well as observational and epidemiological data in humans. These show that there is enough circumstantial evidence for a protective role of carotenoids in the retina to justify further research. Some emphasis will be put on age-related macular degeneration (AMD), a multifactorial degenerative retinal disease for which the exposure to light and thus photochemical damage has been suggested as one of the etiological factors. Recent attempts at nutritional intervention in this condition will also be reviewed.


Oxidative stress in diabetic retina.

The authors describe the alterations usually associated with diabetic retinopathy. They concern the classical thickening of the basal membrane of retinal capillaries and the associated modification of retinal vessel permeability. These alterations correspond to the blood-retinal barrier disruption. The authors then discuss the participation of oxygenated free radicals in the pathogenesis of diabetic retinopathy. They report several experimental studies establishing such a participation and finally describe their own results obtained on a model of retinas isolated from alloxan-induced diabetic rats. After one month of evolution, the electroretinograms (ERG) recorded on isolated retinas from diabetic rats had an amplitude about 20% lower than the controls, whereas after two months of diabetes, this decrease was about 60%. Under these conditions, the authors tested the protective properties of Ginkgo biloba extract (EGb 761) on their model. They observed that in EGb-treated animals (100 mg/kg/day), the ERG had a significantly (p less than 0.001) greater amplitude than untreated animals after two months of diabetes evolution. In conclusion, the authors discuss the possible utilization of a free radical scavenger, such as EGb 761, in the prevention of the retinal impairment in diabetes.


Genetic stability and oxidative stress: common mechanisms in aging and cancer.

Much evidence indicates that age-related diseases as well as the aging process itself may be a result of genetic instability, resulting in cells changing their proper state of differentiation. Such genetic changes could be of an epigenetic or genetic nature (or both) and suggest that mechanisms acting to stabilize the differentiated state of cells could be major determinants of animal species' general health and longevity. There is also much data indicating that a causative factor in cancer is genetic instability, suggesting the importance of genetic stabilized factors governing the frequency of this disease. Possibly related to these two observations is the good positive correlation between rate of cancer and the rate of aging of different mammalian species. In addition, cells from longer-lived species appear to have a higher intrinsic ability to maintain their proper differentiated state and animals whose life span has been extended by food restriction also have a postponed onset of cancer. These data suggest the possibility that genetic instability may represent a common causative mechanism for both aging and cancer. This suggestion is supported by evidence indicating that oxidative stress can increase cancer frequency and that longer-lived species appear to have a lower oxidative stress state. However, there appears to be a serious lack of evidence indicating that physical and chemical mutagenic agents accelerate aging as they do cancer. For these and other considerations, it is proposed that aging is not a result of genetic instability as are many different age-related diseases. Thus, physiological aging and some age-related diseases could have separate and distinct mechanisms of causation and control.


Free radicals and aging of the skin.

Cutaneous aging is the result of genetically determined or intrinsic aging superimposed by degenerative changes due to actinic irradiation, also called photoaging. The manifestations of cutaneous aging, as it relates to the perception of age, is caused by ultraviolet light, in particular in those parts of the body exposed daily to solar radiation. Free radical generation in the skin by UV light and from other sources, such as cellular infiltrations or the xanthine oxidase reaction, may be detected by direct and indirect methods. The decrease in antioxidant enzymes and small molecular weight antioxidants such as glutathione, vitamin E and ubiquinone upon exposure to UV light is an indication that the pro-antioxidant balance can be overwhelmed by acute or chronic photo-oxidative stress. Antioxidant supplementation is therefore a means for prevention or at least retardation of premature cutaneous aging.


Modulators of free radical activity in diabetes mellitus: role of ascorbic acid.

Free radical mechanisms are increasingly being implicated in the pathogenesis of tissue damage in diabetes. Various sources of free radicals may modulate oxidative stress in diabetes, including non-enzymatic glycosylation of proteins and monosaccharide autooxidation, polyol pathway activity, indirect production of free radicals through cell damage from other causes, and reduced antioxidant reserve. Ascorbic acid, which may be a principal modulator of free radical activity in diabetes, is shown to be consumed, presumably through free radical scavenging, thus preserving levels of other antioxidants such as glutathione.


Possible role of free radicals in the chronic inflammation of the gut.

Recent studies have demonstrated that intracolonic administration of trinitrobenzenesulfonic acid (TNB) dissolved in ethanol produces chronic colitis in rats, and that this model shares many features of human inflammatory bowel disease (IBD), particularly Crohn's disease. We investigated the role of free radicals in the pathogenesis of this colitis model. In the early stage of this colitis, antioxidant enzymes (such as superoxide dismutase, glutathione peroxidase) and an antioxidant, alpha-tocopherol, were significantly decreased with the severity of colonic damage. Mn-SOD at a dose of 50000 U/kg attenuated this colitis when preadministered subcutaneously one hour before the induction of colitis. These results suggest that oxygen-derived free radicals may play an important role in this colitis.


Age-related variations of enzymatic defenses against free radicals and peroxides.

Superoxide dismutase, glutathione peroxidase and catalase are the three main enzymatic systems of defense of the organism against free radicals and peroxides. A survey of the literature shows that no general tendency of evolution of these systems in aging emerges, even if some recent studies in humans demonstrate the existence of a concomitant decrease in most of the antioxidant enzymes in blood of the elderly. The study of the antioxidant systems and their interrelations in the elderly represents a large field of future investigations.


Antioxidant status (selenium, vitamins A and E) and aging.

Antioxidant status can be evaluated by blood selenium, vitamins A and E. The level of selenium was determined in whole blood, erythrocytes and plasma of 170 French people (70-95 years old) healthy and with intercurrent illness, by using PIXE (proton-induced X-ray emission analysis). These results are discussed with other values from the literature. Plasma levels of vitamins A and E have been measured by HPLC. All data were compared with those obtained for younger subjects. Healthy elderly people residing in a geriatric home received selenium supplements during 1 month. The influence of this supplementation brought to light a deficiency for this trace element. The correlation of aging and nutritional requirements with immune function, heart and cancer death rate is presented and discussed.


Inverse correlation between essential antioxidants in plasma and subsequent risk to develop cancer, ischemic heart disease and stroke respectively: 12-year follow-up of the Prospective Basel Study.

There is accumulating evidence that free radicals may contribute to various diseases such as cancer or cardiovascular disease. Possible health hazards can to some extent be prevented by the body's multilevel defense system against free radicals, which comprises, besides others, antioxidant vitamins. The 12-year mortality follow-up of 2,974 participants of the Basal Study allowed to test the hypothesis that low antioxidant vitamin plasma concentrations (vitamin A, C, E and carotene) were associated with increased death from cancer of various sites and death from atherosclerosis such as ischemic heart disease and stroke, respectively. For the analysis 204 cancer cases, 132 fatalities from ischemic heart disease (IHD) and 31 deaths from cerebral vascular disease were available. Cancer mortality. Overall mortality from cancer was associated with low mean plasma levels of carotene adjusted for cholesterol (p less than 0.01) and of vitamin C (p less than 0.01). Bronchus and stomach cancers were associated with a low mean plasma carotene level (p less than 0.01). Subjects with subsequent stomach cancer had also lower mean vitamin C and lipid-adjusted vitamin A levels than survivors (p less than 0.05). Calculating the relative risk with exclusion of mortality during the first two years of follow-up, low plasma carotene was associated with an increased risk for bronchus cancer (RR 1.8, p less than 0.05), and the small number of stomach cancer cases (RR 2.95, p less than 0.05) low plasma levels of carotene and vitamin A with all cancer types (RR 2.47, p less than 0.01), and low plasma retinol in older subjects (greater than 60 years) with lung cancer (RR 2.17, p less than 0.05). Studies in other cohorts with a poor vitamin E status revealed an increased risk of subsequent cancer at low vitamin E levels as well. It is concluded that low plasma levels of all major essential antioxidants are associated with an increased risk of subsequent cancer mortality. Cardio-vascular mortality. Plasma carotene concentration below quartile 1 was associated with an increased risk for IHD (RR 1.53, p = 0.02). The same was true for low levels of both carotene and vitamin C (RR = 1.96, p = 0.022). The risk of cerebrovascular death was elevated in subjects with low carotene in the presence of low vitamin C plasma concentration (RR 4.17, p less than 0.01). These data confirm and extend recent findings on an inverse correlation of beta-carotene and vitamin C respectively to CVD.(ABSTRACT TRUNCATED AT 400 WORDS)


Vitamin E requirement in relation to dietary fish oil and oxidative stress in elderly.

A growing body of evidence shows that oxygen radicals and other products of free radical reactions are involved in aging and age-related degenerative diseases. Recent studies have suggested that fish oils (FO) have a potentially beneficial effect on age-associated diseases. Consumption of FO may increase requirement for vitamin E, especially under conditions where oxidative stress is increased. Vitamin E requirement increases with increased intake of dietary polyunsaturated fatty acids (PUFA). This relationship may be exaggerated in elderly subjects. Our studies, as well as those of others, have shown that plasma lipid peroxides are significantly higher in older subjects compared to young subjects. Thus, in conditions where the percentage of highly unsaturated fatty acid increases in the membrane, older subjects may be more susceptible to oxidative damage. In a series of human studies, we found that older women, receiving FO supplements for 3 months exhibited a greater increase in plasma PUFA compared to young subjects. By substituting membrane fatty acids with the potentially unstable (n-3) fatty acids of FO, older subjects were found to be at greater risk of oxidative stress than young subjects. In addition, when exposed to eccentric exercise-induced oxidative stress, older men, receiving vitamin E supplements for 48 days, exhibited significantly lower levels of lipid peroxides in urine compared to placebo control. These data indicate that older subjects are more susceptible to oxidative stress and may benefit from the antioxidant protection provided by vitamin E.


