The presence of circulating tumor cells (CTCs) in peripheral blood is associated with metastasis and prognosis in hepatocellular carcinoma (HCC) patients. The epithelial–mesenchymal transition (EMT) has a pivotal role in tumor invasion and dissemination. To identify more sensitive biomarkers for evaluating metastasis and prognosis, we investigated the expression of EMT markers, including vimentin, twist, ZEB1, ZEB2, snail, slug and E-cadherin in CTCs, primary HCC tumors and adjacent non-tumoral liver tissues. After isolating viable CTCs from the peripheral blood of HCC patients using asialoglycoprotein receptors (ASGPRs), the CTCs were identified with immunofluorescence staining. CTCs were detected in the peripheral blood obtained from 46 of 60 (76.7%) HCC patients. Triple-immunofluorescence staining showed that twist and vimentin expression could be detected in CTCs obtained from 39 (84.8%) and 37 (80.4%) of the 46 patients, respectively. The expression of both twist and vimentin in CTCs was significantly correlated with portal vein tumor thrombus. Coexpression of twist and vimentin in CTCs could be detected in 32 (69.6%) of the 46 patients and was highly correlated with portal vein tumor thrombus, TNM classification and tumor size. Quantitative fluorescence western blot analysis revealed that the expression levels of E-cadherin, vimentin and twist in HCC tumors were significantly associated with the positivity of isolated CTCs (P=0.013, P=0.012, P=0.009, respectively). However, there was no significant difference in ZEB1, ZEB2, snail and slug expression levels in CTCs, primary HCC tumors and adjacent non-tumoral liver tissues across samples with regard to the clinicopathological parameters. Our results demonstrate that the EMT has a role in promoting the blood-borne dissemination of primary HCC cells, and the twist and vimentin expression levels in CTCs could serve as promising biomarkers for evaluating metastasis and prognosis in HCC patients.
circulating tumor cells; epithelial–mesenchymal transition; hepatocellular carcinoma; metastasis; biomarkers
Alcohol consumption is one of the world's major risk factors for disease development. But underlying mechanisms by which moderate-to-heavy alcohol intake causes damage are poorly understood and biomarkers are sub-optimal. Here, we investigated metabolite concentration differences in relation to alcohol intake in 2090 individuals of the KORA F4 and replicated results in 261 KORA F3 and up to 629 females of the TwinsUK adult bioresource. Using logistic regression analysis adjusted for age, body mass index, smoking, high-density lipoproteins and triglycerides, we identified 40/18 significant metabolites in males/females with P-values <3.8E−04 (Bonferroni corrected) that differed in concentrations between moderate-to-heavy drinkers (MHD) and light drinkers (LD) in the KORA F4 study. We further identified specific profiles of the 10/5 metabolites in males/females that clearly separated LD from MHD in the KORA F4 cohort. For those metabolites, the respective area under the receiver operating characteristic curves were 0.812/0.679, respectively, thus providing moderate-to-high sensitivity and specificity for the discrimination of LD to MHD. A number of alcohol-related metabolites could be replicated in the KORA F3 and TwinsUK studies. Our data suggests that metabolomic profiles based on diacylphosphatidylcholines, lysophosphatidylcholines, ether lipids and sphingolipids form a new class of biomarkers for excess alcohol intake and have potential for future epidemiological and clinical studies.
