Normal Human Blood Lymphocytes Research Articles

Studies on human T lymphocyte rosette formation with human and sheep erythrocytes

A large proportion of normal human blood lymphocytes forms rosettes with neuraminidase-treated autologous and allogeneic human erythrocytes (nHRBC) or sheep red blood cells (SRBC). In contrast, the percentage of rosette-forming cells is extremely reduced in the peripheral blood of patients with chronic lymphocytic leukemia. The formation of mixed rosettes after addition of nHRBC and SRBC suggests that most normal rosette-forming lymphocytes have receptor sites for both types of erythrocytes. Rosette formation with neuraminidase-untreated HRBC occurs only with a small fraction of normal lymphocytes, but can be increased by preincubating the lymphocytes with neuraminidase. Similar to rosette formation with SRBC. the binding of nHRBC is temperature-dependent, is prevented by pretreatment of the lymphocytes with proteolytic enzymes, but is not inhibited by EDTA. Rosette dissociation during incubation at 37 degree C is accompanied by conversion of ring- or morula-like rosettes into horseshoes or caps.

Distinguishing normal human blood lymphocytes from lymphocytes in dengue and typhoid fever.

Blood smears of clinical cases of dengue and typhoid fever were obtained from the Philippines and stained by the Feulgen method. Scanning-microscopic densitometry was used to digitize cell images for analysis by principles of pattern recognition.

Comparison of Normal and CLL Lymphocyte Surface Ig Determinants Using Peroxidase-labeled Antibodies. I. Detection and Quantitation of Light Chain Determinants

Abstract Chronic lymphocytic leukemia (CLL) and normal human blood lymphocytes were investigated with regard to their membrane-associated light (L) chains. Peroxidase-labeled anti-kappa or anti-lambda antibodies were used to visualize by light microscopy the cells bearing L chain determinants. In addition, the activity of the enzyme coupled to the antibody was measured spectrophotometricly in order to determine the number of antigenic determinants on each positive cell. The results demonstrated that 16%-25% of lymphocytes from six normal control subjects were positive for kappa chains, and 4%-10% were positive for lambda chains. The average number of antigenic sites on the surface of each positive cell was calculated to be 90,000. Lymphocytes of each of the 14 CLL patients studied carried either kappa or lambda antigenic determinants on their surface, but not both; 50%-70% of the lymphocytes were stained. The average number of antigenic sites per positive CLL was calculated to be 9000. These results confirm previously reported studies indicating that CLL is a monoclonal proliferative disease. In addition, the quantitative results demonstrate that the CLL lymphocyte surface membrane bears, on the average, only 10% of the L chain determinants present on the normal lymphocyte.

DNA-directed and RNA-primed DNA synthesis in microsomal and mitochondrial fractions of normal human lymphocytes

Nascent DNA synthesized from RNase-sensitive endogenous DNA polymerase activities of microsomal and mitochondrial fractions from phytohemagglutinin-stimulated normal human blood lymphocytes is covalently attached to a small RNA molecule (<4S in the case of the microsomal fraction). This complex is in turn originally hydrogen bonded to a much larger preexistent DNA molecule, indicating that this synthesis of DNA is directed by DNA (template) and that the RNA is utilized solely as an initiator molecule (primer). This endogenous RNase-sensitive DNA synthesis should not be confused with the endogenous RNase-sensitive DNA synthesis in type-C RNA tumor viruses and in related cytoplasmic particles from human leukemic cells catalyzed by reverse transcriptase which utilizes RNA as both template and primer. These findings are consistent with the concept that some DNA synthesis in mammalian cell organelles may be analogous to that of bacteria in utilizing RNA primers with DNA templates.

The Effect of Methotrexate on Transformation and Mitosis of Normal Human Blood Lymphocytes In Vitro

Abstract The effect of methotrexate (MTX) on transformation and mitosis of normal lymphocyte cultures was investigated. Mitosis was 1000-fold more sensitive to MTX than was transformation; mitosis could be prevented without any observable effect on transformation. The antimitotic effect of MTX depended on its concentration, duration of contact with lymphocytes, as well as on the developmental stage of the lymphocytes at the time of exposure. Incorporation of labeled precursors into protein, DNA, and RNA proceeded at normal rates unaffected by the presence of MTX. Transformation was partially inhibited by high concentrations of MTX (above 75 µg/ml). Folinic acid abolished the antimitotic effect of MTX, with the number of cells undergoing division directly proportional to the quantity of folinic acid added.

Relation of lysosomal fragility in CLL lymphocytes to PHA reactivity.