Vitamin C and vitamin E--synergistic interactions in vivo?

The synergistic relationship between ascorbic acid (vitamin C) and alpha-tocopherol (vitamin E) in the inhibition of lipid peroxidation has been known for some time and is now well established in vitro systems. The possibility that ascorbic acid may also reduce tocopheroxyl radicals in vivo is a subject of some interest and speculation. Although not all experiments have failed to suggest a synergistic antioxidant interaction, recent data indicate that the postulated synergism between these vitamins might be relatively unimportant compared with other metabolic processes.


The threshold of age in exercise and antioxidants action.

Physical activity and exercise are important factors in determining the quality of life in old animals and humans. With age there is a slow but significant reduction in muscle mass and ability to perform certain physical activities. This may be due to changes with the age of muscle composition and protein turnover, as well as decrease of trophic influence in neural control of muscles of old individuals. Exercise in general was shown to improve muscle performance even in old age. However a concept of threshold of age in exercise was advanced forward in the 1970s. Accordingly, the idea was that for a given exercise of a particular duration and intensity there is a certain age beyond which this exercise may not have a positive influence, but can become detrimental to the exercising animal or human. Recent studies on the effect of antioxidants such as Vitamins C and E and selenium have shown that these agents could decrease the free radical associated muscle damage caused by extensive exercise. Thus, administration of these antioxidants especially vitamins C and E may reduce the oxidative damage due to exercise, and may alter the threshold of age by delaying it to an older age.


Antioxidant therapy in the aging process.

A total of 1,265 patients with age-related diseases such as diabetes, arthritis, vascular disease and hypertension as well as 1,100 persons in diminished health without apparent disease, were treated with the metal chelator EDTA and antioxidants such as vitamin C, E, beta-carotene, selenium, zinc and chromium. Good results were observed in the majority of patients. This is encouraging for the initiation of controlled clinical trials.


Oxygen-induced mitochondrial damage and aging.

Although the views of Harman and Gerschman provide a reasonable explanation for many of the effects of aging, they fail to explain why many cell types, from amoebae to mammalian spermatogonia, do not show a time-related involution, while other cells (especially the neurons) change with age. We feel that a better understanding of senescence (from the molecular to the organ and organismic levels) can be gained by integrating the free radical theory of aging with the classic concepts of Minot and Pearl on the role of cell differentiation and metabolic rate in, respectively, triggering and pacing senescence. In agreement with the above, we maintain that aging is the non-programmed but unavoidable "side effect" of oxy-radical damage to the membrane and genome of the mitochondria of irreversibly differentiated cells. If oxy-radical damage to mtDNA occurs, it will block the rejuvenation of the mitochondrial population by the process of organelle division, thus leading to bioenergetic decline and cellular death.


Instabilities of metabolic regulations in aging.

1) That non-invasive NMR and optical methods can a) quantify the work stress on mitochondria for ATP production, and b) indicate the tissue O2 tension in the capillary bed that is responsible for the rate of radical generation. 2) That free radical damage to mitochondrial function can be quantified by reciprocal plots of inverse slope giving the extrapolated Vm of mitochondria. 3) That a particular genetically deficient individual requiring high dosages of menadione has survived over 9 years. 4) That mitochondrial deficiency leads to an exercise hyperoxia.


Lipofuscinogenesis in a model system of cultured cardiac myocytes.

Lipofuscin, or age pigment, is a yellowish-brown, autofluorescent, protein, and lipid containing pigment that accumulates in the lysosomal vacuome of a variety of post-mitotic cells in man and animals during aging. Lipofuscin seems to be an end product of peroxidation, fragmentation and polymerization of proteins and lipids. Protein and lipid containing materials are regularly sequestered to the lysosomal system by means of endocytosis and autophagocytosis. Lysosomes and acidified endosomes constitute cellular compartments where iron for short periods of time may exist in reactive form allowing for peroxidative processes. The importance was demonstrated in model systems of cultured cells subjected to oxidative stress and augmented amounts of FeCl3 in the culture medium. This combination was considered to result in increased intracellular, mainly mitochondrial, production of hydrogen peroxide as well as increased intralysosomal concentration of iron. One of the results of this situation was dramatically increased lipofuscinogenesis.


Cellular clones and transgenic mice overexpressing copper-zinc superoxide dismutase: models for the study of free radical metabolism and aging.

Down's Syndrome (DS), the most frequent of congenital birth defects, results from the trisomy of the chromosome numbered 21 in all cells of affected patients. This disease is characterized by developmental anomalies, mental retardation and features of rapid aging, particularly in the brain where the occurrence of Alzheimer's disease (AD) is observed in all trisomy 21 patients over the age of 35. Elucidation of the biological mechanisms leading to brain aging in DS might provide new insight into the understanding of brain aging and AD in normal people. Copper-zinc superoxide dismutase (CuZnSOD) is one of the genes encoded by chromosome 21. As a consequence of gene dosage excess, CuZnSOD activity and protein are increased by 50% in all DS tissues. The level of CuZnSOD protein and mRNA is particularly high in hippocampal pyramidal neurons susceptible to degenerative processes in AD and in dopaminergic melanized-neurons vulnerable in Parkinson's disease. Increased CuZnSOD activity in these age-related neurodegenerative disorders might result in H2O2 overproduction and subsequently promote peroxidative damages within cells. Increase of seleno-dependent glutathione peroxidase (Se-GPx) in DS cells supports this concept. In order to test this hypothesis, cell and animal models of CuZnSOD overexpression have been designed. In cells transfected with the human CuZnSOD gene, and increased Se-GPx activity is observed, a situation which mimics DS. In mice transgenic for the human CuZnSOD, the expression pattern of the transgene in the brain is similar to that in humans, and we can observe an increased peroxidation in this tissue. These data, like others in the literature, support the hypothesis that excess CuZnSOD induces an imbalance in the regulation of oxygen-derived free radical production which might result in peroxidative brain damage and possibly contribute to accelerated aging and age-related neuropathology.


The importance of antioxidant enzymes in cellular aging and degeneration.

Aerobic cells contain various amounts of the three main antioxidant enzymes: superoxide dismutase (SOD), catalase and GSH peroxidase. These three enzymes are necessary for cell survival since inhibition of their activity leads to the arrest of cell mitosis and to cell death. Amongst them, GSH peroxidase was shown to be more efficient than catalase and much more than SOD. This result was obtained by comparing the cell protection against oxidative stress after their microinjection in the cytoplasm. With age, the level of these antioxidant enzymes does not change in several experimental models, so that it is not possible to explain the aging process by a lack of protection due to a decrease in the activity of these three enzymes. However, tissues and cells are more susceptible to free radical attacks with age. In order to understand the importance of free radicals in this process, we have to distinguish between their respective effects on cell mitosis, cell death and cell aging. The effects on mitosis and cell death are well described, and the results clearly show a threshold of response which is determined by the antioxidant content of the cell. There is now evidence that short free radical stresses can also speed up the aging of in vitro cultured human fibroblasts. However, such effects are not typical of free radicals but are also obtained with many other deleterious substances so that free radicals have to be considered as one amongst other factors responsible for influencing the evolution of a cell to an older stage or to cell death. The lowering of the general metabolism and of the free energy in old cells are probably the main factors responsible for the increased susceptibility of these cells to stresses such as oxidative stresses.


Increased serum N-acetyl-beta-D-glucosaminidase and alpha-D-mannosidase activities in obese subjects.

We have studied N-acetyl-beta-D-glucosaminidase and alpha-D-mannosidase activities in human sera from 35 control subjects, 47 normo- and hyperinsulinemic obese persons, and 12 diabetic patients after a fasting period of 12 h and at 30, 60, 90, and 120 min after an oral glucose overload. The results show a significantly higher activity of these 2 enzymes in obese subjects and diabetic patients, of similar magnitude, especially in those obese persons with a higher grade of obesity. Moreover, the activity of these glycosidases decreases in a similar way in all these 3 groups after the oral glucose overload.


Effects of calcium channel blocking and atrial stimulation on QT time during hyper- and normocalcemia in man.

We investigated how calcium channel blocking agents modify the known decrease of QT time with increasing heart rate and calcium level. Furthermore, we examined how the influence of calcium channel blocking agents is modulated by atrial stimulation. To answer these questions, we measured the QT time both at a spontaneous heart rate and during atrial stimulation with 90 and 110 beats/min before and after intravenous administration of 5 mg verapamil in 23 patients with primary hyperparathyroidism, pre- and postoperatively. Atrial stimulation increased the frequency-related standard values of QT time. The spontaneous heart rate (HR) was influenced by neither the calcium level nor verapamil. The statistically significant correlations QT vs. HR and QT vs. Ca were reduced by verapamil, which indicated its influence. However, this effect did not achieve statistical significance.


Comparison of dietary intakes in four selected European populations.

