Mean Telomere Restriction Fragment Research Articles

Short Leukocyte Telomere Length is Associated with Abdominal Aortic Aneurysm (AAA)

Objective Telomeres are specialised DNA structures present at the ends of linear chromosomes, which shorten with each successive cell division and the length of which represents cellular biological age. The aim of this study was to determine the relationship between abdominal aortic aneurysm (AAA) and white cell telomere length. Methods Peripheral blood samples were collected from 190 patients with AAA and 183 controls. Genomic DNA was extracted from white cells and telomere lengths determined using a chemiluminescence technique. Results The mean white cell telomere length was significantly lower in AAA patients compared to controls (median age 66 years in both groups), with a mean difference of 189 base pairs (bp) (95% confidence interval 77 bp to 301 bp, P = 0.005). This relationship between case–control status and mean telomere restriction fragment (TRF) length did not change after adding other risk factors into a multiple regression model. The risk of having AAA doubled in patients with a mean TRF length in the lowest quartile compared to patients with a mean TRF length in the highest quartile (odds ratio 2.30, 95% Confidence Interval 1.28–4.13, P = 0.005). Conclusion Our data show that patients with AAA have shorter leukocyte telomere length compared to controls. This suggests that vascular biological aging may have a role in the pathogenesis of AAA.

Molecular Distinctions between Stasis and Telomere Attrition Senescence Barriers Shown by Long-term Culture of Normal Human Mammary Epithelial Cells

Normal human epithelial cells in culture have generally shown a limited proliferative potential of approximately 10 to 40 population doublings before encountering a stress-associated senescence barrier (stasis) associated with elevated levels of cyclin-dependent kinase inhibitors p16 and/or p21. We now show that simple changes in medium composition can expand the proliferative potential of human mammary epithelial cells (HMEC) initiated as primary cultures to 50 to 60 population doublings followed by p16-positive, senescence-associated beta-galactosidase-positive stasis. We compared the properties of growing and senescent pre-stasis HMEC with growing and senescent post-selection HMEC, that is, cells grown in a serum-free medium that overcame stasis via silencing of p16 expression and that display senescence associated with telomere dysfunction. Cultured pre-stasis populations contained cells expressing markers associated with luminal and myoepithelial HMEC lineages in vivo in contrast to the basal-like phenotype of the post-selection HMEC. Gene transcript and protein expression, DNA damage-associated markers, mean telomere restriction fragment length, and genomic stability differed significantly between HMEC populations at the stasis versus telomere dysfunction senescence barriers. Senescent isogenic fibroblasts showed greater similarity to HMEC at stasis than at telomere dysfunction, although their gene transcript profile was distinct from HMEC at both senescence barriers. These studies support our model of the senescence barriers encountered by cultured HMEC in which the first barrier, stasis, is retinoblastoma-mediated and independent of telomere length, whereas a second barrier (agonescence or crisis) results from telomere attrition leading to telomere dysfunction. Additionally, the ability to maintain long-term growth of genomically stable multilineage pre-stasis HMEC populations can greatly enhance experimentation with normal HMEC.

Short leucocyte telomere length is associated with abdominal aortic aneurysm

1078-5884/$36 a 2010 European Socie doi:10.1016/j.ejvs.2010.01.013 Abstract Objective: Telomeres are specialised DNA structures present at the ends of linear chromosomes, which shorten with each successive cell division and the length of which represents cellular biological age. The aim of this study was to determine the relationship between abdominal aortic aneurysm (AAA) and white cell telomere length. Methods: Peripheral blood samples were collected from 190 patients with AAA and 183 controls. Genomic DNA was extracted from white cells and telomere lengths determined using a chemiluminescence technique. Results: The mean white cell telomere length was significantly lower in AAA patients compared to controls (median age 66 years in both groups), with a mean difference of 189 base pairs (bp) (95% confidence interval 77 bp to 301 bp, P Z 0.005). This relationship between caseecontrol status and mean telomere restriction fragment (TRF) length did not change after adding other risk factors into a multiple regression model. The risk of having AAA doubled in patients with a mean TRF length in the lowest quartile compared to patients with a mean TRF length in the highest quartile (odds ratio 2.30, 95% Confidence Interval 1.28e4.13, P Z 0.005). Conclusion: Our data show that patients with AAA have shorter leukocyte telomere length compared to controls. This suggests that vascular biological aging may have a role in the pathogenesis of AAA. a 2010 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

