Immunomodulatory effect of canine periodontal ligament stem cells on allogenic and xenogenic peripheral blood mononuclear cells

Purpose: The aim of this study was to investigate the immunomodulatory effects of canine periodontal ligament stem cells on allogenic and xenogenic immune cells in vitro. Methods: Mixed cell cultures consisting of canine stem cells (periodontal ligament stem cells and bone marrow stem cells) and allogenic canine/xenogenic human peripheral blood mononuclear cells (PBMCs) were established following the addition of phytohemagglutinin. The proliferation of PBMCs was evaluated using the MTS assay. The cell division of PBMCs was analyzed using the CFSE assay. The apoptosis of PBMCs was assessed using the trypan blue uptake method. Results: Periodontal ligament stem cells and bone marrow stem cells inhibited the proliferation of allogenic and xenogenic PBMCs. Both periodontal ligament stem cells and bone marrow stem cells suppressed the cell division of PBMCs despite the existence of a mitogen. No significant differences in the percentages of apoptotic PBMCs were found among the groups. Conclusions: Canine periodontal ligament stem cells have an immunomodulatory effect on allogenic and xenogenic PBMCs. This effect is not a product of apoptosis of PBMCs but is caused by the inhibition of cell division of PBMCs.

Additive effects of LPL, APOA5 and APOE variant combinations on triglyceride levels and hypertriglyceridemia: results of the ICARIA genetic sub-study

<p>Abstract</p> <p>Background</p> <p>Hypertriglyceridemia (HTG) is a well-established independent risk factor for cardiovascular disease and the influence of several genetic variants in genes related with triglyceride (TG) metabolism has been described, including <it>LPL</it>, <it>APOA5 </it>and <it>APOE</it>. The combined analysis of these polymorphisms could produce clinically meaningful complementary information.</p> <p>Methods</p> <p>A subgroup of the ICARIA study comprising 1825 Spanish subjects (80% men, mean age 36 years) was genotyped for the <it>LPL</it>-HindIII (rs320), S447X (rs328), D9N (rs1801177) and N291S (rs268) polymorphisms, the <it>APOA5</it>-S19W (rs3135506) and -1131T/C (rs662799) variants, and the <it>APOE </it>polymorphism (rs429358; rs7412) using PCR and restriction analysis and TaqMan assays. We used regression analyses to examine their combined effects on TG levels (with the log-transformed variable) and the association of variant combinations with TG levels and hypertriglyceridemia (TG ≥ 1.69 mmol/L), including the covariates: gender, age, waist circumference, blood glucose, blood pressure, smoking and alcohol consumption.</p> <p>Results</p> <p>We found a significant lowering effect of the <it>LPL</it>-HindIII and S447X polymorphisms (<it>p </it>< 0.0001). In addition, the D9N, N291S, S19W and -1131T/C variants and the <it>APOE</it>-ε4 allele were significantly associated with an independent additive TG-raising effect (<it>p </it>< 0.05, <it>p </it>< 0.01, <it>p </it>< 0.001, <it>p </it>< 0.0001 and <it>p </it>< 0.001, respectively). Grouping individuals according to the presence of TG-lowering or TG-raising polymorphisms showed significant differences in TG levels (<it>p </it>< 0.0001), with the lowest levels exhibited by carriers of two lowering variants (10.2% reduction in TG geometric mean with respect to individuals who were homozygous for the frequent alleles of all the variants), and the highest levels in carriers of raising combinations (25.1% mean TG increase). Thus, carrying two lowering variants was protective against HTG (OR = 0.62; 95% CI, 0.39-0.98; <it>p </it>= 0.042) and having one single raising polymorphism (OR = 1.20; 95% CI, 1.39-2.87; <it>p </it>< 0.001) or more (2 or 3 raising variants; OR = 2.90; 95% CI, 1.56-5.41; <it>p </it>< 0.001) were associated with HTG.</p> <p>Conclusion</p> <p>Our results showed a significant independent additive effect on TG levels of the <it>LPL </it>polymorphisms HindIII, S447X, D9N and N291S; the S19W and -1131T/C variants of <it>APOA5</it>, and the ε4 allele of <it>APOE </it>in our study population. Moreover, some of the variant combinations studied were significantly associated with the absence or the presence of hypertriglyceridemia.</p

Analysis of genetic background in familiar hypercholesterolemia patients. Genotype-Phenotype association

