Serum soluble interleukin-2 (IL-2) receptor levels in women with breast carcinoma and its correlation with IL-2 receptor expression on blood lymphocytes and lymphocytic infiltration within the tumour

Summary This study describes serum levels of soluble interleukin-2 receptor (sIL-2R) in 13 healthy women, 10 women with breast dysplasia and 37 patients with breast carcinoma. A difference was found between sIL-2R levels in normal women and cancer patients. sIL-2R increased with the advance in stage of cancer but the extent of increase fell from stage III to stage IV as compared to the change from stage II to stage III. Of the 15 patients who were followed after surgery and/or therapy, 10 (67%) showed a fall in the serum sIL-2R levels. A negative correlation was found between sIL-2R levels and lymphocytic infiltration only within the malignant tissue. These findings probably indicate that sIL-2R exerts an immunomodulatory effect on blood lymphocytes by preventing their infiltration into the tumour tissue. To our knowledge, this is the first report of its kind in immunology of breast carcinoma.     

Enhancement of lipid peroxidation and its amelioration by vitamin E in a subject with mutations in the SBP2 gene

Selenocysteine (Sec) insertion sequence-binding protein 2 (SBP2) is essential for the biosynthesis of Sec-containing proteins, termed selenoproteins. Subjects with mutations in the SBP2 gene have decreased levels of several selenoproteins, resulting in a complex phenotype. Selenoproteins play a significant role in antioxidative defense, and deficiencies in these proteins can lead to increased oxidative stress. However, lipid peroxidation and the effects of antioxidants in subjects with SBP2 gene mutations have not been studied. In the present study, we evaluated the lipid peroxidation products in the blood of a subject (the proband) with mutations in the SBP2 gene. We found that the proband had higher levels of free radical-mediated lipid peroxidation products, such as 7β-hydroxycholesterol, than the control subjects. Treatment of the proband with vitamin E (α-tocopherol acetate, 100 mg/day), a lipid-soluble antioxidant, for 2 years reduced lipid peroxidation product levels to those of control subjects. Withdrawal of vitamin E treatment for 7 months resulted in an increase in lipid peroxidation products. Collectively, these results clearly indicate that free radical-mediated oxidative stress is increased in the subject with SBP2 gene mutations and that vitamin E treatment effectively inhibits the generation of lipid peroxidation products.     

Apolipoprotein B gene variants are involved in the determination of blood glucose and lipid levels in patients with non-insulin dependent diabetes mellitus

We have examined the frequency of the EcoRI, XbaI and MspI RFLPs of the apolipoprotein B (apo B) gene in 110 type 2 diabetic patients and 91 healthy control subjects in order to ascertain whether variation in this gene may influence the development of non-insulin dependent diabetes mellitus (type 2 diabetes). Serum lipids including total-cholesterol (T-Chol), triacylglycerol (TAG), apolipoprotein E (apo E), apolipoprotein AI (apo AI), apolipoprotein B and lipoprotein (a) (Lp(a)) were analysed. Genomic DNA was extracted and the apo B polymorphic regions amplified by the polymerase chain reaction. Regions carrying EcoRI, XbaI, and MspI restriction sites present in the apo B gene were amplified and digested separately by the respective enzymes. No significant difference for genotypic frequencies was observed for the EcoRI, XbaI and MspI restriction sites in type 2 diabetic patients as compared to controls. Type 2 diabetic patients and controls with EcoRI +/+ and XbaI +/+ genotypes had higher apo E levels. The MspI +/+ genotype is more frequent in the patient and control groups with elevated T-Chol. Furthermore, the EcoRI -/-, XbaI -/-, and MspI +/+ genotypes were found to be significantly more frequent in type 2 diabetic patients with higher blood glucose levels. This study identifies the apo B gene polymorphisms in modulating plasma lipid/lipoprotein and glucose levels in patients with type 2 diabetes.     

Ochratoxin A levels in blood serum of Czech women in the first trimester of pregnancy and its correspondence with dietary intake of the mycotoxin contaminant

Abstract

The mycotoxin ochratoxin A (OTA) can elicit a wide range of toxic properties including embryotoxicity and teratogenicity. OTA crosses the placenta at early gestation rather than in late gestation, maternal OTA exposure may represent a risk for the developing fetus. The study focuses on the assessment of OTA intake of pregnant women (aged 19–40 years) in the first trimester of pregnancy by means OTA levels in 100 blood serum samples by high-performance liquid chromotography with fluorescence detection (HPLC-FD) method and comparison with dietary OTA exposure in pregnant women. Of all, 96% tested serum samples were positive with values ranging from 0.1 to 0.35?µg/l with a mean value of 0.15?µg/l.     

