Prevention of transcriptional gamma-globin gene silencing by inducing the hereditary persistence of fetal hemoglobin point mutation using chimeraplast-mediated gene targeting

Objective: Hemoglobin F [HbF] augmentation is considered a clinically beneficial phenomenon in beta-hemoglobinopathies. Prevention of gamma-globin gene silencing, inspired by the hereditary persistence of fetal hemoglobin, may be a suitable strategy to upregulate HbF expression in these patients. Therefore, our objective was to assess the potential feasibility of induced -117 G?A substitution in HBG promoter in prevention of transcriptional silencing of the gamma-globin

Materials and Methods: In this experimental study, human peripheral blood-derived hematopoietic stem cells [HSCs] and the K562 cell line were differentiated to erythroid cells. Erythroid maturation was examined using cell morphology parameters and flow cytometry analysis of CD235a expression. A synthesised chimeraplast was transfected to differentiating cells. The efficiency of chimeraplast delivery into target cells was assessed by flow cytometry. Restriction-fragment length polymorphism and DNA sequencing verified oligonucleotide-directed mutagenesis. Gene conversion frequency and globin genes expression was quantified through Allele specific-quantitaive polymerase chain reaction [AS-qPCR] and quantitative-PCR respectively

Results: Increase in CD235a-expressing cells along with observations made for different stages of erythroid maturation confirmed erythroid differentiation in HSCs and K562 cells. gamma to beta-globin gene switching was estimated to be on days 18-21 of HSC differentiation. Flow cytometry analysis showed that more than 70% of erythroid progenitor cells [EPCs] were transfected with the chimeraplast. The highest gene conversion efficiency was 7.2 and 11.1% in EPCs and K562 cells respectively. The induced mutation led to a 1.97-fold decrease in beta/gamma-globin gene expression in transfected EPCs at the experimental end point [day 28] whereas, due to the absence of beta-globin gene expression following K562 differentiation, this rate was not evaluable

Conclusion: Our results suggest the effectiveness of chimeraplasty in induction of the mutation of interest in both EPCs and K562 cells. We also demonstrate that the single nucleotide promoter variant was able to significantly inhibit gamma-globin gene silencing during erythroid differentiation

Melatonin ameliorates the production of COX-2, iNOS, and the formation of 8-ohdg in non-targeted lung tissue after pelvic irradiation

2 [COX-2], inducible nitric oxide synthase [iNOS], and 8-hydroxydeoxyguanosine [8-OHdG] in lung tissues of Sprague-Dawley rats with and without pre-administration of melatonin. A 2x2 cm[2] area of the pelvis of male Sprague-Dawley rats with and without pre-administration of melatonin [100 mg/kg] by oral and intraperitoneal injection was irradiated with a 3 Gy dose of 1.25 MeV gamma-rays. Alterations in the levels of COX-2, iNOS, and 8-OHdG in the out-of-field lung areas of the animals were detected by enzyme immunoassay. The bystander effect significantly increased COX-2, iNOS, and 8-OHdG levels in non-targeted lung tissues [P<0.05]. Melatonin ameliorated the bystander effect of radiation and significantly reduced the level of all examined biomarkers [P<0.05]. The results indicated that the ameliorating effect of a pre-intraperitoneal [IP] injection of melatonin was noticeably greater compared to oral pre-administration. Our findings revealed that the bystander effect of radiation could induce oxidative DNA damage and increase the levels of imperative COX-2 and iNOS in non-targeted lung tissues. Interestingly, melatonin could modulate the indirect destructive effect of radiation and reduce DNA damage in non-targeted cells

Development of flow cytometry-fluorescent in situ hybridization [Flow-FISH] method for detection of PML/RARa chromosomal translocation in acute promyelocytic leukemia cell line

Background: acute Promyelocytic Leukemia [APL] is a subclass of acute myeloid leukemia. The chromosomal aberration in 95% of APL cases is t[15; 17] [q22; q21], which prevents cell differentiation. Characterization of the underlying molecular lesion is valuable in determining optimal treatment strategy. The goal of this study was to develop a new and powerful Flow- FISH technique to detect the long isoform [L] of PML-RARa fusion transcript in NB4 cell line

Methods: to achieve the best condition for fixation, two different fixatives including 2% paraformaldehyde and 75% ethanol were used. 0.2% Triton X-100 and 0.2% saponin were used for the permeabilization step .In hybridization, a wide range of times and temperatures were used and probe was designed in FRET system. Results were confirmed by fluorescent microscope assay and reverse transcription PCR

Results: in the present study, a novel technique was successfully optimized that combines in situ hybridization with flow cytometry to detect the presence of PML-RARa transcript. Using standard fixation and permeabilization protocol of 2% PFA and 0.2% saponin gave the best fluorescent results in flow cytometry. Also, results indicated that the optimum time and temperature for hybridization was 2 hr at 42degreeC. The results of reverse transcription PCR and fluorescent microscopy confirmed the presence of PML-RARa transcript

Conclusion: the concordance between the results of Flow-FISH and those of two other techniques including reverse transcription PCR and FISH indicated that this method would be applicable as a diagnostic test for APL in clinical samples and MRD monitoring

Expression of basophil activation markers in pediatric asthma

Background: asthma is very common in children and its diagnosis is based on clinical manifestations, which can be misdiagnosed as other respiratory diseases with similar signs and symptoms

