Detection of lipid peroxidation in light-exposed mouse retina assessed by oxidative stress markers, total hydroxyoctadecadienoic acid and 8-iso-prostaglandin F2alpha

Exposure to excessive light induces retinal photoreceptor cell damage, which may involve lipid peroxidation. Morphological changes and the detection of internucleosomal DNA fragmentation confirmed the retinal damage caused by exposure of the retina of Balb/c mice to white fluorescent light (5000 lux, 2 h). The total amounts of hydroxyoctadecadienoic acid (tHODE) and 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha) in the retinas obtained from light-exposed mice were assessed after reduction and saponification. In this method, both the free and ester forms of hydroperoxides, hydroxides, and ketones of linoleic acid are measured as tHODE by gas chromatography-mass spectrometry (GC-MS) analysis. When compared with controls, a significant increase in the concentrations of tHODE and 8-iso-PGF2alpha was observed 24 h after light exposure. Furthermore, the stereoisomeric ratio (Z,E)-HODE/(E,E)-HODE decreased after light exposure, suggesting the involvement of free-radical-mediated peroxidation. By the immunohistochemical technique, it was confirmed that 8-iso-PGF2alpha increased in the inner plexiform layer (IPL), outer plexiform layer (OPL), rod outer segment, and choroidal layer, while 13-HODE increased in the OPL and rod inner segment after light exposure. These results demonstrate that tHODE and 8-iso-PGF2alpha assessed by the present method are appropriate biomarkers responding to retinal photooxidative stress in vivo.     

The effect of intra-abdominal pressure on the generation of 8-iso prostaglandin F2alpha during laparoscopy in rabbits

BACKGROUND: Carbon dioxide pneumoperitoneum induces peritoneal oxidative stress. The aim of this study was to verify the effect of intra-abdominal pressure on oxidative stress in the peritoneum and on post-operative adhesion formation. METHODS: Forty-one rabbits underwent laparoscopic surgery: either gasless, or with CO(2)-pneumoperitoneum at pressures of 5, 10 or 15 mmHg. Serial parietal peritoneal biopsies were taken at various time-points: immediately after reaching the abdominal cavity, 30, 60, 90 and 120 min afterwards, and 15 min after abdominal desufflation. 8-iso prostaglandin F(2alpha) (8-iso PGF(2alpha)), a marker of oxidative stresss, was assayed by enzyme immunoassay and adhesion formation was scored by second-look laparoscopy on day 14. RESULTS: The gasless group showed no significant changes in 8-iso PGF(2alpha). Conversely, significant changes occurred in CO(2)-pneumoperitoneum in a time- and pressure-dependent manner. Adhesions developed only in the CO(2)-pneumoperitoneum groups, and total adhesion score was correlated with the amount of CO(2) insufflated and intra-abdominal pressure, but not with 8-iso PGF(2alpha), which was correlated with intra-abdominal pressure. CONCLUSION: Intra-abdominal pressure increased 8-iso PGF(2alpha) in the parietal peritoneum in a graded fashion, whilst gasless laparoscopy had no impact. It also influenced the frequency and severity of adhesion formation, but no causal link was found between 8-iso PGF(2alpha) and post-operative adhesion formation.     

Hematocrit and blood osmolality in developing chicken embryos (Gallus gallus): in vivo and in vitro regulation

