Simple in vitro generation of human leukocyte antigen-G-expressing T-regulatory cells through pharmacological hypomethylation for adoptive cellular immunotherapy against graft-versus-host disease.

BACKGROUND: Major barriers in using classical FOXP3+ regulatory T cells (Tregs) in clinical practice are their low numbers in the circulation, the lack of specific cell surface markers for efficient purification and the loss of expression of Treg signature molecules and suppressive function after in vitro expansion or in a pro-inflammatory microenviroment. A surface molecule with potent immunosuppressive function is the human leukocyte antigen-G (HLA-G), which is normally expressed in placenta protecting the "semi-allogeneic" fetus from maternal immune attack. Because HLA-G expression is strongly regulated by methylation, we asked whether hypomethylating agents (HA) may be used in vitro to induce HLA-G expression on conventional T cells and convert them to Tregs. METHODS: Human peripheral blood T cells were exposed to azacytidine/decitabine and analyzed for HLA-G expression and their in vitro suppressor properties. RESULTS: HA treatment induces de novo expression of HLA-G on T cells through hypomethylation of the HLA-G proximal promoter. The HA-induced CD4+HLA-Gpos T cells are FOXP3 negative and have potent in vitro suppression function, which is dependent to a large extent, but not exclusively, on the HLA-G molecule. Converted HLA-Gpos suppressors retain their suppressor function in the presence of tumor necrosis factor (TNF) and preserve hypomethylated the HLA-G promoter for at least 2 days after azacytidine exposure. Decitabine-treated T cells suppressed ex vivo the proliferation of T cells isolated from patients suffering from graft-versus-host disease (GVHD). DISCUSSION: We propose, in vitro generation of HLA-G-expressing T cells through pharmacological hypomethylation as a simple, Good Manufacturing Practice (GMP)-compatible and efficient strategy to produce a stable Treg subset of a defined phenotype that can be easily purified for adoptive immunotherapy.

Impact of estrogen receptor α gene and oxytocin receptor gene polymorphisms on female sexuality.

Over the past decades, research attention has increasingly been paid to the neurobiological component of sexual behavior. The aim of the present study was to investigate the correlation of estrogen receptor α (ERA) gene polymorphism (rs2234693-PvuII) (T→C substitution) and oxytocin receptor gene polymorphism (rs53576) (G→A substitution) with sexuality parameters of young, healthy women. One hundred thirty-three Greek heterosexual women, students in higher education institutions, 20-25 years of age, sexually active, with normal menstrual cycles (28-35 days), were recruited in the study. Exclusion criteria were chronic and/or major psychiatric diseases, use of oral contraceptive pills (OCs), polycystic ovary syndrome (PCOS), thyroid diseases as well as drugs that are implicated in hypothalamus-pituitary-gonadal axis. T allele (wildtype) of rs2234693 (PvuII) polymorphism of ERA gene was correlated with increased levels of arousal and lubrication, whereas A allele (polymorphic) of rs53576 (OXTR) polymorphism was correlated with increased arousal levels. The simultaneous presence of both T allele of rs2234693 (PvuII) and A allele of rs53576 (OXTR) polymorphisms (T + A group) was correlated with increased arousal, orgasm levels as well as female sexual function index full score. To our knowledge, this is the first study to investigate the interaction between ERA and OXTR with regard to sexual function in women. Female sexuality is a complex behavioral trait that encompasses both biological and psychological components. It seems that variability in female sexual response stems from genetic variability that characterizes endocrine, neurotransmitter and central nervous system influences.

Association of Calpain (CAPN) 10 (UCSNP-43, rs3792267) gene polymorphism with elevated serum androgens in young women with the most severe phenotype of polycystic ovary syndrome (PCOS).

