Histology of the healing tympanic membrane following perforation in rats

Objectives/Hypothesis: 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. Study Design: The study is a prospective cohort study of 19 male Sprague-Dawley (Rattus norvegicus) rats. Methods: Rat TMs were perforated using a sterile needle and sacrificed at time points during the 14 days following perforation. Results: The healing of the TM resembles cutaneous wound healing except that the TM is unique in the lack of a supportive matrix beneath the regenerating epithelia. This prevents the influx of reparative cells and nutrients and the in growth of the usual fibroblastic reaction. Conclusions: TM wound healing contrasts with cutaneous wound healing in that keratinocytes are the first cells to close the wound and not the last. A keratin scaffold may not be important in the healing process. The malleus plays a crucial role in the healing of the TM and is the site of significant mitotic activity during the healing process. Migration across layers of the TM appears to account for the closure of the perforation. © 2010 The American Laryngological

Keratinocyte growth factor 1, fibroblast growth factor 2 and 10 in the healing tympanic membrane following perforation in rats

The aim of this study was to provide a transcriptome profile of Keratinocyte Growth Factor (KGF)-1, Fibroblast Growth Factor (FGF) 2 and FGF10 (KGF2) in the healing rat tympanic membrane (TM) over 7 days and an immunohistochemical account over 14 days following perforation. KGF1, FGF2, and FGF10 play important roles in TM wound healing. The tympanic membranes of rats were perforated and sacrificed at time points over a 14-day period following perforation. The normalized signal intensities and immunohistochemical protein expression patterns at each time point for KGF1, FGF2, and FGF10 are presented. The primary role of both KGF1 and FGF2 appeared to be in the proliferation and migration of keratinocytes. Whereas the role of KGF1 appeared to be exclusively concerned with increased proliferation and migration at the perforation site, the continued expression of FGF2, beyond perforation closure, suggested it has an additional role to play. FGF10 (KGF2), whilst possessing the highest sequence homologous to KGF1, has a different role in TM wound healing. The effect of FGF10 on keratinocytes in wound healing appeared to emanate from the connective tissue layer. © 2010 Springer Science+Business Media B.V

Experimental Characterization of Hb Flurlingen (HBA2: c.177 C > G, p.His > Gln) and Hb Boghé (HBA2: c.177 C > A, p.His > Gln) Reveals Contradictory HBA2 Expression and Translation Patterns Despite Identical Amino Acid Substitutions

In this study, we describe the clinical features and provide experimental analyses of Hb Flurlingen (HBA2: c.177 C > G, p.His > Gln) that contrasted with Hb Boghé (HBA2: c.177 C > A, p.His > Gln). Despite the identical amino acid substitution in both variants, Hb Flurlingen shows the phenotype of α-thalassemia (α-thal), whereas Hb Boghé has no impact on α2-globin (HBA2) production. For in vitro transcription analysis, HBA2 expression constructs carrying the HBA2-WT (wild type), Hb Flurlingen and Hb Boghé sequences were generated and expressed in human bladder carcinoma 5637 cells for downstream analyses by quantitative real time-polymerase chain reaction (qReTi-PCR) and immunofluorochemistry (IFC). In silico analysis of secondary folding structures of the HBA2-WT, Hb Flurlingen and Hb Boghé mRNA sequences was performed using Mfold software. The gene transcription and translation analyses revealed that cells transfected with the Hb Flurlingen construct had significantly lower HBA2 transcription (−55.4%, p ≤ 0.01) and reduced protein synthesis when compared to the wild type group. In contrast, cells transfected with the Hb Boghé construct showed no significant changes in HBA2 transcription or translation activities when compared to the wild type group. The in silico prediction of possible effects of these mutations on the folding structures of the HBA2 transcripts showed a change of secondary folding pattern in the Hb Flurlingen transcript when compared to those of HBA2-WT and Hb Boghé. Our experimental findings support the clinical presentation of an α-thalassemic phenotype for Hb Flurlingen in contrast with Hb Boghé, despite identical amino acid substitutions. The results confirm the importance of experimental analysis in establishing the impact of novel base substitutions

The role of epidermal growth factor in the healing tympanic membrane following perforation in rats

Epidermal Growth Factor (EGF) has been identified as playing a critical role in the wound healing process. The objective of this study is to investigate the role that EGF plays in rat tympanic membrane (TM) wound healing using two techniques, microarray and immunohistochemistry. The tympanic membranes of rats were perforated using a sterile needle and sacrificed at time points during 2 weeks following perforation. The normalized signal intensities at the time points for EGF and associated genes are presented. The rat EGF mRNA did not change significantly between time points. Five associated proteins, including heparin-binding EGF-like growth factor were found to be differentially expressed above a two fold threshold at 12 h following perforation. EGF staining was found at low levels in the uninjured TM. Levels of EGF staining increased at 24 h in the basal keratinocyte layer, became diffusely elevated in the specimen at 36 h, before a second peak in staining of the keratinocyte layer at Day 4. The staining of EGF corresponds to its multiple roles in TM wound healing. © 2010 Springer Science+Business Media B.V

