Reza Ghassemifar

Histology of the healing tympanic membrane following perforation in rats

The aim of this study was to provide a detailed cytological account on the healing tympanic membrane (TM) over 14 days and to complement existing research into TM wound healing.

Chronic tympanic membrane perforation: a better animal model is needed

Developments in the treatment of chronic tympanic membrane perforation have been hindered by the lack of an ideal animal model. It is not appropriate to test such treatments on acute perforations as the majority of these heal spontaneously. An ideal animal model would be one that most closely resembles the human clinical situation. It should be inexpensive, readily available, and easy to create. There have been a number of attempts to create a chronic tympanic membrane perforation model with limited success. All published attempts at chronic tympanic membrane perforations have been reviewed and the limitations of each model are discussed. A number of areas for research exist for further developing a chronic tympanic membrane perforation model. These areas include a perforation model in the presence of bacteria and eustachian tube dysfunction. Understanding the molecular and genetic mechanisms of chronic otitis media and potential treatments will also be useful.

Advancing Towards a Tissue-engineered Tympanic Membrane: Silk Fibroin as a Substratum for Growing Human Eardrum Keratinocytes

Human tympanic membrane cells (hTMCs), harvested from tympanic membrane (TM) explants, were grown in culture and then seeded on membranes prepared from silkworm (Bombyx mori) silk fibroin (BMSF) and on tissue-culture plastic membranes (PET). Fibroin was isolated from silk cast into membranes with a thickness of 10—15 μm. The hTMCs were cultured on both materials for 15 days in a serum-containing culture medium. The cells grown on both substrata were subjected to nuclear staining (DAPI) and counted. Further, the cultures were immunostained for a number of protein markers related to the epithelial/keratinocyte phenotype and cell adhesion complexes. The BMSF membranes supported levels of hTMC growth higher than that observed on the PET membranes. The immunofluorochemical analysis indicated unequivocally that BMSF is a more suitable substratum than PET with respect to the growth patterns, proliferation, and cell—cell contact and adhesion. BMSF appear as a promising substratum in the tissue-engineered constructs for the replacement of TM in case of nonhealing perforations.

Tympanic membrane wound healing in rats assessed by transcriptome profiling

The aim of this study is to elucidate transcriptional changes that occur in response to tympanic membrane (TM) perforation in rats and to infer key genes and molecular events in the healing process.

In vitro characterization of the α-thalassemia point mutation HBA2:c.95+1G>A [IVS-I-1(G>A)(α2)]

The α-thalassemias are a group of disorders occurring as a result of decreased synthesis of α-globin chains, most commonly due to deletions of α-globin genes. Detection of α-thalassemia (α-thal) caused by point mutations has increased during the past few years and more than 70 different point mutations have been reported for the α1- and α2-globin genes. The mutation at the splice donor site of the first intervening sequence [IVS-I-1 (G>A)] of the α2-globin gene, HBA2:c.95+1G>A, is thought to cause a thalassemic phenotype by interfering with and preventing the normal splicing of pre-mRNA. We developed an in vitro expression system to study α-globin gene point mutations at the molecular and cellular levels. The expression vector carrying the HBA2:c.95+1G>A mutation (α2GIVS-I-1G>A) was created using site-directed mutagenesis of a wild type (WT) construct of the α2-globin gene (α2G2034WT). Gene expression experiments in human bladder carcinoma 5637 cells were carried out using sequence verified WT and mutated clones. Complementary DNA synthesis and polymerase chain reaction (PCR) analysis showed normal α2-globin transcripts from cells transfected with the WT vector, but aberrant transcripts from cells transfected with the mutated vector carrying the splice donor site mutation. In the presence of the G>A mutation, normal splicing does not occur, and a cryptic splice site 49 bp upstream of the normal site is used. The translation of this product produces a premature termination codon, thus resulting in a thalassemic phenotype.

Green tea polyphenol “epigallocatechin-3-gallate”, differentially induces apoptosis in CLL B-and T-Cells but not in healthy B-and T-Cells in a dose dependant manner

B-cell chronic lymphocytic leukaemia (CLL) is characterized by an accumulation of CD5-positive monoclonal B-cells due in large part to a failure of apoptosis. The ability to study CLL B-cells in vitro has always been a challenge and hampered by the low viability of the CLL B-cells in cell culture systems. In this study, we present a multicellular cell culture system to maintain CLL B-cells viable in culture for 60h in the presence of a stromal cell feeder layer in combination with a whole white blood cell preparation. Using this optimized system, we tested and showed that the addition of epigallocatechin-3-gallate (EGCG) at concentrations ranging from 25 to 100μg/ml induced apoptosis in CLL B-cells whilst not affecting healthy control B-cells. Moreover, the results showed that in contrast to healthy controls, T-cells from CLL patients underwent apoptosis in the presence of EGCG. This study demonstrated that the combination of a cell feeder layer with a whole white blood cell preparation maintained B-cell viability in vitro over an extended period of time. In addition, the study showed that EGCG differentially induces apoptosis in CLL B-and T-Cells but not in healthy B-and T-Cells in a dose dependent manner.