Hematocrit (Hct) regulation is a complex process involving potentially many factors. How such regulation develops in vertebrate embryos is still poorly understood. Thus, we investigated the role of blood pH in the regulation of Hct across developmental time in chicken embryos. We hypothesized that blood pH alterations in vitro (i.e., in a test tube) would affect Hct far more than in vivo because of in vivo compensatory regulatory processes for Hct. Large changes in Hct (through mean corpuscular volume (MCV)) and blood osmolality (Osm) occur when the blood was exposed to varying ambient temperatures (T(a)'s) and P(CO2) in vitro alongside an experimentally induced blood pH change from ~7.3 to 8.2. However, homeostatic regulatory mechanisms apparently limited these alterations in vivo. Changes in blood pH in vitro were accompanied by hydration or dehydration of red blood cells depending on embryonic age, resulting in changes in Hct that also were specific to developmental stage, due likely to initial blood gas and [HCO(3)(-)](v) values. Significant linear relationships between Hct and pH (Hct/ΔpH=-21.4%/(pH unit)), Hct and [HCO(3)(-)] (ΔHct/Δ[HCO(3)(-)]=1.6%/(mEq L(-1))) and the mean buffer value (Δ[HCO(3)(-)]/ΔpH=-13.4 (mEq L(-1))/(pH unit)) demonstrate that both pH and [HCO(3)(-)] likely play a role in the regulation of Hct through MCV at least in vitro. Low T(a) (24°C) resulted in relatively large changes in pH with small changes in Hct and Osm in vitro with increased T(a) (42°C) conversely resulting in larger changes in both Hct and Osm. In vivo exposure to altered T(a) caused age-dependent changes in Hct, demonstrating a trend towards increased Hct at higher T(a). Further, exposing embryos to a gas mixture where P(CO2) = 5.1 kPa for >4 h period at T(a) of 37 or 42°C also did not elicit a change in Hct or Osm. Presumably, homeostatic mechanisms ensured that in vivo Hct was stable during a 4-6 h temperature and/or hypercapnic stress. Thus, although blood pH decreases (induced by acute T(a) increase and exposure to CO(2)) increase MCV and, consequently, Hct in vitro, homeostatic mechanisms operating in vivo are adequate to ensure that such environmental perturbations have little effect in vivo.     

Associations between urinary phthalate monoesters and thyroid hormones in pregnant women

BACKGROUND: Maternal hypothyroidism during pregnancy can cause adverse effects in the fetus. Scientific evidence has shown that probable thyroid-like function of some phthalates in vitro and in vivo, and phthalates exposure, can begin in utero. This study investigated the association between phthalate exposure and thyroid hormones in pregnant women. METHODS: Serum and spot urine samples were collected from 76 Taiwanese pregnant women at second trimester. Thyroid hormones, including thyroid-stimulating hormone (TSH), triiodothyronine (T(3)), thyroxine (T(4)) and free T(4) (FT(4)) were analysed in serum samples, and five urinary phthalate monoesters, including mono butyl phthalate (MBP), monoethyl phthalate (MEP) and mono ethylhexyl phthalate (MEHP), were measured. RESULTS: Urinary MBP, MEP and MEHP, the median levels of which were 81.8, 27.7 and 20.6 ng/ml, respectively, were the predominant substances in the urinary phthalate monoesters. Significant mild negative correlations were found between T(4) and urinary MBP (R = -0.248, P < 0.05), and between FT(4) and urinary MBP (R = -0.368, P < 0.05). After adjusting for age, BMI and gestation, urinary MBP levels showed negative associations with FT(4) and T(4) (FT(4): beta = -0.110, P < 0.001; T(4): beta=-0.112, P = 0.003). CONCLUSIONS: Exposure to di-n-butyl phthalate (DBP) may affect thyroid activity in pregnant women, but how DBP affects thyroid function is unclear. Further studies are needed to elucidate the mechanism of action and to investigate whether any other factors related to DBP exposure alter the thyroid function.     

Low rates of DNA fragmentation in selected motile human spermatozoa assessed by the TUNEL assay

