Autosensitization to DNA: evidence for an immunologic basis.

A 59-year-old female with spontaneous painful ecchymoses developed ecchymoses after intracutaneous injection of washed autologous whole blood cells and calf thymus DNA. Immunofluorescent studies of the spontaneous lesions revealed granular deposits of IgM, C3, factor B and properdin at the dermal-epidermal junction but no deposits in her normal skin. T cells were decreased in number but responded normally to polyclonal mitogens and did not transform in response to DNA containing antigens. Repair of UV-damaged DNA by her lymphocytes appeared to be depressed. The findings presented here are the first immunologic abnormalities uncovered in this disorder and may help in understanding the pathogenesis of the inflammatory lesions seen in autosensitization to DNA.

Glycosylation enhances malondialdehyde binding to proteins.

To determine whether glycosylation of proteins increases their susceptibility to modification with malondialdehyde (MDA), bovine serum albumin, which was pretreated with 500 mg/dl dextrose at 37 degrees C for 0, 1, 2, and 4 weeks, were incubated with 100 mM MDA at 37 degrees C for 24 h. The MDA content of the protein samples were determined after dialysis using thiobarbituric acid (TBA) assay. In addition, a specific anti-MDA protein antiserum was used to demonstrate MDA proteins with immunoblotting technique. The MDA content of BSA preincubated with dextrose for 4 weeks and reincubated with MDA (0.0649 +/- 0.0019 microgram MDA/mg protein) was significantly higher (p < .001) than the MDA content of BSA preincubated with dextrose for only one (0.0227 +/- 0.0031 microgram/mg) or two (0.0347 +/- 0.0034 microgram/mg) weeks or the MDA content of nonglysolated BSA incubated with MDA at the same experimental conditions (0.0201 +/- 0.0029 microgram/mg). These differences could also be found in the immunoblots. However, the correlation of TBA assay with the estimates on immunoblots was poor. It is likely that the immunoblotting assay is more of an estimate of the number of BSA molecules modified with MDA, rather than MDA content of each BSA molecule. It is concluded that in vitro glycosylation of proteins increases their susceptibility to MDA-modification. This may well be an additional pathway of diabetes-related modification of proteins.

Immunochemical properties of malondialdehyde-protein adducts.

Malondialdehyde (MDA), a product of lipid peroxidation, can bind to and modify proteins by adduct formation. To determine whether MDA adducts were immunogenic, MDA was added to rabbit serum albumin (RSA) in order to characterize MDA-proteins and to immunize rabbits. Bound MDA was proportional to the concentration of MDA added in the range of 2.5-20 mM as measured by thiobarbituric acid reactivity. MDA adducts of RSA migrated further toward the anode than native serum protein in zone and immunoelectrophoresis indicating increased negative charge. Rabbits immunized with MDA-RSA produced high titers of IgG antibodies to MDA-RSA as measured by enzyme-linked immunosorbent assay (ELISA). Hapten specificity of the antibody was demonstrated by antisera reactivity with MDA-RSA but not with unaltered RSA. Our findings support the possibility that MDA may serve as a hapten to form neoantigens which may represent a pathway by which lipid peroxidation could produce tissue damage via an immunologic mechanism.

Antigen enhances neuronally induced contraction of intrapulmonary bronchi from IgE-producing rabbits.

The effect of in vitro antigen exposure on contraction induced by electrical field stimulation (EFS) was examined in bronchial rings isolated from rabbits producing specific IgE antibodies. After exposure to antigen, tissues showed an enhanced isometric contractile response to EFS especially at low frequencies, leading to a significant change in the mean slope factor (p less than 0.05) derived from modeling the log frequency response curve using a 4-parameter logistic function. Also, the mean log EF20 +/- SEM decreased from 1.03 +/- 0.05 to 0.88 +/- 0.07 Hz (p less than 0.02). This antigen-induced effect was blocked by pretreatment with 3 microM chlorpheniramine and not observed in unsensitized tissues. Antigen challenge of tissues passively sensitized with IgE (but not IgG) antibodies led to a similar EFS-enhancing effect, significantly reducing the mean slope factor (p less than 0.025). Substituting EFS with exogenous acetylcholine resulted in no antigen-induced enhancement of contraction. The data suggest that antigen-IgE interaction leads to local histamine release sufficient to enhance the function of excitatory airway neurons.

