Cell Fraction Of Peripheral Blood Research Articles

Generation of dendritic cells from patients with chronic myelogenous leukemia.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) specialized to internalize, process, and present antigen. They have the capacity to stimulate the primary immune response of resting T-cells. We generated DCs from the adherent cell fraction of peripheral blood, as well as from purified CD34+ cells from CML patients. Characterizing DCs from ten CML patients by flow cytometry, we found that these cells are highly positive for HLA-DR, CD1a, CD23, and CD80 and negative for CD14, CD15, and CD16. The yield of DCs ranged from 19.5 to 68%. In addition, we used a functional test of FITC-dextran uptake to verify that early DCs take up large particles (0.5-3 microm) by macropinocytosis while monocytes do not. FITC-dextran uptake was detected by flow cytometry, showing that DCs had accumulated these fluorescent particles. Electron-microscopic analysis showed no major morphological differences between normal and CML-derived DCs. Furthermore, cultured DCs were isolated by FAC sorting for CD1a and HLA-DR expression. In these highly purified cells the Ph chromosome was detected by interphase fluorescence in situ hybridization (FISH) and by fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION); 30-85% of DCs generated were Ph-chromosome positive. It might therefore be possible not only to prime T-cells with bcr/abl-specific synthetic peptides, but also to stimulate T-cells directly with Ph-positive DCs. Use of DCs might serve as a novel therapeutic approach in CML patients, due to their ability to induce highly specific T-cell responses in an autologous system.

Increase in a heat-shock protein from blood cells in response of nestling house martins (Delichon urbica) to parasitism: an experimental approach.

Heat-shock proteins (HSPs) are synthesized by animals and plants in response to various stressors. The level of the HSP60 stress protein was measured from the cell fraction of peripheral blood obtained from nestling house martins (Delichon urbica) to test whether ectoparasitism increased the concentration of stress protein. We assessed HSP from nestlings raised in nests previously treated with an insecticide or infested with 50 martin bugs (Oeciacus hirundinis). In addition, haematozoa infections were checked in blood smears. Nestlings from parasite-infested nests, or nestlings infected with trypanosomes, had increased levels of HSP in their blood cells. Nestling growth as determined from wing length was negatively related to HSP60 levels and within-brood variation in wing length increased with increasing levels of the stress protein independently of treatment and infection by trypanosomes. These results suggest HSPs may play a role in host-parasite interactions, and that they can be used reliably for measuring physiological responses to parasites.

Characterization of Titanium Alloy Particulates to Study the in Vitro Release of Inflammatory Mediators by Human Peripheral Blood Monocytes.

Particulate wear debris found around joint arthroplasties has been linked to the premature failure of artificial joints. Cytokines and eicosanoids released by macrophages are known mediators of inflammation and osteolysis. Previous in vitro studies of wear debris-mediated inflammation have relied upon poorly characterized particulates and peripheral blood cell fractions of mixed cellular composition. The objective of this study was to utilize titanium particulates that closely match wear debris found around joint arthroplasties and to study their effects on the biosynthesis of inflammatory mediators by isolated monocytes.To mimic wear debris that surrounds artificial joints, titanium alloy particulates (1-3 μm in diameter) were obtained. A submicron fraction of these particulates was isolated by differential sedimentation. The morphology, size and concentration of the resulting population was characterized utilizing scanning electron microscopy. Titanium particulate size was estimated from photomicrographs of calibrated latex microspheres mixed with titanium particles (Figure 1). A known volume of titanium particulates in suspension was applied to a carbon planchette and allowed to dry.

Peripheral Blood Platelets Express VEGF-C and VEGF which Are Released during Platelet Activation

VEGF-C is a recently characterised endothelial growth factor structurally related to vascular endothelial growth factor (VEGF). We studied the expression of VEGF-C and VEGF in the cells of peripheral blood and in the umbilical cord blood CD 34+ cells, representing haematopoietic progenitor cells. Expression of VEGF-C was detected in the CD34+ cells. In peripheral blood VEGF-C mRNA was restricted to platelets and T-cells. In contrast to the expression pattern of VEGF-C, VEGF mRNA was detected in all peripheral blood cell fractions studied, and also in CD34+ cells. VEGF-C mRNA was also detected in fresh bone marrow samples of acute leukaemia patients, but the expression did not show lineage specificity. VEGF-C and VEGF polypeptides were present in platelets and they were released from activated platelets together with the release of beta-thromboglobulin, suggesting that VEGF-C and VEGF reside in the alpha-granules of platelets. VEGF-C and VEGF, released from activated platelets, may have a role in angiogenesis during wound healing, and possibly also in other pathological conditions, such as atherosclerosis, tumour growth, and metastasis formation.

