Host genetic and epigenetic factors in toxoplasmosis

Analysing human genetic variation provides a powerful tool in understanding risk factors for disease. Toxoplasma gondii acquired by the mother can be transmitted to the fetus. Infants with the most severe clinical signs in brain and eye are those infected early in pregnancy when fetal immunity is least well developed. Genetic analysis could provide unique insight into events in utero that are otherwise difficult to determine. We tested the hypothesis that propensity for T. gondii to cause eye disease is associated with genes previously implicated in congenital or juvenile onset ocular disease. Using mother-child pairs from Europe (EMSCOT) and child/parent trios from North America (NCCCTS), we demonstrated that ocular and brain disease in congenital toxoplasmosis associate with polymorphisms in ABCA4 encoding ATP-binding cassette transporter, subfamily A, member 4 previously associated with juvenile onset retinal dystrophies including Stargardt's disease. Polymorphisms at COL2A1 encoding type II collagen, previously associated with Stickler syndrome, associated only with ocular disease in congenital toxoplasmosis. Experimental studies showed that both ABCA4 and COL2A1 show isoform-specific epigenetic modifications consistent with imprinting, which provided an explanation for the patterns of inheritance observed. These genetic and epigenetic risk factors provide unique insight into molecular pathways in the pathogenesis of disease.

Toxoplasma gondii infection induces lipid metabolism alterations in the murine host

Host lipids have been implicated in the pathogenesis of Toxoplasma gondiiinfection. To determine if Toxoplasmainfection influences the lipid status in the normal host, we assessed serum lipids of Swiss-Webster mice during infection with the BGD-1 strain (type-2) at a series of time points. Mice were bled at days zero and 42 post-infection, and subgroups were additionally bled on alternating weeks (model 1), or sacrificed at days zero, 14 and 42 (model 2) for the measurement of total cholesterol (Chl), high density lipoproteins (HDL), low density lipoproteins (LDL) and triglycerides and adiponectin. At day 42, brains were harvested for cyst enumeration. A significant decrease (p = 0.02) in HDL and total Chl was first noted in infected vs. control mice at day 14 and persisted to day 42 (p = 0.013). Conversely, LDL was unaltered until day 42, when it increased (p = 0.043). Serum LDL levels at day 42 correlated only with cyst counts of above 300 (found in 44% mice), while the change in HDL between days zero and 42 correlated with both the overall mean cyst count (p = 0.041) and cyst counts above 300 (p = 0.044). Calculated per cyst, this decrease in HDL in individual animals ranged from 0.1-17 µmol/L, with a mean of 2.43 ± 4.14 µmol/L. Serum adiponectin levels remained similar between infected and control mice throughout the experiment.

Decreased PM10 exposure attenuates age-related lung function decline: genetic variants in p53, p21, and CCND1 modify this effect.

BACKGROUND: Decreasing exposure to airborne particulates was previously associated with reduced age-related decline in lung function. However, whether the benefit from improved air quality depends on genetic background is not known. Recent evidence points to the involvement of the genes p53 and p21 and of the cell cycle control gene cyclin D1 (CCND1) in the response of bronchial cells to air pollution. OBJECTIVE: We determined in 4,326 participants of the Swiss Cohort Study on Air Pollution and Lung and Heart Diseases in Adults (SAPALDIA) whether four single-nucleotide polymorphisms in three genes [CCND1 (rs9344 [P242P], rs667515), p53 (rs1042522 [R72P]), and p21 (rs1801270 [S31R])] modified the previously observed attenuation of the decline in the forced expiratory flow between 25% and 75% of the forced vital capacity (FEF(25-75)) associated with improved air quality. METHODS: Subjects of the prospective population-based SAPALDIA cohort were assessed in 1991 and 2002 by spirometry, questionnaires, and biological sample collection for genotyping. We assigned spatially resolved concentrations of particulate matter with aerodynamic diameter < or = 10 microm (PM(10)) to each participant's residential history 12 months before the baseline and follow-up assessments. RESULTS: The effect of diminishing PM(10) exposure on FEF(25-75) decline appeared to be modified by p53 R72P, CCND1 P242P, and CCND1 rs667515. For example, a 10-microg/m(3) decline in average PM(10) exposure over an 11-year period attenuated the average annual decline in FEF(25-75) by 21.33 mL/year (95% confidence interval, 10.57-32.08) among participants homozygous for the CCND1 (P242P) GG genotype, by 13.72 mL/year (5.38-22.06) among GA genotypes, and by 6.00 mL/year (-4.54 to 16.54) among AA genotypes. CONCLUSIONS: Our results suggest that cell cycle control genes may modify the degree to which improved air quality may benefit respiratory function in adults.