BACKGROUND: In this study we present the physiological changes observed in ejaculated spermatozoa of normospermic men after exposure to hydrogen peroxide (H(2)O(2)) or gamma irradiation. METHODS: Motility changes as well as membrane and DNA-damage were determined in spermatozoa after incubation with 25 micromol/l of H(2)O(2) during increasing intervals of time (0--60 min and after 24 h) or after irradiation of cells using alpha rays. Annexin V-binding in combination with propidium iodide was used for the assessment of membrane changes after each incubation time. TdT-mediated-dUTP nick-end labelling (TUNEL) was used to evaluate DNA damage. RESULTS: After 1 h incubation of the spermatozoa with H(2)O(2), almost all cells were positive for Annexin-V, while no significantly increase in TUNEL positivity was observed. TUNEL results were significantly higher 24 h after incubation with H(2)O(2) (10--16.3%, P = 0.03). In the control group (cumulus cells), an increase in the percentage of TUNEL positive cells was observed after 15 min of incubation with H(2)O(2) and showed a five-fold increase after 24 h (from 8.1-72.1%, P < 0.001). TUNEL positive cells after alpha irradiation increased with the doses and post-irradiation time (from 10.8--47.2%). Interestingly, when only motile spermatozoa from irradiated samples were analysed, only 0.5% were TUNEL positive. CONCLUSION: Motility may be a relevant physiological marker for DNA-intact sperm after exposure of spermatozoa to H(2)O(2) and alpha irradiation.     

Rapid and sensitive quantification of 8-isoprostaglandin F2alpha in human plasma and urine by liquid chromatography-electrospray ionization mass spectrometry

The isoprostane, 8-isoprostaglandin F2alpha (8-iso-PGF2alpha), is produced non-enzymatically by direct oxidation of arachidonic acid on the cell surface by oxygen radicals. We developed a new assay method for 8-iso-PGF2alpha using 2H4-8-iso-PGF2alpha as the internal standard (I.S.) by high-performance liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). For this assay, we established a very simple and rapid pretreatment method using a membrane filter-type solid-phase extraction column (Empore disk cartridge) for human urine extracts or intact plasma. LC-ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 353.24 (8-iso-PGF2alpha) and m/z 357.26 (I.S.) with a resolution of 1,500. The imprecision for this method was below 13.7%. Mean inaccuracy was 8.7% for added levels of 8-iso-PGF2alpha up to 5,000 pg/ml of urine and 500 pg/ml of plasma. Determination of plasma and urinary 8-iso-PGF2alpha concentrations in healthy subjects by the present method revealed that its urinary concentration in smokers tends to be higher than that in nonsmokers.     

Tumour necrosis factor-alpha, interleukin-6 and interleukin-8 do not promote adhesion of human endometrial epithelial cells to mesothelial cells in a quantitative in vitro model

BACKGROUND: A key factor in the pathogenesis of endometriosis is the endometrial-peritoneal adhesion. To study the pathogenesis of endometriosis, a quantitative in vitro assay (QIVA) was developed to measure in vitro adhesion between human endometrial epithelial cells and mesothelial cells using commercially available cell lines. Using the QIVA, the hypothesis was tested that tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-8 (IL-8) promote adhesion of endometrial epithelial cells to mesothelial cells. METHODS: Mesothelial cells were pre-treated with TNF-alpha, IL-6 or IL-8 in various concentrations (ranging from 0 to 1000 IU/ml) for 24 h. Confluent endometrial epithelial cells were labelled with [35S]methionine, added to the confluent mesothelial cells and incubated for 1 h. After incubation, non-adhering cells were removed and adherent cells were solubilized and their [35S]methionine radioactivity was counted to quantify the adherence of endometrial epithelial cells to mesothelial cells. RESULTS: The in vitro adhesion of human endometrial epithelial cells to human mesothelial cells was inhibited in a dose-dependent manner by TNF-alpha (P=0.0007), IL-6 (P<0.0001) and IL-8 (P=0.0004). CONCLUSIONS: Using a quantitative in vitro adhesion assay, we were unable to confirm our hypothesis that TNF-alpha, IL-6 and IL-8 promote the in vitro adhesion between endometrial epithelial cells and mesothelial cells.     