OBJECTIVES: To highlight a possible association of Calpain (CAPN 10) gene UCSNP-43 polymorphism with hormonal and metabolic traits of young women with different phenotypes of polycystic ovary syndrome (PCOS). DESIGN: PCOS women were genotyped for the CAPN 10 gene UCSNP-43 polymorphism. A comparison of clinical and biochemical features of women with PCOS stratified on the basis of the CAPN 10 gene UCSNP-43 variants was assessed. METHODS: Anthropometric, hormonal and biochemical measurements were carried out in 668 PCOS women and 200 healthy controls. Subjects were also genotyped for the CAPN 10 gene UCSNP-43 polymorphism. The genotype frequency distributions between groups and controls were compared using the chi-square test. The association of the polymorphism with the clinical and biochemical features of the study cohort was estimated as well. RESULTS: No association of the frequency of CAPN 10 gene UCSNP-43 polymorphism with PCOS was detected. No association of the polymorphism with the anthropometric, biochemical and hormonal features was detected both in PCOS and control women. The polymorphism was associated with serum Δ4 androstenedione (p = 0.018), as well as with 17-OH progesterone (17-hydroxyprogesterone) among women with PCOS phenotype A (p = 0.012). CONCLUSIONS: CAPN 10 gene polymorphism UCSNP-43 is deprived of a metabolic contribution to cardiovascular disease (CVD). However, due to its association with androgen excess in phenotype A, CAPN 10 gene polymorphism UCSNP-43 could be used as a genetic marker for CVD in young PCOS women.

Complement C3, C2, and factor B gene polymorphisms and age-related macular degeneration in a Greek cohort study.

PURPOSE: To elucidate whether polymorphisms of C2, C3, and CFB genes are major genetic determinants of age-related macular degeneration (AMD) in a Greek population. METHODS: This was a case-control association study comprising 120 Greek patients with early and late-stage AMD and 140 independent controls of Caucasian origin. All participants were genotyped for rs547154, rs2230199, rs641153, and rs12614 polymorphisms by a combination of PCR and direct DNA sequencing assays. RESULTS: The frequency of the rs2230199 G allele (minor allele) was significantly higher in patients with AMD in comparison with controls (0.34 vs 0.22, p = 0.0031) and similar to the frequency of other reported populations. There was a significant difference in the frequencies of the rs2230199 genotypes among cases and controls (p = 0.0055). rs2230199 was found to be a significant predictor of advanced AMD status (odds ratio 6.41, confidence interval [CI] 2.72-15.09, p<0.0001; area under the curve 0.706, CI 0.61-0.78, p<0.0001]). For the other single nucleotide polymorphism (SNP) loci, the allele and genotype frequencies did not reach statistical significance. The minor allele frequencies in controls and cases were similar and still much lower than the frequencies reported in other populations. CONCLUSIONS: The rs547154, rs641153, and rs12614 SNPs were not associated with AMD development in Greek patients. However, this finding should be viewed with caution as the particular polymorphisms presented with very low frequencies in the Greek population. Finally, the replication of the reported associations of C3 with AMD suggests that the presence of the C3 G allele could serve as a high-risk genetic marker for the development of AMD and the progression of the disease to the advanced clinical stage.

Increased frequency of the anti-mullerian-inhibiting hormone receptor 2 (AMHR2) 482 A>G polymorphism in women with polycystic ovary syndrome: relationship to luteinizing hormone levels.

CONTEXT: The polycystic ovary syndrome (PCOS) is a common and complex disease without a clear pattern of inheritance. Anti-Müllerian hormone (AMH) has an inhibitory effect on FSH-stimulated follicle growth. Serum AMH levels are higher in women with PCOS than in normo-ovulatory women. The elevated AMH levels may reflect abnormalities in AMH signaling. OBJECTIVE: The purpose of this study was to evaluate the association of the anti-Müllerian hormone receptor 2 (AMHR2) -482 A>G polymorphism (rs2002555) with the pathophysiology of PCOS. DESIGN: AMHR2 -482 A>G polymorphism genotyping were performed in a large cohort of women with PCOS and in a healthy control group. SETTING/SUBJECTS: A total of 858 Caucasian Greek women with PCOS and 309 healthy control women were studied. INTERVENTIONS: Genotyping and hormonal measurements were preformed. MAIN OUTCOME MEASURES: Hormone levels in women with PCOS were analyzed. RESULTS: The AMHR2 polymorphism was more common in women with PCOS than in control women (P = .026). Homozygous AMHR2 -482 A>G gene polymorphisms (GG) were associated with decreased levels of LH (P = .003) and lower LH to FSH ratios (P = .01) in women with PCOS, as well as with lower prolactin levels (P = .004). No other associations related to AMHR2 -482 A>G polymorphisms were observed in women with PCOS or control women. CONCLUSION: In this study, the role of the AMHR2 -482 A>G gene polymorphism in the pathogenesis of PCOS was suggested by the association of the variant with PCOS risk. Thus, further research is needed to elucidate a possible association of the AMHR2 -482 A>G gene polymorphism with AMH signaling and impaired ovarian function and its clinical significance in women with PCOS.

