Augmentation of human neutrophil and alveolar macrophage LTB4 production by N-acetylcysteine: role of hydrogen peroxide

1. The actions of N-acetylcysteine (NAC) on hydrogen peroxide (H2O2) and leukotriene B4 (LTB4) production by human resting and stimulated peripheral blood neutrophils and alveolar macrophages were investigated. 2. At a concentration of 100 microM, NAC significantly (P < 0.01) suppressed the accumulation of H2O2 in the incubation medium of resting and opsonized zymosan (OZ; 0.5 mg ml[-1])- or N-formylmethionyl-leucyl-phenylalanine (fMLP; 1 microM)-stimulated neutrophils and of resting and OZ-stimulated macrophages. At concentrations of 10 microM and above, NAC augmented significantly the level of LTB4 in the supernatants of OZ- and fMLP-stimulated neutrophils (P < 0.01 and P < 0.05, respectively) and OZ-stimulated macrophages (P < 0.05 at 10 microM, P < 0.01 at 100 microM NAC). 3. NAC (100 microM) caused a significant (P < 0.01) reduction in the quantity of measurable H2O2 when incubated with exogenous H2O2 concentrations equivalent to those released from OZ-stimulated neutrophils and macrophages. At no concentration did NAC affect quantitites of measurable LTB4 when incubated with exogenous LTB4. 4. Superoxide dismutase (SOD), which catalyzes the conversion of superoxide anion to H2O2 had no significant effect on LTB4 production by human neutrophils. In contrast, catalase, which catalyzes the conversion of H2O2 to H2O and O2, caused a pronounced, statistically significant (P < 0.01) increase in the levels of LTB4 measured in the supernatants of OZ- and fMLP-stimulated neutrophils. 5. H2O2 (12.5 microM and 25 microM, concentrations equivalent to those measured in the supernatants of activated neutrophils and alveolar macrophages, respectively) caused a small (13%) decrease in the quantity of measurable LTB4 (P = 0.051 and P < 0.05 at 12.5 microM and 25 microM, respectively) that was inhibited by NAC (100 microM) but not by catalase (400 u ml[-1]). 6. In conclusion, the anti-oxidant drug, NAC, increases LTB4 production by human neutrophils and alveolar macrophages, probably through the elimination of cell-derived H2O2. LTB4 undergoes a H2O2-dependent oxidation that is inhibited by NAC but this is unlikely to account fully for the increased levels of LTB4, suggesting that NAC may increase LTB4 production by blocking the H2O2-dependent inhibition of a synthetic enzyme, such as 5-lipoxygenase.     

Inclusion of oxidized vegetable oil in broiler diets. Its influence on nutrient balance and on the antioxidative status of broilers

Over a period of 4 wk, 24 10-d-old broiler hens were fed diets containing 11% vegetable oil (9% rapeseed oil, 2% soybean oil), which was added either fresh (1 meq O2/kg oil) or oxidized (156 meq O2/kg oil). The effects of the dietary treatments on nutrient digestibility were examined in a balance experiment. The antioxidative status of the animals was evaluated using plasma concentrations of thiobarbituric acid-reactive substances (TBARS), erythrocyte hemolysis in vitro, selenium-dependent and selenium-independent activity of glutathione peroxidase in liver cell cytosolic fractions, and concentrations of tocopherols and other fat-soluble compounds with antioxidative properties (lutein, beta-carotene, and retinol) in plasma and various tissues (skeletal muscle, cardiac muscle, liver, and abdominal fat). Compared to the fresh oil, the concentrations of linoleic and linolenic acid were slightly lower in oxidized oil. The concentration of alpha-tocopherol in the diet with fresh oil was an average of 80.8 mg/kg diet, whereas the diet with oxidized oil only provided 44 mg/kg. The dietary selenium content averaged 0.48 mg/kg in both diets. During the experiment, none of the animals showed symptoms of diarrhea or vitamin E deficiency. The intake of oxidized oil caused a growth depression after 2 wk. The retention of fat (P = 0.07), energy (P = 0.09), and alpha-tocopherol (P < 0.01) was lower in the group fed oxidized fat. Furthermore, these animals showed significantly higher plasma concentrations of TBARS (P < 0.01), and lower concentrations of tocopherols, lutein, beta-carotene, and retinol in plasma and tissues.     