Differential gene expression analysis in early and late erythroid progenitor cells in β-thalassaemia

β‐ thalassaemia is a disorder of globin gene synthesis resulting in reduced or absent production of the β‐globin chain in red blood cells. In this study, haematopoietic stem cells were isolated from the peripheral blood of six transfusion dependent β‐thalassaemia patients and six healthy controls. Following 7 and 14 d in culture, early‐ and late‐ erythroblasts were isolated and purified. No morphological difference in maturation was observed following 7 d in culture, while a delayed maturation was observed in the patient group after 14 d. Following RNA isolation and linear amplification, gene expression analyses were performed using microarray technology. The generated data were analysed by two methods: the BRB‐ArrayTools platform and the Bioconductor platform using bead level data. Following 7 d culture, there was no difference in gene expression between the control and patient groups. Following 14 d culture, 384 differentially expressed genes were identified by either analysis. A subset of 90 genes was selected and the results were confirmed by Quantitative‐Real‐Time‐polymerase chain reaction. Pathways shown to be significantly altered in the patient group include apoptosis, MAPKinase and the nuclear factor‐κB pathway.

Alpha thalassaemia due to non-deletional mutations on the -3.7 alpha globin fusion gene: laboratory diagnosis and clinical importance

Aims: Alpha (&agr;) thalassaemia may be caused by large deletions of the &agr; globin gene(s), or rarely, non-deletional mutations. Both types of mutations may co-exist, and if located on the same allele (&agr;0), produce a reproductive risk of hydrops fetalis. We illustrate how clinical-laboratory correlation and accurate &agr; gene sequencing are essential in identifying such patients. Method: Nine asymptomatic patients with -&agr;3.7 thalassaemia trait were noted to have significant microcytosis that was insufficiently explained by a single &agr; deletion. Hence &agr;1 and &agr;2 globin gene sequencing were performed, which detected a non-deletional mutation in all patients. A new set of &agr;1 specific primers were designed for separate sequencing of the &agr;1 gene and the -&agr;3.7 fusion gene, respectively, so that the non-deletional mutation could be localised to the correct allele. Results: In six of nine patients tested, the non-deletional mutation was on the &agr;1 globin gene. In three patients the mutation was located on the -&agr;3.7 fusion gene. The latter group functionally has an &agr;0 allele (&agr;&agr;/–) with a reproductive risk for Hb Barts hydrops fetalis. Conclusion: Non-deletional mutations can occur on the &agr; globin gene or a fusion gene such as the -&agr;3.7 allele. Identification and accurate localisation of these mutations is important as this can have significant reproductive implications.

A molecular tool to assess the pathological relevance of alpha-globin DNA variants.

Aim: While the phenotype for heterozygous beta-thalassaemia is straightforward, it is more difficult to confirm a causative relationship for mutations in the alpha-globin genes. The aim of this study was to generate an in vitro system to evaluate the pathological relevance of &agr;-globin mutations. Methods: The novel variant HBA1:c.301-3C>G was used as a model. In silico analysis predicted an aberrant acceptor splice site in the mutant sequence. Subsequent in vitro studies included generation of and transfection of an expression vector carrying the HBA1:c.301-3C>G mutation, RNA purification, reverse-transcription polymerase chain reaction (RT-PCR) and cDNA sequencing. Immunofluorochemistry (IFC) with antibodies specific to the N- and or C- terminal of the &agr;-globin protein was used in protein detection. Results: In vitro molecular characterisation of this point mutation confirmed the preferential utilisation of a cryptic splice site at intron 2 of the pre-mRNA, resulting in a shift in the reading frame causing a premature termination codon (PTC) at codons 101/102 and generation of a truncated protein. Conclusion: We have described here a molecular tool to study mutations that affect &agr;-globin pre-mRNA splicing and translation. We confirm in silico predictions of the consequences of the HBA1:c.301-3C>G mutation, proving aberrant RNA splicing and the production of a truncated &agr;-globin protein.

Differential expression of the Bcl-2 and Bax isoforms in CD19 positive B-lymphocytes isolated from patients diagnosed with chronic lymphocytic leukaemia

Aim: To examine the relative gene expression levels of the anti-apoptotic Bcl-2&agr; and &bgr; isoforms and the pro-apoptotic Bax&agr; and &bgr; isoforms in patients with chronic lymphocytic leukaemia (CLL) and healthy controls (HC). Methods: Peripheral blood was obtained from 36 patients diagnosed with CLL and 10 HC. CD19+ B-lymphocytes were isolated using an antibody coupled magnetic bead isolation system; from these cells the total RNA was isolated and purified. The relative levels of gene expression were examined by quantitative real-time polymerase chain reaction (qReTi-PCR) using primers specific for each isoform. Results: Bcl-2&agr; and Bax&agr; are expressed at higher levels than their &bgr;-isoforms in CLL and HC. Bcl-2&agr;, Bcl-2&bgr; and Bax&bgr; expression is increased in CLL while Bax-&agr; is expressed at similar levels to HC. The Bcl-2&agr;/Bcl-2&bgr; ratio is similar in CLL and HC. The Bcl-2&agr;/Bax&agr; ratio is increased in CLL when compared with HC. Conclusion: Bcl-2&agr; and Bax&agr; appear to be the dominant anti- and pro-apoptotic isoforms in CLL. The Bcl-2&agr;/Bax&agr; ratio is increased in CLL while the Bcl-2&agr;/Bcl-2&bgr; ratio is similar to HC.