Early and prolonged intake of partially hydrogenated fat alters the expression of genes in rat adipose tissue

ObjectiveOur previous study indicated that partially hydrogenated fat (PHF) diets, rich in trans-isomers, alter plasma lipids and increase the lipogenesis rate on adipose tissue in rats at a young age. In the present study we investigated the effects of dietary PHF on the expression of genes associated with glucose and lipid metabolism in rat adipose tissue.MethodsFemale Wistar rats were fed normolipidic diets containing PHF (rich in trans-fatty acids and poor in polyunsaturated fatty acids [PUFAs]), soy oil (rich in ω-6 PUFAs), and fish oil (rich in ω-3 PUFAs) during gestation and lactation; young male pups were fed the same diets from weaning until 120 d of life. The mRNA expression of peroxisome proliferator-activated receptor-γ, tumor necrosis factor-α, resistin, adiponectin, and leptin were analyzed in retroperitoneal adipose tissue (RET) using real time polymerase chain reaction.ResultsThe PHF group showed the highest triacylglycerol, glucose, and insulin levels and the lowest plasma adiponectin level. The RET of PHF incorporated trans-fatty acids, whereas fish oil and soy oil groups had increased ω-3 and ω-6 PUFAs, respectively. In the RET the PHF group had the highest resistin and tumor necrosis factor-α levels and the lowest adiponectin and peroxisome proliferator-activated receptor-γ gene expressions, whereas the fish oil group had the highest peroxisome proliferator-activated receptor-γ and the lowest leptin gene expression.ConclusionProlonged intake of PHF has a negative effect on the expression of genes in RET when compared with diets with ω-6 and ω-3 PUFAs. These changes may be an effect of the smaller proportions of PUFAs in this fat, instead of being only caused by trans-fatty acids.     

Phosphorylated HER-2 tyrosine kinase and Her-2/neu gene amplification as predictive factors of response to trastuzumab in patients with HER-2 overexpressing metastatic breast cancer (MBC)

AimTrastuzumab (T), a humanised monoclonal antibody against HER-2, is active in HER-2-positive MBC patients. However, nearly 60% of the patients do not benefit from T, stressing the need for additional predictive markers. The following markers could be implicated in response to T: (1) the magnitude of Her-2 gene amplification; (2) the co-expression of the other HER family receptors, possibly responsible for HER-2 trans-activation; (3) the activated status of HER-2; (4) the activated status of downstream effectors as mitogen-activated protein kinases (MAPKs), p38 and p27.MethodsMedical files of patients with MBC treated with T either as a single agent or in combination with chemotherapy (CT) were reviewed. HER family members (EGFR, HER-2, HER-3, HER-4), the phosphorylated forms of EGFR (p-EGFR), HER-2 (p-HER-2) and of the downstream effectors were evaluated in the archival tumours. The correlation between clinical outcome and the expression of these markers was investigated.Results(1) Increasing values of Her-2 amplification were associated with a higher probability of achieving an objective response; (2) no statistical significant correlation between the expression of the HER family receptors was found; (3) p-HER-2 was predictive of response in patients treated with T + CT; (4) a statistically significant correlation between p-ERK 1/2, p-p38 and p-HER-2 emerged, pointing to the activated vertical pathway p-HER-2 → p-MAPKs.Conclusionsp-HER-2 and the magnitude of Her-2 amplification were predictive of response to T and their role deserves to be analysed in larger and more homogenous T-treated populations such as those from large phase III trials.     

Changes in the pattern of DNA methylation associate with twin discordance in systemic lupus erythematosus

