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Abelson virus-transformed lymphocytes: null cells that modulate H-2.

A-MuLV-transformed lymphoid cells from Balb/c mice had the properties of null lymphocytes. They did not secrete Ig and all but one did not have detectable cell-associated Ig; one line synthesized, but did not secrete, the mu chain of IgM. The cells expressed H-2D and H-2K, but not H-21 histocompatibility antigens or theta-antigen; they had Fc receptors. Most cell lines grew to form donor cell tumors after inoculation into (Balb/c X C57B1/6)F1 mice. The tumor cells have more H-2Dd than cells passaged in vitro. Cell lines carried in vitro progressively lost H-2Dd. A line in which 5-30% of the cells were lysable by anti-H-2Dd was cloned; all eleven clones had H-2Dd (13-69% lysable) demonstrating that H-2 modulates in vitro. A clone with little H-2Dd (10-15% lysable) was tumorigenic even after treatment with anti-H-2Dd sera; at least 50% of the tumor cells were lysed by anti-H-2Dd. Thus A-MuLV-transformed lymphocytes modulate H-2 in vivo to higher levels and in vitro to lower levels.

Animals

Rosette formation of human null lymphocytes with Rhesus monkey erythrocytes.

Human null lymphocytes which lack B- and T-lymphocyte markers were found to form spontaneous rosettes with uncoated Rhesus monkey erythrocytes. Rosette-forming null lymphocytes were determined in null lymphocyte-enriched preparations of healthy persons and of patients with lymphoproliferative diseases. Quantitative determinations of these rosette-forming lymphocytes and other cells bearing B- or T-cell markers showed that some, and not all, of the null lymphocytes possess this rosette-forming capacity. The possibility of these null lymphocytes being related to T lymphocytes, but at a different cellular stage, is discussed.

Animals

Identification of erythroid colony progenitors in a subset of human peripheral null lymphocytes devoid of Fc receptors.

This study was aimed at purifying the progenitors of erythroid burst units (BFU-E) from human peripheral blood. Human mononuclear leukocytes from five normal donors were fractionated into several mononuclear cell subpopulations, including null lymphocytes with (null Fc+) and without (null Fc-) receptor for the Fc fragment of immunoglobulin G, through a succession of rosetting procedures and discontinuous Ficoll-Hypaque gradient centrifugations. The fractionated cells were separately cultured for 14 days in plasma clots in the presence of erythropoietin. Among fractionated cell subpopulations large and numerous BFU-E derived colonies grew only from the Fc- null lymphocyte subpopulation. This fraction, representing less than 4% of all mononuclear cells, also contains cells (42 + 11%) capable to differentiation towards the B-cell and plasma-cell lineages. The Fc+ null lymphocytes, representing less than 9% of all mononuclear cells, contained 15.2 + 3.3% cells capable of differentiation toward the T-cell lineage. The whole null lymphocyte subpopulation generated half the number of BFU-E colonies expected from its content in Fc- null lymphocytes. These data demonstrate that the progenitor of erythroid cells (BFU-E) resides in a small heterogeneous null Fc- subpopulation of circulating lymphocytes and suggest that its in vitro differentiation, though generally subjected to inhibitory and enhancing influences from other circulating cell subpopulations, does not necessarily require interaction with other peripheral blood cells.

Adult

B and T lymphocytes in man. III. B, t, and "null" lymphocytes in multiple sclerosis.

T, B, and "null" lymphocytes were determined in peripheral blood obtained from 24 control subjects and 15 multiple sclerosis patients. The total number of lymphocytes and T lymphocytes were reduced significantly in multiple sclerosis patients as compared with those of controls. Although the percentage of B lymphocytes increased in these patients, the absolute number of B lymphocytes was similar to controls. The mitotic response of lymphocytes from multiple sclerosis patients to phytohemagglutinin and pokeweed mitogen in 7-day-old cultures was decreased as compared with that of controls.

Adult

B and T lymphocytes in man. IV. Circulating B, T and "null" lymphocytes in aging population.

T, B and "Null" lymphocytes were determined in the peripheral blood of 98 adults, 26 of them were between 20 to 40 years of age and 72 between 60 to 96 years of age. The total number of lymphocytes were about the same in both groups. The percent and absolute number of T cells decreased significantly in the aged. Although the percent of B lymphocytes increased with age, the absolute number of B lymphocytes remained about the same.

Adult

Do human B and null lymphocytes form a single immunoglobulin-bearing population?

Human "null" lymphocytes have been recognised because they lack T-lymphocyte membrane markers and the surface membrane immunoglobulin (smIg), detectable by direct immunofluorescence, which conventionally characterises B lymphocytes. But almost all null lymphocytes express smIg determinants which are demonstrable by the direct antiglobulin and mixed antiglobulin rosette tests. It is argued that the definition of a B lymphocyte should be extended to include the null cells which react in these tests. If the heterogeneity of the B-lymphocyte population is more widely recognized, the precise nature of human natural and antibody-dependent killer cells may become clearer.