New genetic models and mesenchymal cells as tools to ameliorate anti-tumor immunity

Résumé Le but final de ce projet est d'utiliser des cellules T ou des cellules souches mésenchymateuses modifiées génétiquement afin de surexprimer localement les deux chémokines CXCL13 et CCL2 ensemble ou chacune séparément à l'intérieur d'une tumeur solide. CXCL13 est supposé induire des structures lymphoïdes ectopiques. Un niveau élevé de CCL2 est présumé initier une inflammation aiguë. La combinaison des deux effets amène à un nouveau modèle d'étude des mécanismes régulateur de la tolérance périphérique et de l'immunité tumorale. Les connaissances acquises grâce à ce modèle pourraient permettre le développement ou l'amélioration des thérapies immunes du cancer. Le but premier de ce travail a été l'établissement d'un modèle génétique de la souris permettant d'exprimer spécifiquement dans la tumeur les deux chémokines d'intérêt à des niveaux élevés. Pour accomplir cette tâche, qui est en fait une thérapie génétique de tumeurs solides, deux types de cellules porteuses potentielles ont été évaluées. Des cellules CD8+ T et des cellules mésenchymateuses de la moelle osseuse transférées dans des receveurs portant une tumeur. Si on pouvait répondre aux besoins de la thérapie génétique, indépendamment de la thérapie immune envisagée, on posséderait là un outil précieux pour bien d'autres approches thérapeutiques. Plusieurs lignées de souris transgéniques ont été générées comme source de cellules CD8+ T modifiées afin d'exprimer les chémokines d'intérêt. Dans une approche doublement transgénique les propriétés de deux promoteurs spécifiques de cellules T ont été combinées en utilisant la technologie Cre-loxP. Le promoteur de granzyme B confère une dépendance d'activation et le promoteur distal de lck assure une forte expression constitutive dès que les cellules CD8+ T ont été activées. Les transgènes construits ont montré une bonne performance in vivo et des souris qui expriment CCL2 dans des cellules CD8+ T activées ont été obtenues. Ces cellules peuvent maintenant être utilisées avec différents protocoles pour transférer des cellules T cytotoxiques (CTL) dans des receveurs porteur d'une tumeur, permettant ainsi d'évaluer leur capacité en tant que porteuse de chémokine d'infiltrer la tumeur. L'établissement de souris transgéniques, qui expriment pareillement CXCL13 est prévu dans un avenir proche. L'évaluation de cellules mésenchymateuses de la moelle osseuse a démontré que ces cellules se greffent efficacement dans le stroma tumoral suite à la co-injection avec des cellules tumorales. Cela représente un outil précieux pour la recherche, vu qu'il permet d'introduire des cellules manipulées dans un modèle tumoral. Les résultats confirment partiellement d'autres résultats rapportés dans un modèle amélioré. Cependant, l'efficacité et la spécificité suggérées de la migration systémique de cellules mésenchymateuses de la moelle osseuse dans une tumeur n'ont pas été observées dans notre modèle, ce qui indique, que ces cellules ne se prêtent pas à une utilisation thérapeutique. Un autre résultat majeur de ce travail est l'établissement de cultures de cellules mésenchymateuses de la moelle osseuse in vitro conditionnées par des tumeurs, ce qui a permis à ces cellules de s'étendre plus rapidement en gardant leur capacité de migration et de greffe. Cela