The composition of diet and food intakes in four selected European populations were compared. Dietary data (from the World Health Organization Project on Monitoring Trends and Determinants in Cardiovascular Disease) were measured by record methods and were available for middle-aged men in Finland (n = 653), France (n = 1128), Northern Ireland (n = 356), and southern Germany (Augsburg region; n = 899). Nutritional variables--which are independent of the absolute energy intake, such as the percentage supply of energy from macronutrients and the ratio of polyunsaturated to saturated fatty acids--were on the whole similar. However, striking differences were found in food intake and in the percentage supply of fat from different foods. The results indicate that detailed knowledge of consumption figures is necessary to develop prudent and acceptable nutrition intervention programs.


Positive correlation between aortic valve pressure gradient and mitochondrial respiratory chain capacity in hypertrophied human left ventricle.

The effect of chronic left ventricular pressure overload on the activities of mitochondrial respiratory chain enzymes was investigated in myocardial biopsies from the left ventricular apex of 13 patients undergoing aortic valve replacement for aortic valve stenosis. Transvalvular pressure gradients measured by left-sided heart catheterization ranged from 52 to 100 mmHg. The specific activity of mitochondrial respiratory chain enzyme complexes I+III (antimycin A sensitive NADH cytochrome c oxidoreductase) and the myocardial concentrations of coenzyme Q10 (CoQ10) increased significantly (P < 0.05) with increasing aortic valve pressure gradient. In contrast, the specific activities of complex IV (cytochrome c oxidase), succinate dehydrogenase, and citrate synthase, a mitochondrial matrix enzyme, showed no significant correlation with the pressure gradient. Since CoQ10 is the rate-limiting compound of the activity of complexes I+III but not of cytochrome c oxidase, succinate dehydrogenase, or citrate synthase, these data suggest that the increase in the activity of complexes I+III is due to the increase in CoQ10 content.


Induction of the fed pattern of human exocrine pancreatic secretion by nutrients: role of cholecystokinin and neurotensin.

The aim of the present study was to assess the role of cholecystokinin and neurotensin in converting the cyclical interdigestive pattern of pancreatic secretion into the non-cyclical fed pattern. Six healthy male volunteers were studied on 4 separate days. During each experiment a mixed liquid meal or solutions of individual nutrients were perfused intraduodenally for 180 min at 2 ml/min. The mixed meal contained 4.3 g glucose, 2.0 g fractionated soya oil, and 1.7 g casein hydrolysate per 100 ml, which delivered a caloric load of 0.9 kcal/min into the duodenum. The isocaloric and isotonic solutions of individual nutrients contained 44.5 g glucose, 17.8 g fractionated soya oil, or 44.5 g hydrolysed serum bovine albumin per liter and delivered 0.36 kcal/min into the duodenum. Duodenal aspirates and blood samples were collected at regular intervals for determination of pancreatic enzyme outputs and plasma levels of cholecystokinin and neurotensin, respectively. The mixed meal converted the cyclical interdigestive secretory pattern into the noncyclical fed pattern whereas none of the three individual nutrients abolished the interdigestive pattern. Not only the mixed meal but also lipid and protein perfusion consistently stimulated cholecystokinin release. Integrated incremental cholecystokinin release amounted to 32.3 +/- 9.9 pg/ml x 180 min with the mixed meal, 23.2 +/- 6.5 with lipid perfusion (P < 0.05 versus mixed meal) and 13.4 +/- 3.8 with protein perfusion (P < 0.05 versus mixed meal). The carbohydrate solution did not significantly release cholecystokinin. None of the duodenal perfusates raised neurotensin plasma levels.(ABSTRACT TRUNCATED AT 250 WORDS)


Long-term follow-up of renal transplantation in type 1 and type 2 diabetic patients.

This study evaluated the long-term outcome of renal transplantation in type 1 (n = 25) and type 2 (n = 18) diabetic patients. Overall postoperative survival at 1 year was 69% in type 1 diabetes and 75% in type 2; at 5 years it was 62% in type 1 diabetes and 58% in type 2. Death was due mainly to cardiovascular disease (60%) and septic gangrene (20%). Outcome was examined in terms of graft function, which was poor in the majority (86%) of patients who died. Patients with fatal outcome suffered major vascular complications prior to renal transplantation and frequently had impaired graft function. Metabolic control was better in patients with good graft function (HbA1c < 6.2%) than in those with poor or no function of kidney transplant (HbA1c > 9.8%). In the absence of severe vascular complications renal transplantation may be the treatment of choice for both type 1 and type 2 diabetic patients with end-stage renal disease. Otherwise, renal transplantation is not able to improve the prognosis of patients with a history of severe vascular complications prior to renal replacement therapy.


Q fever endocarditis: diagnostic approaches and monitoring of therapeutic effects.

The scope of current diagnostic methods for Q fever endocarditis includes serology, direct demonstration of Coxiella burnetii in the resected heart valve tissue, and animal inoculation studies. Illustrated by a clinical case report, the different methods are presented and discussed. Serology represents the primary method, using the techniques of complement fixation, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). The latter two techniques allow the detection of immunoglobulins G, M, and A to the phase I and II antigens of C. burnetii. After cardiac surgery, we visualized C. burnetii on smears and specifically stained it on histologic sections of the resected heart valve by light and electron microscopic immunohistochemistry. In addition, seroconversion in animals after inoculation with valve specimens confirmed the presence of C. burnetii in the heart valve. The antibody titers determined by ELISA correlated well with the patient's clinical course during the treatment period. Therefore it is suggested that its usefulness for monitoring the efficacy of antimicrobial agents in patients with Q fever endocarditis should be further evaluated.


Successful treatment of primary adrenal insufficiency due to malignant non-Hodgkin's lymphoma.

Although the adrenal glands are frequently the site of tumor metastases, adrenal insufficiency is exceedingly rare. We report on a patient with high-grade B-cell centroblastic lymphoma who initially presented with right axillary lymphadenopathy and bilateral adrenal masses. Four months after axillary lymphadenectomy the patient developed overt signs of Addison's disease. He recovered promptly after initiation of hormone replacement therapy and bilateral adrenalectomy. At present, 16 months after additional chemo- and radiation therapy the patient is considered free of tumor. To our knowledge this is the first report on a patient who presented with adrenal insufficiency in the course of non-Hodgkin's lymphoma and who was successfully treated. Demonstrating this case, we would also like to stress that the development of adrenal insufficiency does not necessarily indicate widespread tumor manifestation in patients with non-Hodgkin's lymphoma.


Renal long-term effects of calcium antagonist treatment in patients with diabetes mellitus.

Although calcium antagonists have long been introduced into antihypertensive treatment, little is known of their renal long-term action in patients with diabetes mellitus, diabetic nephropathy, or hypertension. Much of the current information concerning this issue is from short-term studies. While urinary albumin excretion, a major indicator of glomerular damage in patients with diabetes mellitus, remains unchanged or even increases during short-term calcium antagonist treatment in type 1 diabetic patients, it is mostly reduced in type 2 diabetic patients, especially by diltiazem and nicardipine. There have been discrepant observations in studies lasting 6-months or longer. In many of the studies, urinary albumin excretion is not decreased by calcium antagonist treatment, although blood pressure is well controlled. Albuminuria is markedly reduced, however, after nitrendipine or diltiazem treatment. While calcium antagonists such as diltiazem, nicardipine, or nitrendipine may be as efficacious as converting enzyme inhibitors in preventing the progression of diabetic kidney disease in diabetic patients, the beneficial efficacy of others is less apparent. Reduced albuminuria may be more difficult to attain in macroalbuminuric patients with advanced nephropathy. However, considerations on the potential effects of calcium antagonist long-term treatment on cardiovascular morbidity or mortality in diabetic patients should not be overlooked when their renal action is under discussion. Thus, further studies are needed to define the role of calcium antagonists more precisely in the long-term treatment of diabetic patients with hypertension or diabetic nephropathy.


Three cases of malignant neoplasm, pneumonitis, and pancytopenia during treatment with low-dose methotrexate.

A 77-year-old man with chronic obstructive pulmonary disease was treated with low-dose methotrexate (7.5-15 mg per week). After 15 months a diagnosis of urothelial carcinoma of the bladder was made; after a further 6 months pneumonitis and pancytopenia developed. The patient died due to massive pulmonary hemorrhage. A malignant teratoma was diagnosed in a 65-year-old asthmatic man 16 months after initiation of methotrexate therapy (15 mg per week). The patient died 4 months later due to fulminant progression of the neoplasm. A third malignant neoplasm (dermal squamous cell carcinoma) was seen in a 64-year-old woman with rheumatoid arthritis after 13 months treatment with 7.5 mg methotrexate per week. These three cases, while obviously not proving a causal relationship between long-term treatment with low-dose methotrexate and development of malignant neoplasm, do call for stringent treatment criteria, close surveillance, and prospective studies.


HLA-DRB3 gene alleles in Caucasian patients with Graves' disease.

