Epsilon Haemoglobin Specific Antibodies with Applications in Noninvasive Prenatal Diagnosis

Invasive procedures for prenatal diagnosis are associated with increased risk of abortion; thus, development of noninvasive procedures would be beneficial. Based on the observation that embryonic nucleated red blood cell (NRBC) crosses the placenta and enters the circulation of pregnant women, the ability to identify such cell would allow development of such procedures. Identification of NRBCs in blood samples would be possible provided that specific antibodies are available. Here we have isolated recombinant antibodies using phage display. From the panel of antibody fragments specifically recognising ε-Hb, one was chosen for further characterization, DAb1. DAb1 binds to ε-Hb both in Western blots and immunocytochemistry. Several ε-Hb positive cells were detected in a blood sample taken as postchorionic villus sampling (CVS). To evaluate the sensitivity of the method, K562 cells (which express ε-Hb) were spiked in a blood sample followed by staining in solution and FACS analysis

Are Keratoacanthomas Variants of Squamous Cell Carcinomas? A Comparison of Chromosomal Aberrations by Comparative Genomic Hybridization

Keratoacanthoma (KA) is a benign keratinocytic neoplasm that usually presents as a solitary nodule on sun-exposed areas, develops within 6–8 weeks and spontaneously regresses after 3–6 months. KAs share features such as infiltration and cytological atypia with squamous cell carcinomas (SCCs). Furthermore, there are reports of KAs that have metastasized, invoking the question of whether or not KA is a variant of SCC. To date no reported criteria are sensitive enough to discriminate reliably between KA and SCC, and consequently there is a clinical need for discriminating markers. We screened fresh frozen material from 132 KAs and 37 SCCs for gross chromosomal aberrations by using comparative genomic hybridization (CGH). Forty-nine KAs (37.1%) and 31 SCCs (83.7%) showed genomic aberrations, indicating a higher degree of chromosomal instability in SCCs. Gains of chromosomal material from 1p, 14q, 16q, 20q, and losses from 4p were seen significantly more frequently in SCCs compared with KAs (P-values 0.0033, 0.0198, 0.0301, 0.0017, and 0.0070), whereas loss from 9p was seen significantly more frequently in KAs (P-value 0.0434). The patterns of recurrent aberrations were also different in the two types of neoplasms, pointing to different genetic mechanisms involved in their developments

Localization of the Werner Protein Together with H2AX in γ-Irradiation-Induced Neoplastic Transformed Human Mesenchymal Stem Cells

The H2A histone family, member X (H2AX), and Werner (WRN) are important proteins for genome and telomere maintenance. WRN has a major role in genome stability, particularly during DNA replication, transcription, recombination, and repair of DNA double-stranded breaks (DSBs) via base excision repair, homologous recombination, or nonhomologous end joining. H2AX plays a part in the rapid, sensitive, cellular response to the ionizing radiation or DNA-damaging chemotherapeutic agents that cause DSBs. This occurs when radiation-induced DSBs trigger the activation of H2AX and begin the damage-repair process. In this study, we investigate the role and localization of WRN together with DNA damage marker H2AX at the radiation-induced damaged sides of both the telomere-immortalized human mesenchymal stem cells (hMSCs) and hMSC-telomere 1 (hMSC-telo1) and in control primary hMSCs. Phosphorylated H2AX and WRN immune staining enabled evaluation of overall genomic integrity and damage/repair. We used peptide nucleic acid-fluorescent in situ hybridization to visualize telomeric damage as a short-term effect. A high-level WRN signal was observed in both primary hMSCs and telomerase-immortalized hMSCs after the cells had been subjected to infrared radiation. Afterward, the irradiation level of the WRN signals decreased considerably, especially in later passages, and WRN was nondetectable in the latest passages of the hMSC Telo1 cells. Contrary to this finding, we found that levels of H2AX phosphorylation in hMSC-telo1 cells increased with time, especially at telomere sides, suggesting that cells with long telomeres and high telomerase activity have the advantage of maintaining genomic integrity. Evaluation of localization of WRN signals demonstrated that WRN does not leave the nucleolus after irradiation. We did not detect the WRN signal at the telomere sides, but we could detect H2AX at the telomeric sides. Thus, our overall data suggest that the WRN protein is not involved in irradiation-induced DNA damage/repair, even at telomeric sides in hMSC and hMSC-telo1

Telomere shortening correlates to dysplasia but not to DNA aneuploidy in longstanding ulcerative colitis

Background Ulcerative colitis (UC) is a chronic, inflammatory bowel disease which may lead to dysplasia and adenocarcinoma in patients when long-lasting. Short telomeres have been reported in mucosal cells of UC patients. Telomeres are repetitive base sequences capping the ends of linear chromosomes, and protect them from erosion and subsequent wrongful recombination and end-to-end joining during cell division. Short telomeres are associated with the development of chromosomal instability and aneuploidy, the latter being risk factors for development of dysplasia and cancer. Specifically, the abrupt shortening of one or more telomeres to a critical length, rather than bulk shortening of telomeres, seems to be associated with chromosomal instability. Methods We investigated possible associations between dysplasia, aneuploidy and telomere status in a total of eight lesions from each of ten progressors and four nonprogressors suffering from longstanding UC. We have analyzed mean telomere length by qPCR, as well as the amount of ultra-short telomeres by the Universal STELA method. Results An increased amount of ultra-short telomeres, as well as general shortening of mean telomere length are significantly associated with dysplasia in longstanding UC. Furthermore, levels of ultra-short telomeres are also significantly increased in progressors (colons harbouring cancer/dysplasia and/or aneuploidy) compared to nonprogressors (without cancer/dysplasia/aneuploidy), whereas general shortening of telomeres did not show such associations. Conclusions Our data suggest that ultra-short telomeres may be more tightly linked to colorectal carcinogenesis through development of dysplasia in UC than general telomere shortening. Telomere status was not seen to associate with DNA aneuploidy