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E Roosnek

Publications and source records attributed to E Roosnek.

At least 19 recordsLinked to original sources
PubMed2006

Impact of high-resolution matching in allogeneic unrelated donor stem cell transplantation in Switzerland.

It is currently unknown what degree of human leukocyte antigen (HLA)-mismatching is acceptable in unrelated donor hematopoietic stem cell transplantation (UD-HSCT). Mismatches at some loci may be more permissive than others. We have analyzed the effect of high-resolution HLA-matching on outcome of all 214 consecutive recipients of UD-HSCT carried out in Switzerland. All typing was by the Swiss reference laboratory. Donor-recipient pairs were HLA-10/10 matched (n=130) or mismatched for either HLA-A/-B/-DRB1/multiple loci (n=33; (HLA-A/-B=10); (-DRB1=8); (multiple=15)); HLA-C (n=29) or HLA-DQ/-DRB3 (n=22; (DQ=16); (-DRB1=6)). The median follow-up was 32 months. Survival probabilities (+/-95% confidence interval) at 3 years were 57 (+/-10)% for recipients of HLA 10/10-matched transplants, 53 (+/-22)% for recipients of HLA-DQ/-DRB3-mismatched transplants, 44 (+/-20)% for recipients of HLA-C-mismatched transplants and 0% for recipients of transplants mismatched at HLA-A/-B/-DRB1/multiple loci (P<0.0001). In multivariate analyses, HLA compatibility was the variable most significantly associated with survival and treatment-related mortality. We found important differences in survival in recipients of UD-HSCT with best results for transplants from 10/10 matched donors. Single mismatches at HLA-DQ/-DRB3 were well tolerated, mismatches at HLA-C had intermediate results and mismatches at HLA-A/-B/-DRB1/multiple loci resulted in poor survival.

Adolescent↗
PubMed2004

Isolated HLA-C mismatches in unrelated donor transplantation for CML.

HLA-incompatibility is a major factor associated with outcome of allogeneic stem cell transplantation, but little is known on the impact of isolated HLA-C mismatches. We analyzed the outcome of 114 CML patients transplanted with marrow from unrelated donors of whom 24 were mismatched for HLA-C only (9/10 match). Univariate estimates of 5-year survival (SRV) (median follow-up: 47 months) in the HLA-matched group were 68+/-12 vs 42+/-20% (P=0.03) for the patients mismatched for HLA-C only and 33+/-33% in the mismatched group (non-HLA-C single mismatches and multiple mismatches) (P=0.0004). Disease stage, GVHD-prophylaxis (T-cell depletion), CMV-status and HLA-incompatibility were the risk factors associated (all P< or =0.005) with poor outcome. In the multivariate analysis, patients mismatched for loci other than HLA-C were at high risk of an adverse outcome (death: RR, 2.9; CI, 1.6-5.4, P=0.008, transplant-related mortality (TRM): RR, 3; CI, 1.5-5.9, P=0.0015). For patients mismatched for HLA-C only, the increased risk was of borderline significance (death: RR, 1.9; CI, 1-3.9, P=0.06, TRM: RR, 2.1; CI, 1-4.5, P=0.07). In spite of their lower expression, HLA-C antigens still represent relevant transplantation barriers that should be considered when searching for an unrelated donor.

Adult↗
PubMed2001

Transfer of the human telomerase reverse transcriptase (TERT) gene into T lymphocytes results in extension of replicative potential.

In most human somatic cells telomeres progressively shorten with each cell division eventually leading to chromosomal instability and cell senescence. The loss of telomere repeats with cell divisions may also limit the replicative life span of antigen-specific T lymphocytes. Recent studies have shown that the replicative life span of various primary human cells can be prolonged by induced expression of the telomerase reverse transcriptase (hTERT) gene. To test whether introduction of hTERT can extend the life span of primary human T lymphocytes, naive CD8(+) T lymphocytes were transfected with retroviral vectors containing the hTERT gene. Transduced T-cell clones expressed high levels of telomerase and either maintained or elongated their telomere lengths upon culture for extended periods of time. Two of the transduced subclones retained a normal cloning efficiency for more than 170 population doublings (PDs). In contrast, T-cell clones transfected with control vectors exhibited progressive telomere length shortening and stopped proliferation at around 108 PDs. Telomerase-positive T clones had a normal 46,XY karyotype, maintained their cytotoxic properties, and showed very little staining for the apoptotic marker annexin-V. These results indicate that ectopic hTERT gene expression is capable of extending the replicative life span of primary human CD8(+) cytotoxic T lymphocytes. (Blood. 2001;98:597-603)

CD8-Positive T-Lymphocytes↗
PubMed2001

Accelerated telomere shortening in hematological lineages is limited to the first year following stem cell transplantation.