Graves' disease (GD) is a human leukocyte antigen (HLA) linked organ-specific autoimmune disease. In German GD patients the disease is associated with HLA specificities of the HLA-DRw52 family (HLA-DR3, -DR5, and DR6; HLA-DRB3 positive HLA haplotypes). Recently, a strong association with a HLA-DRB3 restriction fragment length polymorphism gene has been described. To study HLA-DRB3 alleles and their association with the disease, a large cohort of controls (n = 3724) and GD patients (n = 304) was analyzed. HLA-DR allelic combinations revealed an increase in HLA-DR3/DR5 heterozygous patients (relative risk 2.9; P < 0.001). HLA-DRB3 alleles, as defined by DNA typing in HLA-DR matched groups revealed a significant increase in DRB3*0101 homozygosity (relative risk 17.5; P < 0.001) in HLA-DR3 homozygous patients. In GD patients with ophthalmopathy (grade II or higher, according to Werner) DRB3*0101/*0202 heterozygosity revealed an increased relative risk of 5.5 (P < 0.001). Non-HLA-DR3 homozygous, DRB3*0101/*0202 heterozygous patients were at the highest risk for endocrine ophthalmopathy (relative risk 10; P < 0.001). Our data, based on DNA typing methods of HLA-D genes, provide evidence that the susceptibility is strongly associated with HLA-DRB3 genes.


Failure to detect any effect of amalgam restorations on peripheral blood lymphocyte populations.

Dental amalgam has been considered to have adverse side effects on the immune system. Reports have been contradictory, indicating both an increase and a decrease in peripheral blood lymphocyte counts associated with amalgam restorations. We investigated two groups of patients, one of which was treated with amalgam restorations for the first time. In the other group, all existing amalgam fillings were removed. Prior to and after treatment, we determined the absolute and relative numbers of granulocytes, lymphocytes, monocytes, T cells, B cells, cytotoxic T cells, helper T cells and natural killer cells. In addition, functional investigations of T cells were performed. We failed to find any effect of amalgam restorations on the immune system in terms of the parameters investigated.


Caries susceptibility and renal excretion of calcium.

Clearance studies were performed for 2 days in two groups of age-matched young female volunteers: those with low caries prevalence and those with high caries prevalence. Both groups were kept on a low-calcium diet for 1 week and received 0.5 g calcium at the beginning of the second day. In both groups, glomerular filtration rate, urinary flow rate and renal excretions of sodium, calcium, and phosphate were subject to significant circadian variations. In both groups the administration of calcium led to a significant increase in renal excretion of sodium and calcium and a significant decrease in that of phosphate. On the first day, calcium excretion was significantly greater in those with low caries prevalence than in those with high caries prevalence, pointing to altered calcium homeostasis in this group.


Esophageal dysfunction in diabetes mellitus: is there a relation to clinical manifestation of neuropathy?

In a prospective study, we evaluated 33 diabetic patients [type I (n = 8) and type II (n = 25)]. Esophageal motor functions were examined by registering clinical symptoms and by performing esophageal manometry. We also investigated peripheral and autonomic neuropathy. In diabetics, the lower and upper esophageal sphincter pressure and amplitudes of peristaltic waves were reduced. Compared with controls (n = 30), in diabetics the esophageal peristaltic velocity was reduced significantly, and the duration of contractions were decreased as well. Multipeaked waves were uncommon in diabetics, while non-propulsive contractions were seen more often. No correlation was found between esophageal dysfunction and peripheral or autonomic neuropathy. Some 60% of diabetics reported esophageal symptoms; however, no relationship between these symptoms and the extent of dysfunction in esophageal motility was found.


Disturbed calcium metabolism in subjects with elevated diastolic blood pressure.

Essential hypertension has been associated with disturbed calcium metabolism, but the available data are controversial. We measured parameters of calcium metabolism in groups of untreated male subjects (n = 78) with elevated diastolic blood pressure (101 +/- 6 mmHg, mean +/- SD) and age-matched male subjects (n = 79) with low diastolic blood pressure (62 +/- 4 mmHg). The participants of the study were drawn from a random population sample. Subjects with high diastolic blood pressure had significantly higher carboxy-terminal parathyroid hormone (PTH) plasma concentrations than controls with low diastolic blood pressure (median 114 vs. 43 pmol/l, P less than 0.01). The 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D concentrations were comparable in both groups. Individuals with high diastolic blood pressure had significantly lower total serum calcium (2.41 +/- 0.10 vs. 2.47 +/- 0.10 mmol/l, mean +/- SD; P less than 0.01). PTH concentrations were correlated with diastolic pressure (r = -0.39, P less than 0.001). The data are compatible with increased parathyroid activity despite unchanged concentrations of vitamin D metabolites in human hypertension.


Sulfadiazine therapy for toxoplasmosis in heart transplant recipients decreases cyclosporine concentration.

Toxoplasmosis may cause serious problems after organ transplantation. For treatment of active infection, pyrimethamine combined with a sulfonamide is recommended. During oral sulfadiazine therapy, a significant decrease in cyclosporine concentrations was observed in three heart transplant recipients. This interaction has not been reported previously.


Current status of HLA matching in renal transplantation. The Collaborative Transplant Study.

The impact of HLA compatibility on the success rate of kidney transplants was studied in over 80,000 recipients of primary transplants. The transplants were done from 1982 to 1991 at over 300 transplant centers in 43 countries. The results show that matching the HLA chromosomes in related donor transplants has a striking influence. It is also important that matching for individual HLA antigens in cadaver transplants provides a highly significant improvement in graft survival (P less than 0.0001). After 5 years, matched grafts have a survival rate approximately 20% higher than completely mismatched grafts. The matching effect is particularly strong in presensitized and second graft recipients. There is now direct evidence that even if it is necessary to transport well-matched kidneys a long way, they have a significantly higher success rate than locally transplanted poorly matched kidneys. New data based on molecular technology show that the precise identification of HLA-DR antigens by DNA typing further improves the success rate of HLA-matched transplants.


Progress towards a molecular understanding of cyclosporin A-mediated immunosuppression.

Immunosuppressive drugs like cyclosporin A (CsA), FK506 and rapamycin exert their immunosuppressive potential primarily by interfering with the activation and proliferation of T cells. These substances bind to intracellular receptor proteins, called immunophilins. Immunophilins are ubiquitous and abundant proteins, found in all prokaryotic and eukaryotic cells investigated, as well as in many subcellular compartments. Immunophilins display an inherent enzymatic activity, the prolyl-peptidyl cis-trans isomerase (PPIase). The eukaryotic PPIases are inhibited upon the binding of immunosuppressants. In addition, the complex of immunophilins and CsA or FK506 acquires a new activity, namely the binding and inhibition of the cytoplasmic Ca(2+)-binding phosphatase calcineurin. This inhibition is postulated to prevent the proper assembly and nuclear positioning of the transcription factor NF-AT (nuclear factor of activated T cells). The correct DNA binding of NF-AT to regulatory elements of the interleukin 2 (IL-2) promoter normally contributes to the transcriptional activation of this gene. Thus, immunosuppressive drugs prevent T-cell activation by interfering with Ca(2+)-dependent signal transduction pathways which regulate gene activities.


Towards understanding the pathophysiology of chronic rejection.

Chronic allograft rejection is the major reason why allografts are lost. While only 2%-3% of all allografts are lost during the first year to irreversible acute rejection, approximately 6%-7% are lost during each subsequent year to chronic rejection. The major manifestation of chronic rejection in all organs is persistent perivascular inflammation and allograft arteriosclerosis. Bearing this in mind, we have developed a model to investigate the pathophysiology of allograft arteriosclerosis using aortic transplantations between inbred rat strains. The results obtained thus far indicate that several different inflammatory cascades are operative within the vascular wall during allograft arteriosclerosis. The relative importance of these different cascades, and particularly the role of growth factors as final effectors, has not yet been defined. Attempts to suppress allograft arteriosclerosis under experimental conditions have already met with some success: under conditions where no immunosuppression is provided we have been able to delay the process by at least 3 months, though we have not been able to block it indefinitely. It may be expected, however, that once the inflammatory cascades leading to smooth muscle cell replication in the allograft media and their influx into the intima are better defined, more specific approaches to the inhibition of allograft arteriosclerosis will be developed.


Autosomal dominant polycystic kidney disease: from molecular genetics to the patients.

One of the gene loci (PKD1) responsible for autosomal dominant polycystic kidney disease was located in 1985 to the short arm of chromosome 16. The clinical consequences of this finding are analyzed. Genetic heterogeneity has been demonstrated since 5%-15% of the families inherit a non-PKD1 mutation. Progress in molecular genetics allows better classification of patients with some atypical manifestations, e.g., those with early renal failure or those with congenital hepatic fibrosis. Identification of the gene(s) and of their defects will provide further progress.


Autosomal recessive polycystic kidney disease.

Autosomal recessive polycystic kidney disease is a rare inherited disorder which usually becomes clinically manifest in early childhood, whereas autosomal dominant polycystic kidney disease usually is a disorder of adult onset. With increasing knowledge and improving diagnostic techniques, it becomes evident that the spectrum of both entities is much more variable than generally known. The presentation of autosomal recessive polycystic kidney disease at later ages and survival into adulthood have been reported. The diagnostic criteria, clinical course, genetics and differential diagnosis of autosomal recessive polycystic kidney disease will be presented.


The nephronophthisis complex: clinical and genetic aspects.

