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PubMed1988

Lymphocyte proliferative response to PHA and anti-CD3/Ti monoclonal antibodies, T cell surface marker expression, and serum IL-2 receptor levels as biomarkers of age and health.

Alteration of T cell surface marker expression with a decrease of CD3 positive cells relative to the number of CD4 and CD8 positive cells, diminished in vitro proliferative response to mitogenic stimuli like PHA and antibodies to the CD3/Ti complex, and increase in serum IL-2 receptor levels, are among the changes in immunologic parameters that have been associated with advanced age. To distinguish between effects of the primary aging process and diseases of aging not known to be directly related to immune function, we investigated these variables in two well characterized populations of elderly donors (greater than 70 years) and a young adult control group (less than 35 years). The first group of older donors reported no evidence of significant chronic or recent acute illness and saw a physician only for routine medical care. The second group was randomly selected from individuals seen in a geriatric medicine clinic for diagnoses that included osteoarthritis and cardiopulmonary disorders. Altered surface marker expression and increased serum IL-2 receptor levels were seen only in the second group. On the other hand, lymphocyte proliferative responses to PHA, Leu 4 (anti-CD3) and a monoclonal antibody to the beta-chain of the T cell antigen receptor (WT31) were significantly decreased in both populations. Because we would expect primary aging to affect even extremely fit individuals of advanced age, we conclude that decrease in T cell proliferative response may represent a biomarker of primary aging in man. The alteration in surface marker expression and increased IL-2R levels in serum appear to be effects secondary to non-immunologic disease rather than aging.

Adult↗
PubMed1992

Age-related changes of the antigen-specific antibody formation in vitro and PHA-induced T-cell proliferation in individuals who met the health criteria of the Senieur protocol.

The antigen-specific antibody secretion in vitro after immunisation with the primary T-cell dependent antigen Helix pomatia Haemocyanin (HPH) was investigated in both young and elderly individuals, who all met the health admission criteria for immunogerontological studies as detailed in the SENIEUR protocol. In addition, elderly non-Senieur persons were incorporated in this study. Young and elderly Senieur volunteers were fully comparable in terms of the occurrence of anti-HPH antibody secreting cells after in vitro simulation of peripheral blood mononuclear cells with variable doses of the antigen. In contrast, the non-Senieur elderly showed a lower number of anti-HPH antibody secreting cells in vitro. PHA-conditioned medium did enhance this in vitro response, whereas the addition of IL-2 remained ineffective. The PHA-induced T-cell proliferation was found to be somewhat impaired in elderly Senieur individuals and significantly lower in elderly non-Senieur individuals compared to young healthy persons. Using an immunofluorescence double staining technique after BrdU incorporation, the phenotype of the proliferating cells was determined. Again the total number of proliferating cells was impaired in the non-Senieur elderly. No changes in the relative contribution of CD4+ or CD8+ cells to the number of proliferating cells were found in the different age groups. On the other hand, a significantly lower number of proliferating cells with IL-2 receptor expression were detected in the non-Senieur individuals, which could account for the lack of response to IL-2 in this group. Our study clearly shows that so-called age-associated immune deficiency can be the result of disease and not necessarily of the ageing process itself.

Adult↗
PubMed1990

CCA (disodium 4-chloro-2,2'-iminodibenzoate) inhibits progression of human T cell proliferation triggered by PHA.

The effect of the anti-rheumatic drug CCA (disodium 4-chloro-2,2'-iminodibenzoate) on the proliferation of T cells activated by PHA was examined. Cell cycle analysis showed that CCA blocked the transition of the cells from G1 to S (progression), but had little effect on the G0----G1 transition (initiation). CCA had no significant effect on IL-2 receptor expression, an early G1 event, but did inhibit transferrin receptor expression, a late G1 event. CCA did not inhibit IL-2 production by PHA-activated T cells, but did block IFN-gamma production at 72 hr after the stimulation. CCA failed to inhibit c-myc mRNA induction, but did delay the decrease in c-myc mRNA levels that normally occurs with the onset of DNA synthesis. These results indicate that CCA inhibits the progression, but not initiation, of human T cell proliferation.

Cell Cycle↗
PubMed1982

Induction of cALL antigen and terminal deoxynucleotidyltransferase in normal peripheral lymphocytes during PHA stimulation.