Inhibition of deoxyribonucleic acid synthesis during premeiotic stages of spermatogenesis by a factor from testis-associated lymphomyeloid tissue in the dogfish shark (Squalus acanthias).

To investigate factors controlling spermatogonial proliferation, we used premeiotic (PrM) spermatocysts (stage-synchronized germ cell/Sertoli cell clones) derived from the testis of the dogfish shark (Squalus acanthias) as an in vitro test system to estimate DNA synthesis by [3H]thymidine incorporation. Coculture of PrM spermatocysts with spermatocysts of the same (PrM) or more advanced stages (M, meiotic; PoM, postmeiotic) revealed the presence of an increasing gradient of inhibitory bioactivity from immature to mature stages (PoM > M > PrM). An even more potent and effective inhibition was detected when PrM spermatocysts were cocultured with equivalent amounts of epigonal organ, a lymphomyeloid tissue encapsulating the testis adjacent to the mature (PoM) region and immediately upstream in the vascular pathway. Inhibitory bioactivity also was present in spent media from cultured epigonal fragments and in cytosolic subfractions of epigonal homogenates but was undetectable in epididymis, muscle, serum, and red blood cells. Lower but significant inhibition was obtained with the white blood cell fraction of peripheral blood in one of two experiments. Effects of the epigonal growth-inhibitory factor (EGIF) were dose- and time-dependent, had a short response latency (3 h), were completely reversible (< 24 h after washout), and counteracted but did not block the stimulatory effects of insulin on [3H]thymidine incorporation by PrM spermatocysts. EGIF was equally inhibitory when tested on each of five PrM substages (stem cells-->preleptotene). Analysis of the entire series of experiments showed that testis-derived inhibitory activity varied seasonally, with maximum effectiveness correlated with periods of spermatogenic inactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Levels of Retinoblastoma Protein Expression in Newly Diagnosed Acute Myelogenous Leukemia

The relationship between the level of retinoblastoma protein (RB) expression and the survival of 113 newly diagnosed acute myelogenous leukemia (AML) patients was studied. Western blotting was used to determine the level of RB protein present in peripheral blood leukemia cells and results were confirmed in 26 patients by immunohistochemistry. The leukemic cells from 22/113 AML patients (19%) contained RB protein at levels that were equal to or less than the level of RB observed in the mononuclear cell fraction of peripheral blood from normal individuals (Low RB). Levels of RB greater than that of normal blood (Elevated RB) were seen in 91 patients (81%). The median survival of patients with low RB was significantly shorter than that seen in patients with elevated RB, 12 weeks versus 40 weeks (P = .02). Remission induction frequency was 36% in low RB patients compared with 68% in AML patients with elevated RB (P = .01). Multivariate analysis showed that low RB protein level was an independent prognostic factor predictive or poor survival after allowing for other known prognostic factors. These data suggest that a low level of the RB protein at the time of diagnosis is associated with shortened survival in AML patients because of inferior response to conventional therapy. Monitoring of the RB level could identify a subgroup of AML patients with an extremely poor prognosis when treated with chemotherapy alone, who would be eligible for alternative therapeutic strategies.

Levels of retinoblastoma protein expression in newly diagnosed acute myelogenous leukemia

The relationship between the level of retinoblastoma protein (RB) expression and the survival of 113 newly diagnosed acute myelogenous leukemia (AML) patients was studied. Western blotting was used to determine the level of RB protein present in peripheral blood leukemia cells and results were confirmed in 26 patients by immunohistochemistry. The leukemic cells from 22/113 AML patients (19%) contained RB protein at levels that were equal to or less than the level of RB observed in the mononuclear cell fraction of peripheral blood from normal individuals (Low RB). Levels of RB greater than that of normal blood (Elevated RB) were seen in 91 patients (81%). The median survival of patients with low RB was significantly shorter than that seen in patients with elevated RB, 12 weeks versus 40 weeks (P = .02). Remission induction frequency was 36% in low RB patients compared with 68% in AML patients with elevated RB (P = .01). Multivariate analysis showed that low RB protein level was an independent prognostic factor predictive or poor survival after allowing for other known prognostic factors. These data suggest that a low level of the RB protein at the time of diagnosis is associated with shortened survival in AML patients because of inferior response to conventional therapy. Monitoring of the RB level could identify a subgroup of AML patients with an extremely poor prognosis when treated with chemotherapy alone, who would be eligible for alternative therapeutic strategies.