Association of estrogen receptor alpha (ERα) gene polymorphisms with endometrial thickness and lipid profile in women with breast cancer treated with aromatase inhibitors.

Aromatase inhibitors (AIs) provide an alternative to tamoxifen as an adjuvant therapy for post-menopausal, hormone-receptor positive breast cancer patients. The aim of the present study was to evaluate the effect of PvuII and XbaI polymorphisms of the ERα gene at ΑΙs treatment's adverse effects in post-menopausal women with breast cancer. The study included 87 post-menopausal women with ER-positive breast cancer treated with AIs and 80 healthy controls. The overall presence of ERα polymorphisms in all women with breast cancer was not different from the healthy controls. Endometrial thickness under AIs treatment was reduced from (mean value ± SD) 6,404 ± 2,901 mm to 3,666 ± 1,4656 mm. Moreover, the AA XbaI genotype was associated with greater reduction in endometrial thickness during therapy with AIs (p = 0.005). The presence of the CC PvuII and the AA XbaI genotypes were associated with elevated LDL levels and elevated triglycerides. In conclusion, the results of the present study showed that the genotype of women with breast cancer under AIs treatment might influence treatment's adverse effects, as, the presence of the CC PvuII and the AA XbaI genotypes of the ERα were associated with elevated LDL and triglycerides serum levels, while the AA XbaI genotype was associated with a greater reduction in endometrial thickness.

No association of the G972S polymorphism of the insulin receptor substrate-1 gene with polycystic ovary syndrome in lean PCOS women with biochemical hyperandrogenemia.

PURPOSE: The aim of the present study was to determine the prevalence and association of the G972S polymorphism of the insulin receptor substrate-1 gene (IRS-1 G972S SNP) with polycystic ovary syndrome (PCOS) and insulin resistance-related traits in a distinct phenotypic group of lean PCOS women with biochemical hyperandrogenemia, excluding obesity, which is considered to be an aggravating parameter of insulin resistance. METHODS: The study included 162 women with PCOS and 122 regularly menstruating, ovulatory women as controls. Physical measurements included weight, height, fat-free mass, fat mass, systolic and diastolic blood pressure and resting heart rate. Biochemical parameters included the serum testosterone, free testosterone, androstenedione, total cholesterol, triglycerides, HDL and LDL cholesterol and glucose levels. Insulin resistance was assessed by determining fasting insulin levels, fasting glucose levels, the fasting glucose/insulin ratio, as well as the HOMA and QUICKI indexes. All DNA samples were genotyped by a PCR-restriction fragment length polymorphism (RLFP) assay. RESULTS: No association of the genotype frequencies of the G972S polymorphism in insulin receptor substrate-1 gene (IRS-1 G972S SNP) with PCOS phenotype and insulin resistance was detected. CONCLUSION: The G972S polymorphism of the IRS-1 gene should not be viewed as major contributor to the development of PCOS or as a causative variant for insulin resistance.