Cold jet: a method to obtain pure Schwann cell cultures without the need for cytotoxic, apoptosis-inducing drug treatment

Trace gases in exhaled air have been used as a simple means of assessing metabolic reactions. The investigations of trace gases derived from bacteria in human exhalation are usually hydrogen (H2) or methane (CH4). On the other hand, nitrous oxide (N2O) is also derived from microorganisms, especially denitrifying bacteria. Although many kinds of denitrifying bacteria have been isolated on and in the human body, there has been few concerning N2O. We studied 222 healthy people from the age of 5 to 85 years. The analysis of N2O in exhaled air was carried out by a infrared-photoacoustic (IR-PAS) analyzer. It was found that N2O ranged from 0 to 1670 ppbv in exhaled air and that 59% (131) of the subjects were producers of N2O. A highly significant relationship was observed between age and concentrations of N2O (r = 0.40, P < 0.01). The rate of production in young children and in the aged was significantly higher than that in adults aged 20-39 years (P < 0.01), and less than 30% were producers during puberty. The change of normal microflora on and in human body with aging may have caused the significant relationship between age and emissions of N2O.     

Effects of hypothermia in hypercapnia and hypercapnic hypoxemia

Anesthetized, paralyzed and mechanically ventilated pigs were hypoventilated to extreme hypercapnia (PaCO2 approximately 20 kPa) at FiO2 0.5, and allotted to a hypothermic group (31.5 +/- 0.1 degrees C, n = 6) or a control group (39.6 +/- 0.2 degrees C, n = 6). Compared with the controls, the hypothermic animals had higher PaO2 (19.2 vs 15.6 kPa, P < 0.05), SaO2 (97.2 vs 89.3%), SvO2 (78.7 vs 68.2%), end-tidal O2 (34.5 vs 24.8 kPa) and arterial pH (7.01 vs 6.91), (P < 0.01), but lower PvO2 (7.0 vs 10.2 kPa) and PaCO2 (13.2 vs 23.5 kPa), (P < 0.01). Hypothermia reduced O2 delivery (DO2), O2 consumption (VO2) and CO2 production by 40-45% (P < 0.05), but O2 extraction ratio, i.e. VO2.DO(2)-1 x 100(%), did not differ between groups. Hypothermic animals had lower heart rate (127 vs 223 beats.min-1, P < 0.05) and cardiac output (2.5 vs 3.9 l.min-1, P < 0.01). Subsequently, the inspired oxygen fraction (FiO2) was decreased stepwise (0.3, 0.25, 0.21, 0.15, 0.10) at 30-min intervals. At FiO2 0.3, the hypothermic group had higher PaO2 (10.0 vs 5.7 kPa), SaO2 (91.3 vs 28.5%), PvO2 (5.8 vs 3.4 kPa), SvO2 (70.7 vs 10.3%), end-tidal O2 (16.7 vs 8.5 kPa), O2 delivery (344 vs 155 ml.min-1), arterial pH (7.02 vs 6.94) and systemic vascular resistance (3850 vs 1652 dyn.s.cm-5 (38,500 vs 16,520 microN.s.cm-5)) compared with the controls (P < 0.01), while PaCO2 was lower (12.4 vs 22.7 kPa), as well as O2 extraction ratio (23 vs 63%) and O2 half saturation tension (4.3 vs 8.0 kPa) (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of temocapril, an angiotensin converting enzyme inhibitor, on the cyclosporine-induced nephrotoxicity

PURPOSE: We examined the effect of temocapril (Tem), an angiotensin converting enzyme inhibitor (ACEI), on the cyclosporine-induced nephrotoxicity (CsA-NT) in rats. MATERIALS AND METHODS: Male Wistar rats were used. Group 1 (G1) received a medium (i.e. olive oil) only, group 2 (G2) received CsA (30 mg./kg./d) only, group 3 (G3) received both CsA (30 mg./kg./d) and Tem (80 micrograms./kg./d), and group 4 (G4) received Tem (80 micrograms./kg./d) only. Each group consisted of 5 animals. Drugs were given orally for fourteen days. Then, renal cortical blood flow (RCBF) and concentrations of serum creatinine (S-Cr), serum potassium (S-K), whole blood CsA (WB-CsA), serum aldosterone (Ald), and plasma renin activity (PRA) were measured. The creatinine clearance (CCr) was also calculated. Kidneys were processed to the light microscopic examination with Bowie stain, and the size of the renin granules (S-RGs) was estimated by an image analysis system. RESULTS: G2 showed a decrease of RCBF (p < 0.01), an increase of S-Cr (p < 0.01), a decrease of CCr (p < 0.01), an increase of S-K (p < 0.05), and an increase of S-RGs (p < 0.01). Compared with G2, G3 showed significant improvement in RCBF (p < 0.01) and S-Cr (p < 0.05), but still showed significant impairments in all indices (p < 0.01 or p < 0.05, vs control). G4 showed no remarkable changes comparing with G1, except a significant (p < 0.01, vs. control) increase of S-K. G3 showed additional effect on the increase of S-K (p < 0.01, vs. control and G2). Tem showed no significant influence on the WB-CsA level. The serum Ald and PRA levels were not significantly changed by these drugs. CONCLUSIONS: These data are compatible with the hypothesis that the vasoconstriction of the afferent arterioles (AAs) is a principal mechanism of CsA-NT. The increase of RGs may be the result of both increased production and inhibited secretion of renin.     