二甲双胍对老年2型糖尿病患者肠道菌群及血糖血脂的影响

目的 探讨二甲双胍对老年2型糖尿病患者肠道菌群、血糖血脂及炎性因子水平的影响。 方法 选取2018年3月至2019年9月我院收治的120例2型糖尿病患者为研究对象,随机分为观察组和对照组,各60例。两组患者均给予常规胰岛素治疗,观察组在此基础上加用二甲双胍治疗。观察两组患者治疗前后肠道菌群变化,胰岛功能水平[空腹胰岛素(FINS)、餐后2 h胰岛素(2hINS)、空腹C肽(FCP)、餐后2 h C肽(2hCP)、胰岛素抵抗指数(HOMA IR)],血糖血脂水平[糖化血红蛋白(HbAlc)、空腹血糖(FPG)、总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL C)、高密度脂蛋白胆固醇(HDL C)]及炎性因子水平[白细胞介素6(IL 6)、C反应蛋白(CRP)、肿瘤坏死因子α(TNF α)]。 结果 治疗前,两组患者肠道菌群数量比较差异无统计学意义(均P>0.05)。治疗后,对照组患者肠道菌群数量与治疗前比较差异无统计学意义(均P>0.05),而观察组患者肠道双歧杆菌、乳杆菌、拟杆菌数量较治疗前和对照组均显著增加,肠球菌、肠杆菌及酵母菌数量显著降低(均P0.05)。治疗后,两组患者FINS、2hINS、FCP、2hCP水平明显升高,且观察组高于对照组(均Plc、FPG、TG、LDL C、CRP、IL 6和TNF α水平显著降低,且观察组低于对照组(均P结论 二甲双胍可明显增加患者肠道有益菌群数量,调节菌群失衡,控制血糖血脂代谢水平,改善炎症状态。     

Genetic association of ADIPOQ gene variants with type 2 diabetes,obesity and serum adiponectin levels in south Indian population

Objective

To investigate the genetic association of eight variants of the adiponectin gene with type 2 diabetes mellitus (T2DM), obesity and serum adiponectin level in the south Indian population.

Methods

The study comprised of 1100 normal glucose tolerant (NGT) and 1100 type 2 diabetic, unrelated subjects randomly selected from the Chennai Urban Rural Epidemiology Study (CURES), in southern India. Fasting serum adiponectin levels were measured by radioimmunoassay. The variants were screened by polymerase chain reaction-restriction fragment length polymorphism. Linkage disequilibrium was estimated from the estimates of haplotype frequencies.

Results

Of the 8 variants, four SNPs namely, + 276 G/T (rs1501299), − 4522 C/T (rs822393), − 11365 C/G (rs266729), and + 712 G/A (rs3774261) were significantly associated with T2DM in our study population. The −3971 A/G (rs822396) and − 11391 G/A (rs17300539) SNPs' association with T2DM diabetes was mediated through obesity (where the association with type 2 diabetes was lost after adjusting for BMI). There was an independent association of + 276 G/T (rs1501299) and − 3971 A/G (rs822396) SNPs with generalized obesity and + 349 A/G (rs2241767) with central obesity. Four SNPs, −3971 A/G (rs822396), + 276 G/T (rs1501299), − 4522 C/T (rs822393) and Y111H T/C (rs17366743) were significantly associated with hypoadiponectinemia. The haplotypes GCCATGAAT and AGCGTGGGT conferred lower risk of T2DM in this south Indian population.

Conclusion

The adiponectin gene variants and haplotype contribute to the genetic risk towards the development of type 2 diabetes, obesity and hypoadiponectinemia in the south Indian population.     

Induction of lacI mutations in Big Blue Rat-2 cells treated with 1-(2-hydroxyethyl)-1-nitrosourea: a model system for the analysis of mutagenic potential of the hydroxyethyl adducts produced by 1,3-bis (2-chloroethyl)-1-nitrosourea.

