Raffaella Petruzzelli

Increased mucosal nitric oxide production in ulcerative colitis is mediated in part by the enteroglial‐derived S100B protein

Abstract  In the central nervous system glial‐derived S100B protein has been associated with inflammation via nitric oxide (NO) production. As the role of enteroglial cells in inflammatory bowel disease has been poorly investigated in humans, we evaluated the association of S100B and NO production in ulcerative colitis (UC). S100B mRNA and protein expression, inducible NO synthase (iNOS) expression, and NO production were evaluated in rectal biopsies from 30 controls and 35 UC patients. To verify the correlation between S100B and NO production, biopsies were exposed to S100B, in the presence or absence of specific receptor for advanced glycation end‐products (RAGE) blocking antibody, to measure iNOS expression and nitrite production. S100B and iNOS expression were evaluated after incubation of biopsies with lipopolysaccharides (LPS) + interferon‐gamma (IFN‐γ) in the presence of anti‐RAGE or anti‐S100B antibodies or budesonide. S100B mRNA and protein expression, iNOS expression and NO production were significantly higher in the rectal mucosa of patients compared to that of controls. Exogenous S100B induced a significant increase in both iNOS expression and NO production in controls and UC patients; this increase was inhibited by specific anti‐RAGE blocking antibody. Incubation with LPS + IFN‐γ induced a significant increase in S100B mRNA and protein expression, together with increased iNOS expression and NO production. LPS + IFN‐γ‐induced S100B up‐regulation was not affected by budesonide, while iNOS expression and NO production were significantly inhibited by both specific anti‐RAGE and anti‐S100B blocking antibodies. Enteroglial‐derived S100B up‐regulation in UC participates in NO production, involving RAGE in a steroid insensitive pathway.

Allele-specific transcriptional activity of the variable number of tandem repeats of the inducible nitric oxide synthase gene is associated with idiopathic achalasia

Background Polymorphisms of genes involved in the regulation of the immune response are risk factors for achalasia, but their contribution to disease pathogenesis is unknown. Nitric oxide is involved both in immune function and inhibitory neurotransmission. Objective The objective of this article is to assess the association and the functional relevance of the CCTTT-inducible nitric oxide synthase (NOS2) gene promoter polymorphism in achalasia. Methods Genomic DNA was isolated from 181 achalasia patients and 220 controls. Genotyping of the (CCTTT)n repeats was performed by PCR and capillary electrophoresis, and data analyzed by considering the frequency of the different alleles. HT29 cells were transfected with iNOS luciferase promoter-reporter plasmids containing different (CCTTT)n. Results The alleles’ distribution ranged from 7 to 18, with a peak frequency at 12 repeats. Analysis of the allele frequencies revealed that individuals carrying 10 and 13 CCTTT repeats were respectively less and more frequent in achalasia (OR 0.5, 95% CI 0.3–0.5 and OR 1.6, 95% CI 1–2.4, all p < 0.05). Long repeats were also significantly associated with an earlier onset of the disease (OR 1.69, 95% CI 1.13–2.53, p = 0.01). Transfection experiments revealed a similar allele-specific iNOS transcriptional activity. Conclusion The functional polymorphism (CCTTT) of NOS2 promoter is associated with achalasia, likely by an allele-specific modulation of nitric oxide production.

Role of the cold shock domain protein A in the transcriptional regulation of HBG expression

Impaired switching from fetal haemoglobin (HbF) to adult globin gene expression leads to hereditary persistence of fetal haemoglobin (HPFH) in adult life. This is of prime interest because elevated HbF levels ameliorate β‐thalassaemia and sickle cell anaemia. Fetal haemoglobin levels are regulated by complex mechanisms involving factors linked or not to the β‐globin gene (HBB) locus. To search for factors putatively involved in the expression of the γ‐globin genes (HBG1, HBG2), we examined the reticulocyte transcriptome of three siblings who had different HbF levels and different degrees of β‐thalassaemia severity although they had the same ΗBA‐ and ΗΒB cluster genotypes. By mRNA differential display we isolated the cDNA coding for the cold shock domain protein A (CSDA), also known as dbpA, previously reported to interact in vitro with the HBG2 promoter. Expression studies performed in K562 and in primary erythroid cells showed an inverse relationship between HBG and CSDA expression levels. Functional studies performed by Chromatin Immunoprecipitation and reporter gene assays in K562 cells demonstrated that CSDA is able to bind the HBG2 promoter and suppress its expression. Therefore, our study demonstrated that CSDA is a trans‐acting repressor factor of HBG expression and modulates the HPFH phenotype.