T cell responses in coinfection with Onchocerca volvulus and the human immunodeficiency virus type 1

Onchocerca volvulus and the human immunodeficiency virus (HIV) are two immunocompromising infectious agents of major public health concern in Uganda. To examine the effect of coinfection with O. volvulus and HIV on cellular immune responses, lymphocyte proliferative responses and cytokine production of peripheral blood mononuclear cells (PBMC) from persons infected with O. volvulus with and without HIV type 1 infection were compared. Proliferation of PBMC to PHA and tuberculin (PPD) in coinfection was less (P = 0.08, P < 0.01) than in O. volvulus infection. O. volvulus extract stimulated lymphocyte proliferation in microfilaria-negative and HIV-negative O. volvulus infection while only an inconspicuous response was observed in microfilaria-negative coinfection. After stimulation of PBMC with PPD, the production of interferon-gamma (IFN-gamma), interleukin (IL)-4 and IL-5-demonstrated in O. volvulus infection-were reduced in coinfection with HIV (P < 0.01). While both groups failed to produce IFN-gamma in response to O. volvulus extract, only O. volvulus infected persons generated pronounced IL-5 and low IL-4 levels (0.01 > P = 0.02). The cellular immune responses in coinfection suggested an HIV-related lack of specific reactivity to O. volvulus antigen and impairment of IL-4 and IL-5 production in addition to the lack of IFN-gamma response on antigenic stimulation.     

Kallikrein gene expression in human pituitary tissues

Somatostatin has been suggested to influence the somatotrophic axis outside the central nervous system, in reducing GH-induced IGF-I mRNA and IGF-I generation. This study aimed to determine whether such effects were mediated via the GH receptor (GHR). GH-deficient dwarf rats aged 45-47 days (n = 8 per group) received twice daily subcutaneous injections of octreotide (1 mg/kg) (group O), saline (group S), octreotide (1 mg/kg) plus bovine GH (0.25 mg/kg) (group OG), or bovine GH (0.25 mg/kg) plus saline (group G) for 10 days. Octreotide-treated animals had less weight gain compared with saline-treated animals, but not when GH cotreated (group OG vs G). Octreotide had an overall effect on decreasing length gain (P < 0.01). Serum IGF-I (ng/ml) was reduced by octreotide (group O 171 +/- 11, group S 239 +/- 20, P < 0.01; group OG 283 +/- 30, group G 362 +/- 10, P < 0.001), as was serum insulin (P < 0.001). A significant decrease in hepatic and muscle IGF-I mRNA expression was found as expected, yet this was not associated with decreased hepatic GHR expression. Rather, an increase in hepatic 125I-bovine GH specific binding was observed (P < 0.001) and, in GH-cotreated animals (OG), hepatic GHR and GH binding protein (GHBP) mRNA expression were also increased by octreotide by approximately 40%. In muscle, octreotide was associated with an approximately 30% decrease in GHBP mRNA and no effect on GHR mRNA. This study suggests that the suppressive effects of octreotide on IGF-I metabolism, at least in liver, are not mediated via down-regulation of GHR expression, but more likely by direct effects on IGF-I expression.     