We have investigated the genotoxic effects of 1-(2-hydroxyethyl)-1-nitrosourea (HENU). We have chosen this agent because of its demonstrated ability to produce N7-(2-hydroxyethyl) guanine (N7-HOEtG) and O⁶-(2-hydroxyethyl) 2′-deoxyguanosine (O⁶-HOEtdG); two of the DNA alkylation products produced by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). For these studies, we have used the Big Blue Rat-2 cell line that contains a lambda/lacI shuttle vector. Treatment of these cells with HENU produced a dose dependent increase in the levels of N7-HOEtG and O⁶-HOEtdG as quantified by HPLC with electrochemical detection. Treatment of Big Blue Rat-2 cells with either 0, 1 or 5 mM HENU resulted in mutation frequencies of 7.2±2.2×10⁻⁵, 45.2±2.9×10⁻⁵ and 120.3±24.4×10⁻⁵, respectively. Comparison of the mutation frequencies demonstrates that 1 and 5 mM HENU treatments have increased the mutation frequency by 6- and 16-fold, respectively. This increase in mutation frequency was statistically significant (P<0.001). Sequence analysis of HENU-induced mutations have revealed primarily G:C→A:T transitions (52%) and a significant number of A:T→T:A transversions (16%). We propose that the observed G:C→A:T transitions are produced by the DNA alkylation product O⁶-HOEtdG. These results suggest that the formation of O⁶-HOEtdG by BCNU treatment contributes to its observed mutagenic properties.     

双歧杆菌三联活菌胶囊辅助治疗对2型糖尿病患者血糖波动及脂质过氧化损伤的影响

目的探讨双歧杆菌三联活菌胶囊辅助治疗对2型糖尿病(T2DM)患者血糖波动及脂质过氧化损伤的影响,为该类患者的治疗提供参考。方法选取2019年1月至8月我院内科门诊治疗的84例T2DM患者,随机分为观察组和对照组各42例。两组患者均给予饮食控制和适当运动锻炼等基础治疗。对照组患者给予甘精胰岛素与阿卡波糖控制血糖。观察组患者在对照组基础加用双歧杆菌三联活菌胶囊630 mg/次,2次/d,温开水服用。两组患者连用12周。观察两组患者治疗前后血糖波动指标[日平均血糖波动幅度(MAGE)、血糖标准差(SDBG)和最大血糖波动幅度(LAGE)]及脂质过氧化指标[谷胱甘肽过氧化物酶(GSH-PX)、脂质过氧化氢(LHP)、活性氧类物质(ROS)和总抗氧化能力(T-AOC)]水平的变化。结果治疗12周后,两组患者血清MAGE、SDBG和LAGE水平较治疗前明显下降,且观察组下降幅度大于对照组(均P0.05);同时两组患者血清GSH-PX和T-AOC水平较治疗前显著上升,血清LHP和ROS水平均较治疗前显著下降,且观察组变化幅度大于对照组(均P0.05)。结论双歧杆菌三联活菌胶囊辅助治疗T2DM患者可改善其糖代谢指标水平,减少血糖波动程度,机制可能与其能减轻脂质过氧化损伤相关。     

Variations in HDL-carried miR-223 and miR-135a concentrations after consumption of dietary trans fat are associated with changes in blood lipid and inflammatory markers in healthy men - an exploratory study

A high consumption of trans fatty acids (TFAs) is associated with an increased risk of cardiovascular diseases (CVDs). High-density lipoproteins (HDLs) have many cardioprotective properties and transport functional microRNAs (miRNAs) to recipient cells. We hypothesized that dietary TFAs modify the HDL-carried miRNA profile, therefore modulating its cardioprotective properties. We assessed whether consumption of dietary TFAs modifies HDL-carried miR-223-3p and miR-135a-3p concentration and the inter-relationship between diet-induced changes in HDL-carried miRNA concentration and CVD risk markers. In a double blind, randomized, crossover, controlled study, 9 men were fed each of 3 experimental isoenergetic diets: 1) High in industrial TFA (iTFA; 3.7% energy); 2) High in TFA from ruminants (rTFA; 3.7% energy); 3) Low in TFA (control; 0.8% energy) for 4 weeks each. HDLs were isolated by ultracentrifugation and miRNAs were quantified by RT-qPCR. Variations in HDL-miR-223-3p concentration were negatively correlated with variations in HDL-cholesterol after the iTFA diet (r_s = 0.82; P = 0.007), and positively correlated with variations in C-reactive protein concentration after the rTFA diet (r_s = 0.75; P = 0.020). Variations in HDL-miR-135a-3p concentration were positively correlated with variations in total triglyceride (TG) concentration following the iTFA diet (r_s = ?0.82; P = 0.007), and with variations in low-density lipoprotein (LDL)-TG concentration following the rTFA diet (r_s = 0.83; P = 0.005), compared to the control diet. However, the consumption of dietary TFAs has no significant unidirectional impact on HDL-carried miR-223-3p and miR-135a-3p concentrations. Our results suggest that the variability in the HDL-carried miRNAs response to TFA intake, by being associated with variations in CVD risk factors, might reflect physiological changes in HDL functions.     