Tympanic membrane wound healing in rats assessed by transcriptome profiling

Objectives/Hypothesis: 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. Study Design: A prospective cohort study of 393 male Sprague-Dawley (Rattus norvegicus) rats. Methods: Sprague-Dawley rats were randomly allocated into either control or perforation groups spanning a 7-day time period. Perforation groups consisted of 12-hour, 24-hour, 36-hour, 2-day, 3-day, 4-day, 5-day, six-day, and 7-day time points. The left TMs of all perforation groups were perforated and the RNA extracted at the specified time point postperforation. Subsequent analysis was performed using Agilent's 4 × 44 k whole rat genome arrays (40 in total) to assess wound-healing gene expression over a 7-day time period. Results: Over a 7-day time course and at nine time points that encompassed the wounding and progression of healing, a total of 3,262 genes were differentially expressed. In this study the transcripts most upregulated occurred at 12 hours. These were Stefin A2 (344-fold), Stefin 2 (143-fold), and Natriuretic peptide precursor type B (222-fold). Those most downregulated also occurred at 12 hours. These were alcohol dehydrogenase 7 (13.1-fold) and gamma-butyrobetaine hydroxylase (10.4-fold). Results were validated by quantitative real-time polymerase chain reaction. Conclusions: The findings of this study provide a baseline against which to identify disease-related molecular signatures, biomarkers, and to develop new treatments for TM conditions based on molecular evidence. Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc

Occludin TM4(-): an isoform of the tight junction protein present in primates lacking the fourth transmembrane domain

The tight junction protein occludin possesses four transmembrane domains, two extracellular loops, and cytoplasmic N- and C-termini. Reverse transcription-PCR analysis of human tissues, embryos and cells using primers spanning the fourth transmembrane domain (TM4) and adjacent C-terminal region revealed two products. The larger and predominant product corresponded in sequence to canonical occludin (TM4+), while the smaller product exhibited a 162 bp deletion encoding the entire TM4 and immediate C-terminal flanking region (TM4-). Examination of the genomic occludin sequence identified that the 162 bp sequence deleted in TM4- coincided precisely with occludin exon 4, strongly suggesting that TM4- is an alternative splice isoform generated by skipping of exon 4. Indeed, the reading frame of downstream exons is not affected by exclusion of exon 4. The presence of both TM4+ and TM4- occludin isoforms was also identified in monkey epithelial cells but TM4- was undetected in murine and canine tissue and cells, indicating a late evolutionary origin for this alternative splicing event. Conceptual translation of TM4- isoform predicts extracellular localisation of the C-terminus. Immunocytochemical processing of living human Caco-2 cells using a C-terminal occludin antibody revealed weak, discontinuous staining restricted to the periphery of subconfluent islands of cells, or islands generated by wounding confluent layers. In occludin immunoblots, a weak band at 58 kDa, smaller than the predominant band at 65 kDa and corresponding to the predicted mass of TM4- isoform, is evident and upregulated in subconfluent cells. These data suggest that the TM4- isoform may be translated at low levels in specific conditions and may contribute to regulation of occludin function

Gene expression regulating epithelial intercellular junction biogenesis during human blastocyst development in vitro

We investigated gene expression associated with trophectoderm epithelial intercellular junction formation in single human embryos at different stages of cleavage using RT–PCR methods based upon magnetic bead separation of polyA+ RNA. Trophectoderm tight junction (TJ) and desmosome biogenesis contribute to intercellular sealing and tissue integrity, critical for vectorial transport and blastocoel cavity formation. Expression of the various genes throughout human preimplantation development showed differing levels of sensitivity of detection; these genes included claudin-1, occludin (TM4+ and TM4 isoforms), ZO-1 (ZO-1+ and ZO-1– isoforms), ZO-2 and JAM (junction adhesion molecule), and the desmosome junction gene, DSC2 (desmocollin 2). Some transcripts appeared to be expressed throughout preimplantation development (claudin-1, JAM, occludin TM4+ and TM4, ZO-1- isoform) while others tended to be expressed preferentially in later cleavage and associated with blastocyst formation (ZO-2, ZO-1+ isoform, DSC-2), illustrating an expression pattern broadly similar to mouse cleavage stages. Human embryo transcript detection was significantly decreased when reverse transcription was performed in solid phase to generate a bead/cDNA transient library rather than after mRNA elution from beads. Transcript detection tended to be positively correlated with embryo morphological grade using the solid phase method. In blastocysts, occludin TM4–, ZO-1+ and DSC2 transcripts were the most susceptible to failure of detection, indicative of low levels of expression which may impact on trophectoderm differentiation competence. Immunoconfocal microscopy analysis of selected adhesion and TJ proteins in human embryos indicated poor membrane assembly compared with mouse blastocysts, which may further affect embryo viability

a-Thalassemia trait caused by frameshift mutations in exon 2 of the a2-globin gene: HBA2:c.131delT and HBA2:c.143delA

We describe two frameshift mutations associated with an a-thalassemia (a-thal) phenotype, identified in three unrelated individuals investigated for persistent microcytosis. The first mutation, HBA2:c.131delT, is located in codon 43, and the second, HBA2:c.143delA, is located in codon 47. Both are due to single base pair deletions that cause a frameshift and a premature termination codon (PTC) at positions 48/49. The presence of a PTC at this position has been documented to result in nonsense mediated mRNA decay that would account for the thalassemic phenotype

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 α-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 α-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 α-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 α-globin protein

Hb lynwood [a107(G14) (-T) (a2) HBA2:c.323delT)] in conjunction with the a3.7 deletion produces a moderately severe a-thalassemia phenotype

We describe a novel frameshift mutation associated with an a-thalassemia (a-thal) phenotype in a patient of Sudanese origin investigated for persistent microcytosis. In addition to the a3.7 deletion, a novel mutation on the a2 gene was detected: HBA2:c.323delT. This mutation causes a frameshift at codon 107 of the a2 gene. The result is a disturbed amino acid sequence for the following 24 amino acids, and a premature termination codon at position 132. © 2011 Informa Healthcare USA, Inc