AFTER addition of various mitogens such as phytohaemagglutinin (PHA) in vitro lymphocytes shift from a “resting” state to one of rapid enlargement, culminating in DNA synthesis and mitosis. The mechanism initiating transformation and proliferation is not yet fully understood. Some observations, however, suggest that alterations of cellular membranes might be involved in these processes. In their cytochemical studies, Allison and Mallucci1 observed increased permeability of lysosomes in normal human peripheral blood lymphocytes after exposure to PHA for 5 h. Hirschhorn et al.2 demonstrated that during the early phase of stimulation 30–120 min after addition of PHA in normal human blood lymphocytes a redistribution of lysosomal acid hydrolases from a sedimentable into a non-sedimentable form as well as an increased release by agents which disrupt membranes, such as streptolysin S and filipin, of lysosomal enzymes from a subcellular fraction rich in lysosomes are taking place. It has been suggested that these alterations, both reflecting enhanced fragility of lysosomal membranes, might be due to increased endocytosis induced by the mitogen and the hypothesis was raised that lysosomal hydrolases might be involved in intracellular processes leading to lymphocyte activation2. We have therefore studied the fragility of lysosomes in unstimulated and PHA-treated lymphocytes from patients with chronic lymphocytic leukaemia (CLL), because lymphocytes in most patients with this disease show a diminished and/or delayed or even no response to PHA3 and other mitogens4,5.

Stimulated normal human lymphocytes contain a ribonuclease-sensitive DNA polymerase distinct from viral RNA-directed DNA polymerase.

Ribonuclease-sensitive DNA synthesis is demonstrated in a cytoplasmic particulate fraction of normal human blood lymphocytes stimulated with phytohemagglutinin, but not in unstimulated lymphocytes. DNA polymerase purified from this fraction does not transcribe the heteropolymeric regions of 70S RNA from RNA tumor viruses, thus distinguishing this enzyme from the RNA-directed DNA polymerase (reverse transcriptase) found in oncogenic RNA viruses and human leukemic cells.

DNA-dependent DNA polymerases I and II from normal human-blood lymphocytes.

Two DNA polymerases have been purified from blood lymphocytes of normal human individuals. In contrast to a DNA polymerase isolated from lymphoblasts from individuals with leukemia, neither of the DNA polymerases from normal lymphocytes transcribes the heteropolymeric regions of tumor-virus RNA into DNA.

Surface immunoglobulin light chain determinants on normal and pha-stimulated human blood lymphocytes studied by immunofluorescence and electronmicroscopy

Lymphocytes from human peripheral blood were cultured in vitro. The distribution of antigenic determinants with specificity of immunoglobulin light or heavy chains on normal and phytohaemagglutinin (PHA) stimulated cells was examined by membrane immunofluorescence and immunoelectron microscopy. It appeared that a relatively large population of both normal and stimulated lymphocytes carried light chain determinants on their surface. Usually considerably more lymhocytes reacted for ϰ than for λ determinants. In contrast no or only few cells reacted with reagents specific for heavy chain (γ or μ) determinants. At the ultrastructural level, the reactivity to antilight chain antibodies in unstimulated cultures was seen to be confined to discontinuous areas of varying size on the surface of cells with the typical morphology of small to medium sized lymphocytes. In cultures treated with PHA, lymphocytes undergoing blastoid transformation often showed larger patches of antilight chain reactivity than unstimulated lymphocytes. The uropods of stimulated lymphocytes often showed a strong reactivity with antilight chain antibodies. No reactions were obtained with antibodies to heavy chains. In contrast, normal as well as PHA-stimulated lymphocytes stained for human species antigens by means of heterologous antisera from rabbits, exhibited an even distribution of the ferritin label over large areas of their surface.

The radiosensitivity of normal and leukaemic human blood lymphocytes

<h2>Summary</h2> Twenty-three experiments have been performed on normal peripheral blood lymphocytes and nine on lymphocytes from the blood of patients with chronic lymphatic leukaemia. The cells are cultured using a modification of the agar method, and then exposed to x-ray radiation. The radiosensitivity of the lymphocytes is estimated by direct cell counts at hourly intervals after various doses of radiation. In both series, the cells decay exponentially. When the normal and leukaemic cells are compared after irradiation by 0, 60 and 120 r, no significant differences in radiosensitivity are found. At 240 r, however, the leukaemic cells were found to be more sensitive than the normal cells. It is possible that other differences noted between normal and non-mitotic chronic lymphatic leukaemic lymphocytes may be correlated with this altered radiosensitivity. It seems unlikely, though that the results can be applied to therapeutic radiation.