Non-Random Telomere Lengthening at Specific Chromosome Ends as a Novel Clonal Event in Chronic Myeloid Leukemia

It is widely accepted that chromosomal telomere dysfunction caused either by telomere shortening or lesions in the capping machinery is an important factor in carcinogenesis. However, in our recent study using quantitative FISH (Q-FISH), telomere restriction fragment (TRF) analysis and fiber FISH techniques on the measurement of telomere length in 32 cases of chronic myeloid leukemia (CML), we found that telomere lengthening at some specific chromosome ends is apparently a non-random event showing a clonal nature.Methods:TRF: genomic DNA was digested by frequently cutting restriction enzymes. After gel electrophoresis and Southern blotting, the blotted DNA was hybridized to a digoxigenin (DIG)-labeled probe specific for telomeric repeats. A DIG-specific antibody covalently coupled to alkaline phosphate followed by the chemiluminescence detection was used to detect telomere signals. The quantitative measurements of mean TRF length can be reached by scanning the signals on the film and analyzing them with the computer software.Q-FISH: chromosomes on cytogenetic slides were hybridized with a peptide nucleic acid (PNA) telomere probe (Panagene, Korea). The leukemia cells can be traced by the particular chromosome rearrangement presented in the metaphase cells, e.g. t(9;22). The signal intensity, which is proportional to telomere length, of each individual telomere was automatically measured with the software of the imaging system (ISIS 2 MetaSystems, Belmont, MA). The relative telomere length was calculated using the ratio of individual signal intensity to the standard deviation.fiber FISH: fiber FISH was performed with cohybridization of a specific subtelomere probe (with a known size and close to the telomere site of interest) and the telomere probe to the DNA/chromatin fibers preparation on the same slide.Results: The telomere lengthening at short arm of a X chromosome (Xp) was the most frequent event (seen in about 50% of CML cases we studied) in leukemia cells that can be identified by the chromosome 9 and 22 translocation [t(9;22)(q34;q11.2)]. The longest telomere length at Xp end for the CML cases reached 200 kb, which is about 20-fold longer than in normal cells. The other telomeres involved in the non-random lengthening include 18p (7/32), Yp (4/32), 4q (4/32), 5p (3/32), 7q (3/32), and 15p (3/32).Conclusion: While the relationship between the sequence organization for telomere shortening and their function is relatively clear, it has not been well stated if telomere lengthening at specific chromosome ends could be involved in cell proliferation, in maintenance of the genome stability and in evolution of the cancer. Our findings have shown the first evidence that the telomere lengthening at some specific chromosome ends is such a salient clonal event. Further investigation on picking up specific individual telomere lengthening in leukemia cells would greatly aid studies of chromosomal stability, telomerase activity, proliferative capacity and the evaluation of clinical status and thus one can use telomere length as an indicator to follow-up the cancer progression, to evaluate the treatment efficacy and to predict the prognosis in cancer.

Telomere length is shorter in healthy offspring of subjects with coronary artery disease: support for the telomere hypothesis

Background:Telomeres are shorter in subjects with coronary artery disease (CAD) and may indicate premature biological ageing. However, whether shorter telomeres are a primary abnormality or secondary to the disease is...