Immunofluorescence in group B streptococcal infection and idiopathic respiratory distress syndrome.

Immunofluorescence was performed on lung tissue obtained at necropsy from 18 newborn infants, including five with group B streptococcal (GBS) sepsis, seven with idiopathic respiratory distress syndrome (IRDS), and six control infants who died from other causes. Deposits of C3, IgG, and fibrin were found within hyaline membranes of infants who died with GBS sepsis or IRDS within 48 hours after birth. In some cases C4, factor B, and IgM were also observed. In five infants with IRDS who died more than five days after birth, immunofluorescent lung findings were less common and less intense. Hyaline membranes, attributed to mechanical ventilators and oxygen therapy in two infants who did not have GBS infection or IRDS, were negative for complement and immunoglobulins although fibrin was detected in one specimen. These data suggest that immunologic processes may contribute to the pathogenesis of certain types of acute lung injury, particularly in infants who die from GBS infection or IRDS during the early neonatal period.

Deficiency of UV-induced excision repair in human thymocytes.

The capacity of human thymocytes and of differentiated lymphocytes circulating in peripheral blood to perform unscheduled DNA synthesis (a measure of nucleotide excision repair) after UV irradiation was measured by radioautographic analysis. Only 4% of immature T lymphocytes, but 68% of circulating lymphocytes exhibited unscheduled DNA synthesis. When UV sensitivity of peripheral blood lymphocytes and thymocytes from the same donor were compared, the thymocytes, in each case, were significantly more UV sensitive than were the circulating lymphocytes. Peripheral blood lymphocytes from subjects undergoing halothane and morphine anesthesia during surgery showed 56% less excision repair capacity than those from unanesthetized donors. The difference occurred in the number of cells capable of repair rather than in the extent of repair synthesis per cell. Ultraviolet-induced unscheduled DNA synthesis occurred in only 3% of the thymocytes removed from rats killed by cervical dislocation. Therefore, the deficiency of excision repair was observed in rat thymocytes which had not been affected by anesthesia or surgical trauma. Since the thymus contains more than 90% immature T-cells, our results indicate that immature T-cells are deficient in nucleotide excision repair whereas the majority of mature peripheral blood lymphocytes exhibit such repair.

Aerosol formulations of terbutaline and isoproterenol in theophylline-stabilized asthmatic patients.

A metered-dose aerosol formulation of terbutaline sulfate (Brethaire), 0.400 mg four times daily, was compared with an identical formulation of isoproterenol sulfate, 0.150 mg four times daily, in a parallel, double-blind, clinical study completed by 40 adult patients with asthma. All patients had been stabilized on theophylline (serum levels of 10 to 20 micrograms/ml). The effectiveness of isoproterenol peaked between 5 and 15 minutes after administration. The effectiveness of terbutaline peaked between 5 and 120 minutes after administration. In each of five visits spaced over a three-month period, patients receiving terbutaline showed a longer duration of bronchodilatory effect than those receiving isoproterenol, with the greatest difference occurring at 60 and 120 minutes after drug administration.

Dose-response study of nebulized bitolterol mesylate solution in asthmatic patients.