In PHA cultures of peripheral lymphocytes from three healthy donors the extent and time sequence of cALL and TdT positivity was followed over a period of seven days. In all cases significant percentages of cALL+ and TdT+ cells were observed. However, the period of positivity for both these markers tended to be short. In contrast to previous results with the diffusion chamber system which yielded a comparatively uniform pattern of positivity, PHA cultures of the present cases revealed a more heterogeneous pattern concerning the extent as well as the time sequence of positivity. These experiments provide a broader basis for the notion that those markers are expressed during blastogenesis of normal lymphocytes which are generally regarded as indicative of early lymphatic differentiation and of the stage of developmental arrest of acute lymphatic leukemias.

Antigens, Neoplasm↗
PubMed1988

Radioresistant pseudo-colony formation in the PHA-leukocyte feeder colony assay.

In the PHA-leukocyte feeder colony assay--a fluid assay on top of an agar underlayer--colonies might not be the product of clonogenic cells but rather from aggregates, as was already shown for hairy cell leukemia (Leukemia Res. 11, 911 (1987)). To study the role of aggregation in this colony assay in other B-cell malignancies, we irradiated cells from B-chronic lymphocytic leukemia, B-non-Hodgkin's lymphoma and multiple myeloma. In nearly all cases, viable "colonies" were seen after irradiation, albeit in lower numbers. These data indicate that in the PHA-leukocyte feeder colony assay, a considerable percentage of colonies from a large variety of B-cell malignancies originate from aggregating rather than from proliferating cells.

B-Lymphocytes↗
PubMed1988

Neural connections identified with PHA-L anterograde and HRP retrograde tract-tracing techniques.

This paper describes a method of identifying specific input and output elements of a two-neuron projection pathway. Phaseolus vulgaris leucoagglutinin (PHA-L) anterograde tract-tracing was used in combination with the retrograde transport of horseradish peroxidase (HRP) to demonstrate connections between small groups of neurons in the brainstem auditory system. Specifically, the projection from cochlear nucleus to olivary neurons that project to the cochlea were demonstrated. The first neuron in this pathway (the cochlear nucleus neuron) was anterogradely labelled with PHA-L and could be traced via labelled axons and terminals to the second neuron (the olivocochlear neuron) whose soma was labelled with HRP after cochlear injection.

Animals↗
PubMed1995

Effect of renutrition on the proliferation kinetics of PHA stimulated lymphocytes from malnourished children.

The fraction of lymphocytes that responded to phytohemagglutinin (PHA) stimulation and initiated cellular proliferation (stimulation index or SI) was determined in groups of healthy and severely malnourished children. SI was determined again in the latter group after a period of nutritional recovery. The proportion of interphasic cells showing PHA response was assessed adding bromodeoxyuridine to the culture, so proliferative nuclei appear big and stain light blue, with dispersed granular chromatin and apparent nucleoli, while non-proliferative nuclei look small, stain red, and have compact and homogeneous chromatin. In mitotic nuclei, differential staining of sister chromatids made it possible to distinguish cells that had gone through one, two and three or more proliferation cycles. Based on the data obtained from interphase nuclei and mitosis, the SI was estimated at 48 and 72 h of culture. SI were higher in lymphocytes from healthy children than in those from children with severe malnutrition, even after the period of nutritional recovery. However, the SI was significantly higher in lymphocytes from malnourished children after nutritional recovery. Although in these children more cells are stimulated, there seems to be still damage that causes a cycling delay.

Bromodeoxyuridine↗
PubMedDate not supplied

Cytoplasmic expression of a CD24-related epitope in human PHA activated normal T lymphocytes.

In this study we have analysed by immunoperoxidase (IPx) and indirect immunofluorescence (IIF) the intracellular and cell surface reactivity of VIB-E3 mAb, previously clustered as anti-CD24 antigen, on resting and activated normal human T lymphocytes. By IPx assay VIB-E3 mAb did not show reactivity with normal resting T cells. In contrast, the analysis of 11 different samples of PHA activated normal mononuclear cells, showed an intracytoplasmic expression of CD24. Kinetic studies showed that CD24 appears 24 to 48 h after PHA stimulation. To our knowledge, this is the first evidence that a CD24-related epitope is expressed in normal activated T lymphocytes.

Antigens, CD↗
PubMed1984

Comparison of metaphase and interphase nucleolar activity in Hela-CCL2 cells and PHA-stimulated human lymphocytes.