Using quantitative fluorescence in situ hybridization and flow cytometry, the telomere length of telomere repeat sequences after stem cell transplantation (SCT) were measured. The study included the telomeres of peripheral blood monocytes that should reflect the length of telomeres in stem cells and the telomeres of T lymphocytes that could shorten as a result of peripheral expansion. The loss of telomeres in monocytes and in memory T cells, although accelerated initially, became comparable to the loss of telomeres in healthy controls from the second year after transplantation. In addition, the telomere length in the naive T cells that were produced by the thymus was comparable to the telomere length in the naive T cells of the donor. Compared to the total length of telomeres available, the loss of telomere repeats in leukocytes after SCT resembles the accelerated shortening seen in early childhood and remains, therefore, relatively insignificant.

Adult↗
PubMed2001

Human memory T cells: lessons from stem cell transplantation.

Animal models have revealed the rules for the organization of mature T-cell pools. However, in humans, little is known about memory T cells, which differ in lifespan and in the number of times that the same antigen is encountered. Here, Nathalie Rufer and colleagues discuss their findings in stem-cell-transplanted patients, which provide interesting data on the human T-cell compartment.

Animals↗
PubMed2001

Hypothesis for generation of the unstable Hb Bucuresti (beta 42 Phe-->Leu) mutation.

INTRODUCTION: Unstable hemoglobin disorders are characterized by a congenital, mostly familial chronic hemolytic anemia with episodes of severe hemolysis during febrile illnesses. Usually, isopropanol and heat stability tests lead to the diagnosis which is confirmed by protein and gene sequencing. Generation of the mutations is still a subject of controversy. PATIENT, MATERIALS AND METHODS: We describe a 6-year-old Swiss child with congenital hemolytic anemia and a negative family history. Hemoglobin was studied by IEF, HPLC reverse phase chromatography, heat stability and isopropranol tests. DNA was sequenced in both coding and non-coding strands. RESULTS: An unstable Hb was diagnosed on the basis of positive heat stability and isopropranol tests. The TTT-->TTG mutation at codon 42 corresponding to a Phe-->Leu substitution was found on DNA sequencing. Paternity was confirmed indicating that we are dealing with a new mutation. CONCLUSION: So far, three different mutations at codon 42 of the beta-chain, and two at the corresponding position of the alpha-chain have been described, all leading to a hemolytic anemia. These mutations can either represent random phenomena occurring at an important location in the heme pocket, or may be secondary to the two highly homologous zones present in this region. These homologous zones may indicate a hot spot for point mutations created by abnormal crossing over or formation of loops, and an imperfect DNA repair process.

Amino Acid Substitution↗
PubMed2001

Limited telomere shortening in hematopoietic stem cells after transplantation.

The number of cell divisions in hematopoietic stem cells (HSCs) following transplantation of bone marrow or mobilized peripheral blood into myelo-ablated recipients is unknown. This number is expected to depend primarily on the number of transplanted stem cells, assuming that stem cells do not differ in engraftment potential and other functional properties. In a previous study, we found that the telomere length in circulating granulocytes in normal individuals shows a biphasic decline with age, most likely reflecting age-related changes in the turnover of HSCs. In order to study HSCs' proliferation kinetics following stem cells transplantation, we analyzed the telomere length in donor-derived nucleated blood cells in four HLA-matched bone marrow transplant recipients relative to comparable cells from the sibling donors. In each case, the telomeres in granulocytes were shorter in the recipient than in the donor. This difference was established in the first year post transplantation and did not change after that. The telomere length in naïve and memory T cells showed marked differences after transplantation, complicating the interpretation of telomere length data using unseparated nucleated blood cells. Interestingly, the telomere length in naïve T cells that were first observed six months post transplantation was very similar in donor and recipient pairs. Our observations are compatible with a limited number of additional cell divisions in stem cell populations after bone marrow transplantations and support the idea that different populations of stem cells contribute to short-term myeloid and long-term lympho myeloid hematopoiesis.

Bone Marrow Transplantation↗
PubMed2000

Recovery of immune reactivity after T-cell-depleted bone marrow transplantation depends on thymic activity.