Telomere biology in giant cell tumour of bone

Giant cell tumour of bone (GCTB) is a benign bone tumour known for the unpredictable clinical behaviour of recurrences and, in rare instances, distant metastases. It consists of uniformly distributed osteoclastic giant cells in a background of mononuclear rounded and spindle-shaped cells. Cytogenetically, telomeric associations are the most common chromosomal aberrations, which, however, are normally almost exclusively found in high-grade malignancies. GCTB has often been regarded as a polyclonal tumour, but more recently a recurrent specific aberration was reported, which suggests a possible role for disturbed telomere maintenance. Here we further investigate telomere maintenance in GCTB using 19 samples from 19 patients. A combination of immunofluorescence and FISH was performed, applying antibodies directed against promyelocytic leukaemia body-related antigen and hTERT and using telomere peptide nucleic acid probes. The TRAP assay and telomere restriction fragment length analysis were performed for functional detection of telomerase activity and alternative telomere lengthening. Both osteoclastic giant cells and mononuclear cells showed positivity for hTERT and promyelocytic leukaemia body-related antigen. In most mononuclear cells, co-expression was present. The TRAP assay demonstrated heterogeneous telomerase activity, while telomere restriction fragment length analysis showed non-heterogeneous telomere lengths, indicating the absence of alternative telomere lengthening. Confocal microscopy showed stereometric co-localization of nucleolin with promyelocytic leukaemia body-related antigen in association with telomeres in the spindle-shaped cells. hTERT was more diffusely distributed throughout the nucleus. Our results show that GCTB demonstrates remarkable telomere maintenance of activated telomerase and inactivated alternative telomere lengthening in the presence of normal mean telomere restriction fragment lengths. These findings strongly suggest that these aggregates, while activating telomerase, are part of a structural telomere protective-capping mechanism rather than of a telomere-lengthening mechanism. Telomere maintenance could be considered an important key factor in the pathogenesis of GCTB.

Signal modelization for improved precision of assessment of minimum and mean telomere lengths

Telomere length is an important measure of cell and tissue regenerative capacities. The mean telomere length is classically used as global indicator of a tissue telomere length. In skeletal muscle, which is made of postmitotic myonuclei and satellite cells (muscle stem cells), minimum telomere length is also used to assess the telomere length of satellite cells and newly incorporated myonuclei. At present, the estimation of the method reproducibility during the assessment of mean and minimum telomere length using Southern blot analysis has never been documented. The aim of this report is to describe a signal modelization for improved precision of assessment of minimum and mean telomere lengths and to document the method reproducibility. Telomeres are assessed using a Southern technique where the gel is directly hybridized with the specific probe without the membrane-transferring step in order to prevent telomeric low signal loss. We found that the improved signal analysis for determination of telomere length is associated with coefficients of variation ranging from 1.37 to 4.29% for the mean telomeric restriction fragment (TRF) length and from 2.04 to 4.95% for the minimum TRF length. Improved method reproducibility would allow saving time and biological material as duplicate and triplicate measurement of the same sample is no longer required.

Telomere length in normal and neoplastic canine tissues

To determine the mean telomere restriction fragment (TRF) length in normal and neoplastic canine tissues. 57 solid-tissue tumor specimens collected from client-owned dogs, 40 samples of normal tissue collected from 12 clinically normal dogs, and blood samples collected from 4 healthy blood donor dogs. Tumor specimens were collected from client-owned dogs during diagnostic or therapeutic procedures at the University of Illinois Veterinary Medical Teaching Hospital, whereas 40 normal tissue samples were collected from 12 control dogs. Telomere restriction fragment length was determined by use of an assay kit. A histologic diagnosis was provided for each tumor by personnel at the Veterinary Diagnostic Laboratory at the University of Illinois. Mean of the mean TRF length for 44 normal samples was 19.0 kilobases (kb; range, 15.4 to 21.4 kb), and the mean of the mean TRF length for 57 malignant tumors was 19.0 kb (range, 12.9 to 23.5 kb). Although the mean of the mean TRF length for tumors and normal tissues was identical, tumor samples had more variability in TRF length. Telomerase, which represents the main mechanism by which cancer cells achieve immortality, is an attractive therapeutic target. The ability to measure telomere length is crucial to monitoring the efficacy of telomerase inhibition. In contrast to many other mammalian species, the length of canine telomeres and the rate of telomeric DNA loss are similar to those reported in humans, making dogs a compelling choice for use in the study of human anti-telomerase strategies.