Antibody Affinity

Human peripheral blood null lymphocytes stimulated by Staphylococcus aureus Cowan I produce atypical acid-labile interferon in vitro.

Peripheral blood mononuclear leucocytes (PBLs) stimulated in vitro by heat-killed formaldehyde-fixed Staphylococcus aureus Cowan I (SACoI) produced acid-labile alpha interferon (IFN-alpha) and, to various extents, also IFN-gamma. The IFN producers resided in nylon wool-nonadherent cells, and monocytes suppressed SACoI-induced IFN responses. Further separation of nonadherent PBLs in accordance with expression of surface antigenic markers was performed with a 'panning' technique. The SACoI induced production of IFN in cells that carried neither surface immunoglobulins nor OKT3-defined antigens. These cells were also characterized as OKM1- and OKT10-negative. In contrast, cells with natural killer (NK) activity against K562 erythroleukaemia cells were located in both OKM1- and OKT10-positive and -negative cells. At centrifugation on Percoll density gradients, cells with NK activity and IFN response against SACoI were recovered from light gradient fractions that contained mainly large granular lymphocytes (LGL). Furthermore, the IFN producers were enriched by removal of sheep erythrocyte-rosetting T cells from the Percoll fractions. These SACoI-induced IFN-producing PBLs are LGL but lack certain antigens that are frequently found on NK cells.

Antibodies, Monoclonal

A distinct population of nonphagocytic and low level CD4+ null lymphocytes produce IFN-alpha after stimulation by herpes simplex virus-infected cells.

Human PBMC were stimulated for 6 h in vitro by HSV or Sendai virus (SV) and analyzed by flow cytometry. IFN-alpha producing cells (IPC) were identified through their content of IFN-alpha mRNA by in situ hybridization using a 35S-labeled IFN-alpha 2 cRNA probe. The IPC induced by HSV-infected WISH cells lacked capacity to adhere to and phagocytose latex particles. The induction of IFN-alpha by free infectious SV occurring in monocytes was abolished by phagocytosis of latex particles present in the cultures during the induction period. Such latex particles actually enhanced the IFN-alpha response induced by glutaraldehyde-fixed HSV- or SV-infected WISH cells or by free intact HSV. The HSV-induced IPC did not express the CD14 Ag expressed on monocytes. Cell sorting was performed on HSV-induced PBMC labeled with phycoerythrin-conjugated anti-CD3 and FITC-conjugated anti-CD4 mAb. A small population consisting of 1.4% of all PBMC, which was CD3- but expressed low but significant levels of CD4, contained the majority of the IPC with a 50-fold increase of their frequency. This cell population had a forward- and right-angle light scatter different from typical monocytes/macrophages. The results therefore further delineate IPC among PBMC into monocytes, being stimulated by viruses such as SV. Another distinct population of infrequent but highly efficient IPC, tentatively designated natural IFN-alpha producing cells, is activated by stimuli such as HSV.

Antibodies, Monoclonal

Loss of circulating T lymphocytes with normal levels of B and 'null' lymphocytes in Thai adults with malaria.

Peripheral blood mononuclear cells from forty-nine Thai adults infected with either Plasmodium falciparum or Plasmodium vivax were examined in order to determine the percentage of T, B, and Fc-receptor-bearing cells present. In comparison to healthy controls, both the percentage and concentration of peripheral T cells were decreased in the malaria-infected individuals as assessed by formation of rosettes with sheep red blood cells. The percentage of peripheral B cells was increased but their concentration was unchanged, as assessed by two techniques: the presence of surface immunoglobulin and the presence of a complement receptor. Both the percentage and concentration of lymphocytes bearing Fc receptors were unchanged in infected individuals. Finally, calculation of the changes in 'null' cells (defined either as non-T, non-B lymphocytes or as non-T, non-B, non Fc-receptor-bearing lymphocytes) revealed an increase in the 'null' cell percentage but a decrease in the absolute number of 'null' cells. These data indicate that in adult Thai patients naturally infected with malaria, there is a real loss of circulating T lymphocytes with no real change in B, Fc-receptor-bearing, or 'null' lymphocytes.

Adolescent

Human peripheral blood "null-lymphocytes", a highly enriched pool for granulocytic stem cells (CFU-C).

A "Null"-lymphocyte preparation containing a high concentration of granulocytic stem cells (CFU-C) was isolated from human peripheral blood. 3 lymphocyte purification procedures were shown to produce an enrichment of CFU-C: 1. Removal of mononuclear phagocytes by iron phagocytosis and plastic adherence procedures. 2. Passage through Ig anti-Ig columns. 3. Removal of E-rosette-forming T cells. A combination of all 3 purification steps resulted in a 30- to 40-fold enrichment of CFU-C in the final "Null"-lymphocyte population as compared to the initial lymphocyte fraction isolated by Ficoll-Urografin density gradient centrifugation.