To evaluate the importance of the thymus for the reconstitution of immunity in recipients of a T-cell-depleted bone marrow, we measured the appearance of CD4(+)CD45RA(+)RO(-) naive T cells (thymic rebound), restoration of the diversity of the T-cell-receptor (TCR) repertoire and the response to vaccinations with tetanus toxoid (TT). Repopulation by CD4(+)CD45RA(+)RO(-) thymic emigrants varied among patients, starting at approximately 6 months after transplantation. Young patients reconstituted swiftly, whereas in older patients, the recovery of normal numbers of naive CD4(+) T cells could take several years. Restoration of TCR diversity was correlated with the number of naive CD4(+)CD45RA(+)RO(-) T cells. Moreover, the extent of the thymic rebound correlated with the patient's capacity to respond to vaccinations. Patients without a significant thymic rebound at the moment of vaccination (CD4(+)CD45RA(+)RO(-) T cells less than 30 microL) did not respond, or responded only marginally even after 3 boosts with TT. We conclude that during the first year after transplantation, the absence of an immune response is due mainly to the loss of an adequate T-cell repertoire. Restoration of the repertoire can come only from a thymic rebound that can be monitored by measuring the increase of CD4(+)CD45RA(+)RO(-) naive T cells. This will allow postponing revaccinations to a moment when the patient will be able to respond more effectively. This may be particularly useful in the elderly patient who, owing to low thymic activity, might not yet be able to respond 1 year after transplant when revaccinations are usually scheduled.

Adolescent↗
PubMed2000

Bone marrow transplantation with unrelated donors: what is the probability of identifying an HLA-A/B/Cw/DRB1/B3/B5/DQB1-matched donor?

Patients transplanted with marrow from an HLA-ABDR serologically matched unrelated donor suffer from more post-transplant complications than those who are transplanted with marrow from an HLA-identical sibling. This is most likely due to either HLA-ABDR incompatibilities not resolved by standard techniques and/or HLA polymorphisms not tested for by routine tissue typing (HLA-Cw,-DQ). By resolving these incompatibilities by molecular techniques combined with the in vitro cytotoxic T lymphocyte precursor frequency (CTLpf) test, we have shown that a high degree of HLA compatibility is associated with increased patient survival. However, higher requirements for HLA matching decrease the number of available donors. We have estimated the probability of finding an HLA-A/B/Cw/DRB1/DRB3/DRB5/DQB1 compatible donor based on 104 consecutive unrelated bone marrow donor searches initiated between January 1995 and December 1997, with December 1998 as the endpoint. For 96 patients (92.3%), one or more ABDR-identical donors were listed in the Bone Marrow Donor Worldwide Registry (BMDW). After contacting the registries, we obtained at least one (mean, 5.36; range, 1-20; total, 461) blood sample for 86 patients. A highly compatible donor was identified for 33/86 patients (38.4%), after testing an average number of 4.5 donors/patients (range, 1-13). However, by accepting an HLA-DRB3 or -DQB1 or -Cw incompatibility, this number would be as high as 68.6%. Approximately half of the patients (n = 40) for whom a search had been initiated have been transplanted: 22 patients with a perfectly matched donor, 15 patients with an HLA-DRB3 or -DQB1 or -Cw mismatch and three with other mismatches. The average time needed to identify the most compatible donor was 4 months. Extremely long searches seemed to be less useful, because after testing the first seven, a more compatible donor was seldom found. These results show that even when requirements for compatibility are high, the chances of finding a donor remain considerably low.

Bone Marrow Transplantation↗
PubMed2000

HLA-DR4 is associated with a diminished risk of the development of chronic myeloid leukemia (CML). Chronic Leukemia Working Party of the European Blood and Marrow Transplant Registry.

