t-10, c-12 CLA Dietary Supplementation Inhibits Atherosclerotic Lesion Development Despite Adverse Cardiovascular and Hepatic Metabolic Marker Profiles

Animal and human studies have indicated that fatty acids such as the conjugated linoleic acids (CLA) found in milk could potentially alter the risk of developing metabolic disorders including diabetes and cardiovascular disease (CVD). Using susceptible rodent models (apoE^−/− and LDLr^−/− mice) we investigated the interrelationship between mouse strain, dietary conjugated linoleic acids and metabolic markers of CVD. Despite an adverse metabolic risk profile, atherosclerosis (measured directly by lesion area), was significantly reduced with t-10, c-12 CLA and mixed isomer CLA (Mix) supplementation in both apoE^−/− (p<0.05, n = 11) and LDLr^−/− mice (p<0.01, n = 10). Principal component analysis was utilized to delineate the influence of multiple plasma and tissue metabolites on the development of atherosclerosis. Group clustering by dietary supplementation was evident, with the t-10, c-12 CLA supplemented animals having distinct patterns, suggestive of hepatic insulin resistance, regardless of mouse strain. The effect of CLA supplementation on hepatic lipid and fatty acid composition was explored in the LDLr^−/− strain. Dietary supplementation with t-10, c-12 CLA significantly increased liver weight (p<0.05, n = 10), triglyceride (p<0.01, n = 10) and cholesterol ester content (p<0.01, n = 10). Furthermore, t-10, c-12 CLA also increased the ratio of 18∶1 to 18∶0 fatty acid in the liver suggesting an increase in the activity of stearoyl-CoA desaturase. Changes in plasma adiponectin and liver weight with t-10, c-12 CLA supplementation were evident within 3 weeks of initiation of the diet. These observations provide evidence that the individual CLA isomers have divergent mechanisms of action and that t-10, c-12 CLA rapidly changes plasma and liver markers of metabolic syndrome, despite evidence of reduction in atherosclerosis.     

Targeting Serum Glucocorticoid-Regulated Kinase-1 in Squamous Cell Carcinoma of the Head and Neck: A Novel Modality of Local Control

Purpose

The inhibition of serum glucocorticoid-regulated kinase-1 (SGK-1) has been found to decrease growth of colon and prostate cancer cells. The purpose of this study is to evaluate the therapeutic effect of SGK-1 inhibition in head and neck squamous cell carcinoma (SCC).

Experimental Design

Human head and neck tumors (HTB41/43) were established in athymic mice. Growth rates between mice treated with vehicle (PBS) injection (group 1, n = 5), SGK-1 Inhibitor GSK 650394 (group 2, n = 6), systemic cisplatin (group 3, n = 6), and a combination of SGK-1 Inhibitor and cisplatin (group 4, n = 6) were compared using repeated measures one-way ANOVA with Newman-Keuls Multiple Comparison Test. Tumor cells were subsequently submitted to further analyses.

Results

At the end of the experiment mean tumor sizes were 122.33+/−105.86, 76.73+/−36.09, 94.52+/−75.92, and 25.76+/−14.89 mm² (mean +/− SD) for groups 1 to 4. Groups 2 and 3 showed decreased tumor growth compared to controls (p<0.001). Group 4 displayed even greater growth suppression (p<0.0001). Importantly, group 4 fared better than group 3 (p<0.001). CD44 expression was reduced in group 2 (p<0.05), and to an even greater extent in groups 3 and 4 (p<0.0025). A trend towards reduction of HER 2 expression was noted in group 4.

Conclusions

SGK-1 inhibition suppresses tumor growth, and in combination with systemic cisplatin exceeds the effect of cisplatin alone. Decreased expression of CD44 and HER 2 implies depletion of tumor stem cells, and less tumorigenicity. SGK-1 inhibition represents a potential modality of local control for palliation in advanced cases.     