Monozygotic (MZ) twins are partially concordant for most complex diseases, including autoimmune disorders. Whereas phenotypic concordance can be used to study heritability, discordance suggests the role of non-genetic factors. In autoimmune diseases, environmentally driven epigenetic changes are thought to contribute to their etiology. Here we report the first high-throughput and candidate sequence analyses of DNA methylation to investigate discordance for autoimmune disease in twins. We used a cohort of MZ twins discordant for three diseases whose clinical signs often overlap: systemic lupus erythematosus (SLE), rheumatoid arthritis, and dermatomyositis. Only MZ twins discordant for SLE featured widespread changes in the DNA methylation status of a significant number of genes. Gene ontology analysis revealed enrichment in categories associated with immune function. Individual analysis confirmed the existence of DNA methylation and expression changes in genes relevant to SLE pathogenesis. These changes occurred in parallel with a global decrease in the 5-methylcytosine content that was concomitantly accompanied with changes in DNA methylation and expression levels of ribosomal RNA genes, although no changes in repetitive sequences were found. Our findings not only identify potentially relevant DNA methylation markers for the clinical characterization of SLE patients but also support the notion that epigenetic changes may be critical in the clinical manifestations of autoimmune disease.Human monozygotic (MZ) twins exhibit variable degrees of concordance for complex diseases, such as cancer, cardiovascular diseases, or autoimmune disorders. Whereas concordance rates close to 100% in identical twins apply to coinheritance of mutant genes that are dominant and highly penetrant, most diseases or traits show a concordance in identical twins in the broad range of 5%–75% (Nance 1978). Most of the twin-based studies have focused on the concordance between siblings that has led to the identification of trait-specific genes (Hrubec and Robinette 1984), while less attention has been paid to the degree of discordance, which suggests the participation of factors other than pure genetic changes. Recently, interest has shifted toward exploring the molecular mechanisms involved in determining phenotypic differences. The increasing recognition of the influence of epigenetics in phenotypic outcomes continues to open up new lines of research involving twin studies. DNA methylation and histone modifications, the major sources of epigenetic information, regulate gene expression levels and provide an alternative mechanism for modulating gene function to those arising from genetic changes (Esteller 2008). Interestingly, epigenetic changes are highly dependent on environmental factors, and recent data support the notion that diet and other external influences can alter the epigenetic status of genes (Mann et al. 2004; Heijmans et al. 2008).In the case of autoimmune disorders, twin studies have provided evidence of the existence of a genetic component and have been useful for identifying susceptibility genes. One of the best studied autoimmune diseases is systemic lupus erythematosus (SLE), an archetypical systemic, autoimmune inflammatory disease characterized by the production of autoantibodies to multiple nuclear antigens. Well-established risk factors include alleles in the major histocompatibility complex region, IRF5, STAT4, BLK, BANK1, PDCD1, MECP2, and TNFSF4, among others (Graham et al. 2007; International Consortium for Systemic Lupus Erythematosus Genetics 2008; Moser et al. 2009). Many SLE susceptibility genes belong to key pathways that are common to those associated with other autoimmune disorders. In fact, not only genetic factors but also clinical signs and symptoms of SLE overlap with those of other autoimmune diseases such as rheumatoid arthritis (RA) or dermatomyositis (DM) (Encinas and Kuchroo 2000) (a summary of clinical features of these three diseases is presented in Supplemental Table 1).In addition to twin discordance, additional evidence highlights the relevance of epigenetic events in the etiology of SLE and other autoimmune diseases. Lymphocytes from patients with SLE and RA exhibit globally hypomethylated DNA (Richardson et al. 1990; Corvetta et al. 1991). In parallel, DNA methylation analysis of several candidate genes in lymphocytes from SLE individuals has identified promoter demethylation that may contribute to their aberrant overexpression. These changes occur in PRF1 (Kaplan et al. 2004), CD70 (Lu et al. 2005), ITGAL (Lu et al. 2002), and the X chromosome gene CD40LG (Lu et al. 2007) promoters, and are known to be associated with the development of lupus. Additional evidence of the role of DNA methylation changes in the development of SLE comes from studies of DNA demethylating drugs. For instance, CD4⁺ T-cells become autoreactive following treatment with 5-azacytidine (Richardson 1986), which causes genome-wide hypomethylation and aberrant overexpression of many genes.Direct comparison of identical twins is an excellent experimental approach for testing environmental epigenetics, because DNA sequence differences, including single-nucleotide polymorphisms, which would be abundant in singleton-based studies, do not interfere in this analysis. Recent studies with twins (Fraga et al. 2005; Kaminsky et al. 2009) have demonstrated the existence of genome-wide epigenetic differences that potentially could explain differences in phenotype. Here, we used a collection of identical twins discordant for SLE and two other related systemic autoimmune diseases to perform the first high-throughput analysis of DNA methylation changes. Interestingly, only SLE samples exhibited significant changes in DNA methylation status at both the global and sequence-specific levels in comparison with their healthy, discordant twins and compared with unrelated matched normal controls. Our study allowed us to identify differential methylation between SLE and healthy twins in a set of genes with biological functions relevant to SLE pathogenesis. We also found a global decrease in global 5-methylcytosine (5mC) content when comparing SLE twins with their corresponding healthy twins. We also observed a change in DNA methylation status of the CpG-rich region of the ribosomal DNA repeat, which contains the transcribed 18S and 28S genes. Our twin study provides the first evidence that large changes in the DNA methylation profile may be associated with the differential onset of the disease in twins discordant for SLE, suggesting that there is an association with environmental effects. In addition, our study also yields a list of epigenetically deregulated DNA sequences in SLE, which may potentially lead to a better understanding of its pathogenesis.     

The effect of chronic administration of l-arginine on the learning and memory of estradiol-treated ovariectomized rats tested in the morris water maze

OBJECTIVE:

The present study was carried out to evaluate the effect of l-arginine on the learning and memory of estradiol-treated ovariectomized (OVX) rats.

METHODS:

Forty-eight rats were divided into six groups: (1) sham, (2) OVX, (3) sham-Est, (4) OVX-Est, (5) sham-Est-LA, and (6) OVX-Est-LA. The animals of the sham-Est and OVX-Est groups were treated by weekly injection of estradiol valerate (2mg/kg). The sham-Est-LA and OVX-Est-LA groups were treated in the same manner but with an additional daily injection of l-arginine (200mg/kg). After eight weeks, animals of all groups were tested in the Morris water maze. The escape latency and path traveled to reach the platform were compared between groups.