CML is characterized by the chromosomal translocation t(9;22) (q34;q11) resulting in the chimeric bcr-abl oncogene that encodes P210 fusion proteins with novel amino acid sequences in the breakpoint region. If these peptides derived from P210 are presented by HLA molecules on the cell membrane of leukemic cells an immunological response may occur. Recent studies using synthetic peptides identical to the bcr-abl fusion region revealed that some peptides are capable of binding to the class I molecules HLA-A2,-A3,-A11 and -B8 and the class II molecules HLA-DR1, -DR2, -DR3, -DR4 and -DR11. Moreover T cell responses have been induced against bcr-abl-derived synthetic peptides bound to some of these HLA molecules. For HLA class I, we have previously shown that individuals expressing HLA-A3 and -B8 have a diminished risk of development of CML. To assess a similar protective effect of class II molecules we performed a large multi-center study. This study compared the frequencies of HLA-DR1, -DR2, -DR3, -DR4 and -DR11 of patients with CML from the database of the EBMT (n = 1462) with unaffected individuals from the registry of Bone Marrow Donors Worldwide (n = 500 596). Patients and controls were matched per country. This analysis yielded significantly lower odds ratios (ORs) of 0.86 (95% CI 0.75-0.98) for HLA-DR3 and of 0.80 (95% CI 0.71-0.89) for HLA-DR4. The OR was 0.91 (95% CI 0.80-1.04) for HLA-DR1, 1.05 (95% CI 0.94-1.18) for HLA-DR2 and 0.87 (95% CI 0.74-1.01) for HLA-DR11. To assess a possible effect of the linkage disequilibrium between HLA-B8 and HLA-DR3 we found that coexpression of HLA-B8 and HLA-DR3 gave an OR of 0.84 (95% CI 0.72-0.98), whereas HLA-DR3 positive/HLA-B8 negative individuals showed an OR of 1.02 (95% CI 0.84-1.24). This means that the protective effect of HLA-DR3 of the development of CML was probably caused by its linkage disequilibrium with HLA-B8. In contrast, as there is no linkage disequilibrium of HLA-DR4 with HLA-A3 or HLA-B8, the results indicate that HLA-DR4 expression itself is associated with a diminished incidence of CML possibly by the presentation of bcr-abl breakpoint peptides in these HLA molecules on the membrane of the leukemic cells.

Bone Marrow Transplantation↗
PubMed1999

HLA-B8 and HLA-A3 coexpressed with HLA-B8 are associated with a reduced risk of the development of chronic myeloid leukemia. The Chronic Leukemia Working Party of the EBMT.

Chronic myeloid leukemia (CML) is characterized by the chromosomal translocation t(9;22) resulting in the chimeric bcr-abl oncogene that encodes the P210 fusion protein, which contains a unique amino acid sequence. If peptides derived from the leukemia-specific part of P210 are expressed in HLA molecules on the cell membrane of leukemic cells, an immunological response may occur. Recent studies using synthetic peptides identical to the bcr-abl fusion region showed that some peptides are capable of binding to HLA-A3, -A11, and -B8 molecules. Cytotoxic T-cell responses have been induced against bcr-abl-derived synthetic peptides bound to HLA-A3 and -B8. We hypothesized that if antigen processing of the P210 fusion protein leads to presentation of peptides from the fusion region by major histocompatibility complex (MHC) molecules in vivo, this may be reflected in a diminished incidence of CML in individuals expressing HLA-A3, -A11, or -B8. Consequently, lower frequencies of these antigens would be expected in patients with CML compared with unaffected individuals. A case-control study and a meta-analysis were performed to test this hypothesis. The multicenter case-control study compared patients with CML from the data base of the European Group for Blood and Marrow Transplantation (EBMT) with unaffected individuals from the registry of Bone Marrow Donors Worldwide. Patients and controls were matched per country. The meta-analysis consisted of five studies reported in the literature. The multicenter case-control study consisting of 1,899 patients and 512, 363 bone marrow donors as controls yielded odds ratios (ORs) of 0.90 (95% confidence interval [CI], 0.80 to 1.00) for HLA-A3, 1.16 (95% CI, 1.02 to 1.33) for HLA-A11, and an OR of 0.73 (95% CI, 0.65 to 0. 82) for HLA-B8. Coexpression of HLA-A3 and HLA-B8 gave an OR of 0.51 (95% CI, 0.40 to 0.67). This can be translated in a protective effect of 27% for HLA-B8, 10% for HLA-A3, and 49% protection for the combination of HLA-A3 and HLA-B8. The meta-analysis comprising 463 CML patients and 4,912 controls showed a 29% risk reduction for individuals expressing HLA-B8 (OR of 0.71; 95% CI, 0.52 to 0.97), but an OR of 1.19 (95% CI, 0.90 to 1.56) for HLA-A3 and an OR of 1. 09 (95% CI, 0.80 to 1.50) for HLA-A11. In conclusion, these results indicate that HLA-B8 expression, in particular when HLA-A3 is coexpressed, is associated with a diminished incidence of CML. A biological mechanism may be that presentation of bcr-abl breakpoint peptides in these HLA molecules can induce a protective immune response.