offre un autre outil précieux, vu que la culture in vitro est un pas nécessaire pour une manipulation thérapeutique. Abstract The ultimate aim of the presented project is to use genetically modified T cells or mesenchymal stem cells to locally overexpress the two chemokines CXCL13 and CCL2 together or each one alone inside a solid tumor. CXCL13 is supposed to induce ectopic lymphoid structures and a high level of CCL2 is intended to trigger acute inflamation. The combination of these two effects represents a new model for studying mechanisms that regulate peripheral tolerance and tumor immunity. Gained insights may help developing or improving immunotherapy of cancer. The primary goal of the executed work was the establishment of a genetic mouse model that allows tumor-specific expression of high levels of the two chemokines of interest. For accomplishing this task, which represents gene therapy of solid tumors, two types of potentially useful carrier cells were evaluated. CD8+ T cells and mesenchymal bone marrow cells to be used in adoptive cell transfers into tumor-bearing mice. Irrespectively of the envisaged immunotherapy, satisfaction of so far unmet needs of gene therapy would be a highly valuable tool that may be employed by many other therapeutic approaches, too. Several transgenic mouse lines were generated as a source of CD8+ T cells modified to express the chemokines of interest. In a double transgenic approach the properties of two T cell-specific promoters were combined using Cre-loxP technology. The granzyme B promoter confers activation-dependency and the lck distal promoter assures strong constitutive expression once the CD8+ T cell has been activated. The constructed transgenes showed a good performance in vivo and mice expressing CCL2 in activated CD8+ T cells were obtained. These cells can now be used with different protocols for adoptively transferring cytotoxic T cells (CTL) into tumor-bearing recipients, thus allowing to study their capacity as tumor-infiltrating chemokine carrier. The establishment of transgenic mice likewisely expressing CXCL13 is expected in the near future. In addition, T cells from generated single transgenic mice that have high expression of an EGFP reporter in both CD4+ and CD8+ cells can be easily traced in vivo when setting up adoptive transfer conditions. The evaluation of mesenchymal bone marrow cells demonstrated that these cells can efficiently engraft into tumor stroma upon local coinjection with tumor cells. This represents a valuable tool for research purposes as it allows to introduce manipulated stromal cells into a tumor model. Therefore, the established engraftment model is suited for studying the envisaged immunotherapy. These results confirm to some extend previously reported results in an improved model, however, the suggested systemic tumor homing efficiency and specificity of mesenchymal bone marrow cells was not observed in our model indicating that these cells may not be suited for therapeutic use. Another major result of the presented work is the establishment oftumor-conditioned in vitro culture of mesenchymal bone marrow cells, which allowed to more rapidly expand these cells while maintaining their tumor homing and engrafting capacities. This offers another valuable tool as in vitro culture is a necessary step for therapeutic manipulations.