Effects of mouth cleansing on the levels of exhaled nitrous oxide in young and older adults

Nitrous oxide (N2O) is produced by denitrification, i.e. by microbial reduction of nitrate (NO3-). Our previous studies have established an analytical method for demonstrating the existence of N2O in exhaled air, and we showed that levels of N2O in exhaled air increase with age after puberty. However, the source of this change and its biological significance are still unclear. The purpose of this study was to examine whether the oral microorganisms are the main source of N2O. We measured exhaled N2O in 35 young adults (aged 19-29 years) and 34 older adults (aged 61-79 years) before and after mouth cleansing. N2O was measured using an infrared-photoacoustic analyzer equipped with an optical filter (UA0985, 2215 cm-1). Participants were classified as producers and non-producers according to the levels of exhaled N2O relative to the level in the atmosphere. N2O production differed significantly between the young adult producers and the older adult producers. Mouth cleansing resulted in an immediate reduction in exhaled N2O in both groups. We only found seven (20.0%) producers in the young, and 32 (94.1%) producers in the older after mouth washing. The differences before and after mouth cleansing were significant in both groups (P < 0.01 in the young and P < 0.05 in the older). The oral cavity is a major source of N2O. However, since approximately half-levels of N2O were still observed in exhaled air after mouth, cleansing, there may exist another N2O source in the human body.     

Comparisons of the frequencies and molecular spectra of HPRT mutants when human cancer cells were X-irradiated during G1 or S phase

In an attempt to elucidate mechanisms underlying the variation in radiosensitivity during the cell cycle, mutations in the HPRT gene were selected with 6-thioguanine, quantified and characterized in synchronous human bladder carcinoma cells (EJ30-15) that were irradiated in G1 or S phase with 3 or 6 Gy. Synchronous cells were obtained by mitotic selection, with approximately 98% of the cells in G1 phase when they were irradiated after 3 h of incubation, and 75% in S phase when they were irradiated after 14 h of incubation. The mutant frequencies were approximately 4-fold higher (P < 0.01) when cells were irradiated in G1 phase compared with S phase, and the lowest frequency (1.5 x 10(-5) for 3 Gy during S phase) was approximately 10-fold higher than the spontaneous frequency. Exon analysis by multiplex polymerase chain reaction was performed on DNA isolated from each independent mutant. The different types of mutants were categorized as class 1, which consisted of base-pair changes or small deletions less than 20 bp; class 2, which consisted of deletions greater than 20 bp but with one or more HPRT exons present; and class 3, which consisted of deletions encompassing the entire HPRT gene and usually genomic markers located 350-750 kbp from the 5' end of the gene and/or 300-1400 kbp from the 3' end. A "hotspot" for class 2 deletions was observed between exons 6 and 9 (P < 0.01). For cells irradiated during G1 phase, the percentages for the different classes (total of 78 mutants) were similar for 3 and 6 Gy, with a selective induction of class 3 mutants (34-38%) compared with spontaneous mutants (3%, total 20). When S-phase cells were irradiated with 3 Gy, there were fewer class 1 mutants (21%, total 37) than when cells were irradiated in G1 phase with 3 Gy (45%, total 42) (P < 0.01). The greatest change was observed when the dose was increased in S phase from 3 Gy to 6 Gy (total of 43 mutants), with the frequency of class 2 mutants decreasing dramatically from 30% to 1% (P < 0.005). A similar decrease in class 2 mutants with an increase in dose has been observed by others in asynchronous cultures of normal human fibroblasts. We hypothesize that these differences occur because: (a) there is more error-free repair of double-strand breaks (DSBs) during S than G1 phase; (b) a single DSB within the HPRT gene causes a class 2 mutation or a certain percentage of class 1 mutations, while two DSBs, with one in each approximately 1-Mbp region 5' and 3' of the gene, cause a class 3 mutation; and (c) a repair process that is induced when the dose during S phase is increased from 3 to 6 Gy results in a preferential decrease in class 2 mutations.     

Hepatotoxicity of halogenated inhalational anesthetics studied in rats hepatocytes

Effects of 2% halothane, 1.5% sevoflurane, 1.5% enflurane, and 1.2% isoflurane on hepatic dysfunction were studied using rat hepatocytes incubated in media containing 95% or 5% O2. The effects of anesthetics on hepatic perfusion were eliminated by incubation of hepatocytes for 45 minutes with each combination of anesthetic and oxygen concentration. After incubation, viability of hepatocytes was assayed by the LDH latency test. Enzyme (GPT, GOT, LDH) activities, lactate concentration and pyruvate concentration in the incubation medium were measured. The concentrations of adenine nucleotides and inorganic phosphorous in the liver were determined. Anesthetics administered in 95% O2 did not produce significant decreases in viability and enzyme release compared to 95% O2 alone. Halothane, sevoflurane, and isoflurane administered in 5% O2 produced significant decreases in viability and enzyme releases compared to 95% O2 alone. In groups administered 95% O2 there was a significant relationship between viability and energy charge in hepatocytes (P < 0.01). In the 5% O2 groups, there were significant relationships between viability and ATP in hepatocytes (P < 0.01) or L/P ratio in incubation medium (P < 0.01). These results suggest that the combination of anesthetics and hypoxia produce hepatotoxicity. Destruction of energy status might be the cause of hepatotoxicity.     