Familial juvenile nephronophthisis (NPH) and medullary cystic disease (MCD) are hereditary forms of early-onset chronic renal failure caused by the bilateral formation of cysts at the corticomedullary junction of the kidney. Polyuria, polydipsia, anemia, and growth retardation precede end-stage renal failure. The absence of edema and hypertension frequently leads to a delay in the diagnosis and commencement of therapy. The condition is a major cause of end-stage renal disease (ESRD) in children, accounting for 10%-25% of these patients. About 300 cases of NPH or MCD have been described. Although they are almost indistinguishable clinically and pathologically, the two conditions are separated by a characteristic age of onset (11.5 years in NPH vs. 28.5 years in MCD) and by the mode of inheritance (autosomal recessive in NPH vs. autosomal dominant in MCD). An association of NPH with retinitis pigmentosa is known as the Senior-Løken syndrome (SLS). Hepatic fibrosis, skeletal defects, and central nervous system abnormalities have been described in association with NPH but are typically absent in MCD. Since the pathology of NPH and MCD is similar, the term "nephronophthisis complex" has been introduced to summarize the related diseases. At present, there are no means of identifying heterozygotes, conducting prenatal diagnosis, or screening children in affected families. The histologic changes of NPH are characteristic but not specific for the disease. Cysts of 1-15 mm in diameter, located primarily at the corticomedullary junction, are seen in 70% of the patients. Light microscopy reveals a chronic sclerosing tubulo-interstitial nephropathy.(ABSTRACT TRUNCATED AT 250 WORDS)


Molecular aspects of Alport's syndrome.

We review the recent progress achieved on the understanding of the molecular basis of Alport's syndrome. This inherited disease is defined as progressive nephritis with sensorineural hearing loss. In 80%-85% of the families, inheritance is compatible with X-linked dominant transmission, whereas in the remaining cases autosomal dominant transmission is assumed. Histology studies demonstrated that the main defect is within the glomerular basement membrane (GBM). In addition, evidence for an altered GBM antigenicity came from immunofluorescence studies which showed a reduced or absent binding of anti-GBM autoantibodies or monoclonal antibodies to the "Goodpasture antigen" in some families. Subsequent studies added substantial evidence that Alport's syndrome is a type IV collagen disease. Genetic linkage analyses coherently identified an Alport locus at the X-chromosomal region Xq21.3-22. Recently, a previously unknown alpha 5 chain of type IV collagen was identified, and the corresponding gene was also mapped to Xq22. Subsequent studies on Alport families by various groups identified more than 25 COL4A5 lesions. Segregation in linkage with the Alport phenotype could be shown in large kindreds. Mainly deletions and only a few point mutations were described. Most lesions reported so far are heterogeneous. We were able to identify two deletions and one point mutation involving a 3' splice site in 20 Alport families from Germany. One of the patients with a COL4A5 deletion and the patient with the splice site mutation developed anti-GBM antibodies after renal transplantation. In contrast, no COL4A5 lesions have been found in 2 further patients with posttransplant anti-GBM nephritis.(ABSTRACT TRUNCATED AT 250 WORDS)


Oscillations: a key event in transformed renal epithelial cells.

Intracellular pH (pHi) plays a critical role in the entry of cells into the DNA-synthesis phase of the cell cycle. Alterations in pHi may contribute to abnormal proliferative responses such as those seen in tumorigenic cells. We observed that alkaline stress leads to genomic transformation of Madin-Darby canine kidney (MDCK) cells. Transformed cells (F cells) form "foci" in culture, lack contact inhibition, and are able to migrate, typical characteristics of dedifferentiated tumorigenic cells. F cells exhibit spontaneous biorhythmicity. Rhythmic transmembrane Ca2+ flux activates plasma membrane K+ channels and Na+/H+ exchange. This leads to periodic changes of membrane voltage and pHi at about one cycle per minute. We conclude that endogenous oscillatory activity could be a trigger mechanism for DNA synthesis, proliferation, and abnormal growth of renal epithelial cells in culture.


The inflammatory function of renal glomerular mesangial cells and their interaction with the cellular immune system.

The autoimmune nature of chronic progredient glomerular diseases has been well established. Like in other chronic inflammatory diseases, the active role of organ-borne cells has become increasingly apparent--both for the inflammatory process and for the initiation and perpetuation of the immune reaction. In most forms of glomerulonephritis, intrinsic glomerular mesangial cells are likely candidates to come into intimate contact with immune cells such as monocytes or lymphocytes. On the basis of cell culture studies we would like to integrate the current knowledge available about the responsiveness of mesangial cells to inflammatory agents and the resulting secretory capacity and, moreover, their possible role in sustaining chronic inflammatory injury and autoimmune reactions through a direct interaction with lymphocytes. Apart from being responsive to physiological stimuli such as angiotensin II, glomerular mesangial cells are predominantly activated by agents related to inflammation. This includes exogenous substances such as the components of gram-negative bacteria and an array of highly potent immunological stimuli like antigen-antibody complexes, activated complement, or various cytokines. The transformation of resting mesangial cells to proliferating cells with an accompanying expansion of their secretory profile and responsiveness is due to mediators like platelet-derived growth factor, transforming growth factor, and others. Numerous low-molecular-weight substances (O2-, H2O2, NO, platelet-activating factor, eicosanoids), proteins (proteinases, matrix components, interleukins 1 and 6, colony-stimulating factors, growth factors), and cell-surface molecules released or expressed by mesangial cells participate in the inflammatory process. Among these products interleukin 1 and/or 6, class II major histocompatibility antigen and integrins also support an interaction with the cellular immune system. It has been well documented that mesangial cells induced in vitro by recombinant T-cell lymphokines, such as interferon-gamma, do express MHC II and ICAM-1 and could function as antigen-presenting cells. However, and perhaps more interestingly, our own recent experiments with cocultures of syngeneic mouse lymphocytes and mesangial cells have demonstrated that T-cells are directly activated by cultured mesangial cells, thus resembling a mesangial cell-specific autoimmune reaction. In parallel to clinical studies searching for a mesangial autoantigen these experiments might help to elucidate the mechanisms of initiation and perpetuation of mesangial cell-dependent autoimmune glomerulonephritis.


The glomerular mesangium: capillary support function and its failure under experimental conditions.

We present a structural analysis of the ability of the biomechanical unit consisting of mesangium and glomerular basement membrane to maintain normal capillary architecture in the face of mechanical challenges due to high intraglomerular pressures. Capillary support function may be considered in terms of the stabilization of local form (development of wall tension against capillary dilation) and global form (centripetal fixation of capillary loops to maintain higher order form). The pathologic consequences of the loss of this support are illustrated by way of experimental models of mechanical mesangial failure. Such failure may express itself as mesangial widening, increased transmesangial macromolecule "traffic," ballooning of capillary segments, and unfolding of capillary loops. Mechanisms are described by which these structural changes may lead to segmental glomerular sclerosis.


Mesangial cells in the pathogenesis of progressive glomerular disease in animal models.

Increasing evidence supports a role for glomerular mesangial cell proliferation and over-production of extracellular matrix by mesangial cells in the development of focal or diffuse glomerulosclerosis. Experimental data obtained mainly in the chronic progressive remnant kidney model and in the acute mesangioproliferative anti-Thy 1.1 glomerulonephritis in rats have shed some insights into the factors governing mesangial cell proliferation and matrix synthesis in vivo. In these experimental models, mesangial cell activation can be demonstrated early in the course of disease as exemplified by the de novo expression by the mesangial cell of a smooth muscle "specific" actin isotype (i.e., alpha-smooth muscle actin). Following mesangial cell activation, cellular proliferation ensues both in the acute anti-Thy 1.1 model and, to a lesser degree, in the chronic remnant kidney model. While a multitude of mitogens for mesangial cells has been proposed on the basis of in vitro experiments, the factors involved in the regulation of mesangial cell proliferation in vivo remain largely undefined. Three growth factors which may have important roles in the in vivo mesangioproliferative response are platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta (TGF-beta). All three cytokine growth factors are present in various inflammatory cells as well as in mesangial cells themselves, thereby allowing these factors to mediate cell proliferation by either paracrine and/or autocrine pathways. In vivo studies show that PDGF, bFGF, and TGF-beta participate in the mesangial cell proliferation and/or the mesangial matrix expansion that follows mesangial cell injury with anti-Thy 1.1 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)


Pathology of the human mesangium in situ.

Mesangial cells play an important role in the development and progression of human glomerular disease. This article summarizes some important aspects of mesangial properties and behaviour in situ. Intrinsic mesangial cells express alpha-smooth muscle actin and are best characterized as myofibroblasts or glomerular pericytes. The main integrin receptor in the mesangium is the alpha 1 beta 1 integrin. The beta 2 and beta 3 integrins have not been detected. Mesangial cells in situ fail to react with many monoclonal antibodies which stain human mesangial cells in culture, including leukocyte activation antigens. Prominent reactions in glomerular disease are mesangial expansion and progressive glomerular sclerosis, which are preceded by or associated with mesangial cell hypertrophy and/or proliferation. Mesangial enlargement is accompanied by an altered integrin expression and an abnormal composition of extracellular mesangial matrix. From the numerous autocrine and paracrine mediators identified in vitro which stimulate or inhibit mesangial cell growth and extracellular matrix synthesis, up to now only a few factors have been shown to be present in selected human glomerulopathies. These include platelet derived growth factors and platelet derived growth factor receptor beta, transforming growth factors beta, interleukin 1 beta, tumor necrosis factor alpha, and interleukin 6. Further identification of such mediators in situ will improve our understanding of pathological glomerular processes, particularly with respect to the multifunctional properties of the mesangial cell.