Familial hypercholesterolemia is an autosomal dominant disease with a prevalence of 1:500. The disease is mainly caused by mutations in the low density lipoprotein receptor gene (LDLR). It is characterised mainly by high serum total cholesterol levels and LDL-C above 200mg/dL. The great variability in lipid levels and the prevalence of coronary artery disease may not be solely explained by the type of LDLR mutation. Other factors such as the environment and modifying genes have been found to affect the phenotype of the disease. The aims of the study were (a) to identify mutations in LDLR gene (number, type, frequency) in familial hypercholesterolemia patients, (b) to find out their distribution in Greece, (c) to correlate the mutations with the phenotype and (d) to analyse genes involved in the development of coronary artery disease and lipid metabolism for common polymorphisms and correlate them with the phenotype. Using DGGE and DNA sequencing analysis, we found 17 mutations were found on the LDLR gene in 67 probands and 11 relatives. Mutations were located in the promoter and 8 different exons (2, 5, 6, 8, 9, 11, 12 and 14) of the LDLR. Among them 11 were missense mutations, two were nonsense mutations, 3 were splice defects (2140+5G>A and 2140+9C>T, 1706-10 G>A) and 1 one was a nucleotide substitution (-45delT) on the promoter. The mutations V408M, G528D, C6W and S265R, account for 73% of heterozygous familial hypercholesterolemia probands. V408M mutation is more common in Central West, while C6W is more common in Central East. From the above mutations A410T, A519T, and the splice site defects 2140+9 C>T are detected for the first time in the Greek population and -45delT is mentioned for the first time worldwide. Expression studies for the newly reported -45delT mutation revealed that the patient bearing the mutated allele had reduced expression to 5% of normal which resulted in reduction of LDL-R binding properties by 76%. Dividing the patients into two groups, receptor defective and receptor negative carriers it was shown that receptor negative carriers have higher total and LDL cholesterol levels, and 6-fold higher incidence of xanthomas. Apart from the genetic analysis performed in LDLR and ApoB genes, analysis in genes known to be involved in biochemical pathways implicated in the progression and the development of cardiovascular disease. The presence of the E4 allele for the ApoE genotype is not associated with lipid levels or the incidence of coronary disease in familial hypercholesterolemia. Other polymorphisms investigated are PON 2 Ser311Cys, LPL Asn291Ser, PAI-1 T11053G, FGB G-455A, NOS A-922G and ANP T2238C and G644A. The PON2 Cys311 allele was correlated with high total and LDL cholesterol and apolipoprotein B levels, while LPL Asn291, PAI-1 T11053, FGΒ G-455 and NOS A-922 allele were correlated with high apolipoprotein B levels. Only the A allele of the ANP G644A was associated with lower levels of Apo-A and HDL-C. These results suggest the Greek population is very homogenous regarding the LDLR mutations compared to others. The type of LDLR mutation affects the clinical phenotype of familial hypercholesterolemia and other genes seem to be involved in the determination of the lipid profile of patients.