TRAIL/TRAIL receptor system and susceptibility to multiple sclerosis

The TNF-related apoptosis inducing ligand (TRAIL)/TRAIL receptor system participates in crucial steps in immune cell activation or differentiation. It is able to inhibit proliferation and activation of T cells and to induce apoptosis of neurons and oligodendrocytes, and seems to be implicated in autoimmune diseases. Thus, TRAIL and TRAIL receptor genes are potential candidates for involvement in susceptibility to multiple sclerosis (MS). To test whether single-nucleotide polymorphisms (SNPs) in the human genes encoding TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 are associated with MS susceptibility, we performed a candidate gene case-control study in the Spanish population. 59 SNPs in the TRAIL and TRAIL receptor genes were analysed in 628 MS patients and 660 controls, and validated in an additional cohort of 295 MS patients and 233 controls. Despite none of the SNPs withstood the highly conservative Bonferroni correction, three SNPs showing uncorrected p values<0.05 were successfully replicated: rs4894559 in TRAIL gene, p = 9.8×10⁻⁴, OR = 1.34; rs4872077, in TRAILR-1 gene, p = 0.005, OR = 1.72; and rs1001793 in TRAILR-2 gene, p = 0.012, OR = 0.84. The combination of the alleles G/T/A in these SNPs appears to be associated with a reduced risk of developing MS (p = 2.12×10⁻⁵, OR = 0.59). These results suggest that genes of the TRAIL/TRAIL receptor system exerts a genetic influence on MS.     

TNFR1 determines progression of chronic liver injury in the IKKγ/Nemo genetic model

Death receptor-mediated hepatocyte apoptosis is implicated in a wide range of liver diseases including viral and alcoholic hepatitis, ischemia/reperfusion injury, fulminant hepatic failure, cholestatic liver injury, as well as cancer. Deletion of NF-κB essential modulator in hepatocytes (IKKγ/Nemo) causes spontaneous progression of TNF-mediated chronic hepatitis to hepatocellular carcinoma (HCC). Thus, we analyzed the role of death receptors including TNFR1 and TRAIL in the regulation of cell death and the progression of liver injury in IKKγ/Nemo-deleted livers. We crossed hepatocyte-specific IKKγ/Nemo knockout mice (Nemo^Δhepa) with constitutive TNFR1^−/− and TRAIL^−/− mice. Deletion of TNFR1, but not TRAIL, decreased apoptotic cell death, compensatory proliferation, liver fibrogenesis, infiltration of immune cells as well as pro-inflammatory cytokines, and indicators of tumor growth during the progression of chronic liver injury. These events were associated with diminished JNK activation. In contrast, deletion of TNFR1 in bone-marrow-derived cells promoted chronic liver injury. Our data demonstrate that TNF- and not TRAIL signaling determines the progression of IKKγ/Nemo-dependent chronic hepatitis. Additionally, we show that TNFR1 in hepatocytes and immune cells have different roles in chronic liver injury–a finding that has direct implications for treating chronic liver disease.     

Deficiency in Clonogenic Endometrial Mesenchymal Stem Cells in Obese Women with Reproductive Failure – a Pilot Study

Objectives

The mechanisms of obesity associated reproductive complications remain poorly understood. Endometrial mesenchymal stem-cells are critical for cyclic renewal and uterine function. Recently, W5C5⁺ cells, with high clonogenicity, capable of producing endometrial stroma in vivo, have been described. We sought to investigate the abundance and cloning efficiency of W5C5⁺ and W5C5⁻ endometrial cells in relation to Body Mass Index, age and reproductive outcome.

Design

W5C5⁺ and W5C5⁻ cells were purified from mid-luteal endometrial biopsies (n = 54) by magnetic bead separation and subjected to in vitro colony-forming assays.

Results

First trimester pregnancy losses were significantly higher in obese subjects (n = 12) compared to overweight (n = 20) and subjects with normal Body Mass Index (n = 22) (P<0.05, P<0.01, respectively). W5C5⁺ cells (%) were significantly lower in obese subjects compared to subjects with normal Body Mass Index (P<0.05). W5C5⁺ cloning efficiency was significantly lower in obese subjects compared to overweight and subjects with normal Body Mass Index (P<0.05, respectively). W5C5⁻ cloning efficiency was significantly lower in obese subjects compared to subjects with normal Body Mass Index (P<0.05). Body Mass Index was significantly negatively correlated with W5C5⁺ cloning efficiency and W5C5⁻ cloning efficiency (P<0.01, respectively), and positively correlated with first trimester loss (P<0.01). We found no significant results with age (P>0.05).

Conclusions

Our observations suggest that the regenerative capacity and plasticity of the endometrium of obese women is suboptimal, which in turn may account for the increased risk of reproductive complications associated with obesity.     

