Exposure Of Peripheral Blood Research Articles

Analysis of γ-rays induced chromosome aberrations: A fingerprint evaluation with a combination of pan-centromeric and pan-telomeric probes

Purpose: To evaluate the types of induced chromosome aberrations after the exposure of peripheral blood to γ-rays by the simultaneous detection of all centromeres and telomeres; and to analyse the suitability of different radiation fingerprints for the assessment of radiation quality in cases of recent exposures.Material and methods: Peripheral blood samples were irradiated at 2, 4 and 6 Gy of γ-rays. Cytogenetic analysis was carried out by fluorescence in situ hybridization (FISH) technique with pan-centromeric and peptide nucleic acid (PNA)-telomeric DNA probes. Cells were analysed using a Cytovision® FISH workstation, chromosome aberrations and the length of the acentric fragments were recorded.Results: The total number of the incomplete chromosome elements was 276. The ratio between incomplete elements and multicentrics was 0.38. The number of acentrics was 1096, 71% were complete acentrics, 15% incomplete acentrics, and 14% interstitial fragments. The relative length of complete, incomplete and interstitial acentrics fragments were 2.70 ± 0.04, 1.91 ± 0.07, and 1.42 ± 0.04 respectively. The mean value of the F-ratio was 11.5 higher than the one, 5.5, previously obtained for α-particles. For the G-ratio there was no difference between γ-rays and α-particles, 2.8 and 2.8 respectively. The mean value of the H-ratio for γ-rays, 0.25, was lower than for α-particles 0.40.Conclusion: The results support that the percentage of incomplete chromosome aberrations depends on radiation type; low-linear energy transfer (LET) radiation would produces less incomplete aberrations than high-LET radiation. The F- and H-ratios seem to be good indicators of radiation quality, although a real estimation of the H-ratio is only possible using pan-telomeric probes.

A Novel Method for Monitoring Response to Chemotherapy Based on the Detection of Circulating Cancer Cells: A Case Report

We describe a novel method for detecting micrometastasis in the blood stream of cancer patients based on RT-PCR amplification of tumor-associated carcinoembryonic antigen (CEA) mRNA. To increase sensitivity and specificity of RT-PCR, CEA transcript was selectively up-regulated in cancer cells by exposure of peripheral blood to non-toxic concentrations of staurosporine (ST). Thereafter, polyA(+) RNA was extracted from tumor cells captured by means of magnetic beads coated with a monoclonal antibody against a common human epithelial antigen. Finally, RNA was subjected to RT-PCR analysis of CEA transcript. Using this approach, we demonstrated an ST-mediated increase in CEA transcript in blood specimens collected from a patient with metastatic colon cancer before receiving treatment with 5-fluorouracil/leucovorin. After a few cycles of chemotherapy, CEA-positive tumor cells were no longer detected. Clinical follow-up of this patient indicated that treatment with chemotherapy induced a dramatic reduction in liver metastasis. Therefore, it can be hypothesized that lack of CEA transcript detection might be consistent with disappearance or at least marked reduction of circulating tumor cells.

Age-Related Reductions in the Activation of Mitogen-Activated Protein Kinases p44mapk/ERK1 and p42mapk/ERK2 in Human T Cells Stimulated via Ligation of the T Cell Receptor Complex

Age-related changes in the functional properties of human T cells are well described, but less is known about possible changes in T cell signaling pathways. The signaling pathways mediated by the mitogen-activated protein kinases (MAPK) are considered essential for normal cellular growth and function. Several stimuli trigger MAPK activation in human T cells and MEK (MAPK or ERK kinases) are immediate upstream inducers of MAPK activation. The current study investigated if aging might influence the activation and expression of MAPK and MEK in human T cells. Exposure of peripheral blood T cells from young subjects to PHA or cross-linked anti-CD3 monoclonal antibodies stimulated rapid increases in MAPK and MEK enzymatic activity. By contrast, significant reductions of MAPK and MEK activation were observed in stimulated T cells from 7 of 13 elderly subjects. Kinetic studies showed that the age-related impairments represented reductions in both the levels and duration of MAPK activation. In addition, Western immunoblot analysis did not reveal significant age-related differences in T cell expression of p42mapk/ERK2, p44mapk/ERK1, or MEK, suggesting impairments in upstream inducers of MEK/MAPK activation. Other experiments determined if agents that directly stimulate upstream Ras or Raf kinase components of the early MAPK cascade might reverse the age-related impairments of MAPK activation. Treatment of elderly T cells with fluoroaluminate (AlF−4), phorbol esters/Ca2+ionophores, or okadaic acid stimulated increased MAPK activation compared to anti-CD3. However, these agents failed to restore MAPK activation in elderly T cells to the levels seen in young T cells. These results suggest that aberrancies in the MAPK activation cascade may underlie the age-related reductions of MAPK activation in human T cells stimulated via the TCR/CD3 complex.

