Molecular spectrum of ß-thalassemia mutations in the admixed Venezuelan population, and their linkage to ß-globin gene haplotypes.

In order to establish the spectrum of ß-thalassemia (ß-thal) mutations in the Venezuelan population for the first time, 127 unrelated subjects either with a suspicion of ß-thal trait or with a clinically recognized ß-thal syndrome of different degrees of severity, were studied. DNA from these subjects was analyzed by a polymerase chain reaction (PCR)-based reverse dot-blot method or amplification refractory mutation system (ARMS). Prototype ß-globin gene sequencing of relevant DNA was performed to confirm the mutations. Fifteen different mutations were identified accounting for 92.0% of the mutant alleles explored, revealing a significant genetic heterogeneity at the ß-globin gene locus in this population. The most frequent mutations were codon 39 (C >T) 34.1%, IVS-I-1 (G >A) 11.1%, IVS-I-6 (T > C) 6.6%, IVS-I-110 (G >A) 6.6%, IVS-II-849 (A >G) 6.6%, -88 (C >T) 6.0%, -29 (A >G) 5.2%, followed by the less common IVS-I-5 (G >A) 3.7%, the 1,393 bp deletion 3.0%, IVS-II-1 (G >A) 3.0%, -86 (C >G) 2.2%, IVS-II-1 (G >T) 1.5%, codons 41/42 (-TCTT) 1.5%, IVS-II-745 (C >G) 0.7% and deletional δß-thal 0.7%. Overall, these data demonstrate that the major sources of ß-thal alleles in Venezuela, as expected, are of Mediterranean and African origins. This is the first large study defining the molecular spectrum of ß-thal in the highly admixed population of Venezuela and lays the foundation for genetic counseling as well as implementing comprehensive clinical care programs. Diversity of haplotypes associated with some of the ß-thal mutations can be explained by in situ recombination events in Venezuela.

Hb Sheffield [ß58(E2)Pro→His] in Oman: potential pitfall in genetic counseling.

A novel ß-globin structural variant, namely Hb Sheffield [ß58(E2)Pro→His], was recently found as a sporadic event in a British Subject and posted to the HbVar database (ID 2672). Here we describe the same variant in 11 Omani subjects in the heterozygous state and in one Omani woman in compound heterozygosity with Hb S [ß6(A3)Glu→Val]. Hb Sheffield coelutes in the Hb A(2) window in the high performance liquid chromatography (HPLC) system as does Hb E [ß26(B8)Glu→Lys], and might be erroneously diagnosed as Hb E unless additional tests including DNA analyses are done. Indeed, correct diagnosis of Hb E is important because of its association with other ß-thalassemic and variant alleles can result in relevant clinical conditions, while Hb Sheffield will not. In a genetic (premarital) counseling setting, and in regions where both Hb E ad Hb Sheffield are present, failure to distinguish these variants will represent a serious pitfall.

Hb A2' (Hb B2) in the Omani population and diagnostic significance.

Hb A(2)' [δ16(A13)Gly→Arg], also called Hb B2, is a δ-globin chain variant that has been identified in several populations of African origin or ancestry and is easily identifiable in alkaline acetate cellulose electrophoresis as doubling of the Hb A(2) band. However, in high performance liquid chromatography (HPLC), commonly employed nowadays, it elutes in the S window. Over a period of 2 years at the Sultan Qaboos University Hospital, Muscat, Oman, we identified 25 Omanis with this variant. The quantity of Hb A(2) ranged from 0.9 to 1.8% in heterozygotes and was undetectable in the single homozygous case. As both α- and ß-thalassemia (α- and ß-thal) as well as Hb S [ß6(A3)Glu→Val] are common in the Omani population, it is important to be aware of the presence of Hb A(2)' in this population to avoid misinterpretation of the HPLC data in terms of underdiagnosis of ß-thal carriers and overestimation of α-thal based on Hb A(2) levels in sickle cell carriers. The haplotype associated with Hb A(2)' in Oman is identical to that described in African populations, suggesting a common origin for this mutation and its introduction into Oman by gene flow.

Forecasting hemoglobinopathy burden through neonatal screening in Omani neonates.