A Vasoactive Role for Endogenous Relaxin in Mesenteric Arteries of Male Mice

The peptide hormone relaxin has striking effects on the vascular system. Specifically, endogenous relaxin treatment reduces myogenic reactivity through nitric oxide (NO)-mediated vasorelaxation and increases arterial compliance in small resistance arteries. However, less is known about the vascular roles of endogenous relaxin, particularly in males. Therefore, we used male wild-type (Rln ^+/+) and relaxin knockout (Rln ^−/−) mice to test the hypothesis that passive wall properties and vascular reactivity in mesenteric arteries would be compromised in Rln ^−/− mice. Passive compliance was determined in arteries (n = 8–9) mounted on a pressure myograph and in Ca²⁺-free Krebs containing 2 mM EGTA. Passive volume compliance was significantly (P = 0.01) decreased in the mesenteric arteries of Rln ^−/− mice. Vascular reactivity was assessed using wire myography. In mesenteric arteries (n = 5) of Rln ^−/− mice, there was a significant (P<0.03) increase in sensitivity to the vasoconstrictors phenylephrine and thromboxane-mimetic {"type":"entrez-nucleotide","attrs":{"text":"U41669","term_id":"1195477","term_text":"U41669"}}U41669. This enhanced responsiveness to vasoconstrictors was abolished by endothelial denudation, and attributed to impaired NO and prostanoid pathways in Rln ^−/− mice. Sensitivity to the endothelial agonist acetylcholine was significantly (n = 7–9, P≤0.03) decreased, and this was abolished in the presence of the cyclooxygenase inhibitor, indomethacin (2 µM). This indicates that prostanoid vasoconstrictor pathways were upregulated in the mesenteric arteries of Rln ^−/− mice. In summary, we demonstrate endothelial dysfunction and impaired arterial wall remodeling in male mice deficient in relaxin. Thus, our results highlight a role for endogenous relaxin in the maintenance of normal mesenteric artery structure and function in males.     

The Role of CXCR3 in DSS-Induced Colitis

Inflammatory bowel disease (IBD) is a group of disorders that are characterized by chronic, uncontrolled inflammation in the intestinal mucosa. Although the aetiopathogenesis is poorly understood, it is widely believed that IBD stems from a dysregulated immune response towards otherwise harmless commensal bacteria. Chemokines induce and enhance inflammation through their involvement in cellular trafficking. Reducing or limiting the influx of these proinflammatory cells has previously been demonstrated to attenuate inflammation. CXCR3, a chemokine receptor in the CXC family that binds to CXCL9, CXCL10 and CXCL11, is strongly overexpressed in the intestinal mucosa of IBD patients. We hypothesised that CXCR3 KO mice would have impaired cellular trafficking, thereby reducing the inflammatory insult by proinflammatory cell and attenuating the course of colitis. To investigate the role of CXCR3 in the progression of colitis, the development of dextran sulfate sodium (DSS)-induced colitis was investigated in CXCR3^−/− mice over 9 days. This study demonstrated attenuated DSS-induced colitis in CXCR3^−/− mice at both the macroscopic and microscopic level. Reduced colitis correlated with lower recruitment of neutrophils (p = 0.0018), as well as decreased production of IL-6 (p<0.0001), TNF (p = 0.0038), and IFN-γ (p = 0.0478). Overall, our results suggest that CXCR3 plays an important role in recruiting proinflammatory cells to the colon during colitis and that CXCR3 may be a therapeutic target to reduce the influx of proinflammatory cells in the inflamed colon.     

Impaired Chemosensitivity of Mouse Dorsal Raphe Serotonergic Neurons Overexpressing Serotonin 1A (Htr1a) Receptors

Background

Serotonergic system participates in a wide range of physiological processes and behaviors, but its role is generally considered as modulatory and noncrucial, especially concerning life-sustaining functions. We recently created a transgenic mouse line in which a functional deficit in serotonin homeostasis due to excessive serotonin autoinhibition was produced by inducing serotonin 1A receptor (Htr1a) overexpression selectively in serotonergic neurons (Htr1a raphe-overexpressing or Htr1a^RO mice). Htr1a^RO mice exhibit episodes of autonomic dysregulation, cardiovascular crises and death, resembling those of sudden infant death syndrome (SIDS) and revealing a life-supporting role of serotonergic system in autonomic control. Since midbrain serotonergic neurons are chemosensitive and are implicated in arousal we hypothesized that their chemosensitivity might be impaired in Htr1a^RO mice.