High avidity state of leukocyte function-associated antigen-1 on rheumatoid synovial fluid T lymphocytes.

The integrin LFA-1 (CD11a/CD18) is a cell surface adhesion molecule required for leukocyte extravasation and subsequent immune and inflammatory responses. Rapid transition between nonadherent and adherent states of LFA-1 is of key importance to Ag-specific recognition of T lymphocytes. In this paper, LFA-1-mediated adhesiveness of peripheral blood (PB) and synovial fluid (SF) T lymphocytes to affinity-purified ICAM-1-coated plates was studied in patients with rheumatoid arthritis (RA) and in patients with non-RA panels, including osteoarthritis, ankylosing spondylitis, erythema nodosum, pseudogout, and pustulosis. LFA-1-mediated adhesiveness of SF T lymphocytes was not observed in any of the 10 non-RA patients studied, although cross-linking of the TCR on lymphocytes from these patients rapidly converted LFA-1 to an adhesive state. In contrast, SF T lymphocytes from 10 of 12 RA patients exhibited LFA-1-mediated adhesiveness without a requirement for cross-linking of the TCR. No difference was seen in the cell surface density of LFA-1 between non-RA and RA T lymphocytes, suggesting that the difference in adhesiveness was due to a high avidity state of LFA-1 on SF T lymphocytes in RA. Furthermore, exposure of PB T lymphocytes, which showed a low avidity state of LFA-1, to whole SF from RA patients that was depleted of T lymphocytes could induce a high avidity state of LFA-1 in vitro. Cellfree SF from RA patients also could stimulate adhesiveness, although to a lesser extent. These data suggest the existence of a LFA-1-activating environment that is selectively found in SF from RA patients.

IgD-receptor-positive human T lymphocytes. I. Modulation of receptor expression by oligomeric IgD and lymphokines.

Studies with human myeloma-derived IgD have demonstrated the existence of IgD-R on peripheral blood T cells. These receptors, which are detected by rosetting with IgD-coated ox E (IgD-rosette-forming cells), are competitively inhibited by IgD, but not by IgM or IgG. Similar results were obtained with human T cell clones and T hybridomas derived from such clones either by rosetting assays or by staining with biotinylated-IgD. In agreement with studies of murine IgD-R+ cells, human IgD-R can be up-regulated by exposure of peripheral blood T cells, T cell clones, and hybridomas derived from such clones, to oligomeric IgD, but not monomeric IgD. Human IgD-R can also be induced by IL-2, IL-4, and IFN-gamma. In contrast with studies of murine IgD-R, which are expressed primarily by CD4+ cells, phenotyping studies show that both the CD4+ and CD8+ human T cell subsets are capable of expressing IgD-R.

Analysis of transformation with Epstein-Barr virus and phenotypic characteristics of lymphoblastoid cell lines established from patients with hairy cell leukemia.

In order to assess the role of Epstein-Barr virus (EBV) in patients with hairy cell leukemia (HCL), we have sought to characterize 1) the ability of EBV to infect and transform hairy leukemic cells in vitro and 2) the phenotypes of cell lines putatively derived from those leukemic cells. Analysis of EBV-induced transformation and the kinetics of Epstein-Barr nuclear antigen (EBNA) induction in leukemic preparations indicated that most leukemic cells were not susceptible to EBV infection but that at least a small subpopulation of leukemic cells could be infected with EBV. Lymphoblastoid cells lines were established after exposure of peripheral blood or splenic cells from HCL patients to B95-8 or QIMR-WIL EBV. Splenic leukemic cell preparations were more sensitive targets for EBV transformation than were peripheral blood cell samples. The newly established cell lines, but not long-established B lines such as Raji, demonstrated high levels of synthesis of p35, (a protein complex expressed abundantly by cells of a subset of HCL patients) and high levels of tartrate-resistant acid phosphatase (an enzyme relatively diagnostic for HCL). Lymphoblastoid lines from one patient with HCL expressed lambda light chains and no kappa chains as did the patient's leukemic cells. Virus expression in these lines showed that HCL-derived lines had spontaneous early antigen (EA) and viral capsid antigen (VCA) expression. Transforming EBV could be rescued from HCL-derived cell lines but not from cord blood-derived lines.