Cardiopulmonary exercise testing (CPET) in pulmonary emphysema

In patients affected by chronic obstructive pulmonary disease (COPD), cardiopulmonary response to exercise was never related to the severity of emphysema (E) measured by high resolution computed tomography (HRCT). Sixteen patients (age=65±8 yrs; FEV(1)=54±18%pred; RV=160±28%pred) with moderate to severe E (quantified by lung HRCT as % voxels <-910 HU) were exercised on a cycle-ergometer to exhaustion. Oxygen uptake (V˙(O2)), carbon dioxide output (V˙(CO2)), ventilation (V˙(E)), tidal volume (V(T)), and end-tidal P(CO2) (PET(CO2)) derived variables were measured breath-by-breath. The % of E correlated with: (1) the ratio V(Tpeak) (r=0.74; p=0.001); (2) the V˙(E)/V˙(CO2) slope (r=-0.77; p=0.0004); (3) PET(CO2) values at peak exercise (r=0.80; p=0.0001). Also, the %E was strongly predicted by the following exercise equation: %E(EST) = 58.1 + 11.9 × ΔV˙(E)/V˙(CO2) (r=0.94; p<0.0001). A V(Tpeak)/FEV1 ratio>1 is typically observed in severe E patients; furthermore, the V˙(E)/V˙(CO2) slope and the PET(CO2peak) values decrease and increase respectively as more as the emphysema is severe.     

肥胖患者氧化应激和脂肪细胞因子变化

目的:研究男性单纯肥胖患者氧化应激(OS)和脂肪细胞因子变化及相互关系.方法:测定42例男性肥胖患者和32例健康非肥胖男性个体的血8-异前列腺素F2α(8-iso-PCF2α)、血浆超氧化物歧化酶(SOD)、血清丙二醛(MDA)、脂联素、瘦素、抵抗素、人肿瘤坏死因子可溶性受体1(TNF-R1)、白介素-1β(IL-1β)、白介素-6(IL-6).结果:肥胖患者8-iso-PGF2α、MDA、瘦素、TNF-RI、IL-1β和IL-6均明显高于正常对照组(P<0.05,P<0.01),脂联素和SOD均明显低于正常对照组(P<0.05,P<0.01),2组抵抗素水平无统计学意义(P>0.05).肥胖组相关分析:8-iso-PGF2α与BMI(r=0.54,P<0.05)呈正相关,与脂联素(r=-0.56,P<0.05)呈负相关,瘦素与体脂(r=-0.53,P<0.05)呈负相关,脂联素与LDL(r=-0.54,P<0.05)和IL-6(r=-0.41,P<0.05)呈负相关.多元逐步回归分析:脂联素和IL-6是影响肥胖患者OS变化的主要因素.结论:男性单纯肥胖患者存在OS和脂肪细胞因子变化,脂肪细胞因子与OS存在明显的相关性.     

Correlations between the circadian patterns of body temperature, metabolism and breathing in rats

It had been demonstrated previously that the circadian patterns of activity and state of arousal are not essential for the manifestation of the daily patterns of pulmonary ventilation (V(E)), tidal volume (V(T)) and breathing frequency (f). In this study we investigated the extent of the linkage between the circadian pattern of breathing and those of body temperature (T(b)) and metabolic rate (oxygen consumption, V(O2), and carbon dioxide production, V(CO2)). Rats were instrumented for measurements of T(b) (by telemetry), and placed in a chamber for continuous 13-day period of measurement of breathing (by a modification of the barometric methodology), and of V(O2) and V(CO2) (by an open flow method). After the first 4 days in control conditions under a 12 h light:12 h dark (L:D) cycle, a perturbation was introduced on day 4, with an L-phase prolongation of 12 h, and on day 9, with an D-phase prolongation of 12 h. During the control days 1-4, all variables had daily oscillations (higher values in D), in phase with each other. During the perturbations (days 4-13), changes in T(b), V(O2) and V(CO2), averaged over the whole period, correlated significantly better with f than with V(T). Day-by-day X-Y loops indicated that V (E), V(T) and f could lead significantly the changes of T(b), V(O2) and V(CO2), and that these relations changed throughout the perturbation period. In addition, f and V(T) did not change necessarily in phase with each other. It is concluded that neither the oscillation in T(b) nor that in metabolism can be considered the direct cause of the daily oscillation of breathing. Presumably, the circadian pattern of breathing reflects the interplay of the daily patterns of many variables, none acting as the primary guide of the breathing daily rhythm.     