To evaluate the incidence of hemoglobinopathies in Omani subjects and to forecast its future burden on health resources, we initiated a prospective neonatal screening program in two major cities of the Sultanate of Oman. Consecutive cord blood samples from a total of 7,837 neonates were analyzed for complete blood counts and for hemoglobin (Hb) profile by high performance liquid chromatography (HPLC). No case with Hb H (beta4) was detected. We observed that the overall incidence of alpha-thalassemia (alpha-thal) was 48.5% [based on the presence of Hb Bart's (gamma4)] and the beta-globin-related abnormalities accounted for 9.5% of the samples (4.8% sickle cell trait, 2.6% beta-thal trait, 0.9% Hb E trait, 0.8% Hb D trait, 0.08% Hb C trait, 0.3% sickle cell disease and 0.08% homozygous beta-thal). This is also the first large study to establish reference ranges of cord red blood cell (RBC) indices for Omani neonates.

In vitro effects of two silicate-based materials, Biodentine and BioRoot RCS, on dental pulp stem cells in models of reactionary and reparative dentinogenesis.

BACKGROUND: Calcium silicate-based cements are biomaterials with calcium oxide and carbonate filler additives. Their properties are close to those of dentin, making them useful in restorative dentistry and endodontics. The aim of this study was to evaluate the in vitro biological effects of two such calcium silicate cements, Biodentine (BD) and Bioroot (BR), on dental stem cells in both direct and indirect contact models. The two models used aimed to mimic reparative dentin formation (direct contact) and reactionary dentin formation (indirect contact). An original aspect of this study is the use of an interposed thin agarose gel layer to assess the effects of diffusible components from the materials. RESULTS: The two biomaterials were compared and did not modify dental pulp stem cell (DPSC) proliferation. BD and BR showed no significant cytotoxicity, although some cell death occurred in direct contact. No apoptosis or inflammation induction was detected. A striking increase of mineralization induction was observed in the presence of BD and BR, and this effect was greater in direct contact. Surprisingly, biomineralization occurred even in the absence of mineralization medium. This differentiation was accompanied by expression of odontoblast-associated genes. Exposure by indirect contact did not stimulate the induction to such a level. CONCLUSION: These two biomaterials both seem to be bioactive and biocompatible, preserving DPSC proliferation, migration and adhesion. The observed strong mineralization induction through direct contact highlights the potential of these biomaterials for clinical application in dentin-pulp complex regeneration.

Validation of Housekeeping Genes to Study Human Gingival Stem Cells and Their In Vitro Osteogenic Differentiation Using Real-Time RT-qPCR.

Gingival stem cells (GSCs) are recently isolated multipotent cells. Their osteogenic capacity has been validated in vitro and may be transferred to human cell therapy for maxillary large bone defects, as they share a neural crest cell origin with jaw bone cells. RT-qPCR is a widely used technique to study gene expression and may help us to follow osteoblast differentiation of GSCs. For accurate results, the choice of reliable housekeeping genes (HKGs) is crucial. The aim of this study was to select the most reliable HKGs for GSCs study and their osteogenic differentiation (dGSCs). The analysis was performed with ten selected HKGs using four algorithms: ΔCt comparative method, GeNorm, BestKeeper, and NormFinder. This study demonstrated that three HKGs, SDHA, ACTB, and B2M, were the most stable to study GSC, whereas TBP, SDHA, and ALAS1 were the most reliable to study dGSCs. The comparison to stem cells of mesenchymal origin (ASCs) showed that SDHA/HPRT1 were the most appropriate for ASCs study. The choice of suitable HKGs for GSCs is important as it gave access to an accurate analysis of osteogenic differentiation. It will allow further study of this interesting stem cells source for future human therapy.

Effects of High-Temperature-Pressure Polymerized Resin-Infiltrated Ceramic Networks on Oral Stem Cells.