Genetic structure of Aedes aegypti in the city of Córdoba (Argentina), a recently reinfested area

To understand the transmission of a vector-borne disease, knowledge of the magnitude of dispersal among vector populations is essential because of its influence on pathogen transfer. The principal vector of dengue, the most common arboviral disease in the world, is the mosquito Aedes aegypti (L.). This tropical and subtropical species is native to Africa but has dispersed worldwide since the XV century. In Argentina, the species was declared eradicated in 1963, but has reinfested the country in recent years. In the present work, we used RAPD-PCR markers to assess the levels of genetic variability and differentiation among populations of Ae. aegypti (the vector of dengue and yellow fever) in Córdoba, the second largest city in Argentina. We detected similar levels of genetic variability (He between 0.351-0.404) across samples and significant genetic differentiation between most population pairs within the city (F ST between 0.0013-0.0253). Genetic distances indicate that there are three distinct groups, formed predominantly by populations that are connected by, or near, main roads. This suggests that, in addition to other factors such as availability of oviposition sites or step-by-step migration, passive transport plays an important role in gene flow within the city.

ELOVL6 Genetic Variation Is Related to Insulin Sensitivity: A New Candidate Gene in Energy Metabolism

BACKGROUND: The elongase of long chain fatty acids family 6 (ELOVL6) is an enzyme that specifically catalyzes the elongation of saturated and monounsaturated fatty acids with 12, 14 and 16 carbons. ELOVL6 is expressed in lipogenic tissues and it is regulated by sterol regulatory element binding protein 1 (SREBP-1). OBJECTIVE: We investigated whether ELOVL6 genetic variation is associated with insulin sensitivity in a population from southern Spain. DESIGN: We undertook a prospective, population-based study collecting phenotypic, metabolic, nutritional and genetic information. Measurements were made of weight and height and the body mass index (BMI) was calculated. Insulin resistance was measured by homeostasis model assessment. The type of dietary fat was assessed from samples of cooking oil taken from the participants' kitchens and analyzed by gas chromatography. Five SNPs of the ELOVL6 gene were analyzed by SNPlex. RESULTS: Carriers of the minor alleles of the SNPs rs9997926 and rs6824447 had a lower risk of having high HOMA_IR, whereas carriers of the minor allele rs17041272 had a higher risk of being insulin resistant. An interaction was detected between the rs6824447 polymorphism and the intake of oil in relation with insulin resistance, such that carriers of this minor allele who consumed sunflower oil had lower HOMA_IR than those who did not have this allele (P = 0.001). CONCLUSIONS: Genetic variations in the ELOVL6 gene were associated with insulin sensitivity in this population-based study.

Frequency of Fabry disease in male and female haemodialysis patients in Spain

BACKGROUND: Fabry disease (FD), an X-linked lysosomal storage disorder, is caused by a reduced activity of the lysosomal enzyme alpha-galactosidase A. The disorder ultimately leads to organ damage (including renal failure) in males and females. However, heterozygous females usually present a milder phenotype with a later onset and a slower progression. METHODS: A combined enzymatic and genetic strategy was used, measuring the activity of alpha-galactosidase A and genotyping the alpha-galactosidase A gene (GLA) in dried blood samples (DBS) of 911 patients undergoing haemodialysis in centers across Spain. RESULTS: GLA alterations were found in seven unrelated patients (4 males and 3 females). Two novel mutations (p.Gly346AlafsX347 and p.Val199GlyfsX203) were identified as well as a previously described mutation, R118C. The R118C mutation was present in 60% of unrelated patients with GLA causal mutations. The D313Y alteration, considered by some authors as a pseudo-deficiency allele, was also found in two out of seven patients. CONCLUSIONS: Excluding the controversial D313Y alteration, FD presents a frequency of one in 182 individuals (0.55%) within this population of males and females undergoing haemodialysis. Moreover, our findings suggest that a number of patients with unexplained and atypical symptoms of renal disease may have FD. Screening programmes for FD in populations of individuals presenting severe kidney dysfunction, cardiac alterations or cerebrovascular disease may lead to the diagnosis of FD in those patients, the study of their families and eventually the implementation of a specific therapy.

Quantification of the genetic change in the transition of Rhodnius pallescens Barber, 1932 (Hemiptera: Reduviidae) from field to laboratory

Previous studies have reported genetic differences between wild-caught sylvatic, domestic and laboratory pop-ulations of several Triatominae species. The differences between sylvatic and laboratory colonies parallel are similar to the differences observed between sylvatic and domestic populations. Laboratory colonies are frequently used as references for field populations, but the consequences of founder events on the genetic makeup of laboratory or domestic populations are rarely quantified. Our goal was to quantify the genetic change in Rhodnius pallescens populations artificially submitted to founder effects via laboratory colonization. We compared the genetic makeup of two sylvatic populations and their laboratory descendants using a panel of 10 microsatellite markers. Both sylvatic populations were initially collected from palm trees, but the colonies differed in the number of founder insects and amount of time kept in the laboratory. We evaluated allelic polymorphism, differences between expected and observed heterozygosity, estimates of population differentiation (Fst) and inbreeding (Fis, Fit) and cluster analyses based on Nei's distances. We found a unique genetic structure for each sample population, with significant differentiation between the field insects and each of the laboratory generations. These analyses showed strong founder effects and showed that genetic drift had led to a genetic equilibrium over several generations of isolation. Our results suggest that laboratory colonies of R. pallescens have a different genetic structure than their wild relatives and similar processes likely affect other Triatominae laboratory stocks.