Case-Control Studies↗
PubMed1999

Sequencing of two HLA-A blank alleles: implications in unrelated bone marrow donor matching.

BACKGROUND: DNA-based methods can type for HLA antigens with no or very low cell surface expression. The role of such serological blank antigens in bone marrow donor selection is unknown. METHODS: HLA-A serological blank antigens detected in two leukemic patients were cloned and sequenced from genomic DNA. mRNA expression and exon 3 splicing were determined by reverse transcriptase-polymerase chain reaction (PCR). RESULTS: Two patients typed A2/x and A3/x by serology were revealed to be A*01/A*0201 and A*03/A*24 by DNA typing. The HLA-A*O1 blank antigen was identified as the A*0104N allele with a C insertion in exon 4, which results in a stop codon. The HLA-A*24 blank antigen was identified as the A*2402102L allele characterized by a mutation in intron 2 leading to impaired splicing of mainly exon 3. As a consequence, functional A24 mRNA was reduced to less than 5% compared with the other HLA-A allele. For both patients, a search for an unrelated donor was initiated on the basis of the functional absence of the serologically blank allele. The second patient with the A*24 blank antigen could undergo transplantation with marrow from a "fully A/B/C/DRB1/DRB5/DQB1-compatible" donor, homozygous for the A antigen. Donor T cells did not react against the patient in a pretransplantation cytotoxic T lymphocyte precursor frequency test known to detect all class I incompatibilities. However, despite this functional pretransplantation compatibility, the patient died 44 days after bone marrow transplantation, suffering from graft-versus-host disease grade IV. The presence of potentially functional A*2402 mRNA could be demonstrated by reverse transcriptase-PCR and sequencing: 50% of the A24 mRNA molecules were estimated to contain exon 3 with a correct splice site. CONCLUSIONS: These findings show that serological blank antigens with low mRNA expression might still be recognized after bone marrow transplantation. The determination of such alleles by molecular methods and the assessment of mRNA expression should therefore be included in the unrelated bone marrow donor search protocol.

Alleles↗
PubMed1999

CD45 isoform phenotypes of human T cells: CD4(+)CD45RA(-)RO(+) memory T cells re-acquire CD45RA without losing CD45RO.

We have studied the alterations in CD45R phenotypes of CD4(+)CD45RA(-)RO(+) T cells in recipients of T cell-depleted bone marrow grafts. These patients are convenient models because early after transplantation, their T cell compartment is repopulated through expansion of mature T cells and contains only cells with a memory phenotype. In addition, re-expression of CD45RA by former CD4(+)CD45RA(-) T cells can be accurately monitored in the pool of recipient T cells that, in the absence of recipient stem cells, can not be replenished with CD45RA(+) T cells through the thymic pathway. We found that CD4(+)CD45RA(-)RO(+) recipient T cells could re-express CD45RA but never reverted to a genuine CD4(+)CD45RA(+)RO(-) naive phenotype. Even 5 years after transplantation, they still co-expressed CD45RO. In addition, the level of CD45RA and CD45RC expression was lower ( approximately 35 %) than that of naive cells. In contrast, the level of CD45RB expression was comparable to that of naive cells. We conclude that CD4(+)CD45RA(-)RO(+) T cells may re-express CD45(high) isoforms but remain distinguishable from naive cells by their lower expression of CD45RA / RC and co-expression of CD45RO. Therefore, it is likely that the long-lived memory T cell will be found in the population expressing both low and high molecular CD45 isoforms.

Adult↗
PubMed1999

Search for an unrelated HLA-compatible stem cell donor.

Through recent advancements in tissue-typing techniques, the true extent of HLA polymorphism has become evident. This has allowed a better assessment of the degree of matching of donor and recipient, which has reiterated the pivotal importance of HLA compatibility for the outcome of a stem cell transplantation. Now that most tissue typing laboratories are technically able to provide a flawless, high-resolution HLA typing, the challenge of the future will be to develop tissue typing-based search strategies that give an optimal chance to find the best donor within the time limits set by the disease of the patient.

HLA Antigens↗
PubMed1998

Reconstitution of the T-cell compartment after bone marrow transplantation: restoration of the repertoire by thymic emigrants.