Principal findings

Loose-seal cell-attached recordings in brainstem slices revealed that serotonergic neurons in dorsal raphe nucleus of Htr1a^RO mice have dramatically reduced responses to hypercapnic challenge as compared with control littermates. In control mice, application of 9% CO₂ produced an increase in firing rate of serotonergic neurons (0.260±0.041 Hz, n = 20, p = 0.0001) and application of 3% CO₂ decreased their firing rate (−0.142±0.025 Hz, n = 17, p = 0.0008). In contrast, in Htr1a^RO mice, firing rate of serotonergic neurons was not significantly changed by 9% CO₂ (0.021±0.034 Hz, n = 16, p = 0.49) and by 3% CO₂ (0.012±0.046 Hz, n = 12, p = 0.97).

Conclusions

Our findings support the hypothesis that chemosensitivity of midbrain serotonergic neurons provides a physiological mechanism for arousal responses to life-threatening episodes of hypercapnia and that functional impairment, such as excessive autoinhibition, of midbrain serotonergic neuron responses to hypercapnia may contribute to sudden death.     

Attenuation of Reactive Gliosis Does Not Affect Infarct Volume in Neonatal Hypoxic-Ischemic Brain Injury in Mice

Background

Astroglial cells are activated following injury and up-regulate the expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Adult mice lacking the intermediate filament proteins GFAP and vimentin (GFAP^−/−Vim^−/−) show attenuated reactive gliosis, reduced glial scar formation and improved regeneration of neuronal synapses after neurotrauma. GFAP^−/−Vim^−/− mice exhibit larger brain infarcts after middle cerebral artery occlusion suggesting protective role of reactive gliosis after adult focal brain ischemia. However, the role of astrocyte activation and reactive gliosis in the injured developing brain is unknown.

Methodology/Principal Findings

We subjected GFAP^−/−Vim^−/− and wild-type mice to unilateral hypoxia-ischemia (HI) at postnatal day 9 (P9). Bromodeoxyuridine (BrdU; 25 mg/kg) was injected intraperitoneally twice daily from P9 to P12. On P12 and P31, the animals were perfused intracardially. Immunohistochemistry with MAP-2, BrdU, NeuN, and S100 antibodies was performed on coronal sections. We found no difference in the hemisphere or infarct volume between GFAP^−/−Vim^−/− and wild-type mice at P12 and P31, i.e. 3 and 22 days after HI. At P31, the number of NeuN⁺ neurons in the ischemic and contralateral hemisphere was comparable between GFAP^−/−Vim^−/− and wild-type mice. In wild-type mice, the number of S100⁺ astrocytes was lower in the ipsilateral compared to contralateral hemisphere (65.0±50.1 vs. 85.6±34.0, p<0.05). In the GFAP^−/−Vim^−/− mice, the number of S100⁺ astrocytes did not differ between the ischemic and contralateral hemisphere at P31. At P31, GFAP^−/−Vim^−/− mice showed an increase in NeuN⁺BrdU⁺ (surviving newly born) neurons in the ischemic cortex compared to wild-type mice (6.7±7.7; n = 29 versus 2.9±3.6; n = 28, respectively, p<0.05), but a comparable number of S100⁺BrdU⁺ (surviving newly born) astrocytes.

Conclusions/Significance

Our results suggest that attenuation of reactive gliosis in the developing brain does not affect the hemisphere or infarct volume after HI, but increases the number of surviving newborn neurons.     

Whole-Body Insulin Sensitivity Rather than Body-Mass-Index Determines Fasting and Post-Glucose-Load Growth Hormone Concentrations

Background

Obese, non-acromegalic persons show lower growth hormone (GH) concentrations at fasting and reduced GH nadir during an oral glucose tolerance test (OGTT). However, this finding has never been studied with regard to whole-body insulin-sensitivity as a possible regulator.

Methods

In this retrospective analysis, non-acromegalic (NonACRO, n = 161) and acromegalic (ACRO, n = 35), non-diabetic subjects were subdivided into insulin-sensitive (IS) and –resistant (IR) groups according to the Clamp-like Index (CLIX)-threshold of 5 mg·kg⁻¹·min⁻¹ from the OGTT.