Bitolterol mesylate, a new beta 2 adrenergic bronchodilator, is a "pro-drug" which is activated by esterases in the lung. In order to determine the optimal bronchodilator dose of bitolterol, six doses, (0.5 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg and 3.0 mg), were administered by closed-port, intermittent-flow nebulization (CPIF) to asthmatic patients on different days. For most patients, the onset of bronchodilator activity (FEV1 increase of at least 15 percent above baseline) occurred within 5 minutes and lasted at least 8 hours. Maximum mean increases in FEV1 were 46-50 percent at the 1.0 mg to 3.0 mg doses. Beyond the 1.0 mg dose, there was no significant improvement in bronchodilator effect, but adverse effects, particularly tremor, increased at higher doses. The optimal dose of bitolterol administered by CPIF was determined to be 1.0 mg which is similar to the dose of bitolterol recommended for use by metered-dose inhaler (MDI) which is 0.7 mg to 1.1 mg. If continuous-flow nebulization is used, two-three times more drug may be needed for a comparable effect. Bitolterol appears to be a safe, effective and long-lasting bronchodilator when administered by jet nebulization.

Adrenal function in adult asthmatics during long-term daily treatment with 800, 1,200, and 1,600 micrograms triamcinolone acetonide. Multicenter study.

A study to assess the effect of the long-term use of triamcinolone acetonide (TA) on adrenal function was conducted with 143 male and female patients with asthma who were randomly assigned to receive 800, 1200, or 1,600 micrograms of TA daily for six months. Adrenal function was assessed prior to treatment and after two weeks and one, three, and six months of TA use. The effect of TA was evaluated by measuring plasma cortisol levels just prior to and 30 min after a bolus IV injection of 0.25 mg cosyntropin. Adrenal suppression was assumed if the plasma concentration of cortisol did not increase by at least 7 micrograms/dl from the prestimulation value, and remained below 18 micrograms/dl 30 min after the cosyntropin injection. Urine collected for 24 h prior to each cosyntropin stimulation was assayed for free cortisol and related metabolites to confirm suppression. Although all treatment regimens caused some reduction in the 24-h excretion of corticosteroid products, none of the mean values was below the normal ranges. The mean data indicate that TA had no significant effect on adrenal function at any dose or at any time for the patients overall. Individually, three patients exhibited some reduction in adrenal function.

Inhaled epinephrine and oral theophylline-ephedrine in the treatment of asthma.

Inhaled and oral over-the-counter bronchodilators are used for self-therapy by asthmatic patients. To evaluate their safety and efficacy, we compared epinephrine and theophylline combined with ephedrine with inhaled metaproterenol and the placebo. Twelve asthmatic patients were studied in a randomized, double-blind, placebo-controlled, crossover trial comparing forced expiratory volume in 1 second (FEV1) after two inhalations of epinephrine (0.2 mg/inh), 1 minute apart, followed in 15 minutes by theophylline (130 mg) with ephedrine (24 mg) versus two inhalations of metaproterenol (0.65 mg/inh), 1 minute apart, versus placebo inhaler and tablets. Onset of FEV1 greater than 15% above baseline values occurred within 15 seconds after inhalations for 100% of epinephrine-treated patients, 92% of metaproterenol-treated patients, and 33% of placebo-treated patients. FEV1 responses were significantly greater (P less than .05) for epinephrine at 0.66 to 1.66 minutes compared with the responses of metaproterenol, and epinephrine and theophylline that was combined with ephedrine compared with metaproterenol beginning at 2 hours. Mean duration of activity was 5.7 hours for the epinephrine- and theophylline with ephedrine-treated patients, 4.9 hours for metaproterenol-treated patients, and 2 hours for the placebo group. There were statistically significant differences for patients receiving epinephrine and theophylline with ephedrine versus the placebo group (P less than .001), metaproterenol patients versus the placebo group (P = .02), and patients receiving epinephrine and theophylline with ephedrine versus metaproterenol-treated patients (P less than .05). Compared with inhaled metaproterenol, inhaled epinephrine followed in 15 minutes by a theophylline-ephedrine tablet had a significantly earlier onset, longer duration of action, numerically greater peak effect, and patient preference.(ABSTRACT TRUNCATED AT 250 WORDS)

Malondialdehyde binding of rat cerebral proteins is reduced in experimental hypothyroidism.