Ventilatory effects of gap junction blockade in the NTS in awake rats

We tested the hypothesis that focally perfusing carbenoxolone, which blocks gap junctions, into the nucleus tractus solitarius (NTS) would reduce the ventilatory response to CO(2). We measured minute ventilation (V(E)), tidal volume (V(T)) and respiratory frequency (F(R)) responses to increasing concentrations of inspired CO(2) ( [formula: see text] ) in rats during wakefulness. Focal perfusion of acetazolamide (10 microM) into the NTS increased V(E) and V(T) during exposure to room air. Carbenoxolone (300 microM) decreased the V(E) and V(T) response to CO(2) when perfused within, but not adjacent to the NTS in animals less than 10 weeks of age. F(R) was decreased at [formula: see text] in these animals. Carbenoxolone did not decrease V(E), V(T) or F(R) in animals 10 weeks of age and older. Carbenoxolone did not decrease V(E), V(T) or F(R) when focally perfused outside the NTS at any age tested. The NTS is an important CO(2) chemosensory site at all ages, and gap junctions amplify the ventilatory response to CO(2) in animals less than 10 weeks of age.     

Peritoneal tissue-oxygen tension during a carbon dioxide pneumoperitoneum in a mouse laparoscopic model with controlled respiratory support

BACKGROUND: Previous animal studies suggested that the peritoneal environment during a carbon dioxide (CO(2)) pneumoperitoneum is hypoxic and that this may contribute to the formation of intra-abdominal adhesions or the growth of malignant cells. There is no study, however, that investigates the relationship between anaesthesia, ventilation and the laparoscopic peritoneal environment to the development of hypoxia. The objective of this study is to monitor the peritoneal tissue-oxygen tension (PitO(2)) under various conditions including anaesthesia alone, during a CO(2) pneumoperitoneum at both low and high intraperitoneal pressure (IPP), and laparotomy, in animal models with controlled respiratory support (CRS). METHODS: C57BL6 mice were divided into eight groups (n = 5) consisting of anaesthesia alone or with CO(2) pneumoperitoneum at low (2 mmHg) or high (8 mmHg) IPP or undergoing laparotomy. Groups were further subdivided into those with or without CRS with endotracheal intubation and mechanical ventilation. Over the course of the 1 h procedure, PitO(2) was continuously monitored. RESULTS: Protocol 1. The PitO(2) levels (104.2 +/- 7.8 mmHg, mean +/- SEM) in non-injured peritoneum during a CO(2) pneumoperitoneum at a low IPP were elevated approximately 2-fold over the levels during laparotomy (49.8 +/- 15.0 mmHg) in ventilated mice. Protocol 2. After insufflation with CO(2), the PitO(2) was immediately elevated and maintained at a higher level. Following laparotomy, it decreased immediately. This elevation was not seen with air insufflation. CONCLUSION: In mice, a significant elevation in PitO(2) occurs during a CO(2) pneumoperitoneum at low IPP with CRS.     

Effects of the insecticide amitraz, an alpha2-adrenergic receptor agonist, on human luteinized granulosa cells

BACKGROUND: Amitraz, an insecticide used to prevent tick and mite infestation of cattle, crops and dogs, is an alpha2-adrenergic receptor agonist that inhibits GnRH release and the ovulatory LH surge in rats. Noradrenalin, the physiological ligand for adrenergic receptors, inhibits progesterone production by IVF-derived granulosa cells, but the effects of amitraz are unknown. METHODS: Luteinized granulosa cells obtained from women undergoing ovarian stimulation were exposed to amitraz (1, 10, 50, 100 microg/ml) for 2-72 h, and to amitraz (50 microg/ml) +/- hCG or the specific alpha2-adrenergic receptor antagonist yohimbine, for 6 h. Cell numbers were determined by 3-(4, 5-dimethylthiazol-(2)-yl)-2, 5-diphenyl tetrazolium bromide(MTT) assay and hormone production by radioimmunoassay. RESULTS: Amitraz 10 microg/ml did not affect cell numbers or estrogen production, but reduced progesterone production to 58 +/- 8% (p < 0.01, 24 h, n = 6) of control values. Amitraz (100 microg/ml) was cytotoxic and caused a corresponding reduction in hormone production. Amitraz 50 microg/ml did not affect cell numbers or estrogen production, but reduced progesterone per cell production to 82 +/- 6% of control values after 6 h. This was prevented by 0.2 mmol/l yohimbine. Exposure to amitraz 50 microg/ml for 6 h exposure abolished hCG-stimulated progesterone production but not estrogen production. CONCLUSIONS: Amitraz inhibited basal and hCG-stimulated progesterone but not estrogen production. The inhibitory action of amitraz and its antagonism by yohimbine suggest that alpha2-adrenergic receptors are expressed by luteinized human granulosa cells.     