RESULTS:

Time latency and path length in the OVX group were significantly higher than in the sham group (P<0.05). The OVX-Est group had a significantly shorter traveled path length and time latency compared to the OVX group (P<0.001). Time latency and path length in the sham-Est group was significantly higher than in the sham group (P<0.001). Time latency and path length in the OVX-Est-LA group were significantly higher than in the OVX-Est group.

CONCLUSIONS:

These results allow us to propose that chronic treatment with estradiol enhances the spatial learning and memory of OVX rats, and that long term l-arginine treatment attenuates the effects of improvement produced by estradiol in OVX rats.     

Short-term treatment of spontaneously hypertensive rats with liver growth factor reduces carotid artery fibrosis, improves vascular function, and lowers blood pressure

OBJECTIVE: Liver growth factor (LGF), a mitogen for liver cells, reduces fibrosis in a rat model of cirrhosis. The present study assesses the possible vascular antifibrotic and antihypertensive effects of LGF treatment on spontaneously hypertensive rats (SHR). METHODS: Six-month-old male SHR and normotensive Wistar Kyoto rats (WKY) were treated with LGF (4.5 microg LGF/rat i.p. twice a week for 2 weeks). Haemodynamic parameters were measured in anaesthetized rats. Vascular structure and function were studied in carotid arteries using optical and confocal microscopy, radioimmunoassay for desmosine, and isometric tension recording. RESULTS: LGF reduced systolic and diastolic blood pressure only in SHR. When compared to those of untreated SHR, carotid arteries from LGF-treated SHR showed: 1) a 50% reduction in collagen area and an increase in vascular smooth muscle cell number in the media, 2) no difference in total elastin content, but an increase in size of fenestrae in the internal elastic lamina, and 3) enhanced relaxation to acetylcholine, sodium nitroprusside, and forskolin. These effects were specific for SHR, since no changes were observed in LGF-treated WKY. CONCLUSION: Short-term treatment with a low dose of LGF induced a large improvement in vascular structure and function and significantly reduced blood pressure in a rat model of essential hypertension. The present results could open future research to explore the vascular effects of this endogenous factor in order to determine its potential as an antifibrotic and antihypertensive agent in humans.     

Prostaglandin production by human osteoclasts in culture

OBJECTIVE: Prostaglandins (PG) are important mediators of bone metabolism with direct and indirect effects on bone cells. They may have important effects on osteoclasts, but it is not known if these cells can synthesize PG. We used 2 experimental models in order (1) to determine the presence and functionality of cyclooxygenase (COX) and phospholipase A2 (PLA2) enzymes in human osteoclasts and (2) to study their role in cell metabolism. METHODS: Experiments were undertaken on authentic human osteoclasts extracted from human fetuses (fhOC) and on human osteoclast-like (hOCL) cells differentiated from peripheral blood mononuclear cells. The presence of COX proteins was determined by immunohistochemistry. COX and PLA2 enzymatic activity was evaluated at the single-cell level by fluorescence microscopy. An enriched population of hOCL cells was used to evaluate total PG production and the influence of COX activity on bone resorption. RESULTS: COX-1 was expressed in the cytoplasm and COX-2 was distributed mainly near the nuclear membrane of osteoclasts. These cells showed a high basal level of COX activity that could be inhibited by pretreatment with COX inhibitors. Cytosolic PLA2 was present in both models. Human osteoclasts actively produced PG, and the COX-1 pathway was implicated in the control of bone resorption. CONCLUSION: These results indicate that PG may be important autacoids for the control of osteoclast biology and that the COX-1 pathway is implicated in the inhibition of bone resorption.     

Transient reticular gallbladder wall thickening in severe dengue fever: a reliable sign of plasma leakage

Background  

Dengue fever (DF) is an acute infection caused by a flavivirus. Although most patients present mild symptoms, some progress to a severe condition characterized by hypovolemic shock and hemorrhagic phenomena. The main feature of this severe form of DF is plasma leakage. Gallbladder wall thickening (GBWT), ascites and pleural effusion represent the sonographic triad of plasma leakage in DF.     

Complication profiles of adult asthmatics requiring paralysis during mechanical ventilation

OBJECTIVE: To assess the characteristics and the incidence of morbidity of intubated asthmatic patients who received long-term paralysis. DESIGN: Retrospective cohort study. SETTING: Five intensive care units (ICUs) in Paris and the surrounding suburbs. PATIENTS AND PARTICIPANTS: The NMB group consisted of patients who received neuromuscular blocking agents for more than 12 h (NMB group) versus sedation alone (SED). INTERVENTIONS: None. MEASUREMENTS AND RESULTS: The incidence of post-extubation muscle weakness and/or myopathy was 18% in the NMB group compared to 2% in the SED group ( p=0.01). The occurrence of ventilator-associated pneumonia was higher in the NMB group (42% versus 4%; p<0.0001). The duration of ICU stay and of mechanical ventilation were significantly greater in the NMB group. Multiple logistic regression analysis showed that inclusion in the NMB group was the only independent predictor of the presence of the overall morbidity [odds ratio 6.4 (2.09; 19.64)]. CONCLUSION: While greater initial severity of respiratory compromise in the NMB group may explain part of the difference, use of NMB agents appears to be strongly related to the presence of significant complications among mechanically-ventilated asthmatic patients.