Animals

Studies of T, B, and null lymphocytes during the course of acute lymphocytic leukemia.

The distribution of T, B and null lymphocytes was studied in the peripheral blood (PB) of 34 children with acute lymphocytic leukemia (ALL), at diagnosis and during the course of their disease. All patients received the same chemotherapy. At diagnosis, the proportion and absolute numbers of T lymphocytes fell into 2 groups, a "low" group (medians 8%, 318/mm3) and a "normal" group (medians 60%, 1405/mm3). Patients with a low proportion of T cells tended to have a high proportion and absolute number of null cells (correl. coeff. r = 0.94). This group has had high white blood cell counts (median = 29,600/mm3) and a high proportion of blasts (84%). During remission, the proportion of T cells returned toward normal (40-75%) in both groups. Four patients in the low T group relapsed; T cells again fell to low levels and returned to normal in response to new therapy. Three interpretations of these observations are suggested: 1) Active leukemia may cause normal lymphocytes (T cells and to a lesser degree B cells) to lose their surface markers; 2) Acute leukemia may inhibit maturation of normal lymphoid cells, and thus T or B markers are not sufficiently developed to be detectable; and 3) Cells without surface markers (null cells) may be a part of the malignant population, even though they are not morphologic blasts. Further studies are necessary to test these hypotheses.

Adolescent

Elevated adenosine deaminase and purine nucleoside phosphorylase activity in peripheral blood null lymphocytes from patients with acquired immune deficiency syndrome.

The purine metabolic enzymes adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) are important in lymphocyte differentiation, and genetic deficiencies of either enzyme have been associated with hereditary immunodeficiency states. Both ADA and PNP activity were measured in null cell-enriched and T cell-enriched peripheral blood lymphocytes from 16 patients with the acquired immune deficiency syndrome (AIDS), seven patients with the AIDS-related symptom complex (ARC), and seven asymptomatic homosexuals. ADA activity in nmol/10(6) lymphocytes/h was significantly elevated in null lymphocytes from AIDS (161 +/- 12) as compared with 23 healthy heterosexual controls (127 +/- 8;P less than .025). PNP activity was also significantly increased in null lymphocytes from AIDS patients (96 +/- 10;P less than .005) as well as those from ARC patients (84 +/- 11:P less than .025) relative to controls (61 +/- 5). No significant differences in enzyme activity were noted in T cell-enriched cells in any group. Along with elevated enzyme activity, AIDS patients had small yet significant increases in the percentages of HLA-DR (P less than .025), terminal deoxynucleotidyl transferase (TdT) (P less than .0001), and peanut agglutinin receptor (P less than .0001) positive lymphocytes in the null fraction compared with controls. TdT-positive cells appeared morphologically as large lymphoblasts with irregular nuclei. The data imply that the cellular immune deficiency in AIDS is not a result of deficiencies in lymphocyte ADA or PNP activity, but is more likely associated with an increase in an immature and/or activated lymphocyte subset.

Acquired Immunodeficiency Syndrome

Cells and mediators which participate in immunoglobulin synthesis by human mononuclear cells. I. The obligatory requirement of null lymphocytes in the synthesis of immunoglobulins in vitro.

The circulating mononuclear cells of normal adult human volunteers were fractionated into T and non-T lymphocytes. The T cells were then fractionated into TM and non-TM cells and the non-T cells were further fractionated, by both positive and negative isolation, into B cells and null cells. Culture of non-T cells, TM cells, and PWM for 7 to 8 days resulted in cytoplasmic Ig (cIg) synthesis by all B cells, as detected by immunofluorescence. However, culture of either purified B or null cells with TM cells and PWM resulted in a marked reduction (75%) of cIg-synthesizing cells. The addition of monocytes did not enhance cIg synthesis. Culture of reconstituted B and null cells with TM cells and PWM restored the capacity to synthesize cIg to the cells. It is concluded that null cells are required, as well as TM cells and PWM, for cIg synthesis by B cells.

B-Lymphocytes

B, T and null lymphocytes in newborn infants and their mothers.

Estimation of B, T and null cells were performed on 29 newborn healthy babies and 16 mothers. The lymphocytes were isolated from peripheral venous blood, which is considered to be more representative of the immune state in the newborn than the cord blood. B lymphocytes were estimated by cytofluorometric measurements, T lymphocytes by sheep red blood cell rosette technique, (SRBC-R). Combined immunofluorescence and SRBC-R technique revealed the null cells. In the newborn babies the amount of B and T cells were found to be diminished. In the mothers the amount of B lymphocytes were low compared with normal adults. The rather high null cell percentage found in the babies might represent immature precursor cells. Mothers seem to be immuno-depressed as reflected in the low amount of B cells.

Adult