Objective: to analyze the expression of ST2L and CD203c in the diagnosis of pediatric asthma

Methods: basophils were purified from whole blood samples of patients and healthy controls using Ficol-Paque gradient and Basophil Isolation Kit. RNA extraction was done by RNX-Plus solution and after synthesis of cDNA, the gene expression was analyzed by means of real time PCR

Results: patients expressed significantly higher levels of CD203c than healthy controls [p=0.01]. Although there was an increase in the transcription level of ST2L gene in patients, the results were not statistically significant compared to those obtained from the healthy controls [p>0.05]. A Specificity of 60% and a sensitivity of 73% were foundusing ROC curve for CD203c expression. Patients with positive family history of asthma exhibited more CD203c and ST2L expression [p<0.05]

Conclusion: it is proposed that determining CD203c expression by real time PCR may be an effective technique for diagnosis of pediatric asthma

Fetal Rhd genotyping using real-time polymerase chain reaction analysis of cell-free fetal DNA in pregnancy of rhd negative women in south of Iran

Background: Maternal-fetal RhD antigen incompatibility causes approximately 50% of clinically significant alloimmunization cases. The routine use of prophylactic anti-D immunoglobulin has dramatically reduced hemolytic disease of the fetus and newborn. Recently, fetal RHD genotyping in RhD negative pregnant women has been suggested for appropriate use of anti-D immunoglobulin antenatal prophylaxis and decrease unnecessary prenatal interventions

Materials and Methods: In this prospective cohort study, in order to develop a reliable and non-invasive method for fetal RHD genotyping, cell free fetal DNA [cffD-NA] was extracted from maternal plasma. Real-time quantitative polymerase chain reaction [qPCR] for detection of RHD exons 7, 5, 10 and intron 4 was performed and the results were compared to the serological results of cord blood cells as the gold standard method. SRY gene and hypermethylated Ras-association domain family member 1 [RASSF1A] gene were used to confirm the presence of fetal DNA in male and female fetuses, respectively

Results: Out of 48 fetuses between 8 and 32 weeks [wks] of gestational age [GA], we correctly diagnosed 45 cases [93.75%] of RHD positive fetuses and 2 cases [4.16%] of the RHD negative one. Exon 7 was amplified in one sample, while three other RHD gene sequences were not detected; the sample was classified as inconclusive, and the RhD serology result after birth showed that the fetus was RhD-negative

Conclusion: Our results showed high accuracy of the qPCR method using cffDNA for fetal RHD genotyping and implicate on the efficiency of this technique to predict the competence of anti-D immunoglobulin administration

Assessment of different permeabilization methods of minimizing damage to the adherent cells for detection of intracellular RNA by flow cytometry

Various fixation and permeabilization techniques have been developed for detection of intracellular antigens by flow cytometry; however, there are few studies using flow cytometry to detect the frequency of intracellular nucleic acids, particularly RNA. We tested six different permeabilization methods in order to gain access to a high quality method with minimal damage to intracellular components focusing on 18S rRNA in HeLa cells. HeLa cells were fixed in 2% paraformaldehyde. A variety of detergents and enzymes including saponin, TritonX-100, Tween-20, NP40, Proteinase K, and streptolysin O were used to optimize a protocol of permeabilization for the flow cytometric enumeration of intracellular 18S rRNA. Treated cells were subjected to standard protocol of flow cytometric in situ hybridization in the presence of FITC-labeled sense and antisense probes to detect 18S ribosomal RNAs. Samples were then analyzed on a FACSCalibur flow cytometer. To evaluate cell morphology, following hybridization the cells were fixed on glass slide, covered with DAPI, and evaluated on a fluorescent microscope with appropriate filter sets. In comparison with other methods, maximum cell frequency in percentage and fluorescent intensity [M1=2.1%, M2=97.9%] were obtained when the cells were treated with 0.2% Tween-20 and incubated for 30 min [p=0.001]. Our study indicated that the highest levels of mean fluorescence could be obtained when the cells were treated with Tween-20. However, it should be taken into consideration that for a successful flow cytometric result, other interfering factors such as hybridization conditions should also be optimized

Production and characterization of a monoclonal antibody against an antigen on the surface of non-small cell carcinoma of the lung

Lung carcinoma is a multiple type cancer comprising of small cell and non-small cell carcinomas [NSCLC]. For therapeutic and diagnostic purposes, serum monoclonal antibodies have been produced against lung cancer. To characterize a murine monoclonal antibody [ME3D11] reactive with human NSCLC. A murine monoclonal antibody [ME3D11] reactive with human NSCLC was selected after immunization of BALB/c mice with a human large cell carcinoma with neuroendocrine differentiation, and was tested by immunofloursence staining and Western blot analysis. Our study showed that the antigen recognized by ME3D11 antibody was a cell surface antigen of 170kDa. This antigen is expressed on the cell surface of all NSCLC and a few carcinoma cell lines. In contrast, this antigen is neither expressed on the cell surface of human sarcoma, nor on the hematopoietic and normal cell lines. This antibody had no effect on spontaneous proliferation of Mehr-80 cell line in vitro. High degree of binding of this monoclonal antibody to NSCLC and some other carcinoma cells warrants further studies on its potential use in diagnosis and therapy of cancer by conjugation to drugs, toxins or radionuclides