An optimized method to separate reticulocytes from peripheral blood for molecular analysis

A method based on immunomagnetic sorting of reticulocytes from peripheral blood was set up and combined to a commercial extraction kit for the isolation of total RNA from whole blood. This procedure resulted in high‐quality RNA samples suitable for molecular analysis. We used this procedure to analyse erythroid‐specific transcripts, starting from peripheral blood samples, to search for differently expressed mRNAs in patients with hereditary persistence of foetal haemoglobin. After erythrocyte lysis, CD15+and CD45+ peripheral cells were negatively sorted to remove leucocyte populations that could have affected the subsequent screening procedure. The cell sorting and RNA extraction procedure was completed within 1–2 h of erythrocyte lysis, which represents a consistent saving of time compared with other procedures. Moreover, it produced 1 μg of total RNA per ml of blood samples, which is sufficient for molecular analysis. Therefore, our method is a reliable and efficient tool to isolate RNA from specific cell subpopulations poorly represented in peripheral blood, particularly when accurate detection and characterization of highly unstable and poorly expressed molecules is required.

research paper: Role of the cold shock domain protein A in the transcriptional regulation of HBG expression

Impaired switching from fetal haemoglobin (HbF) to adult globin gene expression leads to hereditary persistence of fetal haemoglobin (HPFH) in adult life. This is of prime interest because elevated HbF levels ameliorate β‐thalassaemia and sickle cell anaemia. Fetal haemoglobin levels are regulated by complex mechanisms involving factors linked or not to the β‐globin gene (HBB) locus. To search for factors putatively involved in the expression of the γ‐globin genes (HBG1, HBG2), we examined the reticulocyte transcriptome of three siblings who had different HbF levels and different degrees of β‐thalassaemia severity although they had the same ΗBA‐ and ΗΒB cluster genotypes. By mRNA differential display we isolated the cDNA coding for the cold shock domain protein A (CSDA), also known as dbpA, previously reported to interact in vitro with the HBG2 promoter. Expression studies performed in K562 and in primary erythroid cells showed an inverse relationship between HBG and CSDA expression levels. Functional studies performed by Chromatin Immunoprecipitation and reporter gene assays in K562 cells demonstrated that CSDA is able to bind the HBG2 promoter and suppress its expression. Therefore, our study demonstrated that CSDA is a trans‐acting repressor factor of HBG expression and modulates the HPFH phenotype.

Role of the Cold Shock Domain Protein A in the transcriptional regulation of gamma-globin gene expression.

Impaired switching from fetal haemoglobin (HbF) to adult globin gene expression leads to hereditary persistence of fetal haemoglobin (HPFH) in adult life. This is of prime interest because elevated HbF levels ameliorate β‐thalassaemia and sickle cell anaemia. Fetal haemoglobin levels are regulated by complex mechanisms involving factors linked or not to the β‐globin gene (HBB) locus. To search for factors putatively involved in the expression of the γ‐globin genes (HBG1, HBG2), we examined the reticulocyte transcriptome of three siblings who had different HbF levels and different degrees of β‐thalassaemia severity although they had the same ΗBA‐ and ΗΒB cluster genotypes. By mRNA differential display we isolated the cDNA coding for the cold shock domain protein A (CSDA), also known as dbpA, previously reported to interact in vitro with the HBG2 promoter. Expression studies performed in K562 and in primary erythroid cells showed an inverse relationship between HBG and CSDA expression levels. Functional studies performed by Chromatin Immunoprecipitation and reporter gene assays in K562 cells demonstrated that CSDA is able to bind the HBG2 promoter and suppress its expression. Therefore, our study demonstrated that CSDA is a trans‐acting repressor factor of HBG expression and modulates the HPFH phenotype.

T1912 Association Between Ccttt Pentanucleotide Repeat in the Inducible Nitric Oxide Synthase Promoter Polymorphism and Achalasia

averaging successive swallows cancels out extrinsic pressure from HREPT studies, isolating the intrinsic esophageal contraction. Topographic plots of peristalsis can thus be reduced in a coordinate-specific scheme to three sequential zones: inhibition-contraction-quiescence. Consequently, motility disorders can similarly be physiologically defined as impaired inhibition (achalasia), abnormal contraction (hyper or hypotensive peristalsis) or persistent contraction (spastic nutcracker) depending on the length-time coordinates of abnormal contractile activity.

research paper: Role of the cold shock domain protein A in the transcriptional regulation of HBG expression: Role of CSDA in Modulation of Globin Gene Expression

Impaired switching from fetal haemoglobin (HbF) to adult globin gene expression leads to hereditary persistence of fetal haemoglobin (HPFH) in adult life. This is of prime interest because elevated HbF levels ameliorate β‐thalassaemia and sickle cell anaemia. Fetal haemoglobin levels are regulated by complex mechanisms involving factors linked or not to the β‐globin gene (HBB) locus. To search for factors putatively involved in the expression of the γ‐globin genes (HBG1, HBG2), we examined the reticulocyte transcriptome of three siblings who had different HbF levels and different degrees of β‐thalassaemia severity although they had the same ΗBA‐ and ΗΒB cluster genotypes. By mRNA differential display we isolated the cDNA coding for the cold shock domain protein A (CSDA), also known as dbpA, previously reported to interact in vitro with the HBG2 promoter. Expression studies performed in K562 and in primary erythroid cells showed an inverse relationship between HBG and CSDA expression levels. Functional studies performed by Chromatin Immunoprecipitation and reporter gene assays in K562 cells demonstrated that CSDA is able to bind the HBG2 promoter and suppress its expression. Therefore, our study demonstrated that CSDA is a trans‐acting repressor factor of HBG expression and modulates the HPFH phenotype.