Complement factor H and LOC387715 gene polymorphisms in a Greek population with age-related macular degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of severe visual loss among people over 60 years old. The lack of a broadly effective treatment for AMD underscores the need to identify causative biomarkers that could serve as preventive targets. Thus far, two major susceptibility loci for AMD have been identified, CFH T1277C and LOC387715 G270T. The primary goal of the present study was to elucidate whether these polymorphisms are major genetic determinants of AMD in a Greek population. PATIENTS AND METHODS: A clinic-based, case-control association study was conducted, comprising 100 Greek patients with early and late-stage AMD and 115 independent controls of Caucasian origin. All participants underwent clinical examination including best-corrected visual acuity, intraocular pressure, and dilated fundus examination. Moreover, they were genotyped for CFH T1277C and LOC387715 G270T polymorphisms, by direct sequencing and ARMS PCR, respectively. RESULTS: The frequency of the CFH 1277C allele was significantly higher in AMD patients in comparison with controls while the odds ratios (ORs) for AMD were 4.4-5.5. Statistical comparison of early and advanced AMD patients, on the basis of CFH genotype, revealed that the CFH 1277C allele was associated with both subgroups when compared with the controls (P < 0.001). When statistical comparison was performed between early and advanced patients on the basis of CFH genotypic frequencies, the CC genotype was found to be more prevalent in advanced AMD patients (P = 0.008, OR = 2.3). The frequency of the LOC387715 270 T allele was higher in AMD patients in comparison with controls (P < 0.04) while the ORs for AMD were 1.4-2. No statistically significant differences were located between the early AMD patients and controls, on the basis of LOC387715 genotype (P = 0.189). On the contrary, the T270G polymorphism was associated with advanced AMD (P = 0.04). Moreover, the TT genotype was more prevalent in patients with advanced AMD (P = 0.011, OR = 1.7) when compared with early AMD patients. Assessment of the combined contribution of CFH T1277C and LOC387715 G270T SNPs showed an independent manner of action of these polymorphisms in the development of the disease. CONCLUSIONS: The replication of the reported associations of CFH T1277C polymorphism with AMD suggest that the 1277C allele could serve as a high-risk genetic marker for the development of AMD and the progression of the disease to the advanced clinical stage in the Greek population.

Association of the Pro12Ala polymorphism in peroxisome proliferator-activated receptor gamma2 with decreased basic metabolic rate in women with polycystic ovary syndrome.

OBJECTIVE: The peroxisome proliferator-activated receptor (PPAR)gamma is a transcription factor involved in glucose homeostasis and energy metabolism. A missense mutation at codon 12 in the PPARgamma2 has been associated with increased body mass index (BMI) and attenuated insulin resistance (IR) in polycystic ovary syndrome (PCOS). We have recently shown a decreased basic metabolic rate (BMR) in PCOS. The aim of the present study is to determine the prevalence of the Pro12Ala polymorphism of the PPARgamma2 gene and its associations with indices of IR and BMR in lean and slightly overweight PCOS women. DESIGN: Case-control association study involving 156 PCOS women with biochemical hyperandrogenism, chronic anovulation and polycystic ovarian morphology in ultrasound and 56 unrelated healthy controls. METHODS: Hormonal determinations were performed by electrochemiluminescence quantitation or RIA. BMR was measured by indirect calorimetry. All subjects were genotyped by a PCR-restriction fragment length polymorphism assay. RESULTS: Genotype frequencies of the Pro12Ala polymorphism in PPARgamma2 did not differ among PCOS women and control subjects. The presence of Pro12Ala polymorphism of PPARgamma2 was associated with lower BMR (P=0.04). This finding was valid in our subgroup of lean PCOS (BMI<25 kg/m(2)), in which the Ala variant was also associated with higher total testosterone values. CONCLUSION: The Pro12Ala polymorphism in the PPARgamma2 gene is associated with decreased BMR in women with PCOS and biochemical hyperandrogenemia. These young women are therefore at risk to increase their body weight and should restrict their energy intake by diet and enhance their energy expenditure by exercise.

Cloning and characterization of the trout perforin.

The pore-forming protein, perforin is one of the effectors of cell-mediated killing. A perforin cDNA clone was isolated from rainbow trout (Oncorhynchus mykiss) after screening of a spleen cDNA library. The full-length cDNA is 2070 bp in size, encoding for a polypeptide of 589 amino acids. The predicted amino acid sequence of the trout perforin is 64, 58 and 40% identical to those of Japanese flounder, zebrafish and human perforins, respectively. Although its membrane attack complex/perforin (MACPF) domain is conserved, trout perforin shows low homology to human and trout terminal complement components (C6, C7, C8 and C9), ranging from 19 to 26% identity. Expression analysis reveals that the trout perforin gene is expressed in the blood, brain, heart, kidney, intestine and spleen. Phylogenetic analysis of proteins which belong to the MACPF superfamily clusters the trout perforin in the same group with other known perforins.