Conditionally immortalized cell lines with differentiated functions established from temperature-sensitive T-antigen transgenic mice

Seventy-two healthy males and females serially breathed air and 100% O2 at 1.0 atm abs (1.01 bar; AIR and O2, respectively), then 100% O2 at 2.36 atm abs (2.39 bar, HBO) to establish reference values for chest (CH), leg (LG), and foot (FT) PtcO2. Subjects sequentially a) rested supine with legs extended (baseline); b) elevated their monitored leg; c) returned to supine/extended position; d) assumed a seated, both legs dependent posture; and e) returned to supine/extended position. LG and FT PtcO2 decreased during leg elevation and increased when both legs were dependent during AIR, O2, and HBO (P < 0.0001, respectively). LG PtcO2 of females exceeded that for males in all conditions (P < 0.05 to P < 0.0001). Baseline CH PtcO2 also was greater than LG PtcO2 for all subjects in all conditions (P < 0.01 to P < 0.0001) and greater than FT PtcO2 for all conditions (P < 0.01 to P < 0.0001) except AIR. We conclude that: a) position and hyperoxygenation of an extremity significantly affect PtcO2; b) PtcO2 does not follow a decreasing CH to FT gradient in all conditions; c) a gender difference exists for LG PtcO2; and d) PtcO2 reference data are established for the comparative evaluation and clinical management of problem wounds.     

Association of glycerylphosphorylcholine with human sperm and effect of capacitation on their metabolism

The presence and localization of glycerylphosphorylcholine (GPC) on the surface of human sperm, as well as the metabolism of its breakdown product L-glycerol 3 phosphate (G3P), were investigated. GPC was found to be associated with sperm after penetrating cervical mucus and was present after repeated washing of the sperm. GPC was partially released by treatment with 0.4 M NaCl in 0.01 M sodium phosphate buffer (pH 7.4) and localized to the head region after sperm fractionation. G3P did not increase O2 uptake of uncapacitated human sperm. However, under aerobic conditions, lactate accumulated when exogenous G3P or uterine GPC diesterase was added to sperm in suspension. The uptake of O2 by washed capacitated sperm pre-incubated with 1 unit of rat uterine GPC diesterase for 30 min was significant. This effect was inhibited by 2 microM oligomycin indicating that oxidative phosphorylation had occurred. The present study indicates that GPC may play a role in the metabolism of human sperm after capacitation.     

The effects of epidermal growth factor, interleukin-1, and phenytoin, alone and in combination, on C19 steroid conversions in fibroblasts

The formation of the biologically active metabolite 5 alpha-dihydrotestosterone (DHT) from testosterone in response to phenytoin (Ph), interleukin-1 (IL-1), and epidermal growth factor (EGF) was investigated. The androgen DHT stimulates matrix synthesis in connective tissue and bone. Duplicate incubations were performed with confluent human gingival fibroblasts, 14C-testosterone, and optimal stimulatory concentrations of IL-1 (5 IU/ml), EGF (10 ng/ml), Ph (5 micrograms/ml), Ph + EGF, and Ph + IL-1 respectively for 24 hours in Eagle's MEM at 37 degrees C. The medium was then analyzed for radioactive metabolites. Similar incubations were performed with human gingival tissue using 14C-4-androstenedione as substrate in the presence or absence of EGF, Ph, and EGF + Ph. In the cell lines studied, EGF stimulated DHT and 4-androstenedione synthesis by 20% (n = 5; P < 0.01; Wilcoxon signed rank statistic for paired observations). IL-1 stimulated DHT and 4-androstenedione synthesis by 2-fold (n = 6; P < 0.01). Ph stimulated DHT and 4-androstenedione synthesis by 2-fold increases (n = 3; P < 0.01). Combinations of phenytoin and EGF stimulated DHT and 4-androstenedione synthesis by 33% and 37% greater than the effect of phenytoin alone (n = 3; P < 0.01). Combinations of Ph and IL-1 caused a 45% increase in the amount of DHT formed and a 66% increase in 4-androstenedione when compared to the effect of phenytoin alone (n = 3; P < 0.01). 14C-4-androstenedione was converted to DHT and testosterone by human gingival tissue. There were 2-fold, 4-fold, and 2.5-fold increases in DHT synthesis and 5-fold, 2-fold, and 6-fold increases in the formation of testosterone in response to EGF, Ph, and EGF + Ph respectively (n = 3; P < 0.01). EGF and IL-1 present in inflammatory exudate may have implications on phenytoin-induced overgrowth via the steroid metabolic pathway.     