Metaphase and interphase nucleolar activity in cultured Hela-CCL2 cells and PHA-stimulated human lymphocytes have been studied with silver nitrate staining. In metaphase, we examined the relationship between the actual number of active NORs (AgNORs) and total number of NOR-bearing acrocentrics. Interphase silver staining over the nucleus was analyzed cytodensitometrically and morphologically. From all investigations, Hela-CCL2 cells and lymphocytes were shown to have similar levels of nucleolar activity. Our results suggest that there is a form of regulation of nucleolar activity in malignant Hela-CCL2 cells as compared to PHA-stimulated human lymphocytes.

Cell Nucleolus↗
PubMed1987

Relative proportions of mitotic T and B cells in PHA-stimulated lymphocyte cultures.

Peripheral blood lymphocytes from six healthy adults were stimulated with phytohemagglutinin (PHA) and cultured for 3 days. The relative proportions of mitotic lymphocyte subsets were determined by a method that allows both cytogenetic and immunologic characterization of a mitotic cell. The results directly indicated that not only T cells but also B cells undergo mitosis in PHA-stimulated lymphocyte cultures. The frequency of the mitotic B cells varied between 6% and 20%.

Adult↗
PubMed1981

OKT3 induces suppressor cells for mixed lymphocyte and PHA mitogenic responses in human peripheral lymphocytes.

Human peripheral lymphocytes pretreated with the Orthoclone monoclonal anti-T cell antibody OKT3 for 48 h markedly suppressed the proliferative response of autologous lymphocytes in one-way MLC and the mitogenic response to PHA. The ability to induce suppression is specific to OKT3 since other monoclonal antibodies to human T cells (OKT1, OKT4 and OKT8) did not elicit similar responses, OKT3 is mitogenic but further proliferation of OKT3 pretreated lymphocytes was not required for the suppression of autologous lymphocytes since mitomycin-C treated cells were fully effective. Kinetic studies indicated that pretreatment of lymphocytes with OKT3 for 24 h was sufficient to induce marked inhibition of the mitogenic response of autologous lymphocytes to PHA whereas suppression in MLC was not observed until lymphocytes were pretreated for 48 h. These studies support the previous observations that OKT3 may be reacting with an important molecule on the T cell surface and that interaction of OKT3 with this molecule induces profound functional changes.

Antibodies, Monoclonal↗
PubMed1983

HLA antigens and the reactivity of lymphocytes to some drugs and PHA.

Peripheral blood lymphocytes from 15 healthy subjects, selected on the basis of their HLA types, were cultured in vitro in the presence of PHA and of a number of drugs which bind adrenergic or dopaminergic receptors (dopamine, norepinephrine, chlorpromazine, haloperidol, propranolol and apomorphine). The results obtained are consistent with an interference between HLA-A1 and cross-reacting specificities and the effect explained by these drugs on the lymphocyte activation by PHA. The possibility is suggested that HLA-A1 and cross-reacting antigens interfere with the binding of such drugs to the cell membrane receptors.

Adult↗
PubMed1980

Frequency of alloantibodies reacting with PHA-activated T lymphocytes, unexplainable by known HLA activities.

Two hundred and three sera obtained from multiparous women were screened to determine the frequency of the appearance of antibodies reactive with PHA-activated T lymphocytes, but not with resting B or T cells from the same donor. The panel of cells on which the sera were tested consisted of lymphocytes from 38 unrelated donors known to be homozygous at the HLA-D locus. Forty-two sera were shown to react uniquely with PHA-activated T cells. The reactivities of these sera could not be explained in terms of the defined HLA-A,-B,-C, or DR antigen specificities on the panel cells, nor on the basis of known cross-reactivities among these antigens. Most of the antisera showed strong cytotoxicity with the activated T cells. Such sera may define antigens presented to the maternal immune system during differentiation and development, which are not expressed on adult T cells unless they are activated.

Animals↗
PubMed1981

Rapid matching for transplantation: PHA primed lymphocytes.

Human lymphocytes, 10 days after treatment with phytohemagglutinin P (PHA-P), show PLT (secondary in vitro restimulation) reaction patterns which correspond to their cellular (HLA-D) type. Lymphocytes with shared cellular type stimulate PHA primed cells to a lesser degree than the majority of lymphocytes with unshared HLA-D type, which stimulate a strong response. Using a standard 3H thymidine incorporation technique, it is possible to detect responses in as early as 12 hr total incubation time, although usually a 22-48 hr total incubation time is normally required. This method is simple, gives results that correspond to the primary mixed leukocyte response, and thus may be useful as a cross-matching technique for cadaveric renal transplantation.