Genetic overlap in kallmann syndrome, combined pituitary hormone deficiency, and septo-optic dysplasia.

Context: Kallmann syndrome (KS), combined pituitary hormone deficiency (CPHD), and septo-optic dysplasia (SOD) all result from development defects of the anterior midline in the human forebrain. Objective: The objective of the study was to investigate whether KS, CPHD, and SOD have shared genetic origins. Design and Participants: A total of 103 patients with either CPHD (n = 35) or SOD (n = 68) were investigated for mutations in genes implicated in the etiology of KS (FGFR1, FGF8, PROKR2, PROK2, and KAL1). Consequences of identified FGFR1, FGF8, and PROKR2 mutations were investigated in vitro. Results: Three patients with SOD had heterozygous mutations in FGFR1; these were either shown to alter receptor signaling (p.S450F, p.P483S) or predicted to affect splicing (c.336C>T, p.T112T). One patient had a synonymous change in FGF8 (c.216G>A, p.T72T) that was shown to affect splicing and ligand signaling activity. Four patients with CPHD/SOD were found to harbor heterozygous rare loss-of-function variants in PROKR2 (p.R85G, p.R85H, p.R268C). Conclusions: Mutations in FGFR1/FGF8/PROKR2 contributed to 7.8% of our patients with CPHD/SOD. These data suggest a significant genetic overlap between conditions affecting the development of anterior midline in the human forebrain.

Microsatellite markers for population genetic studies of the blowfly Chrysomya putoria (Diptera: Calliphoridae)

The investigation of the genetic variation and population structure of Chrysomya species is of great interest for both basic and applied research. However, very limited genetic information is available for this genus across its geographical distribution. Here, we describe 12 polymorphic microsatellite loci isolated from Chrysomya putoria with expected heterozygosities ranging from 0.1402-0.8312. These markers are of potential applied interest for forensic entomologists and for the characterisation of the genetic structure of C. putoria from recently colonised regions, with great promise for understanding the colonisation dynamics and spread of the genus Chrysomya in the New World.

Genetic male infertility and mutation of CATSPER ion channels.

A clinically significant proportion of couples experience difficulty in conceiving a child. In about half of these cases male infertility is the cause and often genetic factors are involved. Despite advances in clinical diagnostics ∼50% of male infertility cases remain idiopathic. Based on this, further analysis of infertile males is required to identify new genetic factors involved in male infertility. This review focuses on cation channel of sperm (CATSPER)-related male infertility. It is based on PubMed literature searches using the keywords 'CATSPER', 'male infertility', 'male contraception', 'immunocontraception' and 'pharmacologic contraception' (publication dates from January 1979 to December 2009). Previously, contiguous gene deletions including the CATSPER2 gene implicated the sperm-specific CATSPER channel in syndromic male infertility (SMI). Recently, we identified insertion mutations of the CATSPER1 gene in families with recessively inherited nonsyndromic male infertility (NSMI). The CATSPER channel therefore represents a novel human male fertility factor. In this review we summarize the genetic and clinical data showing the role of CATSPER mutation in human forms of NSMI and SMI. In addition, we discuss clinical management and therapeutic options for these patients. Finally, we describe how the CATSPER channel could be used as a target for development of a male contraceptive.

Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines

BACKGROUND The inability of cancer cells to present antigen on the cell surface via MHC class I molecules is one of the mechanisms by which tumor cells evade anti-tumor immunity. Alterations of Jak-STAT components of interferon (IFN)-mediated signaling can contribute to the mechanism of cell resistance to IFN, leading to lack of MHC class I inducibility. Hence, the identification of IFN-gamma-resistant tumors may have prognostic and/or therapeutic relevance. In the present study, we investigated a mechanism of MHC class I inducibility in response to IFN-gamma treatment in human melanoma cell lines. METHODS Basal and IFN-induced expression of HLA class I antigens was analyzed by means of indirect immunofluorescence flow cytometry, Western Blot, RT-PCR, and quantitative real-time RT-PCR (TaqMan(R) Gene Expression Assays). In demethylation studies cells were cultured with 5-aza-2'-deoxycytidine. Electrophoretic Mobility Shift Assay (EMSA) was used to assay whether IRF-1 promoter binding activity is induced in IFN-gamma-treated cells. RESULTS Altered IFN-gamma mediated HLA-class I induction was observed in two melanoma cells lines (ESTDAB-004 and ESTDAB-159) out of 57 studied, while treatment of these two cell lines with IFN-alpha led to normal induction of HLA class I antigen expression. Examination of STAT-1 in ESTDAB-004 after IFN-gamma treatment demonstrated that the STAT-1 protein was expressed but not phosphorylated. Interestingly, IFN-alpha treatment induced normal STAT-1 phosphorylation and HLA class I expression. In contrast, the absence of response to IFN-gamma in ESTDAB-159 was found to be associated with alterations in downstream components of the IFN-gamma signaling pathway. CONCLUSION We observed two distinct mechanisms of loss of IFN-gamma inducibility of HLA class I antigens in two melanoma cell lines. Our findings suggest that loss of HLA class I induction in ESTDAB-004 cells results from a defect in the earliest steps of the IFN-gamma signaling pathway due to absence of STAT-1 tyrosine-phosphorylation, while absence of IFN-gamma-mediated HLA class I expression in ESTDAB-159 cells is due to epigenetic blocking of IFN-regulatory factor 1 (IRF-1) transactivation.

Genetic polymorphisms of RANTES, IL1-A, MCP-1 and TNF-A genes in patients with prostate cancer

BACKGROUND Inflammation has been implicated as an etiological factor in several human cancers, including prostate cancer. Allelic variants of the genes involved in inflammatory pathways are logical candidates as genetic determinants of prostate cancer risk. The purpose of this study was to investigate whether single nucleotide polymorphisms of genes that lead to increased levels of pro-inflammatory cytokines and chemokines are associated with an increased prostate cancer risk. METHODS A case-control study design was used to test the association between prostate cancer risk and the polymorphisms TNF-A-308 A/G (rs 1800629), RANTES-403 G/A (rs 2107538), IL1-A-889 C/T (rs 1800587) and MCP-1 2518 G/A (rs 1024611) in 296 patients diagnosed with prostate cancer and in 311 healthy controls from the same area. RESULTS Diagnosis of prostate cancer was significantly associated with TNF-A GA + AA genotype (OR, 1.61; 95% CI, 1.09-2.64) and RANTES GA + AA genotype (OR, 1.44; 95% CI, 1.09-2.38). A alleles in TNF-A and RANTES influenced prostate cancer susceptibility and acted independently of each other in these subjects. No epistatic effect was found for the combination of different polymorphisms studied. Finally, no overall association was found between prostate cancer risk and IL1-A or MCP-1 polymorphisms. CONCLUSION Our results and previously published findings on genes associated with innate immunity support the hypothesis that polymorphisms in proinflammatory genes may be important in prostate cancer development.

Association study of genetic variants of pro-inflammatory chemokine and cytokine genes in systemic lupus erythematosus

BACKGROUND. Several lines of evidence suggest that chemokines and cytokines play an important role in the inflammatory development and progression of systemic lupus erythematosus. The aim of this study was to evaluate the relevance of functional genetic variations of RANTES, IL-8, IL-1alpha, and MCP-1 for systemic lupus erythematosus. METHODS. The study was conducted on 500 SLE patients and 481 ethnically matched healthy controls. Genotyping of polymorphisms in the RANTES, IL-8, IL-1alpha, and MCP-1 genes were performed using a real-time polymerase chain reaction (PCR) system with pre-developed TaqMan allelic discrimination assay. RESULTS. No significant differences between SLE patients and healthy controls were observed when comparing genotype, allele or haplotype frequencies of the RANTES, IL-8, IL-1alpha, and MCP-1 polymorphisms. In addition, no evidence for association with clinical sub-features of SLE was found. CONCLUSION. These results suggest that the tested functional variation of RANTES, IL-8, IL-1alpha, and MCP-1 genes do not confer a relevant role in the susceptibility or severity of SLE in the Spanish population.