地塞米松对大鼠胸膜间皮细胞水通道蛋白1表达的调节

 目的：观察胸膜间皮细胞水通道蛋白1（AQP-1）的表达及地塞米松（Dex）对其表达的影响，为进一步研究胸水治疗的机制提供实验依据。方法:体外培养大鼠胸膜间皮细胞，细胞鉴定后，采用免疫细胞化学、RT-PCR方法检测AQP-1表达；应用Western blotting检测以不同浓度Dex（10^-8、10^-7、10^-6、10^-5、10^-4 mmol/L）处理细胞 24 h 及以10^-4 mmol/L Dex处理细胞 6 h、12 h、24 h、36 h、48 h、72 h后AQP-1表达情况。结果:发现胸膜间皮细胞存在AQP-1表达， 10^-8、10^-7、10^-6、10^-5、10^-4 mmol/ L Dex干预胸膜间皮细胞 24 h 后，AQP-1蛋白表达量(积分吸光度)分别为755.04±19.81、843.72±19.41、862.96±26.53、694.80±32.00、938.08±13.32，分别高于正常对照组（372.90±16.46） 2.02、2.26、2.31、1.86、2.52倍 (P<0.01)，AQP-1表达升高与地塞米松浓度无关；以10-4 mmol/L Dex干预细胞 6 h、12 h、24 h、36 h、48 h、72 h 后，AQP-1蛋白表达量分别为：554.14±23.57、917.78±38.62、1 587.20±61.22、1 322.09±28.65、918.40±26.62、1 117.60±51.32，分别高于正常对照组（495.91±23.12）1.12、1.85、3.20、2.67、1.85、2.25倍(P<0.01)，AQP-1表达与地塞米松作用时间有关。结论:胸膜间皮细胞存在AQP-1表达，地塞米松对胸膜间皮细胞AQP-1表达有明显的增强性调节作用，具有时间依赖性。     

The role of arterial oxygen tension in the respiratory response to localized heating of the hypothalamus and to hyperthermia

1. Rectal temperatures, respiratory rates, arterial blood gas tensions, arterial pH and the percentage of red cells in arterial blood have been measured in the unanaesthetized ox in a cool environment (15/12 degrees C, dry bulb/wet bulb [DB/WB]), in a hot, dry environment (40/21 degrees C, DB/WB), during hyperthermia, during infra-red irradiation, and during localized heating of the anterior hypothalamus. In some experiments the gas tensions and pH of mixed venous blood, and the percentage saturation of the arterial blood with oxygen, were also measured.2. In the cool environment at a mean rectal temperature (T(r)) of 38.8 degrees C and a respiratory rate (f) of 28/min the mean values obtained from six animals were: arterial oxygen tension (P(a, O) (2)), 93 mm Hg; arterial carbon dioxide tension (P(a, CO) (2)) 42 mm Hg; arterial pH 7.49; arterial oxygen saturation (S(a, O) (2)) 94%; arterial oxygen capacity (Cap(a, O) (2)) 13.6 vol.%; arterial packed cell volume (P.C.V.) 29%.3. Exposure to the hot, dry environment resulted in a small increase in the rectal temperature and thermal polypnoea, but there were no statistically significant changes in the blood gas tensions.4. During hyperthermia statistically significant increases occurred in rectal temperature, respiratory rate, P(a, O) (2), pH and arterial haematocrit, while the P(a, CO) (2) decreased. The venous oxygen tension (P(v, O) (2)) decreased also, and the tentative conclusion was made that although the oxygenation of arterial blood remained unimpaired during hyperthermia, tissue hypoxia may supervene. At very high levels of deep body temperature, some evidence for a secondary decrease in P(a, O) (2) was obtained.5. Localized heating of the anterior hypothalamus caused an increase in respiratory rate and in P(a, O) (2). The P(v, O) (2) increased also. These changes were considered to be due to increased cardiac output and diversion of blood to the skin.6. During infra-red irradiation of three animals at an environmental temperature of 40/21 degrees C, the respiratory rate increased, but the P(a, O) (2) decreased.     