Expression of the p16INK4A Gene Is Associated Closely with Senescence of Human Mesenchymal Stem Cells and Is Potentially Silenced by DNA Methylation During In Vitro Expansion

The precise biological characteristics of human mesenchymal stem cells (hMSCs), including growth regulatory mechanisms, have not yet been defined. Using 29 strains of hMSCs isolated from bone marrow, we have performed extensive analyses of the growth profiles of hMSCs in vitro. All 29 strains stopped proliferating with a mean population doubling (PD) of 28, although there was a considerable difference among strains. The mean telomere restriction fragment length of the cells passaged twice correlated well with the final number of PDs in each strain, suggesting the value of this measurement to be predictive of the growth potential of hMSCs. The expression level of the p16INK4A gene was associated closely with the PD number of each strain (p = .00000001). Most of the p16INK4A-positive cells were Ki67-negative and senescence associated beta-galactosidase-positive, and the suppression of p16INK4A gene expression by small interfering RNA in senescent hMSCs reduced the number of senescent cells and endowed them with the ability to proliferate. Twenty-five of the 29 strains showed a steady gradual increase in the expression of p16INK4A. The remaining four strains (13.8%) showed different profiles, in which DNA methylation in the promoter region occurred in vitro. One of the four strains continued to proliferate for much longer than the others and showed chromosomal aberrations in the later stages. These results indicated p16INK4A to be a key factor in the regulation of hMSC growth, and, most importantly, careful monitoring of DNA methylation should be considered during the culture of hMSCs, particularly when a prolonged and extended propagation is required.

Change in the telomere length distribution with age in the Japanese population

Telomeres play a role in cellular aging and they may also contribute to the genetic basis of human aging and longevity. A gradual loss of the telomeric repeat sequences has been reported in adult tissue specimens. This study determined the percentage of telomere restriction fragment in various molecular-sized regions in addition to measuring the average telomere length. Mean telomere restriction fragment (TRF) length was determined by Southern blot analysis using a longer telomeric repeat probe with higher sensitivity. A significant decrease in longer telomere fragments and a quick increase in the shortest fragments were observed, especially in male subjects. There was a tendency that the age-adjusted telomere length was longer in females than that observed in males, while males lose the telomeric sequence faster than females. These data indicated that the percentage of longer telomeres fragments decreased, while the shortest fragments increased quickly with age. In addition, the longest telomere fragments decreased and the short fragments increased with a relatively stable frequency with age. There was also a significant difference in the longest telomere fragment percentage between males and female in their 40s and 50s, whereas no difference was observed in the mean TRF length. Interestingly, the changing rate of the longest and the shortest range group of TRF percentage associated with aging seemed quite different between before and after 50-year old with a gender-related contrast. This contrast implies a drastic change around the age of 50 of unknown factors that affect telomere attrition.

Telomere length and cardiovascular risk factors in a middle‐aged population free of overt cardiovascular disease

Evidence assembled over the last decade shows that average telomere length (TL) acts as a biomarker for biological aging and cardiovascular disease (CVD) in particular. Although essential for a more profound understanding of the underlying mechanisms, little reference information is available on TL. We therefore sought to provide baseline TL information and assess the association of prevalent CVD risk factors with TL in subjects free of overt CVD within a small age range. We measured mean telomere restriction fragment length of peripheral blood leukocytes in a large, representative Asklepios study cohort of 2509 community-dwelling, Caucasian female and male volunteers aged approximately 35-55 years and free of overt CVD. We found a manifest age-dependent telomere attrition, at a significantly faster rate in men as compared to women. No significant associations were established with classical CVD risk factors such as cholesterol status and blood pressure, yet shorter TL was associated with increased levels of several inflammation and oxidative stress markers. Importantly, shorter telomere length was associated with an increasingly unhealthy lifestyle, particularly in men. All findings were age and gender adjusted where appropriate. With these cross-sectional results we show that TL of peripheral blood leukocytes primarily reflects the burden of increased oxidative stress and inflammation, whether or not determined by an increasingly unhealthy lifestyle, while the association with classical CVD risk factors is limited. This further clarifies the added value of TL as a biomarker for biological aging and might improve our understanding of how TL is associated with CVD.