A review of nursing strategies to reduce patient anxiety in coronary care. Part 2.

Illness and associated hospitalisation are stressful events for any individual. The stress associated with an admission to a coronary care unit may be related to the severity and acuteness of the illness, the effects of hospitalisation on the individual, their family, their job and financial position, and often manifests itself as anxiety, fear or depression. This paper highlights the use of anxiety reduction strategies such as muscle relaxation, music, education and counseling. The literature reviewed does not provide any clear messages regarding the effectiveness of these strategies in the coronary care unit setting. As a consequence, there is a need to continue researching this important area of nursing practice.


Cerebral aneurysms and subarachnoid haemorrhage.

Up to 2000 Australians each year rupture a cerebral aneurysm. This is immediately fatal in some, and others remain disabled. The main risks after a first haemorrhage are recurrent bleeding, prevented by early surgery, and delayed cerebral ischaemia due to vasospasm. This is minimised by keeping a good fluid balance, calcium antagonists, and possibly newer treatments such as aminosteroids or plasminogen activator. An important problem is that of the missed haemorrhage, which often leads to delay in treatment and a fatal or disabling recurrence.


RITA--Registry of Industrial Toxicology Animal-data--progress of the working group.

A data base for the collection of histopathological diagnoses of neoplastic and pre-neoplastic lesions and related data from control animals involved in long-term carcinogenicity or toxicity studies has been operated since 1988. Factors which may have an influence on the spontaneous tumor rate are integrated in the data base. Systematized nomenclature and standardized diagnostic criteria have been established as well as the applied data acquisition and data validation procedure.


Ultrastructure and biochemistry of ischemic damages of small intestinal epithelial cells.

Ischemia (one hour) and following reperfusion (up to one hour) of the small intestine induce biochemical changes which are indices for the formation and action of oxygen free radicals and which occur predominantly during the reperfusion period. But the villi and the epithelial cells show different patterns of damage, occurring both at the end of the ischemic period and during the reperfusion period. Although the quantitative morphological changes are increased during the reperfusion in comparison with the ischemic phase the quality of the pattern of structural damage is the same in both periods of the experiment. This pattern of the damage includes: 1. the neighbourhood of groups of villi with total ischemic-lytic dissolution of the villi, of villi with damage of the epithelial cells at the tip and at the lateral area and of normal villi; 2. the different degree of structural damage of neighbouring epithelial cells within one villus whose cells are either of regular structural or damaged at subcellular organelles including the plasma membrane or of those being necrotically destroyed and on the way of release into the luminal space; 3. a differentiation of the structural changes of the microvilli and other organelles within single and neighbouring epithelial cells. The biochemical findings on purine nucleotide metabolism and on the formation of oxygen free radicals as "mean values" of a homogenate from a large group of cells cannot reflect the morphological-ultrastructural changes of single villi or even single epithelial cells. The possible reasons for the mosaicism of the morphological changes during ischemia and reperfusion are discussed.


A new simulation approach for the determination of the error of parameters of biological importance describing the growth of the Ehrlich ascites tumor of the mouse.

Some aspects of the growth of the Ehrlich ascites tumor of the mouse can be described by the logistic function. The parameters used are fitted to the experimental data in a nonlinear way. It is important to determine the error of these parameters of biological importance in order to discriminate between different consequences induced by various experimental situations by means of statistics. But often there is a gap between the data that are available and the stronger assumptions necessary for statistics. We propose a simulation approach which also works in a much larger range of similar situations to close this gap.


The influence of systemic hypoxia and reoxygenation on the glutathione redox system of brain, liver, lung and plasma in newborn rats.

The concentrations of reduced (GSH) and oxidized glutathione (glutathione disulfide, GSSG) in lung, liver, brain and plasma of newborn rats were investigated under the condition of reversible hypoxia. Brain and lung of newborn rats seem to be susceptible to reversible hypoxia. We found an increase in GSSG concentration after hypoxia in these organs. This alteration of the GSH-GSSG redox system was reversible within 2 hours of reoxygenation. A second increase in cerebral GSSG concentration after 4 hours of reoxygenation was connected with fasting during the experiment. In the liver we found a hypoxia dependent decrease in the GSH level, followed by a decrease in GSSG concentration. The increased GSSG concentrations in lung and brain are accompanied by an enhancement of plasma GSSG concentration.


Bile salt-associated electrolyte secretion. A study following a short-term biliary obstruction in the rat.

The mechanisms involved in bile salt-induced choleresis are poorly known. To give an insight in this physiological process, bile salt-associated electrolyte secretion was studied following relief of a short-term (2h) biliary obstruction in the rat, an experimental model that shows an important diminution of bile salt choleretic efficiency. For this purpose, biliary excretion of total bile salts and electrolytes (sodium, chloride and bicarbonate) were studied in such a model during taurocholate infusion at increasing rates. The results showed that bile flow, bile salt output and electrolyte secretion stimulated by taurocholate administration were decreased in the rats that were subjected to biliary obstruction. Besides, the choleretic efficiency of the excreted bile salts, as estimated by the slope of the regression line of bile flow vs. bile salt output, was diminished by 46% (p < 0.005). Multiple regression analysis of bile flow vs. bile salt and electrolyte outputs allowed to detect a selective diminution of the fraction of bile flow related to bile salt-associated electrolyte secretion ("secretory fraction" of the choleretic efficiency of bile salts) (3.2 +/- 0.3 vs. 2.5 +/- 0.2 L/mol, p < 0.05) whereas the "osmotic fraction" of the choleretic efficiency of bile salts was not modified by the treatment (5.0 +/- 0.4 vs. 5.1 +/- 0.3 L/mol, p > 0.05). Since both chloride and bicarbonate biliary concentrations in the volume of bile stimulated by taurocholate were reduced by 53% and 52% respectively, a role of these anions in the generation of bile salt-induced choleresis was suggested. Possible mechanisms involved in such a process and in its early impairment during cholestasis are discussed.


Laser treatment of diffuse retinal pigment epitheliopathy.

The Authors report laser treatment of 18 eyes (14 patients) with a variant of central serous chorioretinopathy associated with widespread decompensation of the retinal pigment epithelium (RPE) in the macula, diffuse retinal pigment epitheliopathy (DRPE). All eighteen eyes with DRPE were treated with Krypton red laser photocoagulation in a grid pattern to leaking RPE in areas of neurosensory retinal detachment. All eyes showed anatomic improvement with resolution of the serous macular detachment and lipid exudation. The visual acuity was stabilized in 15 eyes (83%) at 6 months' follow-up, but only 3 eyes (17%) demonstrated improved visual acuity in the same time interval. At eighteen months, 2 of 15 eyes (13%) retained improved visual acuity, 11 eyes (74%) remained unchanged, and 2 eyes (13%) had visual loss. In this series of patients with DRPE, grid laser photocoagulation treatment was uniformly successful in producing an anatomic improvement in the exudative manifestations of the macula and in preventing or slowing progression of vision loss.


Surgical management of exudative maculopathies: preliminary report.

A new surgical approach to the management of exudative maculopathies has been developed including the transvitreal route. As a result of surgery the subretinal spaces are drained and their viscous content removed. A special tool was designed for this operation. The method has been clinically tested on 32 patients (32 eyes) with different forms of age-related macular degeneration in the phase of complicated pigment epithelium detachment and on six patients (six eyes) with myopic exudative maculopathy. The follow-up ranged from 6 to 32 months. No fluid reappeared in the macular zone in patients with age-related macular degeneration, except one; visual function improved in 26 out of 32 cases (81.3%). Similar results were obtained in all six patients with myopic exudative maculopathy.


Ophthalmic aid to eastern Europe: can the individual ophthalmologist help?

The problems of ophthalmic care in the newly independent countries of Central and Eastern Europe may not be fully appreciated by Western ophthalmologists. Information that has been received as a result of a questionnaire is discussed and some suggestions are made by which help can be given to countries that have been disadvantaged by political and economic influences for nearly half a century.


Fundus angiography with fluorescein-labelled peptide fraction from bovine factor VIII: correlation with histologic findings.

A peptide fraction (VUEFFE) obtained from bovine factor VIII, with a high affinity for vessel endothelium, was bound to fluorescein and used for angiography in rabbits. Several eyes had undergone laser photocoagulation on the retina. The location of the dye was studied histologically with fluorescence microscopy on the enucleated, freeze-dried eyes. The angiographic and histologic findings were compared with findings obtained using sodium fluorescein. Fluorescein-labelled VUEFFE gave a longer lasting fluorescence of the retinal vessels with histologic evidence of dye deposition on the vessel wall. It also showed marked affinity for the photocoagulated tissues, giving intense and prolonged staining of the laser burns. Possible clinical applications are discussed.


Randomised double-masked trial of lodoxamide and sodium cromoglycate in allergic eye disease. A multicentre study.