To determine the effect of thyroid hormones (TH) on cerebral tissue malondialdehyde (MDA) content and accumulation of MDA-bound proteins, hyperthyroid rats and hypothyroid rats were compared to euthyroid controls. Hyperthyroidism was induced by daily injection of l-3,5, 3'-triiodothyronine (15 ug (100 g)-1) intraperitoneally daily for 10 days. Hypothyroidism was induced with 0.025% methimazole in the drinking water for 4 weeks. Immunoblot analysis of cerebral and plasma proteins was carried out using a specific anti-MDA-protein antiserum. MDA was measured as thiobarbituric acid reactive substance. Hypothyroidism was associated with significant reduction in MDA-proteins of plasma (59.3+/-9.5% vs. 99.8+/-23.0% of control p<0.05) and cerebral tissue (17.6+/-19.9% vs. 100.2+/-29.0% of control p<0.001). Hyperthyroidism did not significantly alter MDA-protein distribution. These changes did not correlate with cerebral tissue or plasma MDA concentration. It is concluded that hypothyroidism in rats is associated with significant decrease in MDA-bound proteins. This may have some clinical and biological implications.

Cholesterol enriched diet enhances malondialdehyde modification of proteins in cerebral microvessels of rabbits.

To determine the role of dietary cholesterol on malondialdehyde (MDA) modification of proteins, the cerebral microvessels of rabbits fed a cholesterol enriched diet for 8 weeks were compared to control rabbits. The MDA proteins were estimated with immunoblotting using a specific polyclonal antiserum against MDA proteins. Cholesterol-fed rabbits had a significantly increased MDA protein band at 175 kDa compared to control rabbits (65.6 +/- 6.8 OD versus 16.5 +/- 2.5 OD, P < 0.01). An increase in MDA protein was also found in rabbits intravenously injected with 2 mg of MDA-modified low density lipoproteins or MDA-modified rabbit serum albumin (RSA) at 0, 2, and 4 weeks of observation. The MDA proteins were not increased in cerebral tissue of cholesterol-fed rabbits. It is concluded that a high cholesterol diet is associated with increased MDA modification of proteins in cerebral microvessels. The mechanism could be related to increased circulatory MDA proteins since exogenously administered MDA-LDL or MDA-RSA resulted in similar increases in MDA proteins in cerebral microvessels.

Age-related changes in tissue content of malondialdehyde-modified proteins.

One of the possible mechanisms of the age-related modifications of proteins is the result of reaction with malondialdehyde (MDA), a lipid peroxidation byproduct. To determine the effect of age on the extent of MDA derivatization of proteins in plasma and various tissues, male Fisher 344 rats at 4, 12 and 26 months of age were studied. Protein electrophoresis and immunoblotting was carried out using a specific antiserum against MDA-protein complexes. The concentration of MDA-proteins in plasma (mean +/- SD of optical density in 50 micrograms protein) was 201.6 +/- 47.7 in 4 month old rats, 197.4 +/- 67 in 12 month old rats and 101.4 +/- 22.7 in 26 month old rats (P < 0.01). The MDA protein content of testicle, liver and heart was increased in 12 month old rats compared to 4 and 26 months old rats. There were no age-related differences in MDA-protein content of lung, brain, and kidney. Because of the interindividual variability of MDA-protein profiles within an age group distinct age-related changes in the distribution of various MDA protein bands could not be documented.

Tissue-specific distribution of malondialdehyde modified proteins in diabetes mellitus.

A potential mechanism of diabetes-related tissue damage is modification of various proteins by lipid peroxidation by-products such as malondialdehyde (MDA). To determine the extent of MDA derivatization of various proteins in diabetes mellitus, Western blots were carried out using a specific anti-MDA antiserum to study proteins in plasma and various tissues of control and streptozotocin (STZ)-induced diabetic rats. The concentration of MDA-proteins was highest in plasma compared to other tissues tested. Diabetes was associated with a reduction in MDA-protein content of plasma, lung and liver while in the heart, testicle, cerebrum and kidney the MDA-protein concentration was not altered. Insulin treatment of diabetic rats normalized MDA-protein content of plasma but not in the lung or liver. A large interindividual variability in various protein species was observed within a group. This was partly attributed to polymerization of MDA-proteins. It is concluded that although diabetes is associated with increased lipid peroxidation the content of MDA-proteins in plasma and in some tissues is decreased.