The Underlying Mechanism of Telomere Maintenance in Human Bone Marrow-Derived Mesenchymal Stem Cells.

Objective: Human bone marrow-derived mesenchymal stem cells(MSCs) are thought to be promising tools in cell and gene therapy. Unfortunately, the low frequency of MSCs in bone marrow and rapid aging in in vitro expansion, which profoundly compromise their proliferative capacity, give rise to a huge hindrance for their clinical use. Previous study indicated that MSCs would undergo quick telomere shortening as well as reduced replicative capacity during in vitro expansion. These findings suggested that MSCs' telomere loss might be associated with their decreased proliferative and differentiative potentials. However, the mechanisms by which MSCs maintain their telomere homeostasis have not yet been fully addressed to date. In the present study, we compared the telomere length, the distribution pattern of telomeric repeat binding factor 1(TRF1) between MSCs and other telomerase-positive cells or telomerase-negative cells, detected extrachromosomal telomeric repeat DNA (ECTR DNA) in MSCs and the variation of telomerase activity during cell cycle progression in order to unveil the mystery of telomere regulation in MSCs.METHODS: MSCs were isolated from healthy human bone marrow (n=34) by the plastic adherence protocols and identified by flow cytometry with markers of CD14, CD45, CD44, HLA-DR, CD34, CD29 and CD166. Telomere length and ECTR DNA were detected with Southern hybridization. The TRF1 distribution were probed with immunofluorescence staining. Telomeric repeat amplification protocol (TRAP ) and/or semi-quantitive Western blot assay were performed to determine the telomerase activity in MSCs, MSCs-derived adipocytes and telomerase levels during cell cycle progression. MSCs were synchronized by serum starvation and Aphidicolin treatment for the aforementioned assay.RESULTS: The mean telomere restriction fragment (mTRF) in MSCs was 8.0 kbp( range, 2.7 kbp-18.0 kbp), similar to telomerase-positive HeLa cells 6.0 kbp (range, 2.7 kbp-8.6 kbp) and 293T cells 5.0 kbp(range, 2.7 kbp-8.6 kbp); while the mTRF in telomerase-negative cells WI-38–2RA was 21.2 kb (range 2.0 kbp->21.2 kbp). The results indicated that telomere length in MSCs and HeLa cells were shorter and relatively more homogeneous than WI-38–2RA cells. TRF1 did not coincide with promyelocytic leukemia (PML) nuclear body in MSCs and HeLa cells while it exclusively did in WI-38–2RA cells. ECTR DNA was negative in MSCs and HeLa cells but positive in WI-38–2RA cells. Detected by TRAP, telomerase activity in MSCs(n=34) was negative with relative telomerase activity (RTA) of 1.44%±0.77%, but it was positive in MSCs-derived adipocytes (n=3) with RTA of 11.80±2.52%(P<0.001). Moreover, a cell cycle-dependent expression profile of telomerase was found in MSCs when they were synchronized by serum starvation and Aphidicolin treatment. Untreated MSCs expressed extremely low level of telomerase probed by Western blot with the 2C4 mAb, but the telomerase level had significantly increased when these cells were trapped in S phase.CONCLUSION: Since MSCs possessed similar features to telomerase-positive cells in telomere length, TRF1 localization pattern and ECTR DNA which were distinct from telomerase-negative ALT cells, and they had increased telomerase activity following differentiation into adipocytes and entrance into S phase, We postulated that the telomere in MSCs was maintained by telomerase pathway other than ALT pathway. The telomerase expression level of MSCs was tightly regulated with cell cycle progression.