135 patients were entered into a 28-day randomized double-masked multicentre study comparing the efficacy and short-term safety of lodoxamide 0.1% ophthalmic solution (Alomide--Alcon Laboratories), a mast cell stabilizer, with sodium cromoglycate 2% ophthalmic solution (Opticrom--Fisons Pharmaceuticals) in the treatment of allergic eye disease. Patients given lodoxamide 0.1% showed a significantly more rapid and greater improvement in their signs and symptoms of allergic eye disease than patients given sodium cromoglycate 2%. Both treatments were found to be safe, and side-effect profiles were comparable between the two treatment groups, although the overall incidence of side-effects in this study was found to be less frequent in the lodoxamide-treated group.


Sequences within the R region of the long terminal repeat activate basal transcription from the HIV-1 promoter.

The importance of the R region in basal human immunodeficiency virus type 1 (HIV-1) transcription was addressed by comparing a panel of HIV-1 R region mutants using in vitro and in vivo assays. Using deletion, base substitution mutants, and compensatory mutants, the precise R region sequences essential for basal HIV-1 promoter activity in vitro were mapped to sequences between +17 to +21. Within this regulatory domain, nucleotides +19 and +21 appear to be critical. The effect of these mutations on steady state RNA levels in transfected cells has been analyzed by S1 nuclease protection assay using uniformly labeled probes. Two main conclusions may be drawn from these studies. First, HIV-1 basal transcription is abundant, with the majority of correctly initiated transcripts truncated between sequences +57 to +70. Second, analysis of the compensatory mutants indicates the secondary structure of the nascent R region RNA is not an obligate requirement for the production of the truncated transcripts. Mutations in R region primary sequence that selectively abolish the production of the truncated transcripts in vivo also exhibit reduced promoter activity in vitro. The appearance of high levels of truncated transcripts raise the interesting possibility that-similar to c-myc, c-myb, and c-fos--basal HIV-1 expression is regulated by transcription elongation.


Members of the USF family of helix-loop-helix proteins bind DNA as homo- as well as heterodimers.

We have isolated human cDNA clones for USF2, a new member of the upstream stimulatory factor (USF) family of transcription factors. Analysis of these clones revealed the existence of highly conserved elements in the C terminal region of all USF proteins. These include the basic region, helix-loop-helix (HLH) motif, and, in the case of the human proteins, the C-terminal leucine repeat (LR). In addition, a highly conserved USF-specific domain is located immediately upstream of the basic region. Using in vitro translated proteins, we found that all members of the USF family bound DNA as dimers. The N-terminal portion of USF, including the USF-specific domain, was entirely dispensable for dimer formation and DNA-binding. However, deletion mutants of USF2 lacking the LR were deficient in DNA-binding activity. Interestingly, each of the USF proteins could form functional heterodimers with the other family members, including the sea urchin USF, which does not have a LR motif. This indicates that the conserved LR in human USF is not required for dimer formation, and influences only indirectly DNA-binding.


Differential modes of activation define orphan subclasses within the steroid/thyroid receptor superfamily.

We report that three orphan receptors, hERR1, hERR2, and hTR2, members of the steroid/thyroid receptor (SR/TR) superfamily, can be activated by different ligand-independent pathways. hERR1 and hERR2 exhibit constitutive activity in the absence of exogenously added ligands. Furthermore, this constitutive activity is localized in the carboxy terminal domain of both receptors and can be transferred to other members of this superfamily using domain switch strategies. In addition, we show that hERR1 can remain constitutively active in the less evolved eukaryotic cell Saccharomyces cerevisiae. In contrast, hTR2 is not constitutively active. However, a chimera of hTR2 can be activated in a ligand-independent manner through a signal transduction pathway initiated at the cell membrane by the neurotransmitter dopamine. Like hERR1 and hERR2, hTR2 is ligand-independently activated through its carboxy terminal domain. Together, these results suggest the existence of emerging subgroups within the SR/TR superfamily that can regulate gene expression through different modes of activation.


Mutation analysis of the Cys-X2-Cys-X19-Cys-X2-Cys motif in the beta subunit of eukaryotic translation initiation factor 2.

Recessive lethal mutations in the beta subunit of eIF-2 that restore HIS4 expression in the absence of an AUG start codon were isolated from diploid Saccharomyces cerevisiae strains. DNA sequence analysis of these alleles and of eIF-2 beta suppressor alleles isolated from haploid strains, identified point mutations that altered one of six amino acids that map to a Cys-X2-Cys-X19-Cys-X2-Cys "zinc finger" motif and immediately adjacent residues. Five of the affected amino acids are identical in the human and yeast eIF-2 beta protein. Together with earlier studies (Donahue et al., 1988), these point mutations implicate the zinc finger domain of eIF-2 beta in start-site selection during the scanning process. We have supplemented the mutations obtained by genetic selection with an additional set of constructed mutations in this region. Our studies indicate that the cysteine residues and the intervening amino acids of this motif are essential for eIF-2 beta function in translation initiation in vivo. However, the effects observed in cells containing a copy of eIF-2 beta with a deletion of this motif suggest that this mutated form is still able to associate with other components of the initiation complex, imparting defects on translation initiation. Thus, this motif may be required only for later events that lead to initiator codon recognition. Alterations in defined positions, as found in our suppressor alleles, could lead to recognition of non-AUG codons.


Fotemustine: an overview of its clinical activity in disseminated malignant melanoma.

Fotemustine is a new chloronitrosourea which is active against disseminated malignant melanoma (DMM), and especially against cerebral metastases (CM). This efficacy has been widely demonstrated through many phase II studies. A multicentre trial of monotherapy was undertaken in 153 evaluable French patients. A response rate (RR) of 24.2% (25.0% RR in CM; 31.8% in non-visceral metastases (NVM) was obtained. Three other phase II studies confirmed these results with respective objective RR of 16.7%, 20.0% and 47.0% and RR in CM of 8.3%, 14.3% and 60.0%. Fotemustine has also been used in combination with dacarbazine (DTIC), in patients with DMM. The RR among 103 patients was 27.2% (26.3% in CM, 37.5% in NVM), confirming the activity of fotemustine. The two drugs have also been administered sequentially, in order to exploit their synergism in interfering with the O6 alkyltransferase. Impressive RR have been achieved, especially in patients with visceral metastases (VM) but at the expense of a pulmonary toxicity that does not arise with other treatment schedules and which precludes its use outside of strictly conducted clinical trials.


Regulation of E2F/cyclin A-containing complex upon retinoic acid-induced differentiation of teratocarcinoma cells.

Retinoic acid-induced differentiation of mouse P19 teratocarcinoma cells is accompanied by alterations in the level of E2F transcription factor. P19 stem cells contain free, uncomplexed E2F and an E2F complex termed E2F/stem. This stem cell complex is a heterotrimeric protein aggregate consisting of E2F transcription factor, E2F-binding protein (E2F/bp1), and cyclin A. Retinoic acid treatment converts P19 stem cells into differentiated neurons, glial cells, and fibroblasts. The presented experiments clearly show that the level of uncomplexed E2F gradually decreases upon differentiation, and fully differentiated cells do not contain free E2F. In addition, the stem cell-specific E2F aggregate is converted into a smaller complex, termed E2F/diff. This smaller complex, which is specific for differentiated cells, does not contain cyclin A and consists of E2F transcription factor associated with E2F/bp1. Finally, the role of E2F complexes in the cessation of cell proliferation, which accompanies P19 cell differentiation, is discussed.


Tamoxifen as a single agent for advanced melanoma in postmenopausal women. A phase II study of the EORTC Malignant Melanoma Cooperative Group.

Between 1983 and 1989 a phase II study was carried out by the EORTC Malignant Melanoma Cooperative Group in which postmenopausal women with advanced melanoma but a good performance status received tamoxifen, 40 mg per day, as a single agent. From 12 centres, 114 patients were registered of whom 107 appeared to be eligible and 102 evaluable. Seven died of progressive disease within 4 weeks, eight others had early progressive disease (of whom seven died within 7 weeks). The response rate was 4.9%, the complete response rate 1%. Without prior chemotherapy three of 58 responded, with prior chemotherapy two of 44. Except for one of 46 patients with lung metastases who experienced a PR of these metastases, all responders were patients with slowly growing small soft tissue metastases. We conclude that, because of the few side effects, tamoxifen can be recommended for (postmenopausal) patients who have only a small number of slowly growing metastases and who are not yet candidates for treatment with toxic drugs.


Metastatic or dystrophic conjunctival calcification in renal failure?

In renal failure the incidence of pingueculae is significantly higher than in a comparable control group but there is no evidence that the calcific precipitation in renal failure is of a dystrophic nature. The lime salts are not located within the area of elastotic degeneration, a prerequisite for dystrophic degeneration. Moreover there is no association between the magnitude of the conjunctival degeneration and the degree of calcification. This makes it likely that the calcium precipitates represent metastatic calcification even though admittedly support for this assumption is tenuous.


BANS: a discussion of the problem.

The significance of the BANS location (upper Back, posterior Arm, Neck and Scalp) as a prognostic factor in patients with stage I melanoma is controversial. A meta-analysis performed by Weinstock et al. on their own and five comparable studies corroborated the hypothesis that this location is influential in the prognosis of intermediate thickness (0.76-1.69 mm) melanomas. Our study investigated the relationship between BANS subsites, thickness and prognosis in 1,082 stage I melanoma patients from two major Italian centres, Turin and Florence. A BANS primary was observed in 212 (19.5%) patients: recurrences occurred in 85 of them (40.1%) vs 309/870 non-BANS patients (35.5%). Overall survival probabilities were significantly shorter (p less than 0.01) in the BANS group (69.1% vs 76.7% at 5 years; 59% vs 68.5% at 10 years). The prognostic value of the BANS location was confirmed by a multivariate analysis using the Cox proportional hazards model. Stratification of BANS and non-BANS groups by thickness clusters showed a significant difference in both survival (p less than 0.001) and disease-free interval (p less than 0.05) in the 3.01-4.00 mm thickness subset, due to the greater incidence of distant and visceral metastases. In the 0.76-1.69 mm thickness range the significance was p = 0.06.