Η οικογενής υπερχοληστερολαιμία είναι αυτοσωμική επικρατής νόσος που προσβάλει 1 στα 500 άτομα. Η ασθένεια αυτή οφείλεται κυρίως σε μεταλλάξεις στο γονίδιο του υποδοχέα της λιποπρωτεΐνης χαμηλής πυκνότητας (Low Density Lipoprotein Receptor-LDLR). Χαρακτηρίζεται κυρίως από υψηλά επίπεδα ολικής χοληστερόλης ορού και LDL χοληστερόλη μεγαλύτερη από 200mg/dL. Η μεγάλη διακύμανση στα επίπεδα λιπιδίων και στην συχνότητα εμφάνισης στεφανιαίας νόσου αποδίδεται όχι μόνο στον τύπο της μετάλλαξης στον LDLR, αλλά και στην έκφραση άλλων ρυθμιστικών γονιδίων καθώς επίσης και σε περιβαλλοντικούς παράγοντες, όπως η διατροφή και η φυσική δραστηριότητα. Στόχοι της διατριβής ήταν (α) η ανίχνευση μεταλλάξεων στο γονίδιο του LDLR (αριθμός, είδος, συχνότητα) σε ασθενείς με οικογενή υπερχοληστερολαιμία, (β) η κατανομή των μεταλλάξεων στην Ελλάδα, (γ) η συσχέτισή τους με την κλινική εικόνα της νόσου (δ) η ανάλυση γνωστών πολυμορφισμών σε άλλα γονίδια που εμπλέκονται στην εξέλιξη της στεφανιαίας νόσου και στο μεταβολισμό των λιπιδίων και η συσχέτισή τους με τον φαινότυπο. Για την ανάλυση των μεταλλάξεων εφαρμόστηκε η μέθοδος ηλεκτροφόρησης σε κλίση αποδιατακτικών παραγόντων (Denaturing Gradient Gel Electrophoresis-DGGE) ανάγνωση της πρωτοδιάταξης του DNA. Ταυτοποιήσαμε 17 μεταλλάξεις στο γονίδιο του LDLR σε 67 ασθενείς και 11 συγγενείς του. Οι μεταλλάξεις εντοπίστηκαν στον υποκινητή και σε οκτώ εξόνια (2, 5, 6, 8, 9, 11, 12, και 14) του LDLR. Έντεκα από τις μεταλλάξεις είναι παρερμηνεύσιμες, δύο είναι ανερμηνεύσιμες, τρεις βρίσκονται στα όρια μεταξύ εσονίου και εξονίου (splice site) και μία οφείλεται σε έλλειψη ενός νουκλεοτιδίου στον υποκινητή του LDLR (-45delT). Μεταξύ αυτών οι V408M, G528D, C6W και S265R, βρέθηκαν σε ποσοστό 73% στους ασθενείς που ήταν φορείς μεταλλάξεων. Η μετάλλαξη V408M παρατηρήθηκε με μεγάλη συχνότητα στην κεντρική Δυτική Ελλάδα ενώ η C6W είναι πιο συχνή στην κεντρική Ανατολική Ελλάδα. Οι μεταλλάξεις Α410Τ, Α519Τ, 1706-10 G>A και 2140+9 C>T, βρέθηκαν για πρώτη φορά στην Ελλάδα και η -45delT αναφέρθηκε για πρώτη φορά στον κόσμο. Πειράματα έκφρασης για τη μελέτη της πρωτοαναφερόμενης μετάλλαξης -del45T έδειξαν μείωση της έκφρασης του μεταλλαγμένου αλληλομόρφου στο 5% με αποτέλεσμα τη μείωση της ικανότητας πρόσδεσης του LDL-R στην LDL κατά 76%. Χωρίζοντας τους ασθενείς σε φορείς μετάλλαξης ελαττωματικού υποδοχέα και φορείς μετάλλαξης αρνητικού υποδοχέα, παρατηρήθηκε ότι οι φορείς μετάλλαξης αρνητικού υποδοχέα παρουσιάζουν υψηλότερα επίπεδα ολικής και LDL χοληστερόλης, και έξι φορές μεγαλύτερη συχνότητα εμφάνισης ξανθωμάτων. Η ύπαρξη του αλληλομόρφου Ε4 για τον γονότυπο της ΑpoE δεν συνδέεται με τα επίπεδα λιπιδίων αλλά ούτε και με τη συχνότητα εμφάνισης στεφανιαίας νόσου. Άλλοι πολυμορφισμοί που μελετήθηκαν είναι οι PON 2 Ser311Cys, LPL Asn291Ser, PAI-1 T11053G, FGB G-455A, NOS A-922G και ANP T2238C και G644A. Για τον πολυμορφισμό PON 2 Ser311Cys, οι φορείς του αλληλομόρφου Cys παρουσίασαν υψηλότερα επίπεδα ολικής και LDL χοληστερόλης και Apo-B. Υψηλότερα επίπεδα Apo-B παρουσίασαν επίσης οι ομοζυγώτες για το αλληλόμορφο Asn στη θέση 291 της LPL, οι φορείς του αλληλομόρφου T στη θέση 11053 του γονιδίου PAI, οι φορείς του G αλληλομόρφου στη θέση -455 του γονιδίου FGB και οι φορείς του αλληλομόρφου A για τον πολυμορφισμό NOS A-922G. Από τους πολυμορφισμούς G644A και C2238T στο γονίδιο ANP το αλληλόμορφο Α στη θέση 644 συνδέεται με χαμηλότερα επίπεδα Apo-A και HDL-C. Τα αποτελέσματα δείχνουν ότι ο Ελληνικός πληθυσμός είναι αρκετά ομοιογενής σε σύγκριση με άλλους, ως προς τις μεταλλάξεις στον LDLR. Ο τύπος της μετάλλαξης του LDLR επηρεάζει τον φαινότυπο της οικογενούς υπερχοληστερολαιμίας ενώ στην διαμόρφωση λιπιδαιμικού προφίλ των ασθενών φαίνεται να εμπλέκονται και άλλα γονίδια