alcohol; alcoholism; biomarkers; gender; lipids; metabolomics
The effect of bisphenol A (BPA) on the reproductive system is highly debated but has been associated with meiotic abnormalities. However, evidence is lacking with regard to the mechanisms involved. In order to explore the underlying mechanisms of BPA-induced meiotic abnormalities in adult male rats, we exposed 9-week-old male Wistar rats to BPA by gavage at 0, 2, 20 or 200 μg/kg body weight (bw)/day for 60 consecutive days. 17β-Estradiol (E2) was administered at 10 μg/kg bw/day as the estrogenic positive control. Treatments with 200 μg/kg bw/day of BPA and E2 significantly decreased sperm counts and inhibited spermiation, characterized by an increase in stage VII and decrease in stage VIII in the seminiferous epithelium. This was concomitant with a disruption in the progression of meiosis I and the persistence of meiotic DNA strand breaks in pachytene spermatocytes,and the ataxia–telangiectasia-mutated and checkpoint kinase 2 signal pathway was also activated; Eventually, germ cell apoptosis was triggered as evaluated by terminal dUTP nick-end labeling assay and western blot for caspase 3. Using the estrogen receptor (ER) antagonist ICI 182780, we determined that ER signaling mediated BPA-induced meiotic disruption and reproductive impairment. Our results suggest that ER signaling-mediated meiotic disruption may be a major contributor to the molecular events leading to BPA-related male reproductive disorders. These rodent data support the growing association between BPA exposure and the rapid increase in the incidence of male reproductive disorders.
bisphenol A; meiocyte spreading; meiosis; spermatogenesis; stage of seminiferous epithelium
Multiple studies have shown that cerebral tissue oxygen saturation () is decreased after phenylephrine treatment. We hypothesized that the negative impact of phenylephrine administration on is affected by arterial blood carbon dioxide partial pressure () because CO2 is a powerful modulator of cerebrovascular tone.
In 14 anaesthetized healthy patients, i.v. phenylephrine bolus was administered to increase the mean arterial pressure ∼20–30% during hypocapnia, normocapnia, and hypercapnia. and cerebral blood volume (CBV) were measured using frequency domain near-infrared spectroscopy, a quantitative technology. Data collection occurred before and after each treatment.
Phenylephrine caused a significant decrease in during hypocapnia [=−3.4 (1.5)%, P<0.001], normocapnia [=−2.4 (1.5)%, P<0.001], and hypercapnia [=−1.4 (1.5)%, P<0.01]. Decreases in were significantly different between hypocapnia, normocapnia, and hypercapnia (P<0.001). Phenylephrine also caused a significant decrease in CBV during hypocapnia (P<0.01), but not during normocapnia or hypercapnia.
The negative impact of phenylephrine treatment on and CBV is intensified during hypocapnia while blunted during hypercapnia.
carbon dioxide; cerebral blood volume; cerebral tissue oxygen saturation; modulation; phenylephrine
Mitochondria are critical targets in the hepatotoxicity of cadmium (Cd). Abnormal mitochondrial dynamics have been increasingly implicated in mitochondrial dysfunction in pathophysiological conditions. Therefore, our study aimed to investigate the effects and underlying mechanism of Cd on mitochondrial dynamics during hepatotoxicity. In the L02 liver cell lines, 12 μM cadmium chloride (CdCl2) exposure induced excessive mitochondrial fragmentation as early as 3 h post-treatment with Cd, which preceded the mitochondrial dysfunction such as reactive oxygen species (ROS) overproduction, mitochondrial membrane potential (ΔΨm) loss and ATP reduction. Concurrent to mitochondrial fragmentation, CdCl2 treatment increased the protein levels of dynamin-related protein (Drp1) and promoted the recruitment of Drp1 into mitochondria. Strikingly, mitochondrial fragmentation also occurred in the liver tissue of rats exposed to CdCl2, accompanied by enhanced recruitment of Drp1 into mitochondria. Moreover, in L02 cells, Drp1 silencing could effectively reverse Cd-induced mitochondrial fragmentation and mitochondrial dysfunction. Furthermore, the increased expression and mitochondrial recruitment of Drp1 were tightly related to the disturbance of calcium homeostasis, which could be prevented by both chelating [Ca2+]i and inhibiting [Ca2+]m uptake. Overall, our study indicated that Cd induced Drp1-dependent mitochondrial fragmentation by disturbing calcium homeostasis to promote hepatotoxicity. Manipulation of Drp1 may be the potential avenue for developing novel strategies to protect against cadmium-induced hepatotoxicity.
cadmium; hepatotoxicity; mitochondrial fragmentation; mitochondrial dysfunction; dynamin-related protein (Drp1)
How phenylephrine and ephedrine treatments affect global and regional haemodynamics is of major clinical relevance. Cerebral tissue oxygen saturation ()-guided management may improve postoperative outcome. The physiological variables responsible for changes induced by phenylephrine and ephedrine bolus treatment in anaesthetized patients need to be defined.