Ventilation and respiratory mechanics during exercise in younger subjects breathing CO2 or HeO2

To determine if ventilation (VE) during maximal exercise would be increased as much by 3% CO2 loading as by resistive unloading of the airways, we studied seven subjects (39 +/- 5 years; mean +/- S.D.) during graded-cycle ergometry to exhaustion while breathing: (1) room air (RA); (2) 3% CO2, 21% O2, and 76% N2; or (3) 79% He and 21% O2). VE and respiratory mechanics were measured during each 1-min increment (20 or 30 W) in work rate. VE during maximal exercise was increased 21 +/- 17% when breathing 3% CO2 and 23 +/- 16% when breathing HeO2 (P < 0.01). Further, the ventilatory response to exercise above ventilatory threshold (VTh) was increased (P < 0.05) when breathing HeO2 (0.89 +/- 0.26 L/min/W) as compared with breathing RA (0.65 +/- 0.12). When breathing HeO2, end-expiratory lung volume (% total lung capacity, TLC) was lower during maximal exercise (46 +/- 7) when compared with RA (53 +/- 6, P < 0.01). In conclusion, VE during maximal exercise can be augmented equally by 3% CO2 loading as by resistive unloading of the airways in younger subjects. This suggests that in younger subjects with normal lung function there are minimal mechanical ventilatory constraints on VE during maximal exercise.     

Augmented placental production of leptin in preeclampsia: possible involvement of placental hypoxia

Preeclampsia (PE) is a hypertensive disorder, which develops in late pregnancy and is usually associated with placental hypoxia and dysfunction. We have recently demonstrated that leptin is a novel placenta-derived hormone in humans and suggested its significance in human pregnancy (see Ref. 19). To explore the changes in the leptin production in placenta in PE, we measured the plasma leptin level and placental leptin messenger RNA expression in pregnant women with PE. Plasma leptin levels in preeclamptic women were elevated significantly, compared with gestational age- and body mass index-matched normal pregnant women (P < 0.0001). Plasma leptin levels in the severe PE group were significantly higher than those in the mild PE group (P < 0.0001). Plasma leptin levels in preeclamptic women were reduced, soon after the placental delivery, to those expected for their body mass indices. Northern blot analysis revealed that leptin messenger RNA levels are increased in the placentas from preeclamptic women, compared with normal pregnant women. Leptin secretion was increased significantly in a human trophoblastic cell line (BeWo cells) cultured under hypoxic conditions (5% O2), compared with those cultured under standard conditions (20% O2; P < 0.01). The present study demonstrated that placental production of leptin is augmented in severe PE, probably because of placental hypoxia, thereby suggesting the possible significance of leptin as a marker of placental hypoxia in severe PE.     

Aminoguanidine inhibits reactive oxygen species formation, lipid peroxidation, and oxidant-induced apoptosis

Aminoguanidine (AG) treatment, like nerve growth factor (NGF) treatment, prevents diabetes-induced apoptosis of retinal Müller cells in the rat eye, but the mechanism involved is unknown. In this study, the effects of preincubation with AG on oxidant-induced apoptosis, oxidant-induced intracellular reactive oxygen species (ROS) production, and lipid peroxidation were determined in rat retinal Müller cells and compared with the effects of NGF, a protein that protects neuronal cells from oxidative stress. The effect of AG on rabbit vitreous lipid peroxide levels was also determined. After exposure to increasing concentrations of H2O2, there was a corresponding increase in the percentage of apoptotic Müller cells. Preincubation with AG for 48 h completely inhibited oxidant-induced apoptosis in response to 10 micromol/l H2O2 (+AG 0 vs. 10 micromol/l, NS), and reduced the percentage of apoptotic cells in response to 50 micromol/l H2O2 by 50% (+AG vs. -AG, P < 0.01). Longer preincubation did not increase the antiapoptotic effect of AG. The effect of AG was dose-dependent. Similar results were obtained after preincubation with NGF. Both AG and NGF preincubation prevented the twofold increase in oxidant-induced lipid peroxides. The fivefold increase in oxidant-induced ROS production was decreased 100% by NGF, but only 61% by AG preincubation. The twofold increase in vitreous lipid peroxide level in diabetic rabbits was completely prevented by AG treatment. AG reduced H2O2-induced benzoate hydroxylation in a dose-dependent manner. Intracellular glutathione content was unchanged. These data demonstrate that AG can act as an antioxidant in vivo, quenching hydroxyl radicals and lipid peroxidation in cells and tissues and preventing oxidant-induced apoptosis.     