Determination of 6,4′-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyridine in plasma and whole blood by high-performance liquid chromatography

A selective and sensitive HPLC assay for the quantitative determination of a new antifilarial drug, 6,4′-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyridine (CDR 101) is described. After extraction from plasma and blood, CDR 101 was analysed using a C₁₈ Nucleosil ODS column (250×4.6 mm, 5 μm particle size) and mobile phase of acetonitrile-0.05 M ammonium acetate adjusted to pH 3.0, with UV detection at 318 nm. The mean recoveries of CDR 101 in plasma and blood over a concentration range of 25–500 ng/ml were 95.5±2.01% and 83.3±1.87%, respectively. The within-day and day-to-day coefficient of variations for plasma were 3.23-6.21% and 2.59-9.90%, respectively, those for blood were 2.59-5.92% and 2.89-6.82%, respectively. The minimum detectable concentration for CDR 101 was 1 ng/ml in plasma and 2.5 ng/ml in whole blood. This method was found to be suitable for clinical pharmacokinetic studies.     

Enhanced pathogenicity and transmissibility of H9N2 avian influenza virus in mammals by hemagglutinin mutations combined with PB2-627K

H9N2 avian influenza viruses (AIVs) circulate globally in poultry and have become the dominant AIV subtype in China in recent years. Previously, we demonstrated that the H9N2 virus (A/chicken/Eastern China/SDKD1/2015) naturally harbors a mammalian-adaptive molecular factor (627K) in the PB2 protein and is weakly pathogenic in mice. Here, we focused on new markers for virulence in mammals. A mouse-adapted H9N2 virus was serially passaged in mice by infecting their lungs. As expected, infected mice showed clinical symptoms and died at passage six. A comparison between the wild-type and mouse-adapted virus sequences identified amino acid substitutions in the hemagglutinin (HA) protein. H9N2 viruses with the T187P ?+ ?M227L double mutation exhibited an increased affinity to human-type (SAα2,6Gal) receptors and significantly enhanced viral attachment to mouse lung tissues, which contributed to enhancing viral replication and virulence in mice. Additionally, HA with the T187P ?+ ?M227L mutation enabled H9N2 viral transmission in guinea pigs via direct contact. AIV pathogenicity in mice is a polygenic trait. Our results demonstrated that these HA mutations might be combined with PB2-627K to significantly increase H9N2 virulence in mice, and this enhanced virulence was achieved in other H9N2 AIVs by generating the same combination of mutations. In summary, our study identified novel key elements in the HA protein that are required for H9N2 pathogenicity in mice and provided valuable insights into pandemic preparedness against emerging H9N2 strains.     

Effects of dietary supplementation of DL-methionine or 2-hydroxy-4-(methylthio)-butanoic acid on food intake,nutrient digestibility,nitrogen balance,and urinary and blood metabolites in healthy,growing dogs

Abstract

The aim of this study was to evaluate effects on nutritional responses of supplemental DL-methionine and 2-hydroxy-4-(methylthio) butanoic acid (HMTBA) in a commercial-type diet in growing dogs. A nitrogen balance study was conducted as a randomized complete block design using 30 Pointer puppies (72-d-old; 5.5 kg). A corn and poultry byproduct meal based diet was supplemented with 0.1 or 0.2% DL-methionine or HMTBA on an equimolar basis. Organic matter and gross energy tended (p < 0.10) to be less digestible by dogs fed the 0.1% HMTBA diet compared with the 0.2% DL-methionine diet, but other nutrients were unaffected. Postprandial urinary calcium tended (p < 0.10) to be lower for the basal and HMTBA treatments. Fecal ammonia tended (p < 0.10) to be lower for the 0.1% HMTBA diet than for the 0.2% DL-methionine diet. At the levels tested, DL-methionine and HMTBA appear to act similarly when included in a corn and poultry by-product meal diet fed to young dogs.     

Measurement of both protein-bound and total S-2-(3-aminopropylamino)ethanethiol (WR-1065) in blood by high-performance liquid chromatography

A high-performance liquid chromatographic method for automated analysis of both protein-bound and total S-2-(3-aminopropylamino)ethanethiol (WR-1065) in blood has been developed in our laboratory. WR-1065 is the active thiol metabolite of the radio- and chemo-protector drug amifostine (WR-2721). Using WR-1065 quality control levels over the experimental range: 7.0, 45.0 and 85.0 μmol/l spiked into plasma, method validation for total WR-1065 included between-run assessment of imprecision (SD/C.V.%: 1.11/16.7%, 6.58/15.5% and 9.24/11.3%, respectively) and % accuracy (94.7, 106.0 and 97.2%).     