A randomized two-treatment cross-over trial was conducted: one bolus dose of phenylephrine (100–200 µg) and one bolus dose of ephedrine (5–20 mg) were given to 29 ASA I–III patients anaesthetized with propofol and remifentanil. , mean arterial pressure (MAP), cardiac output (CO), and other physiological variables were recorded before and after treatments. The associations of changes were analysed using linear-mixed models.
The CO decreased significantly after phenylephrine treatment [▵CO=−2.1 (1.4) litre min−1, P<0.001], but was preserved after ephedrine treatment [▵CO=0.5 (1.4) litre min−1, P>0.05]. The was significantly decreased after phenylephrine treatment [▵=−3.2 (3.0)%, P<0.01] but preserved after ephedrine treatment [▵=0.04 (1.9)%, P>0.05]. CO was identified to have the most significant association with (P<0.001). After taking CO into consideration, the other physiological variables, including MAP, were not significantly associated with (P>0.05).
Associated with changes in CO, decreased after phenylephrine treatment, but remained unchanged after ephedrine treatment. The significant correlation between CO and implies a cause–effect relationship between global and regional haemodynamics.
cardiac output; cerebral tissue oxygen saturation; ephedrine; mean arterial pressure; phenylephrine
Schizophrenia is a severe complex mental disorder affecting 0.5–1% of the world population. To date, diagnosis of the disease is mainly based on personal and thus subjective interviews. The underlying molecular mechanism of schizophrenia is poorly understood. Using targeted metabolomics we quantified and compared 103 metabolites in plasma samples from 216 healthy controls and 265 schizophrenic patients, including 52 cases that do not take antipsychotic medication. Compared with healthy controls, levels of five metabolites were found significantly altered in schizophrenic patients (P-values ranged from 2.9 × 10−8 to 2.5 × 10−4) and in neuroleptics-free probands (P-values ranging between 0.006 and 0.03), respectively. These metabolites include four amino acids (arginine, glutamine, histidine and ornithine) and one lipid (PC ae C38:6) and are suggested as candidate biomarkers for schizophrenia. To explore the genetic susceptibility on the associated metabolic pathways, we constructed a molecular network connecting these five aberrant metabolites with 13 schizophrenia risk genes. Our result implicated aberrations in biosynthetic pathways linked to glutamine and arginine metabolism and associated signaling pathways as genetic risk factors, which may contribute to patho-mechanisms and memory deficits associated with schizophrenia. This study illustrated that the metabolic deviations detected in plasma may serve as potential biomarkers to aid diagnosis of schizophrenia.
metabolic pathway; metabolomics; neuroleptics-free; schizophrenia
Postnatal lung growth and development has primarily been evaluated from a very limited number of autopsied lungs; however, it still remains unclear whether alveolarization of the lung is complete during infancy and whether the conducting airways grow proportionately. The purpose of our study was to evaluate lung growth and development in vivo in infants and toddlers using multi-slice computed tomography.
Thirty-eight subjects (14 male, 24 female) aged 24–142 weeks had low-dose volumetric HRCT imaging at an inflation pressure of 20 cmH2O during an induced respiratory pause. Lung volume and weight were determined, as well as airway dimensions (inner and outer area, and wall area) for the trachea and next 3–4 generations. Lung volume, air volume, and tissue volume increased linearly with body length. The air and tissue components of the lung parenchyma increased at a constant rate with each other. In addition, airway caliber decreased with increasing generation from the trachea into each lobe. Airway caliber also correlated with body length; however, there was no interaction effect between airway generation and body length on transformed airway size. Our in vivo assessment suggests that growth of the lung parenchyma in infants and toddlers occurred with a constant relationship between air volume and lung tissue, which is consistent with lung growth occurring primarily by the addition of alveoli, rather than expansion of alveoli. In addition, the central conducting airways grow proportionately in infants and toddlers. This information may be important for evaluating subjects with arrested lung development.