Results

Non-acromegalic IS (CLIX: 8.8±0.4 mg·kg⁻¹·min⁻¹) persons with similar age and sex distribution, but lower (p<0.001) body-mass-index (BMI = 25±0 kg/m², 84% females, 56±1 years) had 59% and 70%, respectively, higher (p<0.03) fasting GH and OGTT GH area under the curve concentrations than IR (CLIX: 3.5±0.1 mg·kg⁻¹·min⁻¹, p<0.001) subjects (BMI = 29±1 kg/m², 73% females, 58±1 years). When comparing on average overweight non-acromegalic IS and IR with similar anthropometry (IS: BMI: 27±0 kg/m², 82% females, 58±2 years; IR: BMI: 27±0 kg/m², 71% females, 60±1 years), but different CLIX (IS: 8.7±0.9 vs. IR: 3.8±0.1 mg·kg⁻¹·min⁻¹, p<0.001), the results remained almost the same. In addition, when adjusted for OGTT-mediated glucose rise, GH fall was less pronounced in IR. In contrast, in acromegalic subjects, no difference was found between IS and IR patients with regard to fasting and post-glucose-load GH concentrations.

Conclusions

Circulating GH concentrations at fasting and during the OGTT are lower in non-acromegalic insulin-resistant subjects. This study seems the first to demonstrate that insulin sensitivity rather than body-mass modulates fasting and post-glucose-load GH concentrations in non-diabetic non–acromegalic subjects.     

Neutrophil-Derived Myeloperoxidase Aggravates Non-Alcoholic Steatohepatitis in Low-Density Lipoprotein Receptor-Deficient Mice

Background

Chronic inflammation and oxidative stress play fundamental roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). Previously, we reported that myeloperoxidase (MPO), an aggressive oxidant-generating neutrophil enzyme, is associated with NASH severity in man. We now investigated the hypothesis that MPO contributes to the development and progression of NASH.

Methodology

Low-density lipoprotein receptor-deficient mice with an MPO-deficient hematopoietic system (LDLR^−/−/MPO^−/−tp mice) were generated and compared with LDLR^−/−/MPO^+/+tp mice after induction of NASH by high-fat feeding.

Results

High-fat feeding caused a ∼4-fold induction of liver MPO in LDLR^−/−/MPO^+/+ mice which was associated with hepatic sequestration of MPO-positive neutrophils and high levels of nitrotyrosine, a marker of MPO activity. Importantly, LDLR^−/−/MPO^−/−tp mice displayed markedly reduced hepatic neutrophil and T-lymphocyte infiltration (p<0.05), and strong down regulation of pro-inflammatory genes such as TNF-α and IL-6 (p<0.05, p<0.01) in comparison with LDLR^−/−/MPO^+/+tp mice. Next to the generalized reduction of inflammation, liver cholesterol accumulation was significantly diminished in LDLR^−/−/MPO^−/−tp mice (p = 0.01). Moreover, MPO deficiency appeared to attenuate the development of hepatic fibrosis as evident from reduced hydroxyproline levels (p<0.01). Interestingly, visceral adipose tissue inflammation was markedly reduced in LDLR^−/−/MPO^−/−tp mice, with a complete lack of macrophage crown-like structures. In conclusion, MPO deficiency attenuates the development of NASH and diminishes adipose tissue inflammation in response to a high fat diet, supporting an important role for neutrophils in the pathogenesis of metabolic disease.     

Prenatal Endotoxemia and Placental Drug Transport in The Mouse: Placental Size-Specific Effects

Lipopolysaccharide (LPS) in high doses inhibits placental multidrug resistance P-glycoprotein (P-gp - Abcb1a/b) and breast cancer resistance protein (BCRP - Abcg2). This potentially impairs fetal protection against harmful factors in the maternal circulation. However, it is unknown whether LPS exposure, at doses that mimic sub-lethal clinical infection, alters placental multidrug resistance. We hypothesized that sub-lethal (fetal) LPS exposure reduces placental P-gp activity. Acute LPS (n = 19;150 µg/kg; ip) or vehicle (n = 19) were given to C57BL/6 mice at E15.5 and E17.5. Placentas and fetal-units were collected 4 and 24 h following injection. Chronic LPS (n = 6; 5 µg/kg/day; ip) or vehicle (n = 5) were administered from E11.5–15.5 and tissues were collected 4 h after final treatment. P-gp activity was assessed by [³H]digoxin accumulation. Placental Abcb1a/b, Abcg2, interleukin-6 (Il-6), Tnf-α, Il-10 and toll-like receptor-4 (Tlr-4) mRNA were measured by qPCR. Maternal plasma IL-6 was determined. At E15.5, maternal IL-6 was elevated 4 h after single (p<0.001) and chronic (p<0.05) LPS, but levels had returned to baseline by 24 h. Placental Il-6 mRNA was also increased after acute and chronic LPS treatments (p<0.05), whereas Abcb1a/b and Abcg2 mRNA were unaffected. However, fetal [³H]digoxin accumulation was increased (p<0.05) 4 h after acute LPS, and maternal [³H]digoxin myocardial accumulation was increased (p<0.05) in mice exposed to chronic LPS treatments. There was a negative correlation between fetal [³H]digoxin accumulation and placental size (p<0.0001). Acute and chronic sub-lethal LPS exposure resulted in a robust inflammatory response in the maternal systemic circulation and placenta. Acute infection decreased placental P-gp activity in a time- and gestational age-dependent manner. Chronic LPS decreased P-gp activity in the maternal myocardium and there was a trend for fetuses with smaller placentas to accumulate more P-gp substrate than their larger counterparts. Collectively, we demonstrate that acute sub-lethal LPS exposure during pregnancy impairs fetal protection against potentially harmful xenobiotics in the maternal circulation.     