Elevated urinary levels of 8-oxo-2′-deoxyguanosine, (5′R)- and (5′S)-8,5′-cyclo-2′-deoxyadenosines,and 8-iso-prostaglandin F2α as potential biomarkers of oxidative stress in patients with prediabetes

Prediabetes is the preclinical stage of type 2 diabetes mellitus (T2DM) with intermediate state of hyperglycemia. Hyperglycemia results in a state of oxidative stress, which may contribute to the production of insulin resistance, β-cell dysfunction and long-term complications of diabetes. Novel approaches are required for prevention and treatment of diabetes. New biomarkers that can be used in risk stratification and therapy control as supplementary to current parameters are needed. These biomarkers may facilitate a more individualized and sufficient treatment of diabetes. Therefore, the aim of this study was to investigate the levels of oxidatively induced DNA damage products, 8-oxo-2′-deoxyguanosine (8-oxo-dG) (also known as 8-OH-dG), (5′R)- and (5′S)-8,5′-cyclo-2′-deoxyadenosines (R-cdA and S-cdA), and the lipid peroxidation product 8-iso-prostaglandin F_2α (8-iso-PGF_2α) as reliable oxidative stress markers in patients with prediabetes or T2DM in comparison with healthy volunteers. Urine samples were collected from these subjects. Absolute quantification of 8-oxo-dG, R-cdA, S-cdA and 8-iso-PGF_2α was achieved by liquid chromatography-isotope dilution tandem mass spectrometry. The levels of 8-oxo-dG, S-cdA and 8-iso-PGF_2α were significantly greater in prediabetes patients than those in healthy volunteers. T2DM patients also had higher levels of 8-oxo-dG than healthy volunteers. No statistically significant difference was observed for R-cdA levels. 8-Oxo-dG levels positively correlated with R-cdA and S-cdA levels for prediabetes and newly diagnosed T2DM. S-cdA levels and HbA1c were found negatively correlated in prediabetes patients. Also 8-iso-PGF_2α levels and HbA1c were found negatively correlated in prediabetes patients. These results indicate that oxidatively induced macromolecular damage appears before the establishment of T2DM. Thus, our data suggest that oxidatively induced DNA damage and lipid peroxidation products that were found to be elevated in prediabetic stage may be used as early disease markers in patients at risk for T2DM.     

Ubiquilin-2 (UBQLN2) binds with high affinity to the C-terminal region of TDP-43 and modulates TDP-43 levels in H4 cells: Characterization of inhibition by nucleic acids and 4-aminoquinolines

Recently, it was reported that mutations in the ubiquitin-like protein ubiquilin-2 (UBQLN2) are associated with X-linked amyotrophic lateral sclerosis (ALS), and that both wild-type and mutant UBQLN2 can co-localize with aggregates of C-terminal fragments of TAR DNA binding protein (TDP-43). Here, we describe a high affinity interaction between UBQLN2 and TDP-43 and demonstrate that overexpression of both UBQLN2 and TDP-43 reduces levels of both exogenous and endogenous TDP-43 in human H4 cells. UBQLN2 bound with high affinity to both full length TDP-43 and a C-terminal TDP-43 fragment (261–414 aa) with K_D values of 6.2 nM and 8.7 nM, respectively. Both DNA oligonucleotides and 4-aminoquinolines, which bind to TDP-43, also inhibited UBQLN2 binding to TDP-43 with similar rank order affinities compared to inhibition of oligonucleotide binding to TDP-43. Inhibitor characterization experiments demonstrated that the DNA oligonucleotides noncompetitively inhibited UBQLN2 binding to TDP-43, which is consistent with UBQLN2 binding to the C-terminal region of TDP-43. Interestingly, the 4-aminoquinolines were competitive inhibitors of UBQLN2 binding to TDP-43, suggesting that these compounds also bind to the C-terminal region of TDP-43. In support of the biochemical data, co-immunoprecipitation experiments demonstrated that both TDP-43 and UBQLN2 interact in human neuroglioma H4 cells. Finally, overexpression of UBQLN2 in the presence of overexpressed full length TDP-43 or C-terminal TDP-43 (170–414) dramatically lowered levels of both full length TDP-43 and C-terminal TDP-43 fragments (CTFs). Consequently, these data suggest that UBQLN2 enhances the clearance of TDP-43 and TDP-43 CTFs and therefore may play a role in the development of TDP-43 associated neurotoxicity.