Direct damage to DNA is generally accepted as the main initiator of mutation and cancer induced by environmental carcinogens or ionising radiation. However, there is accumulating evidence suggesting that extracellular/extranuclear targets may also have a key role in mediating the genotoxic effects of ionising radiation. As the possibility of a particle traversal through the cytoplasm is much higher than through the nuclei in environmental radiation exposure, the contribution to genotoxic damage from cytoplasmic irradiation should not be ignored in radiation risk estimation. Although targeted cytoplasmic irradiation has been shown to induce mutations in mammalian cells, the precise mechanism(s) underlying the mutagenic process is largely unknown.
A microbeam that can target the cytoplasm of cells with high precision was used to study mechanisms involved in mediating the genotoxic effects in irradiated human–hamster hybrid (AL) cells.
Targeted cytoplasmic irradiation induces oxidative DNA damages and reactive nitrogen species (RNS) in AL cells. Lipid peroxidation, as determined by the induction of 4-hydroxynonenal was enhanced in irradiated cells, which could be suppressed by butylated hydroxyl toluene treatment. Moreover, cytoplasmic irradiation of AL cells increased expression of cyclooxygenase-2 (COX-2) and activation of extracellular signal-related kinase (ERK) pathway.
We herein proposed a possible signalling pathway involving reactive oxygen/nitrogen species and COX-2 in the cytoplasmic irradiation-induced genotoxicity effect.
cytoplasmic irradiation; genotoxicity; reactive oxygen/nitrogen species; 4-hydroxynonenal; cyclooxygenase-2
Bystander effects induced by cytoplasmic irradiation have been reported recently. However, the mechanism(s) underlying, such as the functional role of mitochondria, is not clear. In the present study, we used either mtDNA-depleted (ρ0) AL or normal (ρ+) AL cells as irradiated donor cells and normal human skin fibroblasts as receptor cells in a series of medium transfer experiments to investigate the mitochondria-related signal process. Our results indicated that mtDNA-depleted cells or normal AL cells treated with mitochondrial respiratory chain function inhibitors had an attenuated γ-H2AX induction, which indicates that mitochondria play a functional role in bystander effects. Moreover, it was found that treatment of normal AL donor cells with specific inhibitors of NOS, or inhibitor of mitochondrial calcium uptake (ruthenium red) significantly decreased γ-H2AX induction and that radiation could stimulate cellular NO and O2•− production in irradiated ρ+ AL cells, but not in ρ0 AL cells. These observations, together with the findings that ruthenium red treatment significantly reduced the NO and O2•− levels in irradiated ρ+ AL cells, suggest that radiation-induced NO derived from mitochondria might be an intracellular bystander factor and calcium-dependent mitochondrial NOS might play an essential role in the process.
radiation-induced bystander effects; signalling pathway; mitochondrion; nitric oxide synthase
In human gene therapy applications, lentiviral vectors may have advantages over γ-retroviral vectors in several areas, including the ability to transduce nondividing cells, resistance to gene silencing and a potentially safer integration site profile. However, unlike γ-retroviral vectors it has been problematic to drive the expression of multiple genes efficiently and coordinately with approaches such as internal ribosome entry sites or dual promoters. Using different 2A peptides, lentiviral vectors expressing two-gene T-cell receptors directed against the melanoma differentiation antigens gp100 and MART-1 were constructed. We demonstrated that addition of amino-acid spacer sequences (GSG or SGSG) before the 2A sequence is a prerequisite for efficient synthesis of biologically active T-cell receptors and that addition of a furin cleavage site followed by a V5 peptide tag yielded optimal T-cell receptor gene expression. Furthermore, we determined that the furin cleavage site was recognized in lymphocytes and accounted for removal of residual 2A peptides at the post-translational level with an efficiency of 20–30%, which could not be increased by addition of multiple furin cleavage sites. The novel bicistronic lentiviral vector developed herein afforded robust anti-melanoma activities to engineered peripheral blood lymphocytes, including cytokine secretion, cell proliferation and lytic activity. Such optimal vectors may have immediate applications in cancer gene therapy.