Accelerated In Vivo Proliferation of Memory Phenotype CD4+ T-cells in Human HIV-1 Infection Irrespective of Viral Chemokine Co-receptor Tropism

CD4⁺ T-cell loss is the hallmark of HIV-1 infection. CD4 counts fall more rapidly in advanced disease when CCR5-tropic viral strains tend to be replaced by X4-tropic viruses. We hypothesized: (i) that the early dominance of CCR5-tropic viruses results from faster turnover rates of CCR5⁺ cells, and (ii) that X4-tropic strains exert greater pathogenicity by preferentially increasing turnover rates within the CXCR4⁺ compartment. To test these hypotheses we measured in vivo turnover rates of CD4⁺ T-cell subpopulations sorted by chemokine receptor expression, using in vivo deuterium-glucose labeling. Deuterium enrichment was modeled to derive in vivo proliferation (p) and disappearance (d*) rates which were related to viral tropism data. 13 healthy controls and 13 treatment-naive HIV-1-infected subjects (CD4 143–569 cells/ul) participated. CCR5-expression defined a CD4⁺ subpopulation of predominantly CD45R0⁺ memory cells with accelerated in vivo proliferation (p = 2.50 vs 1.60%/d, CCR5⁺ vs CCR5⁻; healthy controls; P<0.01). Conversely, CXCR4 expression defined CD4⁺ T-cells (predominantly CD45RA⁺ naive cells) with low turnover rates. The dominant effect of HIV infection was accelerated turnover of CCR5⁺CD45R0⁺CD4⁺ memory T-cells (p = 5.16 vs 2.50%/d, HIV vs controls; P<0.05), naïve cells being relatively unaffected. Similar patterns were observed whether the dominant circulating HIV-1 strain was R5-tropic (n = 9) or X4-tropic (n = 4). Although numbers were small, X4-tropic viruses did not appear to specifically drive turnover of CXCR4-expressing cells (p = 0.54 vs 0.72 vs 0.44%/d in control, R5-tropic, and X4-tropic groups respectively). Our data are most consistent with models in which CD4⁺ T-cell loss is primarily driven by non-specific immune activation.     

SLC22A1-ABCB1 Haplotype Profiles Predict Imatinib Pharmacokinetics in Asian Patients with Chronic Myeloid Leukemia

Objective

This study aimed to explore the influence of SLC22A1, PXR, ABCG2, ABCB1 and CYP3A5*3 genetic polymorphisms on imatinib mesylate (IM) pharmacokinetics in Asian patients with chronic myeloid leukemia (CML).

Patients and Methods

Healthy subjects belonging to three Asian populations (Chinese, Malay, Indian; n = 70 each) and CML patients (n = 38) were enrolled in a prospective pharmacogenetics study. Imatinib trough (C_0h) and clearance (CL) were determined in the patients at steady state. Haplowalk method was applied to infer the haplotypes and generalized linear model (GLM) to estimate haplotypic effects on IM pharmacokinetics. Association of haplotype copy numbers with IM pharmacokinetics was defined by Mann-Whitney U test.