CR3 complement receptor: cloning and characterization in rainbow trout.

The beta 2 integrin CR3 is a leukocyte adhesion heterodimeric glycoprotein which functions both as receptor for iC3b and in several cell-cell and cell-substrate adhesion interactions. In order to elucidate the molecular evolution of the CR3 receptor, here we report the cloning and characterization of its beta2 (CD18) and aM (CD11b) subunits in rainbow trout (Oncorhynchus mykiss). The predicted polypeptide sequences of trout CD18 and CD11b-like exhibit 50, 49, or 61% and 25, 25, or 30% identity with human, mouse, and zebrafish orthologs, respectively. The 'domain' architecture of trout CD18 and CD11b-like subunits retains several characteristics of the mammalian ortholog proteins, such as cysteine-rich regions, N-linked glycosylation sites and several proposed domains and signal sequences (von Willebrand factor type A, Integrin alpha, Integrin B tail, EGF, and Transmembrane domain). The tissue expression profiles of trout CR3 subunits diverge from those of mammalian counterparts, showing the kidney as the main source of the trout CD18 and CD11b-like mRNA transcripts. This is the first report of cloning and characterization of the CR3 receptor in low vertebrates.

Association of the 17-hydroxysteroid dehydrogenase type 5 gene polymorphism (-71A/G HSD17B5 SNP) with hyperandrogenemia in polycystic ovary syndrome (PCOS).

OBJECTIVE: To evaluate the association of an activating single-nucleotide polymorphism (SNP) at position -71 of the promoter of 17beta-hydroxysteroid dehydrogenase type 5 gene (-71A/G HSD17B5 SNP) and polycystic ovary syndrome (PCOS) in a well characterized cohort of caucasian PCOS women with biochemical hyperandrogenemia. DESIGN: The PCOS patients and unrelated healthy control subjects were genotyped for the -71A/G HSD17B5 SNP. The acquired genotypic data was tested for association with PCOS and other quantitative phenotypic traits of the syndrome in PCOS patients. SETTING: Subjects were recruited from the Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, at the University Hospital of Patras, Greece. Genotyping and biochemical determinations took place at the Laboratory of Molecular Endrocinology, University of Patras Medical School, Rion, Greece. PATIENT(S): Participants comprised 150 caucasian Greek PCOS women with biochemical hyperandrogenism and chronic anovulation and polycystic ovarian morphology on ultrasound and 51 healthy control subjects. MAIN OUTCOME MEASURE(S): HSD17B5 genotype, serum testosterone, serum androstenedione. RESULT(S): No association of the -71A/G HSD17B5 SNP with PCOS was detected. However, the -71G HSD17B5 variant was associated with increased serum testosterone levels and decreased androstenedione/testosterone ratio. CONCLUSION(S): The -71G HSD17B5 variant is not a major component of the molecular pathogenetic mechanisms of PCOS, although it might contribute to the severity of hyperandrogenemia in women with PCOS and biochemical hyperandrogenism.

The vitronectin gene in rainbow trout: cloning, expression and phylogenetic analysis.

Vitronectin is a major cell adhesion glycoprotein that is found in plasma and the extracellular matrix. Vitronectin consists of an N-terminal somatomedin B domain and two hemopexin-like domains and controls functions including cell adhesion, migration, haemostasis and immune defence. In order to study the molecular evolution of the complement lytic pathway regulation, we have cloned and characterized the vitronectin gene from rainbow trout (Oncorhynchus mykiss). The deduced amino acid sequence of trout vitronectin exhibits 45%, 46%, 47% and 63% identity with human, chicken, Xenopus and zebrafish orthologs, respectively. The domain architecture of the trout vitronectin, consisting of a somatomedin B domain and two hemopexin-like domains, resembles that of mammalian vitronectins. Analysis of partial genomic clones shows that trout vitronectin gene exhibits the same exon-intron organization profile as the human ortholog gene. The trout vitronectin gene is probably present as a single copy in the trout genome, showing a differential expression pattern among tissues investigated.