T-cell receptor; adoptive immunotherapy; tumor immunity; lentivirus; 2A peptide
This study was designed to evaluate the functional and radiological outcomes of patients with complex tibial plateau fractures treated with double-buttress plate fixation.
Sixty five cases of complex (Schatzker type V and VI) tibial plateau fractures were treated with double-buttress plate fixation in our centre from September 2001 to September 2006 through two separate plate incisions. Fifty four patients were followed up for a period ranging from 12 to 48 months and evaluated for the functional and radiological outcomes by a series of standard questionnaire and measurement.
Due to the good exposure without any extensive soft-tissue dissection of the double-buttress plate fixation, the fractures in all 54 patients were healed and the treatment achieved greater than 90% of satisfactory-to-excellent rates of reduction. The mean time of bone union was 15.4 weeks (range, 12-30 weeks), and the mean time of full weight-bearing was 18.7 weeks (range, 14-26 weeks). At the final follow-up visit, no patients showed knee instability; the mean range of motion was 107.6° (range, 85°-130°). For all patients, no statistically significant difference in the functional outcomes was observed between their 6-months and final follow-up visits; or in the radiological findings between their immediate postoperative and final follow-up examinations.
Double-buttress plate fixation is a feasible treatment option for bilcondylar and complex tibial plateau fractures. Although technically demanding, it offers reliable stability without additional postoperative adjuvant external fixation, and at the same time avoids extensive soft tissue dissection, allowing the early painless range of motion.
Functional Outcome; Radiological Outcome; Tibial Plateau Fracture; Fracture Fixation; Double Buttress Plate
A variety of factors contribute to the development of cognitive impairment in elderly people. Previous studies have focused upon a single or a few risk factors. In this study we assessed and compared the significance of a wide variety of potential risk factors for cognitive impairment in postmenopausal women.
A total of 208 pairs of elderly women (mean age = 73.2 years) were examined in a cross-sectional case-control study. Each pair consisted of a case (with impaired cognition) and a control subject matched by age and educational status. Cognitive functions were determined using a modified version of the Blessed test. Participants were also subjected to a general clinical examination and they were interviewed to collect information on lifestyle practices and comorbid disorders. Genotypes for the apolipoprotein E (APOE) epsilon4, catechol-O-methyltransferase (COMT) Val/Met, and brain-derived neurotropic growth factor (BDNF) Val/Met polymorphisms were determined. Data were analyzed by conditional logistic regression.
We identified a set of risk factors for age-related cognitive impairment. A statistical model for assessment of the importance of these factors was constructed. The factors in this model were physical exercise (odds ratio [OR] = 0.50, 95% confidence interval [CI] = 0.32–0.78), regular alcohol consumption (OR = 0.49, 95% CI = 0.29–0.83), metabolic syndrome (OR = 2.83, 95% CI = 1.26–6.39), depression (OR = 3.24, 95% CI = 1.28–8.22), and the APOE epsilon4 allele (OR = 1.76, 95% CI = 1.09–2.83). Also COMT genotype was present as a risk factor in the statistical model (p = 0.08).
Lifestyle risk factors, comorbid disorders, and genetic factors contribute to development of age-related cognitive impairment. The two former groups of risk factors appear to be particular important in this respect.
age-related cognitive impairment; risk factors; lifestyle choices; comorbid disorders; genetic susceptibility
retinoid; tazarotenic acid; oral
Objective To determine the factors associated with difference in prevalence of asthma in children in different regions of China.