We have studied the reconstitution of the T-cell compartment after bone marrow transplantation (BMT) in five patients who received a graft-versus-host disease (GVHD) prophylaxis consisting of methotrexate, cyclosporin, and 10 daily injections (day -4 to day +5) of Campath-1G. This treatment eliminated virtually all T cells (7 +/- 8 T cells/microL at day 14) which facilitated the analysis of the thymus-dependent and independent pathways of T-cell regeneration. During the first 6 months, the peripheral T-cell pool was repopulated exclusively through expansion of residual T cells with all CD4(+) T cells expressing the CD45RO-memory marker. In two patients, the expansion was extensive and within 2 months, the total number of T cells (CD8>>CD4) exceeded 1,000/microL. In the other three patients, T cells remained low (87 +/- 64 T cells/microL at 6 months) and remained below normal values during the 2 years of the study. In all patients, the first CD4(+)CD45RA+RO- T cells appeared after 6 months and accumulated thereafter. In the youngest patient (age 13), the increase was relatively fast and naive CD4(+) T cells reached normal levels (600 T cells/microL) 1 year later. In the four adult patients (age 25 +/- 5), the levels reached at that time-point were significantly lower (71 +/- 50 T cells/microL). In all patients, the T-cell repertoire that had been very limited, diversified with the advent of the CD4(+)CD45RA+RO- T cells. Cell sorting experiments showed that this could be attributed to the complexity of the T-cell repertoire of the CD4(+)CD45RA+RO- T cells that was comparable to that of a normal individual and that, therefore, it is likely that these cells are thymic emigrants. We conclude that after BMT, the thymus is essential for the restoration of the T-cell repertoire. Because the thymic activity is restored with a lag time of approximately 6 months, this might explain why, in particular in recipients of a T-cell-depleted graft, immune recovery is delayed.

Adolescent↗
PubMed1998

HA-1 and the SMCY-derived peptide FIDSYICQV (H-Y) are immunodominant minor histocompatibility antigens after bone marrow transplantation.

BACKGROUND: Allogeneic bone marrow donors can be incompatible at different levels. Even HLA-identical pairs will be still incompatible for numerous minor histocompatibility antigens (mHag). Nevertheless, some incompatibilities are found to be associated with an increased risk of graft-versus-host disease (GVHD), which could be related to the way the immune system recognizes these antigens. METHODS: We determined the specificity of cytotoxic T-cell clones isolated during acute GVHD or during bone marrow graft rejection in patients (n=14) transplanted with marrow from donors who were histoincompatible for different minor and/or major histocompatibility antigens. RESULTS: We found a clear hierarchy among the different types of histoincompatibilities. In three combinations mismatched for a class I allele, all 27 clones isolated during GVHD were specific for the incompatible HLA molecule. In the 11 class I-identical combinations, 14 different mHags were recognized. The mHag HA-1, known to have a significant impact on the development of GVHD, was recognized in the two HA-1-incompatible combinations. In one of these combinations, which was sex mismatched, all 56 clones analyzed were directed against HA-1, demonstrating the dominance of this mHag. In the four HA-1-compatible, sex-mismatched combinations, the anti-H-Y response was directed against one immunodominant epitope rather than against multiple Y-chromosome-encoded epitopes. All male specific cytotoxic T lymphocytes (n=15) recognized the same high-performance liquid chromatography-purified peptide fraction presented by T2 cells. Moreover, all cytotoxic T lymphocytes tested (n=6) were specific for the SMCY-derived peptide FIDSYICQV, originally described as being the H-Y epitope recognized in the context of HLA-A*0201. CONCLUSIONS: Some histocompatibility antigens are recognized in an immunodominant fashion and will therefore be recognized in the majority of mismatched combinations. Only for such antigens, correlations between mismatches and the occurrence of GVHD or graft rejections will be found.

Bone Marrow Transplantation↗
PubMed1998

Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry.

To measure the average length of telomere repeats at chromosome ends in individual cells we developed a flow cytometry method using fluorescence in situ hybridization (flow FISH) with labeled peptide nucleic acid (PNA) probes. Results of flow FISH measurements correlated with results of conventional telomere length measurements by Southern blot analysis (R = 0.9). Consistent differences in telomere length in CD8+ T-cell subsets were identified. Naive and memory CD4+ T lymphocytes in normal adults differed by around 2.5 kb in telomere length, in agreement with known replicative shortening of telomeres in lymphocytes in vivo. T-cell clones grown in vitro showed stabilization of telomere length after an initial decline and rare clones capable of growing beyond 100 population doublings showed variable telomere length. These results show that flow FISH can be used to measure specific nucleotide repeat sequences in single cells and indicate that the very large replicative potential of lymphocytes is only indirectly related to telomere length.

Adult↗