Relationship between telomere length and radiosensitivity in various human cancer cell lines

Objective: To investigate the relationship between telomere length and radiosensitivity in various human cancer cell lines with the expectation to find a valid and common predictor of radiosensitivity for different cancers. Methods: Eight human cancer cell lines were used, including five human breast cancer cell lines (ZR-75-30, MCF-7, MDA-MB-435S, T-47-D, F539-1590), two human larynx squamous carcinoma cell lines (Hep-2 and Hep-2R) and a human malignant glioma cell line (U251). Among them, the radioresistant cell line Hep-2R was isolated and established from a radiosensitive human larynx squamous carcinoma cell line Hep-2 by our center. The radiobiological characteristics of the eight lines were analyzed by the method of colony-forming assay and the radiosensitivity parameters were calculated. Telomere length was analyzed by TRF (mean Telomere Restriction Fragments) length assay. Results: The radioresistance of Hep-2R cell line proved to be stable in long-term passaged cultures as well as in frozen samples. Radiosensitivity parameters are different among those lines. The SF2 values of Hep-2 and U251 are 0.4148 and 0.7520, respectively; The SF2 values of breast cancer cell lines are between those of Hep-2 and U251. The TRF of Hep-2R is 11.12Kb, longer than three times that of its parental counterpart. There is a positive correlation both between SF2 and TRF (r=0.786, P<0.05), and between D0 and TRF (r=0.905, P<0.01). Conclusion: It is concluded that radiosensitivity and telomere length (TRF) are negatively correlated, TRF could be a valid predictor for radiosensitivity.

Telomere length versus hormonal and bone mineral status in healthy elderly men

Telomeres, the termini of linear chromosomes, exert a key role in the process of cellular ageing. Progressive telomere shortening is implicated in senescence in vitro and ample evidence exists to support the hypothesis that telomere length is correlated with chronological age and ageing phenotypes in vivo. In this study, we assessed whether mean telomere length of peripheral blood leukocytes predicts age-associated bone loss and/or is related to sex steroid status in an elderly healthy male population (71–86 years). Out of this population, we selected 110 samples for telomere restriction fragment (TRF) length analysis. Fasting blood was analysed for testosterone, estradiol, sex hormone binding globulin and biochemical markers of bone turnover. Also, the bioavailable fractions of sex steroids were calculated. Bone mineral density was measured at baseline and longitudinal follow-up was available for 84 men. We found that mean TRF length was inversely correlated with age ( r = −0.19; P = 0.049). Although no correlations were found with sex steroids or BMD at baseline, age corrected mean TRF length was associated with longitudinal bone loss for different distal forearm sites ( P < 0.05). Further studies are required to confirm our results, yet in this study, the predictive value of telomere length for bone loss appears to be substantial, hence underscoring the role of telomere length as a biomarker of ageing phenotypes.

Mapping of a Major Locus that Determines Telomere Length in Humans

Telomere length is a crucial factor for both normal chromosomal function and senescence. Mean telomere length in humans shows considerable interindividual variation and strong genetic determination. To see if a locus (or loci) affecting telomere length in humans could be mapped, we performed a quantitative-trait linkage analysis of mean leukocyte telomere-restriction-fragment (TRF) lengths, measured by Southern blotting, in 383 adult subjects comprising 258 sib pairs. Heritability of mean (+/-SE) TRF was 81.9%+/-11.8%. There was significant linkage (LOD score 3.20) of mean TRF length to a locus on chromosome 12, which explained 49% of the overall variability in mean TRF length. We present preliminary analysis of a strong candidate gene in the region, the DNA helicase DDX11. In conclusion, we report mapping of the first locus that determines mean telomere length in humans. Identification of the gene involved and elucidation of its mechanism of action could have important implications for our understanding of chromosomal assembly, telomere biology, and susceptibility to age-related diseases.

Telomere attrition in white blood cell correlating with cardiovascular damage.