Tandem mass spectrometric quantification of 8-iso-prostaglandin F2alpha and its metabolite 2,3-dinor-5,6-dihydro-8-iso-prostaglandin F2alpha in human urine

Whole body synthesis of F2-isoprostanes, a family of cyclooxygenase-independent eicosanoids formed by free-radical catalysed peroxidation, should be best assessed by quantifying their urinary metabolites. Two methods for the quantitative determination of F2-isoprostane metabolites in human urine performing either thin-layer chromatography (TLC) (method A) or high-performance liquid chromatography (HPLC) (method B) prior to GC-tandem MS are described. Method A allows for simultaneous quantification of 8-iso-PGF2alpha, one prominent member of the F2-isoprostane family, and its major urinary metabolite, 2,3-dinor-5,6-dihydro-8-iso-PGF2alpha. Mean excretion was found to be 223 and 506 pg/mg creatinine of 8-iso-PGF2alpha and 2,3-dinor-5,6-dihydro-8-iso-PGF2alpha, respectively (n=14). A tight correlation existed between the urinary excretion of these two isoprostanes (r=0.86). Method B enables quantification of dinor-dihydro metabolites of various F2-isoprostanes including 8-iso-PGF2alpha. 2,3-Dinor-5,6-dihydro-8-iso-PGF2alpha was found to be an abundant dinor-dihydro F2-isoprostane metabolite. Validity of method A was proven by a combination of HPLC with TLC prior to GC-tandem MS analysis. A correlation was observed between the urinary concentrations of 2,3-dinor-5,6-dihydro-8-iso-PGF2alpha measured by GC-MS and GC-tandem MS (r=0.84).     

Ventilatory chemosensitivity and thermogenesis of the chicken hatchling after embryonic hypercapnia

Hypoxia during incubation results in hatchlings with a reduced thermogenic capacity and a blunted ventilatory (V (E)) chemosensitivity (Szdzuy, K., Mortola, J.P., 2007b. Ventilatory chemosensitivity of the 1-day-old chicken hatchling after embryonic hypoxia. Am. J. Physiol. (Regul. Integr. Comp. Physiol.) 293, R1640-R1649). We asked if similar effects occurred with embryonic hypercapnia, that is, with a non-hypoxic sustained stimulation of the chemoreceptors. White Leghorn chicken eggs were incubated at 38 degrees C either in air (controls, C) or in 4% CO(2) from embryonic day 5 (4% CO(2)), hatching included. The 4% CO(2) embryos hatched about 12h later than C, with similar body weight. On the day of hatching the thermogenic capacity, assessed from the changes in oxygen consumption (.V(O2)) during 1h at 30 degrees C, increased from the early (about 3h old) to the late hours (about 20 h old), and was similar between 4% CO(2) and C. Ventilatory chemosensitivity was evaluated from the changes in (.V(E)) and in ventilatory equivalent (.V(E)/.V(O2)) during acute hypoxia (15 and 10% O(2), 20 min each) or hypercapnia (2 and 4% CO(2), 20 min each). Both at the early and late hours (.V(E)) chemosensitivity was lower in 4% CO(2) than in C. The .V(E)/.V(O2) responses of 4% CO(2) in hypoxia and hypercapnia averaged, respectively, about 45 and 60% of C. A separate set of eggs incubated in 2% CO(2) gave results qualitatively intermediate between C and 4% CO(2). We conclude that prenatal hypercapnia does not compromise the newborn's thermogenesis, but, like hypoxia, affects the development of respiratory control, resulting in a blunted chemosensitivity.     

