ObjectiveWe evaluated the influence of the renin–angiotensin system (RAS) on intestinal inflammation and fibrosis.DesignCultured human colonic myofibroblast proliferation and collagen secretion were assessed following treatment with angiotensin (Ang) II and Ang (1–7), their receptor antagonists candesartan and A779, and the ACE inhibitor captopril. Circulating and intestinal RAS components were evaluated in patients with and without IBD. Disease outcomes in patients with IBD treated with ACE inhibitors and angiotensin receptor blockers (ARBs) were assessed in retrospective studies.ResultsHuman colonic myofibroblast proliferation was reduced by Ang (1–7) in a dose-dependent manner (p<0.05). Ang II marginally but not significantly increased proliferation, an effect reversed by candesartan (p<0.001). Colonic myofibroblast collagen secretion was reduced by Ang (1–7) (p<0.05) and captopril (p<0.001), and was increased by Ang II (p<0.001). Patients with IBD had higher circulating renin (mean 25.4 vs 18.6 mIU/L, p=0.026) and ACE2:ACE ratio (mean 0.92 vs 0.69, p=0.015) than controls without IBD. RAS gene transcripts and peptides were identified in healthy and diseased bowels. Colonic mucosal Masson’s trichrome staining correlated with Ang II (r=0.346, p=0.010) and inversely with ACE2 activity (r=−0.373, p=0.006). Patients with IBD who required surgery (1/37 vs 12/75, p=0.034) and hospitalisation (0/34 vs 8/68, p=0.049) over 2 years were less often treated with ACE inhibitors and ARBs than patients not requiring surgery or hospitalisation.ConclusionsThe RAS mediates fibrosis in human cell cultures, is expressed in the intestine and perturbed in intestinal inflammation, and agents targeting this system are associated with improved disease outcomes.

Expression studies with FRDA-EGFP fusion constructs will facilitate delineation of regulatory elements determining the tissue and developmental specificity of FRDA gene expression. These constructs should also facilitate screening for pharmacological compounds that can modulate the expression of the FRDA gene in a clinically relevant manner.

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A greater understanding of the regulatory mechanisms that govern γ-globin expression in humans, especially the switching from γ- to β-globin, which occurs after birth, would help to identify new therapeutic targets for patients with β-hemoglobinopathy. To further elucidate the mechanisms involved in γ-globin expression, a novel fluorescent-based cellular reporter assay system was developed. Using homologous recombination, two reporter genes, DsRed and EGFP, were inserted into a 183-kb intact human β-globin locus under the control of (G)γ- or (A)γ-globin promoter and β-globin promoter, respectively. The modified constructs were stably transfected into adult murine erythroleukaemic (MEL) cells and human embryonic or fetal erythroleukemic (K562) cells, allowing for rapid and simultaneous analysis of fetal and adult globin gene expression according to their developmental stage-specific expression. To demonstrate the utility of this system, we performed RNA interference (RNAi)-mediated knockdown of BCL11A in the presence or absence of known fetal hemoglobin inducers and demonstrated functional derepression of a γ-globin-linked reporter in an adult erythroid environment. Our results demonstrate that the cellular assay system represents a promising approach to perform genetic and functional genomic studies to identify and evaluate key factors associated with γ-globin gene suppression.