Design Multicentre epidemiological survey.
Setting Three cities in China.
Participants 10 902 schoolchildren aged 10 years.
Main outcome measures Asthma and atopic symptoms, atopic sensitisation, and early and current exposure to environmental factors.
Results Children from Hong Kong had a significantly higher prevalence of wheeze in the past year than those from Guangzhou and Beijing (odds ratio 1.64, 95% confidence interval 1.35 to 1.99). Factors during the first year of life and currently that were significantly associated with wheeze were cooking with gas (odds ratio 2.04, 1.34 to 3.13), foam pillows (2.58, 1.66 to 3.99), and damp housing (1.89, 1.26 to 2.83). Factors protecting against wheeze were cotton quilts and the consumption of fruit and raw vegetables.
Conclusion Environmental factors and diet may explain the differences in prevalence of asthma between children living in different regions of China.
STUDY OBJECTIVE—To analyse trends in socioeconomic differences in cardiovascular disease risk factors among an urban Chinese population using educational attainment as the socioeconomic indicator.
DESIGN—Population surveys with randomly selected independent samples were carried out in 1989 and in 1996. Educational attainment, blood pressure, body mass index, cigarette smoking and lack of leisure time physical activity were determined.
SETTING—Urban areas of the city of Tianjin, China.
PARTICIPANTS—A total of 14 275 respondents aged 25-64 years.
MAIN RESULTS—Diastolic blood pressure increased and the proportion of people without leisure time physical activity decreased in both sexes during the study period. The prevalence of smoking and the number of cigarettes smoked daily increased significantly among men. Smoking decreased in the least educated men and increased in those who had studied at least to college level. Body mass index decreased across all educational strata in women, but blood pressure increased in women with at least college level education.
CONCLUSIONS—These data reveal a different picture in trends in the association of education and cardiovascular risk factors from those depicted in developed countries. This highlights the need for an effective intervention programme in the study population.
Keywords: trends; socioeconomic status; cardiovascular disease risk factor
breast cancer; ataxia telangiectasia; protein truncation test (PTT); cancer predisposition
Characteristics probably associated with the fetal hormonal milieu have recently been shown to increase (birth size indicators, prematurity, neonatal jaundice) or decrease (pregnancy toxaemia) breast cancer risk in the female offspring. However, it is unknown whether differences in pregnancy hormone levels may contribute to the marked geographical variation in breast cancer incidence. We have compared, in a highly standardized manner, pregnancy hormone levels in a population with high incidence and one with low incidence of breast cancer. Three hundred and four pregnant Caucasian women in Boston and 334 pregnant Chinese women in Shanghai were enrolled from March 1994 to October 1995. Levels of oestradiol, oestriol, prolactin, progesterone, human growth hormone, albumin and sex hormone-binding globulin were measured in maternal blood at weeks 16 and 27 of gestation and compared between the two study sites using non-parametric Wilcoxon's rank-sum test. Demographical, anthropometrical and pregnancy characteristics were ascertained through interview, and relevant variables concerning delivery and the newborn were abstracted from medical records and paediatric charts. During the first visit, median serum levels of all studied hormones were statistically significant, and in most instances substantially, higher among Chinese women, who have a low incidence of breast cancer, compared with American women, who have a high incidence of breast cancer. An analogous pattern was evident during the second visit, although the relative differences tended to be smaller. Further research is needed to identify lifestyle or other exogenous determinants of pregnancy hormone levels, as well as possible mechanisms by which they may influence carcinogenic processes in the breast and possibly other organs. © 1999 Cancer Research Campaign
epidemiology; pregnancy steroids; breast cancer
The mutant Z form of alpha1-antitrypsin (alpha1AT) is responsible for > 95% of all individuals with alpha1AT deficiency, an important inherited cause of emphysema and liver disease. Since secreted Z alpha1AT is a functional antiprotease, we hypothesized that interrupting catabolism of retained Z alpha1AT might increase its transport out of cells, causing an increase in extracellular protease protection. Both the protein translation inhibitor cycloheximide and the specific inhibitor of proteasome function, lactacystin, prevented intracellular degradation of Z alpha1AT. Moreover, this inhibition of degradation was associated with partial restoration of Z alpha1AT vesicular transport. This effect was observed in a model system of transfected CHO cells as well as in human alveolar macrophages synthesizing Z alpha1AT. This study supports the hypothesis that altering the intracellular fate of a mutant protein may be an option in the treatment of diseases associated with misfolded but potentially functional proteins.