OBJECTIVES: The development of CAD-CAM techniques called for new materials suited to this technique and offering a safe and sustainable clinical implementation. The infiltration of resin in a ceramic network under high pressure and high temperature defines a new class of hybrid materials, namely polymer infiltrated ceramics network (PICN), for this purpose which requires to be evaluated biologically. We used oral stem cells (gingival and pulpal) as an in vitro experimental model. METHODS: Four biomaterials were grinded, immersed in a culture medium and deposed on stem cells from dental pulp (DPSC) and gingiva (GSC): Enamic (VITA®), Experimental Hybrid Material (EHM), EHM with initiator (EHMi) and polymerized Z100™ composite material (3M®). After 7 days of incubation; viability, apoptosis, proliferation, cytoskeleton, inflammatory response and morphology were evaluated in vitro. RESULTS: Proliferation was insignificantly delayed by all the tested materials. Significant cytotoxicity was observed in presence of resin based composites (MTT assay), however no detectable apoptosis and some dead cells were detected like in PICN materials. Cell morphology, major cytoskeleton and extracellular matrix components were not altered. An intimate contact appeared between the materials and cells. CLINICAL SIGNIFICANCE: The three new tested biomaterials did not exhibit adverse effects on oral stem cells in our experimental conditions and may be an interesting alternative to ceramics or composite based CAD-CAM blocks.

Characterization and functionality of CXCR4 chemokine receptor and SDF-1 in human corneal fibroblasts.

PURPOSE: The aim of this study was to investigate whether cultured human corneal fibroblasts express functional chemokine CXCR4 receptors on their cell surface and to determine the presence of its specific ligand, SDF-1 (CXCL12), in human corneal fibroblasts. METHODS: Human corneal fibroblast cultures were obtained using human donor corneas. CXCR4 receptors were characterized using binding studies and autoradiography with [125I]SDF-1. The functionality of CXCR4 receptors was assessed by intracellular calcium measurement using a dynamic imaging microscopy system. CXCR4 and SDF-1 mRNA were detected in human corneal fibroblasts using reverse transcriptase polymerase chain reaction (RT-PCR). The CXCR4 protein was detected by western blot analysis. RESULTS: [125I]SDF-1 specifically bound to cultured corneal fibroblasts with a KD value of 8.3+/-1.2 nM. The presence of CXCR4 was confirmed by autoradiography of the radioligand on slices of corneal stroma. SDF-1 induced a rapid and transient intracellular calcium increase in cultured corneal fibroblasts that was blocked by the specific antagonist bicyclam. Moreover, a 48 kDa protein was detected by western blot analysis of corneal fibroblast extracts, using a specific CXCR4 polyclonal antibody. RT-PCR showed the expression of both CXCR4 and SDF-1 mRNAs in human corneal fibroblasts. CONCLUSIONS: These results indicate for the first time that cultured human corneal fibroblasts express the chemokine receptors CXCR4 and its ligand SDF-1. This latter might exert physiological effects on the cornea and could be involved in pathological conditions such as corneal angiogenesis.

Formation of cartilage and synovial tissue by human gingival stem cells.

Human gingival stem cells (HGSCs) can be easily isolated and manipulated in culture to investigate their multipotency. Osteogenic differentiation of bone-marrow-derived mesenchymal stem/stromal cells has been well documented. HGSCs derive from neural crests, however, and their differentiation capacity has not been fully established. The aim of the present report was to investigate whether HGSCs can be induced to differentiate to osteoblasts and chondrocytes. HGSCs were cultured either in a classical monolayer culture or in three-dimensional floating micromass pellet cultures in specific differentiation media. HGSC differentiation to osteogenic and chondrogenic lineages was determined by protein and gene expression analyses, and also by specific staining of cells and tissue pellets. HGSCs cultured in osteogenic differentiation medium showed induction of Runx2, alkaline phosphatase (ALPL), and osterix expression, and subsequently formed mineralized nodules consistent with osteogenic differentiation. Interestingly, HGSC micromass cultures maintained in chondrogenic differentiation medium showed SOX9-dependent differentiation to both chondrocyte and synoviocyte lineages. Chondrocytes at different stages of differentiation were identified by gene expression profiles and by histochemical and immunohistochemical staining. In 3-week-old cultures, peripheral cells in the micromass cultures organized in layers of cuboidal cells with villous structures facing the medium. These cells were strongly positive for cadherin-11, a marker of synoviocytes. In summary, the findings indicate that HGSCs have the capacity to differentiate to osteogenic, chondrogenic, and synoviocyte lineages. Therefore, HGSCs could serve as an alternative source for stem cell therapies in regenerative medicine for patients with cartilage and joint destructions, such as observed in rheumatoid arthritis.