Responses of synovial fluid and peripheral blood mononuclear cells to bacterial antigens and autologous antigen presenting cells.

The specificity of T cells in the inflamed joints of patients with rheumatoid arthritis (RA) has been the subject of much study. Bacterial antigens are suspect in the aetiology of rheumatic diseases. The responsiveness of the mononuclear cell fraction of peripheral blood and synovial fluid of patients with RA and of patients with rheumatic diseases other than RA to bacterial antigens such as cell wall fragments of the anaerobic intestinal flora, cell wall fragments of Streptococcus pyogenes, intestinal flora derived peptidoglycan polysaccharide complexes, the 65 kilodalton protein of Mycobacterium tuberculosis, and muramyldipeptide was investigated. No significant difference in response was found to all these bacterial antigens in the synovial fluid of patients with RA compared with the responses in patients with other rheumatic diseases. The highest responsiveness in the synovial fluid of the patients with RA was to the streptococcal cell wall fragments and to the 65 kilodalton protein. Higher responses to several bacterial antigens in the synovial fluid of patients with RA were found compared with peripheral blood from the same patient group. The antigen presenting cell population of the synovial fluid in patients with RA and the patients with other rheumatic diseases was found to be stimulatory for autologous peripheral blood T cells even in the absence of antigen. This suggests an important role for the synovial antigen presenting cell in the aetiology of inflammatory joint diseases.

A method for the rapid screening of human blood samples for the presence of HIV-1 sequences: the probe-shift assay.

In theory, screening directly for the human immunodeficiency virus (HIV) DNA has advantages over screening for HIV-related antibodies; however, the HIV genome appears to be present only in a small fraction of peripheral blood cells from infected individuals. The polymerase chain reaction (PCR) specifically amplifies defined DNA segments, permitting the identification of specific DNA segments even if they are present in a fraction of cells. HIV-derived PCR-amplified segments have been detected by Southern transfer, "dot-blot" hybridization, and digestion with restriction endonucleases. Unfortunately, PCR may amplify not only the single segment of interest but also irrelevant segments, which can be incorrectly identified as HIV related. We describe here an alternative approach to specific detection of PCR-amplified DNA segments, the probe-shift assay, that is simpler, faster, and less subject to erroneous identification than the previously described techniques. The probe-shift assay relies on the hybridization of an appropriately labeled oligonucleotide probe to the amplified segment in solution. The hemiduplex formed between the amplified DNA and the probe is detected following fractionation on nondenaturing gels. This procedure is sufficiently sensitive to detect a single infected cell in the presence of more than 10(5) uninfected cells and has been applied directly to clinical samples.

Immunogold cell staining using monoclonal antibodies: Application to lymphoid cells on coated slides

The immunogold staining technique was presented for light microscopic detection of cell surface antigens with monoclonal antibodies. The lymphoid cells were adhered to glass slides precoated with the adhesive poly-(dimethyl-diallyl-)ammonium chloride. The staining with the monoclonal mouse antibodies followed by the gold labelled sheep anti-mouse antibody was carried out on the cells adhered to the slides. In spite of the adherence, the patching of the gold marker as the prerequisite for the light microscopic detection occurred and the positively stained cells were visualized. On basis of this method the content of T lymphocytes (BL-T2+), B lymphocytes (BL-Ig-L/1+) and monocytes (BL-M/G+) in the mononuclear cell fraction of peripheral blood was determined in a population of healthy donors and of patients. The content of T lymphocytes of 29 healthy donors was determined using the immunogold staining technique (66.7 +/- 10.4%) and the immunofluorescence technique (67.6 +/- 9.5%). The correlation between the results obtained with both methods was highly significant (p less than 0.001).

Release of granulocyte-macrophage colony-inhibiting activity by normal human postthymic precursor cells.