Results obtained by using a computerized image analysis system designed as an aid to diagnosis of cutaneous melanoma.

Results obtained using a computerized image analysis system as an aid to clinical diagnosis of melanoma are reported. The system comprises a colour television camera connected through a digitizing board to a 386 personal computer. By means of original algorithms able to measure the shape, the colours and texture of a pigmented lesion of the skin, the system provides eight on/off indicators that are matched with the histological diagnosis to identify benign and malignant pigmented lesions. The chances that a given lesion is malignant increase with the increasing number of positive indicators. The training field of the system was constituted of images and data of 169 cutaneous lesions in 165 patients. Taking two positive indicators as the threshold between pigmented benign and malignant lesions, the efficiency of the system is 0.98, the positive predictive value is 0.45 and the negative predictive value is 0.95. These values were confirmed in a series of 44 pigmented lesions, 10 of which were melanoma, that constitute the present test series. The authors conclude that this computerized image analysis system should be regarded as a useful aid to diagnosis for a non-expert clinician. The system limit is transformation within a naevus.


A unique in vivo assessment of 4-[10B]borono-L-phenylalanine in tumour tissues for boron neutron capture therapy of malignant melanomas using positron emission tomography and 4-borono-2-[18F]fluoro-L-phenylalanine.

A unique in vivo approach to assessing the concentrations of 4-[10B]borono-L-phenylalanine (L-BPA), a melanoma targeting compound for boron neutron capture therapy (BNCT), was investigated using L-BPA labelled with positron-emitting 18F (half-life = 110 min), i.e., 4-[10B]borono-2-[18F]fluoro-L-phenylalanine (L-[18F]FBPA). High melanoma uptake of L-[18F]FBPA was reduced slightly by competition with L-BPA in the two animal models of the murine B16 melanoma and the melanotic Greene's melanoma No. 179 in hamsters. In mice given L-[18F]FBPA and L-BPA, the concentrations of 10B in B16 estimated from 18F radioactivity were lower than those measured by inductively coupled plasma-atomic emission spectroscopy. Lower estimated values were dependent on the time after injection and on the loading dose of L-BPA. The estimated 10B concentrations for Green's melanomas were comparable to the measured values. Positron emission tomography (PET) using L-[18F]FBPA allowed Greene's melanomas to be clearly visualized. In conclusion, when L-[18F]FBPA is used as a probe for L-BPA in BNCT of malignant melanomas, the melanoma can be localized and the 10B concentrations in tissues can be assessed in vivo using 18F radioactivity by PET.


Expression of cytokine genes, including IL-6, in human malignant melanoma cell lines.

As a preliminary to transducing human melanoma cells with lymphokine genes, we sought for constitutive gene expression and production of eight interleukins, tumour necrosis factors and granulocyte-colony stimulating factor in 19 human melanoma cell lines. Conversion of RNA into cDNA by reverse transcriptase and polymerase chain reaction (RT-PCR) were employed to evaluate gene expression while enzyme-linked immunosorbent assays (ELISA) or biological assays were used to assess the presence of proteins. No expression of interleukins (IL) 3, 4, and 5 or interferon-gamma RNA was found, while the other cytokines were variably expressed in melanoma lines, with IL-1 alpha, IL-1 beta, IL-6, IL-8, being detectable in most of the lines. At protein level, 10 melanoma cells were tested with ELISA and all were found to produce IL-8, five produced IL-6, two tumour necrosis factor (TNF)-alpha, one IL-1 alpha and two TNF beta. The levels of TNF beta were at the limit of test sensitivity. The amount of various cytokines released by the different lines varied widely. Biological assay with the D10-G4 clone confirmed the presence of IL-1 alpha in the supernatant of melanoma (ME) 10221 and revealed an IL-1 activity in the supernatant of Me 4024/1. The proliferating activity of melanoma supernatants on D10-G4 was inhibited by treatment with polyclonal antibodies against IL-1 alpha but not with antibodies against IL-1 beta. TNF biological activity was tested against the TNF-susceptible fibrosarcoma WEHI 164 clone 13.(ABSTRACT TRUNCATED AT 250 WORDS)


High sensitivity of human melanoma cell lines to the growth inhibitory activity of mycoplasmal arginine deiminase in vitro.

Arginine deiminase (AD) is a potent growth inhibitor for some but not all tumour cell lines in vitro. As AD catalyses the direct conversion of L-arginine to L-citrulline, the AD-sensitivity of various tumour cells might be attributed to the levels of urea cycle enzymes involved in L-arginine biosynthesis. This study demonstrated that human melanoma cells were highly sensitive to the growth inhibitory activity of AD. Five melanoma cell lines tested also exhibited reduced argininosuccinate synthetase (ASS) gene expression--being almost absent in four cell lines and at low level in one cell line. This resulted in an inability of the cells to utilize L-citrulline for growth. Based on the tissue-specific regulation of ASS gene, the feature of melanomas suggests that it might be possible to develop agents with strong AD activity for chemotherapeutic use for human melanomas in vivo.


Febrile infections and malignant melanoma: results of a case-control study.

Several reports have described an inverse relationship between the frequency of infections and various malignancies. In this paper results of a hospital-based case control study on 139 melanoma patients and 271 suitable selected controls are presented, addressing the question of whether this relationship exists with respect to malignant melanoma while simultaneously controlling for the effects of other risk factors. Data on childhood diseases (group I), febrile diseases of adulthood (group II) and common febrile infections within a 5-year period prior to the diagnosis of melanoma (group III) were collected using a standardized interview. Group I diseases did not show a marked influence on the risk of malignant melanoma. Considering group II diseases, a significant protective effect was determined for chronic infectious diseases (OR = 0.32) and also for wound infections, abscesses and furunculosis (OR = 0.21). In group III, herpes simplex infections (OR = 0.45) and influenza/common cold (OR = 0.32) substantially reduced the melanoma risk. This effect was less pronounced for gastroenteritis (OR = 0.52). Analysis of the cumulative influence of infections pointed to a strong dose-response relationship between the frequency of febrile infections in adulthood and malignant melanoma. In particular, the risk reduction was striking when two or more febrile infections were compared to no febrile infections in group II (OR = 0.09) and group III (OR = 0.20). The study confirms the hypothesis that an inverse relationship exists between febrile infections and malignant melanoma, but these results have to be interpreted cautiously due to the inherent limitations of the case-control design.(ABSTRACT TRUNCATED AT 250 WORDS)


Immune pathogenesis of hepatitis B.

Available information about the immune pathogenesis of HBV infection in man is very limited. However, the present availability of recombinant sources of the different HBV antigens expressed in the appropriate forms to induce activation of either HLA class I or HLA class II-restricted T cells, provides the necessary tools to investigate directly the mechanisms of liver damage, the role of the different cellular components of the immune system in HBV clearance and the specific nature of the immune defects potentially responsible for the chronic evolution of HBV infection. In addition, improved knowledge of HBV biology suggests a dynamic interpretation of the HBV-immune system interactions, based on which viral mutations as well as direct interferences of HBV with specific immune functions are believed to play a relevant role with respect to the outcome of HBV infection.


PCR analysis of HBV infected sera: relationship between expression of pre-S antigens and viral replication.

In order to determine the biological significance of the pre-S antigens in HBV infection, HBsAg sera were tested for the presence of pre-S1 and pre-S2. HBV DNA was detected by spot-hybridization and PCR. The data show a complete correlation between pre-S antigenemia and HBV DNA replication in anti-HBe positive cases. PCR but not spot-hybridization was adequately sensitive to also detect HBV DNA in roughly half of the preS negative sera as well. Thus PCR appears to be a valuable technique for detection of potentially infectious anti-HBe carriers.


Detection of HBV DNA by PCR in serum from an HBsAg negative blood donor implicated in cases of post-transfusion hepatitis B.

An HBsAg negative blood donor, and three of her recipients, who developed HBsAg positive post-transfusion hepatitis B, were all positive for serum HBV DNA by polymerase chain reaction (PCR), and by subtype discriminating PCR were found to harbour HBV specifying ayw. Thus HBV specifying ayw. Thus HBV DNA may be detected and sub-typed by PCR in infectious HBsAg negative individuals.


Measurement of anti-HBc IgM levels using the Amerlite anti-HBc IgM assay.

The Amerlite anti-HBc IgM assay was evaluated as a tool for determination of antibody levels. With an assay time of 1 hour, the test showed a broad dose response range with high sensitivity and specificity. The software configuration of the analyser can be customized to suit specific research requirements.


Evaluation of a new enhanced luminescence immunoassay for confirming the presence of HBsAg in human serum or plasma.

An enhanced luminescence assay was developed to confirm the presence of HBsAg in weakly reactive sera. The test proved reliable in differentiating between truly reactive and falsely reactive samples.