We demonstrate that adenoviral-mediated gene transfer of a dominant negative rac1 gene product (N17rac1) inhibits the intracellular burst of reactive oxygen species (ROS) that occurs after reoxygenation of vascular smooth muscle cells. In contrast, expression of a dominant negative ras gene (N17ras) had no effect. Challenge of control cells and cells expressing N17rac1 with a direct oxidant stress produced an equivalent increase in intracellular ROS levels and subsequent cell death. This suggests that N17rac1 expression appears to block production of harmful oxygen radicals and does not act directly or indirectly to scavenge ROS generated during reoxygenation. Expression of N17rac1 results in protection from hypoxia/reoxygenation-induced cell death in a variety of cell types including vascular smooth muscle cells, fibroblasts, endothelial cells, and ventricular myocytes. These results suggest that reoxygenation injury requires the activation of rac proteins, and that inhibition of rac-dependent pathways may be a useful strategy for the prevention of reperfusion injury in ischemic tissues.
Neonatal animals were not considered as suitable vaccine recipients either because of immune immaturity or because passively delivered antibody interferes with immune induction. In this report, we evaluated the response of neonatal mice to immunization with naked DNA encoding a herpes simplex virus (HSV) protein, and determined if maternally derived HSV antibody interfered with immunogenicity. Our results show that neonatal mice develop effective humoral and T cell responses after immunization with either DNA or inactivated vaccines. The nature of the responses to HSV immunization, however, was more Th2-like in neonates than in adults. Whereas neonatal mice from HSV-naive mothers responded well to both DNA and inactivated vaccines, only DNA immunization induced effective immunity in neonates born to immune mothers. Our results indicate that DNA vaccines might provide a useful means of immunizing young animals that still possess high levels of potentially interfering maternal antibody.
We demonstrated recently that the arachidonic acid (AA) cascade is involved in cytomegalovirus (CMV)-induced generation of reactive oxygen species (ROS) and the activation of nuclear factor (NF)-kappaB in human smooth muscle cells (SMCs). Since AA release from neutrophils is mediated by pertussis toxin (PTx)-sensitive guanine nucleotide-binding (G) proteins, we hypothesized by analogy that CMV stimulates ROS generation in SMCs and ultimately activates NF-kappaB via a PTx-sensitive G protein-coupled pathway. Our first test of this hypothesis demonstrated that PTx blocked AA release induced by CMV infection of SMCs, as well as blocked the terminal products of this reaction, ROS generation and NF-kappaB activation. More proximal components of the pathway were then examined. CMV infection increased phosphorylation and activity of cytosolic phospholipase A2 (cPLA2), an enzyme causing AA release; these effects were inhibited by PTx. CMV infection activated mitogen-activated protein (MAP) kinase, a key enzyme for cPLA2 phosphorylation, an effect also inhibited by PTx. Finally, inhibition of MAP kinase kinase (MAPKK), which phosphorylates and thereby activates MAP kinase, inhibited CMV-induced ROS generation. These data demonstrate that a PTx-sensitive G protein-dependent signaling pathway mediates cellular effects of CMV infection of SMCs. The downstream events include phosphorylation and activation of MAP kinase by MAPKK and subsequent phosphorylation and activation of cPLA2 (with its translocation to cell membranes), followed by stimulation of the AA cascade, which generates intracellular ROS and thereby activates NF-kappaB.