Study of oncogenic transformation in ex vivo expanded mesenchymal cells, from paediatric bone marrow

OBJECTIVES: Mesenchymal stromal cells (MSCs) have attracted considerable interest in both the scientific and clinical fields. In order to obtain a sufficient cell number for application, their in vitro expansion is necessary, but during this process their characteristics may be altered and cells may acquire oncogenic properties. We have investigated properties of MSC that may be related to oncogenesis, a critical parameter that has to be evaluated prior to MSC clinical use. MATERIALS AND METHODS: We studied the expression of p53, p16, RB, H-RAS and human telomerase reverse transcriptase (hTERT) in MSCs from bone marrow of children diagnosed with idiopathic thrombocytopenic purpura (ITP) and autoimmune neutropenia. The same cells were seeded in soft agar to confirm their anchorage dependence and were karyotypically analysed. Finally, MSCs were subcutaneously transplanted into SCID mice and their ectopic osteogenic as well as tumorigenic potential was evaluated. RESULTS: We have shown that MSCs derived from bone marrow of children with ITP and autoimmune neutropenia do not undergo transformation, the cells expressed normal levels of p53, p16, RB and H-RAS. Expression of hTERT was undetectable, chromosome content remained stable, and their anchorage dependence was confirmed. In an in vivo model, when MSCs were subcutaneously transplanted into SCID mice, no tumorigenesis was observed. CONCLUSIONS: These findings suggest that MSCs from bone marrow of children do not have oncogenic properties and, therefore, represent validate candidates for applications in regenerative medicin

Enhanced levels of the apoptotic BAX/BCL-2 ratio in children with acute lymphoblastic leukemia and high-risk features

It has been suggested that leukemia is characterized by an impaired balance between the proliferation of blood cells and their capacity to undergo apoptosis. The aim of this study was to examine the expression of key molecules related to apoptosis (BCL-2, BAX, FAS, FAS-L) in children with acute lymphoblastic leukemia (ALL). Measurement of BCL-2 and BAX mRNA was performed by quantitative real-time PCR, and membrane expression of FAS and FAS-L was assessed by flow cytometry in bone marrow mononuclear cells, both at diagnosis and at remission following induction chemotherapy. At diagnosis, increased levels of the apoptotic BAX/BCL-2 ratio were observed in children older than 10 years and with higher white blood cell counts. A DNA index < 1.16 was associated with increased BAX/BCL-2, both at diagnosis and at remission, and the del(9p) chromosome abnormality with increased BAX/BCL-2 at remission. The expression of the apoptotic receptor FAS was significantly higher at remission compared to diagnosis, which might reflect enhanced sensitivity of the leukemic clone to apoptosis and response to treatment. Altogether, our results highlight the association of apoptosis-related genes with clinical and cytogenetic prognostic parameters in pediatric ALL. A better understanding of the mechanisms and regulation of apoptosis should enable the design of novel targeted therapies for these patients

Effect of interaction between adherence to a Mediterranean diet and the methylenetetrahydrofolate reductase 677C→T mutation on homocysteine concentrations in healthy adults: The ATTICA Study

Background: Dietary and genetic factors may influence the effect of raised homocysteine concentrations on coronary artery disease risk. Objective: We evaluated the effect of the interaction between adoption of a Mediterranean diet and the methylenetetrahydrofolate reductase gene (MTHFR) 677C→T mutation on homocysteine concentrations in healthy adults participating in the ATTICA study. Design: We studied demographic, lifestyle, clinical, biochemical, and genetic information from 322 men (x- ± SD age: 46 ± 13 y) and 252 women (45 ± 14 y) who had no clinical evidence of cardiovascular or any other chronic disease. We also measured total plasma homocysteine concentrations, the distribution of the MTHFR genotype, and adherence to a Mediterranean diet. Results: The distribution of MTHFR genotypes was as follows: homozygous normal (CC), 41%; heterozygous (CT), 48%; and homozygous mutant (TT), 11%. Homocysteine concentrations were higher in persons with the TT genotype than in those with the CC and CT genotypes (x- ± SD: 15.8 ± 9 compared with 11.3 ± 8 and 10.8 ± 9 μmol/L, respectively; P &amp;lt; 0.001). The Mediterranean diet score was not significantly associated with homocysteine concentrations (P = 0.89). However, after control for potential confounders, the stratified analysis showed that adherence to a Mediterranean diet was associated with reduced homocysteine concentrations in persons with the TT and CTgenotypes (β = -0.21, P = 0.002, and β = -0.14, P = 0.025, respectively) but not in those with the CC genotype (β = -0.03, P = 0.38). Conclusion: The observed association of an MTHFR 677C→T gene-diet interaction on homocysteine concentrations may provide a pathophysiologic explanation for how a Mediterranean diet may influence coronary risk in persons with raised homocysteine concentrations. © 2004 American Society for Clinical Nutrition