Analysis of cytokine and specific antibody profiles in hydatid patients with primary infection and relapse of disease

We studied in vitro cytokine production by peripheral blood mononuclear cells (PBMC) from patients with primary and recurrent hydatid disease when cells were incubated with mitogen (PHA) and antigen from hydatid cyst fluid (HCFAg); levels of specific IgE, IgG4 and eosinophil counts were also measured in sera. When specifically stimulated, PBMC from patients produced higher levels of IL-2 (P < 0.02), IFN-gamma (P < 0.0028) and IL-5 (P < 0.01) than those from uninfected donors, whereas IL-10 levels were comparable. Notably, IL-5 was also produced in higher levels (P < 0.01) by PBMC from patients when incubated with PHA. The IL-5:IFN-gamma ratio was significantly greater (P < 0.02) when measured in response to specific stimulation than it was for PHA-stimulated cultures. These cytokine data suggest a bias towards a Th2-response which is in agreement with the high levels of IgG4 and IgE observed. The polarized response appears to be related to clinical status, as differences between patients with primary infection and those with relapse of disease were demonstrated, with significantly higher levels of IgE (P < 0.003), IgG4 (P < 0.04) titres and eosinophil counts (P < 0.04) in the latter; in addition a tendency to an increased production of IL-5 buy lower IFN-gamma was also observed in this group. These results merit further study as they are suggestive of a putative role of Th2-like responses in susceptibility to reinfection by E. granulosus.     

Proglucagon processing in an islet cell line: effects of PC1 overexpression and PC2 depletion

Proglucagon (proG) is differentially processed in the A cells of the pancreas to yield glucagon, and in the L cells of the intestine to generate glicentin, oxyntomodulin, the incretin glucagon-like peptide (GLP)-1(7-36NH2) and the intestinotropin GLP-2. To establish roles for the prohormone convertases PC1 and PC2 in proG processing within the context of a physiological model, we created stable cell lines from an islet-derived cell line, InR1-G9. These cells express proG and PC2, but not PC1, messenger RNA (mRNA). InR1-G9 cells were stably transfected with PC1 or antisense PC2. Selection was carried out in G418 (InR1-G9/PC1) or Zeocin (InR1-G9/ASPC2). Both PC1 mRNA and protein were highly expressed in InR1-G9/PC1 cells (P < 0.01-0.001) compared with wild-type (WT) cells. Cells transfected with ASPC2 demonstrated significant decreases in both PC2 mRNA (P < 0.001) and protein (P < 0.05) levels. ProG-derived peptides in WT, control, InR1-G9/PC1, and InR1-G9/ASPC2 cells were identified by HPLC and RIA. Overexpression of PC1 in InR1-G9 cells resulted in increased processing to glicentin (P < 0.01), oxyntomodulin (P < 0.05), and GLP-2 (P < 0.05). Interestingly, processing to GLP-1(7-36NH2) did not increase upon transfection of PC1. Transfection of InR1-G9 cells with ASPC2 resulted in the disappearance of glicentin (P < 0.05). However, production of glucagon was not altered by antisense deletion of PC2. Surprisingly, GLP-1(7-36NH2) production appeared to be augmented (P < 0.05) in InR1-G9/ASPC2 cells, whereas GLP-2 production was not altered. In conclusion, these studies establish the role of PC1 in the processing of proG to the intestinal proG-derived peptides. This study also establishes a role for PC2 in the production of glicentin; however, the liberation of glucagon appears to be mediated by another, yet to be identified, convertase.     