Results

Global haplotype score statistics revealed a SLC22A1 sub-haplotypic region encompassing three polymorphisms (rs3798168, rs628031 and IVS7+850C>T), to be significantly associated with IM clearance (p = 0.013). Haplotype-specific GLM estimated that the haplotypes AGT and CGC were both associated with 22% decrease in clearance compared to CAC [CL (*10⁻² L/hr/mg): CAC vs AGT: 4.03 vs 3.16, p = 0.017; CAC vs CGC: 4.03 vs 3.15, p = 0.017]. Patients harboring 2 copies of AGT or CGC haplotypes had 33.4% lower clearance and 50% higher C_0h than patients carrying 0 or 1 copy [CL (*10⁻² L/hr/mg): 2.19 vs 3.29, p = 0.026; C_0h (*10⁻⁶ 1/ml): 4.76 vs 3.17, p = 0.013, respectively]. Further subgroup analysis revealed SLC22A1 and ABCB1 haplotypic combinations to be significantly associated with clearance and C_0h (p = 0.002 and 0.009, respectively).

Conclusion

This exploratory study suggests that SLC22A1-ABCB1 haplotypes may influence IM pharmacokinetics in Asian CML patients.     

Candidate Gene Study of TRAIL and TRAIL Receptors: Association with Response to Interferon Beta Therapy in Multiple Sclerosis Patients

TRAIL and TRAIL Receptor genes have been implicated in Multiple Sclerosis pathology as well as in the response to IFN beta therapy. The objective of our study was to evaluate the association of these genes in relation to the age at disease onset (AAO) and to the clinical response upon IFN beta treatment in Spanish MS patients. We carried out a candidate gene study of TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 genes. A total of 54 SNPs were analysed in 509 MS patients under IFN beta treatment, and an additional cohort of 226 MS patients was used to validate the results. Associations of rs1047275 in TRAILR-2 and rs7011559 in TRAILR-4 genes with AAO under an additive model did not withstand Bonferroni correction. In contrast, patients with the TRAILR-1 rs20576-CC genotype showed a better clinical response to IFN beta therapy compared with patients carrying the A-allele (recessive model: p = 8.88×10⁻⁴, p_c = 0.048, OR = 0.30). This SNP resulted in a non synonymous substitution of Glutamic acid to Alanine in position 228 (E228A), a change previously associated with susceptibility to different cancer types and risk of metastases, suggesting a lack of functionality of TRAILR-1. In order to unravel how this amino acid change in TRAILR-1 would affect to death signal, we performed a molecular modelling with both alleles. Neither TRAIL binding sites in the receptor nor the expression levels of TRAILR-1 in peripheral blood mononuclear cell subsets (monocytes, CD4+ and CD8+ T cells) were modified, suggesting that this SNP may be altering the death signal by some other mechanism. These findings show a role for TRAILR-1 gene variations in the clinical outcome of IFN beta therapy that might have relevance as a biomarker to predict the response to IFN beta in MS.     

Effects of APOA5 ?1131T>C (rs662799) on Fasting Plasma Lipids and Risk of Metabolic Syndrome: Evidence from a Case-Control Study in China and a Meta-Analysis

The apolipoprotein A5 (APOA5) gene −1131T>C (rs662799) has been suggested to be involved in the pathway of lipid homeostasis and the development of metabolic syndrome (MetS). However, the findings are not consistent. To systematically evaluate the associations between −1131T>C polymorphism and fasting lipid parameters and the risk of MetS, we conducted a case-control study in a Chinese population and a meta-analysis. The findings from 1840 Chinese participants indicated that the C allele carriers had significantly higher fasting total cholesterol (TC), triglycerides (TG) and lower HDL-cholesterol (HDL-C) than the TT homozygotes carriers. The −1131C allele was also found to be significantly associated with increased risk of MetS (OR  =  1.40, 95% confidence interval (CI)  =  1.15, 1.69) compared to the TT homozygotes. In the meta-analysis of 51,868 participants from 46 East Asian studies, 26 European studies and 19 studies of other ethnic groups, the −1131C allele was associated with higher fasting TC (weighted mean difference (WMD)  =  0.08 mmol/L, 95% CI  =  0.05, 0.10, P = 1.74×10⁻⁹), TG (WMD  =  0.30 mmol/L, 95% CI  =  0.26, 0.33, P =  1.87×10⁻⁵⁵), LDL-cholesterol (LDL-C) (WMD  =  0.04 mmol/L, 95% CI  =  0.02, 0.07, P = 0.002), and lower HDL-C (WMD  =  −0.05 mmol/L, 95% CI  =  −0.06,−0.04, P = 1.88×10⁻²¹), respectively. Based on 12 studies with 5,573 MetS cases and 8,290 controls from 5 East Asian studies, 5 European studies and 2 studies of other ethnic groups, the −1131C allele was associated with increased risk of MetS with an OR (95% CI)  =  1.33 (1.16, 1.53) in the overall population, 1.43 (1.29, 1.58) in East Asian and 1.30 (0.94, 1.78) in European populations. In conclusion, the −1131C allele may be associated with elevated levels of fasting TG, TC, LDL-C and decreased HDL-C, and increased risk of MetS, especially in East Asians.     