Splicing mutations are common causes of ␤-thalassemia. Some splicing mutations permit normal splicing as well as aberrant splicing, which can give a reduced level of normal ␤-globin synthesis causing mild disease (thalassemia intermedia). For other mutations, normal splicing is reduced to low levels, and patients are transfusiondependent when homozygous for the disease. The development of therapies for ␤-thalassemia will require suitable mouse models for preclinical studies. In this study, we report the generation of a humanized mouse model carrying the common IVSI-110 splicing mutation on a BAC including the human ␤-globin ( hu ␤-globin) locus. We examined heterozygous murine ␤-globin knock-out mice ( mu ␤ th-3/؉ ) carrying either the IVSI-110 or the normal hu ␤-globin locus. Our results show a 90% decrease in hu ␤-globin chain synthesis in the IVSI-110 mouse model compared with the mouse model carrying the normal hu ␤-globin locus. This notable difference is attributed to aberrant splicing. The humanized IVSI-110 mouse model accurately recapitulates the splicing defect found in comparable ␤-thalassemia patients. This mouse model is available as a platform for testing strategies for the restoration of normal splicing.Thalassemia is one of the common inherited genetic disorders affecting hemoglobin synthesis. Approximately 300,000 patients with clinically relevant hemoglobinopathies are born each year (1). In the case of ␤-thalassemia a reduction or absence of ␤-globin chain synthesis results in free or unpaired ␣-globin chains, which aggregate and precipitate within red cells causing ineffective erythropoiesis and severe anemia.Over 200 different mutations have been found that cause ␤-thalassemia, with splicing mutations among the most common. Most of these mutations activate aberrant cryptic 5Ј-donor or 3Ј-acceptor splice sites without completely abolishing normal splicing. These mutations lead to the production of variable amounts of normal transcripts. Some mutations allow a significant level of normal splicing (such as IVSI-6), leading to thalassemia intermedia, whereas others reduce normal splicing to low levels (such as IVSI-110) or very low levels (such as IVSI-5 and IVSII-654), causing a transfusion-dependent disease in homozygotes.Because of the complex pathophysiology associated with the hemoglobinopathies, transgenic mouse models are an essential platform for delineating the pathological mechanisms and, as in vivo model systems, for validating future therapeutic strategies. The humanized mouse model approach, where the transgenic mouse model contains large human genomic fragment(s), has provided valuable insight into the regulatory elements required for the developmental expression of human globin genes (2-6). To date, the YAC transgenic mouse for sickle cell anemia, exclusively expressing human ␤ s -globin, is the only mouse thalassemic model expressing a disease gene from the intact hu ␤-globin locus (7). Other transgenic mice exclusively expressing human HbA, HbC, HbF, and HbS have also been repor...

Background:The intestinal vitamin D receptor (VDR) remains poorly characterized in patients with inflammatory bowel disease (IBD).Methods:Colonoscopic biopsies and intestinal resection specimens from the terminal ileum, ascending and sigmoid colon, from patients with and without IBD, were analyzed for VDR mRNA quantification by polymerase chain reaction, and protein localization and semi-quantification by immunohistochemistry. The relationship between VDR and intestinal inflammation, serum 25(OH)D and oral vitamin D intake was elicited.Results:A total of 725 biopsies from 20 patients with Crohn’s disease (CD), 15 with ulcerative colitis (UC) and 14 non-IBD controls who underwent colonoscopy were studied. VDR gene expression and protein staining intensity was similar across all three groups, and across the intestinal segments. Sigmoid colon VDR mRNA expression inversely correlated with faecal calprotectin (r = −0.64, p = 0.026) and histological score (r = −0.67, p = 0.006) in UC, and histological score (r = −0.58, p = 0.019) in patients with CD. VDR staining intensity was higher in quiescent than diseased segments. No relationship with serum 25(OH)D or oral vitamin D intake was noted. Immunohistochemical staining of 28 intestinal resection specimens from 15 patients (5 each with CD, UC and non-IBD controls) showed diffuse VDR staining in the mucosa, submucosa and circular muscle.Conclusions:VDR transcript expression and protein staining intensity are inversely related to inflammation in IBD, but unrelated to serum 25(OH)D, and similar to non-IBD controls. Strategies to upregulate intestinal VDR, potentially translating to modulation of disease activity, require investigation.

ABSTRACTβ-thalassemia is an inherited hemoglobinopathy caused by defective synthesis of the β-globin chain of hemoglobin, leading to imbalanced globin chain synthesis. Excess α-globin precipitates in erythroid progenitor cells resulting in cell death, ineffective erythropoiesis and severe anemia. Decreased α-globin synthesis leads to milder symptoms, exemplified in individuals who co-inherit α-and β-thalassemia. In this study, we investigated the feasibility of utilizing short-interfering RNA (siRNA) to mediate reductions in α-globin expression. A number of siRNA sequences targeting murine α-globin were tested in hemoglobinized murine erythroleukemic cells. One highly effective siRNA sequence (si-α4) was identified and reduced α-globin by approximately 65% at both the RNA and the protein level. Electroporation of si-α4 into murine thalassemic primary erythroid cultures restored α:β-globin ratios to balanced wild-type levels and resulted in detectable phenotypic correction. These results indicate that siRNA-mediated reduction of α-globin has potential therapeutic applications in the treatment of β-thalassemia.Key words: siRNA-mediated reduction, α-globin, phenotypic, β-thalassemic cells.Citation: Voon HPJ, Wardan H, and Vadolas J. siRNA-mediated reduction of α-globin results in phenotypic improvements in β-thalassemic cells.

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