T(h)1 versus T(h)2 cytokine profile determines the modulation of in vitro T cell-independent type 2 responses by IL-4

We have previously demonstrated that stimulation of B cells by multivalent membrane Ig cross-linking, using dextran-conjugated anti-IgD mAb (alpha delta-dex), in the presence of cytokines, is an in vitro model for T cell-independent type 2 (TI-2) Ig secretory responses. Earlier studies have shown that IL-4 enhances IgM secretion upon stimulation with alpha delta-dex plus IL-5 and induces IgG1 isotype-switching, without altering the proliferative response to alpha delta-dex. Here we show that IL-4 can have both stimulatory and inhibitory effects on alpha delta-dex-induced Ig secretion. Both the kinetics and time of exposure to IL-4, and the nature of the cytokine additions, T(h)1 versus T(h)2, determine whether stimulation or inhibition is observed. Preincubation of sort-purified B cells with IL-4 caused a 6- to 8-fold increase in Ig secretory responses to subsequent stimulation with alpha delta-dex plus IL-1, IL-2 or a combination of both. However, the continued presence of IL-4 during B cell stimulation suppressed responses to all cytokine combinations tested, except for those which included IL-5. Of 11 cytokines tested, only IL-4 showed this dual effect of enhancement and suppression. The stimulatory effect of IL-4 required a minimum of 4 h of preincubation and could be inhibited by the addition of IFN-gamma. Thus stimulation of non-MHC class II-dependent T or non-T cells by multivalent antigens to secrete IL-4 may regulate the response to these antigens, such that early and brief exposure of B cells to IL-4 will enhance a subsequent TI-2 response in the presence of T(h)1-dependent cytokines, while continuous exposure will result in inhibition of the response.     

Assessment of selected co-stimulatory, adhesion and activatory molecules and cytokines of Th(1)/Th(2) balance in acute lymphoblastic leukemia in children

INTRODUCTION: Recent years have seen a rise in the importance of cytokine production and co-stimulatory/activatory molecule expression in the immune response in leukemia. The aim of our study was to assess the function of T lymphocytes in children with acute lymphoblastic leukemia (ALL) during remission induction based on selected cytokine and co-stimulatory/activatory molecule expression. MATERIAL/METHODS: The study group consisted of 50 children with ALL (B cell precursor). Peripheral blood samples were taken before treatment (day 0), after the prednisone prophase (day 8), and during (day 15) and after (day 33) remission induction. The percentages of T cells with interferon (IFN)-gamma (Th(1)), interleukin (IL)-4 (Th(2)) and IL-2 receptor (IL-2R), CD28, CTLA-4, CD38, ICAM-1, and HLA-DR expression were assessed by tricolor flow cytometry. RESULTS: At the time of diagnosis we noted higher percentages of T cells with adhesion molecule ICAM-1, activation molecule CD38 expression, and an increased population of Th(2 )cells (IL-4) compared with the control group. During and after remission induction we observed a decreased population of CD38(+) T cells, elevated percentages of helper T lymphocytes with IL-2R expression, and a rise in helper T lymphocytes producing IFN-gamma (Th(1)). During fever/infection, higher levels of activated T lymphocytes (CD4(+)HLA-DR(+), CD8(+)HLA-DR(+)), a rise in Th1, and no change in Th(2 )populations were observed. CONCLUSIONS: The results suggest T cell activation and Th(2 )predominance at the time of diagnosis and during remission induction in ALL in children. These results confirm the involvement of cellular immunity in the leukemic process and can be used in immune therapy in leukemia.