Determination of telomere length is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH. We here present a quantitative flow cytometric approach, Q-FISHFCM, for evaluation of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3probe and DNA staining with propidium iodide. A simple and rapid protocol with results within 30 h was developed giving high reproducibility. One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for potential differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, blood, lymph nodes and tonsils. A significant correlation was found between Southern blotting and Q-FISHFCMtelomere length values ( P = 0.002). The mean sub-telomeric DNA length of the tested cell lines and clinical samples was estimated to be 3.2 kbp. With the Q-FISHFCMmethod the fluorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase.
SummaryMinimal residual disease (MRD) assessment is a powerful prognostic factor for determining the risk of relapse in childhood acute lymphoblastic leukaemia (ALL). In this Swedish multi-centre study of childhood ALL diagnosed between 2002 and 2006, the MRD levels were analysed in 726 follow-up samples in 228 children using real-time quantitative polymerase chain reaction (RQ-PCR) of rearranged immunoglobulin/T-cell receptor genes and multicolour flow cytometry (FCM). Using an MRD threshold of 0AE1%, which was the sensitivity level reached in all analyses, the concordance between RQ-PCR and FCM MRD values at day 29 was 84%. In B-cell precursor ALL, an MRD level of ‡0AE1% at day 29 predicted a higher risk of bone marrow relapse (BMR) with both methods, although FCM was a better discriminator. However, considering the higher median MRD values achieved with RQ-PCR, a higher MRD cut-off ( ‡0AE2%) improved the predictive capacity of RQ-PCR. In T-ALL, RQ-PCR was notably superior to FCM in predicting risk of BMR. That notwithstanding, MRD levels of ‡0AE1%, detected by either method at day 29, could not predict isolated extramedullary relapse. In conclusion, the concordance between RQ-PCR and FCM was high and hence both methods are valuable clinical tools for identifying childhood ALL cases with increased risk of BMR.
This paper presents a multiproxy high-resolution study of the past 2600 years for Seebergsee, a small Swiss lake with varved sediments at the present tree-line ecotone. The laminae were identified as varves by a numerical analysis of diatom counts in the thin-sections. The hypothesis of two diatom blooms per year was corroborated by the 210Pb and 137Cs chronology. A period of intensive pasturing during the ‘Little Ice Age’ between ad 1346 and ad 1595 is suggested by coprophilous fungal spores, as well as by pollen indicators of grazing, by the diatom-inferred total phosphorus, by geochemistry and by documentary data. The subsequent re-oligotrophication of the lake took about 88 years, as determined by the timelag between the decline of coprophile fungal spores and the restoration of pre-eutrophic nutrient conditions. According to previous studies of latewood densities from the same region, cold summers around ad 1600 limited the pasturing at this altitude. This demonstrated the socio-economic impact of a single climatic event. However, the variance partitioning between the effects of land use and climate, which was applied for the whole core, revealed that climate independent of land use and time explained only 1.32% of the diatom data, while land use independent of climate and time explained 15.7%. Clearly land use in‘ uenced the lake, but land use was not always driven by climate. Other factors beside climate, such as politics or the introduction of fertilizers in the seventeenth and eighteenth centuries also in‘ uenced the development of Alpine pasturing.
The present study reports results of qualitative melissopalynological analyses of Finnish honey between the years 2000-2007 and changes in its pollen content from the period 1960-2007. Altogether the pollen content of 734 honey samples was analysed with an average of 415 pollen grains counted from a sample. Pollen of Trifolium repens type, Rubus spp., Salix spp. and the Brassicaceae family were present in more than 90% of the samples, and these pollen types were also found in the highest proportions. Annual variation in the relative amounts of the most numerous pollen types could be as high as 10%. On the basis of the pollen spectra of the honey samples, four regions of forage plants for bees could be identified in Finland. In the period between 1960 and 2007, the most marked change observed was that the percentage of the Trifolium spp. pollen type had decreased from 70% to 10%, while the proportions of Brassicaceae and Rosaceae pollen types showed a corresponding increase.
Over the last decade, telomere length (TL) has gained attention as a potential biomarker in cancer disease. We previously reported that long blood TL was associated with a poorer outcome in patients with breast cancer and renal cell carcinoma. Based on these findings, we hypothesized that certain immunological components may have an impact on TL dynamics in cancer patients. One aim of the present study was to investigate a possible association between serum cytokines and TL of peripheral blood cells, tumors and corresponding kidney cortex, in patients with clear cell renal cell carcinoma. For this purpose, a multiplex cytokine assay was used. Correlation analysis revealed significant positive correlations between tumor TL and peripheral levels of three cytokines (IL-7, IL-8 and IL-10). In a parallel patient group with various kidney tumors, TL was investigated in whole blood and in immune cell subsets in relation to peripheral levels of regulatory T cells (Tregs). A significant positive association was found between whole blood TL and Treg levels. However, the strongest correlation was found between Tregs and TL of the T lymphocyte fraction. Thus, patients with higher Treg levels displayed longer T cell telomeres, which might reflect a suppressed immune system with fewer cell divisions and hence less telomere shortening. These results are in line with our earlier observation that long blood TL is an unfavorable prognostic factor for cancer-specific survival. In summary, we here show that immunological components are associated with TL in patients with renal cell carcinoma, providing further insight into the field of telomere biology in cancer.
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