Inhibition of the TRAIL Death Receptor by CMV Reveals Its Importance in NK Cell-Mediated Antiviral Defense

TNF-related apoptosis inducing ligand (TRAIL) death receptors (DR) regulate apoptosis and inflammation, but their role in antiviral defense is poorly understood. Cytomegaloviruses (CMV) encode many immune-modulatory genes that shape host immunity, and they utilize multiple strategies to target the TNF-family cytokines. Here we show that the m166 open reading frame (orf) of mouse CMV (MCMV) is strictly required to inhibit expression of TRAIL-DR in infected cells. An MCMV mutant lacking m166 expression (m166^stop) is severely compromised for replication in vivo, most notably in the liver, and depleting natural killer (NK) cells, or infecting TRAIL-DR^−/− mice, restored MCMV-m166^stop replication completely. These results highlight the critical importance for CMV to have evolved a strategy to inhibit TRAIL-DR signaling to thwart NK-mediated defenses.     

Oxidative Stress Induced Age Dependent Meibomian Gland Dysfunction in Cu,Zn-Superoxide Dismutase-1 (Sod1) Knockout Mice

Purpose

The purpose of our study was to investigate alterations in the meibomian gland (MG) in Cu, Zn-Superoxide Dismutase-1 knockout (Sod1 ^−/−) mouse.

Methods

Tear function tests [Break up time (BUT) and cotton thread] and ocular vital staining test were performed on Sod1 ^−/− male mice (n = 24) aged 10 and 50 weeks, and age and sex matched wild–type (+/+) mice (n = 25). Tear and serum samples were collected at sacrifice for inflammatory cytokine assays. MG specimens underwent Hematoxylin and Eosin staining, Mallory staining for fibrosis, Oil Red O lipid staining, TUNEL staining, immunohistochemistry stainings for 4HNE, 8-OHdG and CD45. Transmission electron microscopic examination (TEM) was also performed.

Results

Corneal vital staining scores in the Sod1 ^−/− mice were significantly higher compared with the wild type mice throughout the follow-up. Tear and serum IL-6 and TNF-α levels also showed significant elevations in the 10 to 50 week Sod1 ^−/− mice. Oil Red O staining showed an accumulation of large lipid droplets in the Sod1 ^−/− mice at 50 weeks. Immunohistochemistry revealed both increased TUNEL and oxidative stress marker stainings of the MG acinar epithelium in the Sod1 ^−/− mice compared to the wild type mice. Immunohistochemistry staining for CD45 showed increasing inflammatory cell infiltrates from 10 to 50 weeks in the Sod1 ^−/− mice compared to the wild type mice. TEM revealed prominent mitochondrial changes in 50 week Sod1 ^−/− mice.

Conclusions

Our results suggest that reactive oxygen species might play a vital role in the pathogensis of meibomian gland dysfunction. The Sod1 ^−/− mouse appears to be a promising model for the study of reactive oxygen species associated MG alterations.     

Genome-Wide Scan Identifies Variant in TNFSF13 Associated with Serum IgM in a Healthy Chinese Male Population

IgM provides a first line of defense during microbial infections. Serum IgM levels are detected routinely in clinical practice. And IgM is a genetically complex trait. We conducted a two-stage genome-wide association study (GWAS) to identify genetic variants affecting serum IgM levels in a Chinese population of 3495, including 1999 unrelated subjects in the first stage and 1496 independent individuals in the second stage. Our data show that a common single nucleotide polymorphism (SNP), rs11552708 located in the TNFSF13 gene was significantly associated with IgM levels (p = 5.00×10⁻⁷ in first stage, p = 1.34×10⁻³ in second stage, and p = 4.22×10⁻⁹ when combined). Besides, smoking was identified to be associated with IgM levels in both stages (P<0.05), but there was no significant interaction between smoking and the identified SNP (P>0.05). It is suggested that TNFSF13 may be a susceptibility gene affecting serum IgM levels in Chinese male population.