Decreased expression of mitochondrial-derived H2O2 and hydroxyl radical in cytoresistant proximal tubules

Increased production of reactive oxygen metabolites (ROM) can contribute to the initiation phase of nephrotoxic and ischemic acute renal failure (ARF). However, whether altered ROM expression also exists during the maintenance phase of ARF has not been adequately assessed. Since diverse forms of tubular injury can initiate a "cytoresistant state," this study tested whether a down-regulation of ROM expression might develop in the aftermath of acute tubular damage, potentially limiting renal susceptibility to further attack. To test this hypothesis, rats were subjected to either mild myohemoglobinuria (glycerol injection) or bilateral ureteral obstruction and 24 hours later, cytoresistant proximal tubular segments (PTS) were isolated to assess ROM expression. PTS from sham operated rats were used to establish normal values. Both sets of cytoresistant PTS manifested approximately 75% reductions in H2O2 levels, as assessed by the phenol red/horseradish peroxidase technique (P < 0.01 to 0.001). A 40% reduction in hydroxyl radical (.OH) levels was also observed (salicylate trap method), thereby substantiating decreased oxidant stress in cytoresistant PTS. Catalase, glutathione peroxidase, and free iron levels were comparable in control and cytoresistant PTS, suggesting that decreased H2O2 production (such as by mitochondria) was the cause of the decreased oxidant stress. To test this latter hypothesis, H2O2 expression by control and cytoresistant PTS was assessed in the presence of respiratory chain inhibitors. Although site 1 and site 3 inhibition markedly suppressed H2O2 production in control PTS, they had no impact on H2O2 production in cytoresistant PTS, implying that production at these sites was already maximally suppressed. Correlates of the decreased mitochondrial H2O2 production were improvements in cell energetics (increased ATP/ADP ratios with Na ionophore treatment) and approximately 40 to 90% increases in PTS/renal cortical glutathione content. We conclude that: (1) proximal tubule H2O2/.OH expression can be downregulated during the maintenance phase of ARF; (2) this seemingly reflects a decrease in mitochondrial ROM generation; and (3) the associated improvements in glutathione content and/or cellular energetics could conceivably contribute to a post-injury cytoresistant state.     

Pelviureteral inhibitory reflex and ureteropelvic excitatory reflex: role of the two reflexes in regulation of urine flow from the renal pelvis to the ureter

The mechanism by which the ureteropelvic junction (UPJ) regulates the passage of urine from the renal pelvis to the ureter, and prevents urinary backflow from the the ureter to the renal pelvis, is not completely understood. The current communication studies this mechanism in 18 dogs. With the dogs under anesthesia, nephrostomy was done through which two catheters (one pressure and one balloon-tipped) were introduced into the UPJ and the renal pelvis, respectively. Renal pelvis distension with a balloon filled with 1 ml of saline effected a rise of renal pelvic pressure from a mean basal pressure of 4.8 +/- 1.2 cm H2O to 6.9 +/- 2.3 cm H2O (P < 0.05). The basal UPJ pressure of 12.6 +/- 2.7 cm H2O showed no significant change with 1 ml distention of the renal pelvic balloon (P > 0.05). Renal pelvic distension with 2, 3, and 4 ml caused a significant rise of renal pelvic pressure to 8.4 +/- 2.7 (P < 0.05), 10.6 +/- 2.2 (P < 0.01), and 11.8 +/- 1.9 (P < 0.01) cm H2O, respectively, and a significant drop of UPJ pressure to 4.8 +/- 1.2, 4.7 +/- 1.1, and 4.6 +/- 1.2 cm H2O (P < 0.01), respectively. Ureteric distension with a balloon filled with 0.5 ml of saline significantly raised the ureteric pressure from a mean basal value of 4.3 +/- 1.4 cm H2O to 14.7 +/- 3.3 cm H2O (P < 0.01) and the UPJ pressure to a mean of 20.8 +/- 3.8 (P < 0.05). Ureteric distension with 1 and 1.5 ml of saline led to an elevation of ureteric and UPJ pressure which was not significantly different from that observed with distension with 0.5 ml (P > 0.05). In contrast, the UPJ showed no significant pressure change upon distension of the locally anesthetized renal pelvis or ureter, respectively. Likewise, the locally anesthetized UPJ exhibited no significant pressure response to renal pelvic or ureteric distension. The study demonstrates that urine might have to accumulate in the renal pelvis up to a certain volume and pressure so as to effect UPJ opening, which occurs at its maximum irrespective of the distending volume. UPJ opening upon renal pelvic distension postulates a reflex relationship which we call "pelviureteral inhibitory reflex." This reflex is believed to regulate the passage of urine from the renal pelvis to the ureter. Ureteric distension closes the UPJ; we call this reflex action the "ureteropelvic excitatory reflex" as it seems to prevent reflux of urine through the UPJ and thus protects the kidney. The concept that the UPJ acts as a physiologic sphincter is put forward.