Malondialdehyde modified proteins and their antibodies in the plasma of control and streptozotocin induced diabetic rats.

One of the possible mechanisms of diabetes-related tissue damage is modification of various proteins via lipid peroxidation byproducts such as malondialdehyde (MDA). To determine the extent of MDA derivatization of plasma proteins, Western blots were carried out using anti-MDA antisera to study plasma proteins in control and streptozotocin (STZ)-induced diabetic rats. Since MDA can modify proteins and may alter or enhance their antigenicity, we screened plasma samples for anti-MDA antibodies using enzyme-linked immunosorbent assay (ELISA), and confirmed antibody specificity by inhibition ELISA. Circulating immune complexes containing MDA were also assayed. This study is the first demonstration of the existence in plasma of MDA-modified proteins with a molecular weight of approximately 100 Kd. Both control and diabetic rats have similar concentrations of plasma anti-MDA antibodies and circulating immune complexes. These results do not support the notion that diabetes alters the immune response to MDA modified proteins. Whether MDA modification of proteins participate in immunological processes that lead to tissue injury remains to be demonstrated.

Effect of select antioxidants on malondialdehyde modification of proteins.

To determine whether commonly used antioxidants alter malondialdehyde (MDA) modification of proteins, a known mechanism of free radical-related tissue injury, we studied the effect of adding 1 mg/mL of pycnogenol, 5 mM of alpha-tocopherol, 5 mM of ascorbate, and 0.2 mg/mL of an ethanol equivalent of red and white wine on MDA-protein content of endothelial cells in culture. The addition of pycnogenol but not of the other antioxidants was associated with significant reduction in MDA-protein content compared with controls (0.521 +/- 0.041 in arbritrary units versus 1.011 +/- 0.021, P < 0. 001). To determine whether the observed effect occurs distal to MDA generation, the effect of these antioxidants on the modification of bovine serum albumin with MDA generated in a cell-free system was studied. In this cell-free assay, pycnogenol but not the other antioxidants reduced MDA-BSA generation by approximately 50%. It is concluded that pycnogenol may reduce MDA modification of proteins at a step distal to MDA generation. This may be an additional mechanism of protective effects of pycnogenol against oxidative stress.

Malondialdehyde modification of proteins in vitro is enhanced in the presence of acetaldehyde.

To test the hypothesis that the cardioprotective effect of alcohol is related to the inhibition of malondialdehyde (MDA) modification of proteins by acetaldehyde (AA), we studied the effect of AA on MDA modification of bovine serum albumin (BSA) in vitro. BSA was incubated simultaneously with a fixed concentration of MDA (70 mM) and different concentrations of AA (120, 60, 30, 10, or 0 mM) for 24 h at 37 degrees C. The MDA-modified or AA-modified BSA was quantitated with immunoblotting by using specific anti-MDA and specific anti-AA protein antisera, respectively. In another set of experiments, BSA was incubated sequentially, first with different concentrations of AA and then with 70 mM of MDA. In both incubation protocols, the presence of AA and AA modification of BSA enhanced MDA binding. These in vitro observations suggest that the putative cardioprotective effects of alcohol or wine cannot be ascribed to AA-mediated reduction in MDA protein formation, a possible biochemical pathway of accelerated atherosclerosis.

Ants and bees: whole body extracts vs venom.

Ants, bees, and other stinging hymenoptera can induce serious anaphylactic reactions in susceptible individuals. Certainly, hymenoptera venom contains potent antigens that have been useful in testing and treating hymenoptera-sensitive patients who fail to acquire protection from whole body extract therapy. However, before whole body extracts are forever abandoned, their antigen content and stability should be re-examined by means of modern immunologic methods.