Aging is a major risk factor for cardiovascular disease. Chronological aging does not always parallel biological aging, but there is no reliable biomarker for the latter. In the present study, we tested the hypothesis that telomere attrition in white blood cells is related to endothelial dysfunction and the extent of atherosclerosis, and thus may serve as a useful marker for biological aging. We evaluated telomere lengths in white blood cells by measuring the mean telomere restriction fragment length (mTRFL), as well as endothelial function by flow mediated dilatation (FMD) in the brachial artery, in patients with various degrees of cardiovascular damage and in normal subjects. Cardiovascular damage was assessed by a cardiovascular damage (CVD) score, with 1 point being given for the presence of each cardiovascular risk factor (hypertension, hyperlipidemia and diabetes) and for each event (angina, myocardial infarction, cerebrovascular event and peripheral vascular disease). Subset analysis of CVD score groups revealed that mTRFL and FMD decreased in the rank order of CVD score. Although mTRFL was inversely correlated with age, telomere index, defined as the ratio of TRFL to TRFL predicted by age, also decreased with increase in CVD score. These results indicate that telomere attrition in white blood cells is more closely associated with endothelial damage and atherosclerosis than is chronological aging, supporting the hypothesis that mTRFL in white blood cells is a useful marker for biological aging of the cardiovascular system.

Telomere length study in celiac disease

Objectives Telomeres are important structures that are critical for maintaining chromosomal integrity and cell surveillance. The aim of this study was to analyze telomere length in patients with celiac disease (CD), a multifactorial disorder with a strong genetic component that exhibits genomic instability and cancer predisposition, particularly T-cell lymphomas. Methods Telomere length measured by telomere restriction fragments (TRF) was studied in small intestinal biopsy (SIB) samples and peripheral blood lymphocytes (PBL) from 20 untreated CD patients, distributed according to the clinical form as four asymptomatic, five monosymptomatic, and 11 polysymptomatic individuals. We also analyzed TRF from normal peripheral blood lymphocytes and normal biopsy samples as normal controls. Results TRF evaluation showed a significant telomere shortening in SIB samples from CD patients (4.21 ± 0.29 Kb) compared to PBL from the same individuals (9.17 ± 0.35 Kb) ( p < 0.0001), independently of clinical form. Mean TRF peak values from normal biopsy samples were significantly higher (8.33 ± 0.38 Kb) than those observed in CD biopsy samples ( p < 0.001). No differences between TRF values in CD-PBL and normal peripheral blood lymphocytes (8.89 ± 0.37Kb) were found. Conclusions Our findings in patients with CD, a disorder in which the gluten-induced mucosal injury could accelerate telomere shortening, would increase the process of end-to-end fusions resulting in chromosomal changes, supports the hypothesis that genomic instability and telomere reduction may play a role in the cancer predisposition observed in these patients.

Telomeres in neonates: new insights in fetal hematopoiesis.

Progressive telomere shortening occurs in somatic cells, and with increasing donor age a significant decline in telomere length has been shown in various postnatal tissues. In contrast, little is known about changes in telomere length during human fetal development. Therefore, we measured telomere length in the leukocyte fraction of umbilical cord blood samples from 15 preterm (<37 wk of gestation) and 11 full-term (>37 wk of gestation) neonates using the telomere restriction fragment assay. Whereas no differences in mean (+/- SD) telomere restriction fragment between the groups of preterm neonates (8512 +/- 523 bp) and full-term newborns (8323 +/- 503 bp) could be found, significantly longer telomeres (p = 0.002) were found in very low birth weight preterm neonates when compared with low birth weight preterm neonates. In addition, a rapid and significant decline in mean telomere restriction fragment was observed between 27 and 32 wk of gestation (p = 0.02, r = 0.79) followed by a period of no significant loss of telomere repeats between 33 and 42 wk of gestation. These results are consistent with the known almost maximal proliferation rate of hematopoietic progenitor cells before 32 wk of gestation. The initial decrease in telomere restriction fragment could be caused by ontogeny-related functional alterations of hematopoietic cells or differences in stem cell turnover or the rate of telomere loss per cell division.

