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Persistent Epstein-Barr virus (EBV) infection is controlled tightly by virus-specific T cells. EBV infection is reactivated intermittently over time, even in apparently healthy carriers. Changes in frequency and reactivity of memory T cells, particularly of CD8(+) origin, have not been assessed in this context. It is hypothesized that viral reactivation is facilitated by diminished EBV-specific T-cell immunity. To this end, blood samples from 14 healthy donors were collected at irregular time intervals for a period of about 1 year. Samples were screened for both EBV plasma viremia and increases in viral load in PBMCs as parameters of EBV reactivation. PBMCs were subject to IFN-γ ELISPOT analysis using the autologous EBV-transformed lymphoblastoid cell line (EBV-LCL) or appropriate HLA class I-restricted EBV peptides as stimulators. Frequencies of epitope-specific CD8(+) T cells were monitored further using HLA tetramers and flow cytometry. Twelve of 14 donors exhibited signs of asymptomatic EBV reactivation. Viral reactivation was accompanied by either substantially decreased IFN-γ responses against autologous EBV-LCL (eight of 12 study participants) and/or increased responses against particular EBV peptides (six of 12 donors). In seven persons with HLA-A2 and/or -B8 alleles numbers of HLA tetramer-positive CD8(+) T cells also varied over time, but showed no correlation to episodes of detectable viral activity. In summary, IFN-γ reactivity of EBV-specific T cells is not constant. Viral reactivation is detected preferably at times of diminished EBV-LCL-specific cellular immunity. However, increased reactivity of single immunodominant CD8(+) EBV-specific T-cell clones may occur in response to virus replication., (Copyright © 2011 Wiley Periodicals, Inc.)

Background: We describe a new nonenzymatic methodology that allows the simultaneous detection of DNA replication and other cellular markers such as immunophenotyping. DNA replicating cells are identified by their incorporation of halogenated thymidine analogs, e.g., 5-bromo-deoxyuridine (BrdUrd)., Methods: Irradiation with ultraviolet (UV)-B or UV-A light in the presence of Hoechst 33258 and subsequent treatment with a hypotonic buffer makes BrdUrd accessible to monoclonal antibodies (mAb), thus allowing its sensitive detection., Results: The photolysis of BrdUrd in DNA with UV light is sequence dependent and results in DNA damage, allowing the detection of remaining BrdUrd using hypotonic conditions. However, treatment with other inducers of single or double- strand breaks of DNA such as gamma irradiation or hydrogen peroxide did not allow BrdUrd detection. The new methodology is compatible with both mild crosslinking fixation, i.e., aldehydes, or coagulative fixation, i.e., alcohols. The successful identification of CD34+, CD138+, or CD19+ cells out of heterogeneous cell suspensions and their cell-cycle analysis are described. Results correlated very well with acid denaturation (r = 0.972). The average coefficient of variation (CV) of G(1) in the DNA histogram was smaller than 5%, resulting in good preservation of DNA distribution. Also, the signal-to-noise ratio was almost twice as high as for 2N acid denaturation, facilitating convenient discrimination of BrdUrd-positive cells., Conclusions: In contrast to previous approaches, this methodology eliminates the need for any additional enzymatic treatment such as DNA digestion or strand-break labeling after UV irradiation. The method is fast, convenient, and inexpensive and should be able to promote the use of halogenated pyrimidines in basic and clinical research of cancer, immunology, and pharmacology., (Copyright 2000 Wiley-Liss, Inc.)

To determine the frequency of in vivo activated T(H)1 lymphocytes, T-cell subsets of 9 multiple sclerosis patients with active disease and 17 healthy controls were analyzed by immunostaining for CCR5, CD26, and their expression of interleukin-2, interferon-gamma, and tumor necrosis factor-alpha. The numbers of CCR5+ interferon-gamma- and tumor necrosis factor-alpha-producing T cells were significantly increased in the peripheral blood of multiple sclerosis patients. CCR5 expression may be a useful marker to identify effector cells in multiple sclerosis and could be used as a tool for monitoring disease activity.

Since the introduction of hematopoietic growth factors, the collection of mobilized stem cells via leukapheresis has widely replaced the harvest of bone marrow in both autologous and allogeneic transplantation settings. We investigated the frequency and the extent of anticoagulant-induced electrolyte changes and the cell-separation-related loss of hemoglobin and platelets. In our study a total of 200 leukaphereses were performed on 60 patients with hematological malignancies. The electrolytes (calcium and potassium) were determined photometrically pre- and post-apheresis. Blood counts were analyzed to calculate the relative decline in hemoglobin and platelet counts. Stem cells were collected by processing a mean total blood volume of 11.6+/-3.9 L with a citrate consumption of 1,345+/-126 mL. More than 50% of all patients needed replacement therapy of either potassium or calcium. In non-substituted patients the initial serum potassium concentration dropped by 11.3+/-7.0% to 3.25+/-0.33 mmol/L post apheresis. In 21% of non-substituted patients, clinical relevant hypokalaemia was observed with levels < 3 mmol/L. The mean citrate-induced reduction of the total calcium was 5.5+/-6.0%. In addition the relative loss of hemoglobin and platelet counts amounted to 10.7+/-5.2% and 24.2+/-12.5%, respectively. In addition to the well-documented citrate-induced hypocalcaemia, we observed a considerable reduction in serum potassium during stem cell apheresis. This can result in a clinically relevant, substitution requiring hypokalaemia. The modest decline in hemoglobin and platelet counts suggested that levels of >9 g/dl (Hb) and platelets >30 x 10(9)/L are sufficient for a safe standard leukapheresis., (Copyright 2000 Wiley-Liss, Inc.)

We describe a flow cytometric method using a newly designed product, fluorochrome-containing microspheres (Flow Count fluorospheres), which facilitates the precise quantification of cells in whole blood samples or heterogeneous cell suspensions on a single-cell level. These microparticles are easily distinguishable from other events of interest and can be detected by their light-scattering and fluorescence properties. In contrast to the traditional manual or automated cell-counting techniques, this method offers the opportunity to quantify cells simultaneously with flow cytometric immunophenotyping without additional cell loss or other cell preparation steps. We evaluated the accuracy and reproducibility of this flow cytometric method on the determination of CD45+ leukocyte counts and compared the results with those obtained by conventional techniques. Particular interest was focused on the behavior of cells and fluorospheres regarding their sedimentation rate over the period of analysis. The data from 48 blood samples with low, normal, and high leukocyte counts confirmed the reliability and comparability of the flow cytometric method, permitting the determination of white blood cell concentration at least to a limit of 100 cells/microL. A broad field of applications will benefit from this flow cytometric supplement because it is easy to perform and highly accurate. The results appear to be transferable to clinical decision-making monitoring of CD4+ lymphocytes in patients infected with human immunodeficiency virus or of CD34+ hematopoietic cells, optimizing the harvest for peripheral blood stem cell transplantation.