Oxidative stress and antioxidant status in beta-thalassemia heterozygotes

Background:Several studies have evaluated the oxidant and antioxidant status of thalassemia patients but most focused mainly on the severe and intermediate states of the disease. Moreover, the oxidative status has not been evaluated for the different beta-thalassemia mutations.Objective:To evaluate lipid peroxidation and Trolox equivalent antioxidant capacity in relation to serum iron and ferritin in beta thalassemia resulting from two different mutations (CD39 and IVS-I-110) compared to individuals without beta-thalassemia.Methods:One hundred and thirty subjects were studied, including 49 who were heterozygous for beta-thalassemia and 81 controls. Blood samples were subjected to screening tests for hemoglobin. Allele-specific polymerase chain reaction was used to confirm mutations for beta-thalassemia, an analysis of thiobarbituric acid reactive species was used to determine lipid peroxidation, and Trolox equivalent antioxidant capacity evaluations were performed. The heterozygous beta-thalassemia group was also evaluated for serum iron and ferritin status.Results:Thiobarbituric acid reactive species (486.24 ± 119.64 ng/mL) and Trolox equivalent antioxidant capacity values (2.23 ± 0.11 mM/L) were higher in beta-thalassemia heterozygotes compared to controls (260.86 ± 92.40 ng/mL and 2.12 ± 0.10 mM/L, respectively; p-value < 0.01). Increased thiobarbituric acid reactive species values were observed in subjects with the CD39 mutation compared with those with the IVS-I-110 mutation (529.94 ± 115.60 ng/mL and 453.39 ± 121.10 ng/mL, respectively; p-value = 0.04). However, average Trolox equivalent antioxidant capacity values were similar for both mutations (2.20 ± 0.08 mM/L and 2.23 ± 0.12 mM/L, respectively; p-value = 0.39). There was no influence of serum iron and ferritin levels on thiobarbituric acid reactive species and Trolox equivalent antioxidant capacity values.Conclusion:This study shows an increase of oxidative stress and antioxidant capacity in beta-thalassemia heterozygotes, mainly in carriers of the CD39 mutation.

Determination of neutral polymorphisms (frameworks) of the human beta globin gene in beta thalassaemias by PCR/DGGE

Purpose: Considering the importance of type beta thalassaemias as hereditary syndromes of high significance in different populations of Mediterranean origin and, by extension, in the Brazilian population, the objective of the present study was to determine by PCR/DGGE the gene structures responsible for neutral polymorphisms (frameworks) observed in the human beta globin gene associated with the mutations responsible for type beta thalassaemias in a sample of the Brazilian population and, more specifically, of the population of the State of São Paulo. Patients and methods: Thirty individuals with beta thalassaemic mutations were analyzed: 22 mutations were in codon 39 (C->T), 5 in IVS1-110 (G->A), 2 in IVS1-6 (T->C) and 1 in IVS1-1 (G->A). DNA was extracted and selective amplification was performed by PCR extending from position IVS1 nt 46 to IVS2 nt 126 (474 pb). The product was then analyzed by polyacrylamide gel electrophoresis on a denaturing 10-60% urea/formamide gradient. Results: The results demonstrated that, as expected, the mutations responsible for type beta thalassaemia observed in this population are of Mediterranean origin, with 73% distribution represented by codon 39,17% by IVS1-110, 7% by IVS1-6 and 3% by IVS1-1. In turn, framework distribution seems to indicate a higher frequency of Fr 1-1 in codon 39 and IVS1-110, of Fr 1-3 in IVS1-6 and of Fr 1-2 in IVS1-1. Conclusions: These results permit us to conclude that gene amplification by PCR followed by DGGE is an appropriate method for the separation of DNA molecules that differ even by a single base change and therefore can be utilized to detect the alterations observed in the human beta globin gene. This methodology shows that, using only a pair of primers, it is possible to define the frameworks that are observed in the beta globin gene.

Vitronectin modulates human mesenchymal stem cell response to insulin-like growth factor-I and transforming growth factor beta 1 in a serum-free environment

The use of animal sera for the culture of therapeutically important cells impedes the clinical use of the cells. We sought to characterize the functional response of human mesenchymal stem cells (hMSCs) to specific proteins known to exist in bone tissue with a view to eliminating the requirement of animal sera. Insulin-like growth factor-I (IGF-I), via IGF binding protein-3 or -5 (IGFBP-3 or -5) and transforming growth factor-beta 1 (TGF-beta(1)) are known to associate with the extracellular matrix (ECM) protein vitronectin (VN) and elicit functional responses in a range of cell types in vitro. We found that specific combinations of VN, IGFBP-3 or -5, and IGF-I or TGF-beta(1) could stimulate initial functional responses in hMSCs and that IGF-I or TGF-beta(1) induced hMSC aggregation, but VN concentration modulated this effect. We speculated that the aggregation effect may be due to endogenous protease activity, although we found that neither IGF-I nor TGF-beta(1) affected the functional expression of matrix metalloprotease-2 or -9, two common proteases expressed by hMSCs. In summary, combinations of the ECM and growth factors described herein may form the basis of defined cell culture media supplements, although the effect of endogenous protease expression on the function of such proteins requires investigation.