Analysis of telomere length and thymic output in fast and slow/non-progressors with HIV infection

There are two models for CD4+ T-cell depletion leading to AIDS: a kinetic model and an immune suppression model. In the kinetic model, direct cell killing due to viral replication results in a continuous demand for CD4+ T-cells, which eventually exhausts their capacity for renewal by proliferative mechanisms. In the immune suppression model, CD4+ T-cell decline is due to an indirect global inhibitory effect of the virus on uninfected immune cell function. In order to address differences in the two models, we investigated proliferative history and thymic output in PBMC from the GRIV cohort of fast (FP) and slow/non-progressors (S/NP), and uninfected controls. Proliferative history and thymic output were assessed by measurement of mean telomeric restriction fragment (TRF) length and T-cell receptor Rearrangement Excision Circles (TREC) levels, respectively. Mean TRF lengths were significantly shorter in S/NP (n = 93, 7.59 +/- 0.11 kb) and FP (n = 42, 7.25 +/- 0.15 kb) compared to controls (n = 35, 9.17 +/- 0.19 kb). Mean TRF length in PBMC (n = 9, 7.32 +/- 0.31 kb) and CD4+ enriched fractions (n = 9, 7.41 +/- 0.30 kb) from a subset of non-GRIV HIV-1 infected samples were also significantly smaller than PBMC (n = 8, 9.77 +/- 0.33 kb) and CD4+ fractions (n = 8, 9.41 +/- 0.32 kb) from uninfected controls. Rates of telomeric shortening, however, were similar among S/NP (n = 93, -45 +/- 20 bp/yr), FP (n = 42, -41 +/- 14 bp/yr) and controls (-29 +/- 17 bp/yr). Paralleling differences observed in mean TRF length, TREC levels were significantly reduced in S/NP (n = 10, 3,433 +/- 843 mol/mu and FP (n = 8, 1,193 +/- 413) compared to controls (n = 15, 22,706 +/- 5,089), indicative of a defect in thymopoiesis in HIV-1 infection. When evaluated in the context of reduced thymopoiesis, the difference in mean TRF length between S/NP and controls (1.58 +/- 0.30 kb) is similar to that observed between memory and naïve T-cells (1.4 +/- 0.1 kb), and may reflect perturbations in the peripheral T-cell population due to a decline in thymic output of naïve T-cells rather than increased turnover. Based on the different clinical criteria used to select S/NP and FP, the sight difference in TREC between these two groups suggests the threshold for pathogenesis as a result of naïve T-cell depletion may be quite low, and incremental increases in thymic output may yield substantial clinical results. Future studies regarding therapeutic vaccination, specifically with HIV-1 Tat targeted anti-immunosuppressive vaccines, should address the defect in thymic output in HIV-1 infection by using TREC analysis as a rapid method for biological evaluation.

Telomere loss in peripheral blood mononuclear cells may be moderately accelerated during highly active antiretroviral therapy (HAART).

It has been speculated that infection with HIV-1 may lead to a significant increase in turnover, and subsequent exhaustion, of immune repopulation. Given that telomeric DNA is lost on mitotic replication, telomere lengths can be used as an indirect gauge of this rate. We have analyzed the mean telomere restriction fragment lengths in peripheral blood mononuclear cells (PBMC) from 31 patients with established, though mainly untreated, HIV infection and found them to be no different than those among healthy controls. Our results are in line with several findings in CD4+ cell fractions but contradict a previous report suggesting that telomere shortening contributes to immune failure. Interestingly, after approximately 2 years of subsequent aggressive antiretroviral treatment we found a telomere reduction corresponding to a loss of about 250 base pairs per year; this is roughly tenfold above that predicted from healthy individuals. This could partly result from nucleoside analogue inhibition of the natural telomere replacement enzyme, telomerase-a reverse transcriptase inducible in certain hematopoietic cells. However, this may also indicate accelerated cell replacement on initiation of optimal therapeutic regimes or result from changes in the composition of the PBMC pool. These results suggest careful monitoring of telomere lengths during long-term HAART.