Seven normal human peripheral blood cell fractions (buffy coat, mononuclear cells, non-T, T, Fc-IgM receptor-depleted T-lymphocyte, Fc-IgG receptor-depleted T-lymphocyte, and autologous rosette-forming T-cell-depleted T-lymphocyte subpopulations) treated with phytohemagglutinin (PHA) were examined for the production of granulocyte-macrophage colony-stimulating activity (CSA). It was found that medium conditioned by a T-lymphocyte subpopulation depleted of autologous rosette-forming T-cells (Tar cells, a postthymic precursor subpopulation that inhibits Ig synthesis) stimulated colony-forming units of granulocyte and macrophages (CFU-GM) to a greater extent than did the other conditioned media (CM) analyzed. Based on this finding, CM from an enriched Tar subpopulation was prepared and thus showed that PHA-treated Tar cells release a factor capable of inhibiting CFU-GM growth. The inhibitory activity of this factor persisted-after heat inactivation, suggesting that cause of the colony-inhibiting activity (CIA) is other than interferon. Further studies revealed that Tar-derived inhibitory factor acts either directly upon CFU-GM or via monocytes/macrophages (M phi/Ma), enhancing CIA, and not the level of CSA production by M phi/Ma. The overall data are interpreted as demonstrating the presence of CIA in a specific T-lymphocyte subpopulation that may represent a new relationship between lymphocytic and myelocytic systems in the human.

Heterogeneous accessory cell requirement for human peripheral blood T lymphocyte activation by PHA into IL-2-responsive colony-forming cells

Mitogen-driven T cell proliferation in liquid culture requires accessory cells that cooperate in interleukin 2 production. We have investigated the accessory cell requirement for human lymphocyte colony formation under PHA stimulation. Semisolid medium limits cell-to-cell contact emphasizing the role of cooperating cells both in growth factor production and in triggering events. Culturing at high T cell density demonstrates that accessory cells can be substituted for colony formation by exogenous IL-2. Culturing at low T cell density in the presence of IL-2 also demonstrates that accessory cells are required for activation of a subset of progenitors into IL-2 responsive colony-forming cells. Consequently, T colony progenitors, contained in the E-rosetting cell fraction of peripheral blood, are heterogeneous in their triggering signals: a minor subset is directly inducible by PHA, and a major subset is inducible by PHA in the presence of accessory cells. We found that monocytes and some leukemic B cells support effective accessory function in both colony growth factor production and colony progenitor sensitization.

Human erythroid burst-forming unit: T-cell requirement for proliferation in vitro.

Human mononuclear leukocytes were fractionated into populations of null, T and B cells by immunoabsorbent column chromatography followed by E-rosette formation and purification of T cells by differential centrifugation and osmotic lysis. The unfractionated and fractionated cell populations were first separately cultured for 14 days in plasma clots in the presence of two international units erythropoietin. Typical erythroid burst-forming unit (BFU-E)-derived colonies grew in the unfractionated cell cultures but not from T- or B-cell cultures. BFU-E colonies grew in null cell cultures but most of the colonies were small and variably hemoglobinized with less than three subcolonies. When intact T cells were added to null cells and cocultured, many typical large BFU-E colonies with more than 10 well homogenized subcolonies appeared. Increasing numbers of large BFU-E colonies in null cell cultures were induced by stepwise addition of T cells but not by the addition of B cells. A conditioned medium in which T cells had been induced to divide by tetanus toxoid substituted for intact T cells in this T-cell-dependent BFU-E colony formation observed in null cells. These findings demonstrate that the BFU-E, a committeded erythroid stem cell, resides in the null cell fraction of peripheral blood, but its proliferative capacity and differentiation in vitro requires a soluble product of T cells. Such experiments now permit a new approach to the assessment of various disorders of erythropoiesis. Erythroid hypoplasia in a particular case may be due to dysfunction of the committed precursor cell or to a failure of a helper effect induced by T cells.

An Immunoperoxidase Method for the Identification of B Lymphocytes by Light Microscopy

Peroxidase-conjugated anti-human immunoglobulin was used to determine the percentage of B lymphocytes in the mononuclear cell fraction of peripheral blood of 100 healthy persons (blood donors). Peroxidase activity was revealed by incubation with the usual mixture of 3′3′ diaminobenzidine and hydrogen peroxide. Cells which were peroxidase-positive after incubation with benzidine solution alone were considered to be monocytes. The number of T lymphocytes was estimated by the formation of E rosettes. 19.6% of mononuclear cells could be shown to be B cells, 4.1% were monocytes and 57.0% T cells.The use of peroxidase-conjugated anti-human immunoglobulin gives rise to the same percentage of B cells as the use of fluorescein-conjugated anti-human immunoglobulin. The advantage of the method described here is that B lymphocytes can be counted by conventional light microscopy.