A functional screen identifies lateral transfer of beta-glucuronidase (gus) from bacteria to fungi

Lateral gene transfer (LGT) from prokaryotes to microbial eukaryotes is usually detected by chance through genome-sequencing projects. Here, we explore a different, hypothesis-driven approach. We show that the fitness advantage associated with the transferred gene, typically invoked only in retrospect, can be used to design a functional screen capable of identifying postulated LGT cases. We hypothesized that beta-glucuronidase (gus) genes may be prone to LGT from bacteria to fungi (thought to lack gus) because this would enable fungi to utilize glucuronides in vertebrate urine as a carbon source. Using an enrichment procedure based on a glucose-releasing glucuronide analog (cellobiouronic acid), we isolated two gus(+) ascomycete fungi from soils (Penicillium canescens and Scopulariopsis sp.). A phylogenetic analysis suggested that their gus genes, as well as the gus genes identified in genomic sequences of the ascomycetes Aspergillus nidulans and Gibberella zeae, had been introgressed laterally from high-GC gram(+) bacteria. Two such bacteria (Arthrobacter spp.), isolated together with the gus(+) fungi, appeared to be the descendants of a bacterial donor organism from which gus had been transferred to fungi. This scenario was independently supported by similar substrate affinities of the encoded beta-glucuronidases, the absence of introns from fungal gus genes, and the similarity between the signal peptide-encoding 5' extensions of some fungal gus genes and the Arthrobacter sequences upstream of gus. Differences in the sequences of the fungal 5' extensions suggested at least two separate introgression events after the divergence of the two main Euascomycete classes. We suggest that deposition of glucuronides on soils as a result of the colonization of land by vertebrates may have favored LGT of gus from bacteria to fungi in soils.

Good results for early treatment of clinically isolated syndrome prior to multiple sclerosis with interferon beta-1b and glatiramer

Background: The first sign of developing multiple sclerosis is a clinically isolated syndrome that resembles a multiple sclerosis relapse. Objective/methods: The objective was to review the clinical trials of two medicines in clinically isolated syndromes (interferon β and glatiramer acetate) to determine whether they prevent progression to definite multiple sclerosis. Results: In the BENEFIT trial, after 2 years, 45% of subjects in the placebo group developed clinically definite multiple sclerosis, and the rate was lower in the interferon β-1b group. Then all subjects were offered interferon β-1b, and the original interferon β-1b group became the early treatment group, and the placebo group became the delayed treatment group. After 5 years, the number of subjects with clinical definite multiple sclerosis remained lower in the early treatment than late treatment group. In the PreCISe trial, after 2 years, the time for 25% of the subjects to convert to definite multiple sclerosis was prolonged in the glatiramer group. Conclusions: Interferon β-1b and glatiramer acetate slow the progression of clinically isolated syndromes to definite multiple sclerosis. However, it is not known whether this early treatment slows the progression to the physical disabilities experienced in multiple sclerosis.

A two-compartment mechanochemical model of the roles of transforming growth factor-beta and tissue tension in dermal wound healing

The repair of dermal tissue is a complex process of interconnected phenomena, where cellular, chemical and mechanical aspects all play a role, both in an autocrine and in a paracrine fashion. Recent experimental results have shown that transforming growth factor-beta (TGF-beta) and tissue mechanics play roles in regulating cell proliferation, differentiation and the production of extracellular materials. We have developed a 1D mathematical model that considers the interaction between the cellular, chemical and mechanical phenomena, allowing the combination of TGF-beta and tissue stress to inform the activation of fibroblasts to myofibroblasts. Additionally, our model incorporates the observed feature of residual stress by considering the changing zero-stress state in the formulation for effective strain. Using this model, we predict that the continued presence of TGF-beta in dermal wounds will produce contractures due to the persistence of myofibroblasts; in contrast, early elimination of TGF-beta significantly reduces the myofibroblast numbers resulting in an increase in wound size. Similar results were obtained by varying the rate at which fibroblasts differentiate to myofibroblasts and by changing the myofibroblast apoptotic rate. Taken together, the implication is that elevated levels of myofibroblasts is the key factor behind wounds healing with excessive contraction, suggesting that clinical strategies which aim to reduce the myofibroblast density may reduce the appearance of contractures.