HLA Antigens↗
PubMed1996

Decrease of intracellular fluorescein fluorescence polarization (IFFP) in human peripheral blood lymphocytes undergoing stimulation with phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM) and anti-CD3 antibody.

In the present study we describe the induction of changes in intracellular fluorescein fluorescence polarization (IFFP) in lymphocytes undergoing activation with a variety of stimulants. These stimulants included the lectins phytohaemagglutinin (PHA), concanavalin (ConA), pokeweed mitogen (PWM) and anti-CD3 antibody. Changes in IFFP were detected in individual cells using the Cellscan apparatus. Our results show that by employing mitogenic concentrations of PHA, as revealed in a [3H]-thymidine incorporation assay, a decrease in the IFFP in human peripheral blood lymphocytes (PBL) occurred within 40 min. ConA and anti-CD3 affected similarly IFFP, whereas PWM, a B lymphocyte lectin, had no effect on IFFP at the concentrations employed. Kinetic analysis revealed that changes in IFFP occurred within 20-40 min after exposure to the stimulants and lasted for 24 h. Our results show that stimulants which activate CD3+ lymphocytes caused immediate changes in IFFP, in an enriched population of human PBL. The possible mechanisms involved in IFFP modulation following exposure to selected stimulants are discussed.

Antibody Specificity↗
PubMed1990

Frontal cortical projections from the suprageniculate nucleus in the rat, as demonstrated with the PHA-L method.

Frontal cortical projections from the rat suprageniculate nucleus (SG) were investigated by an anterograde tracing using Phaseolus vulgaris-leucoagglutinin (PHA-L). After PHA-L injection into the SG, labeled terminals were found in the frontal and temporal cortical regions. Labeled terminals in the frontal cortex were almost exclusively localized in the medial agranular area, and those in the temporal cortex were mainly localized in the primary and associational auditory areas. The labeled terminals in cortices were distributed predominantly in layers I, III and IV. The results suggest that ascending information through the SG projects to the medial agranular area in the frontal cortex as well as to the temporal cortex.

Animals↗
PubMed1991

Patterns of corticothalamic terminations following injection of Phaseolus vulgaris leucoagglutinin (PHA-L) in the sensorimotor cortex of the rat.

The morphology and spatial distribution of terminals emitted by corticothalamic axons originating from the rat motor cortex (as defined by intracortical microstimulations) were studied using Phaseolus vulgaris leucoagglutinin (PHA-L) as an anterograde tracer. After PHA-L injection in the face, forelimb or hindlimb motor cortical areas, small and densely packed boutons (about 1 micron in diameter), en passant and terminaux, were seen in the ventrolateral nucleus of the thalamus and, more sparsely, in the reticular nucleus, the nucleus ventrobasalis and the posterior nucleus of the thalamus. A separate projection with giant boutons (5-10 microns in diameter), en passant and terminaux, terminated in the posterior nucleus of the thalamus exclusively. Giant boutons originated from corticothalamic axons distinct from those providing small boutons. The corticothalamic projection originating from the motor cortex has basic organizational properties comparable to previous data obtained in the auditory and somatosensory corticothalamic projection systems.

Afferent Pathways↗
PubMed1993

Inhibition of PHA induced mononuclear cell proliferation by FK506 in combination with cyclosporine, methylprednisolone, 6-mercaptopurine and mycophenolic acid.

In vitro inhibition of human peripheral blood mononuclear cell (PBMC) proliferation by immunosuppressive drugs has been shown to correlate with clinical outcomes in kidney transplant patients. The aim of our study was to analyse the degree of variability of in vitro interactions of FK506 (FK) with combinations of cyclosporin A (CyA), methylprednisolone (MP), 6-mercaptopurine (6ME), and mycophenolic acid (MPA) using phytohaemagglutinin (PHA) stimulated PBMCs. Our hypotheses were: that a wide range of interindividual variation would be detected; and that certain combinations of drugs would have a synergistic inhibitory effect on PHA stimulated PBMCs. Cells were cultured in supplemented RPMI 1640 for 65 hours at 37 degrees C in humidified 5% CO2 - 95% air and proliferative responses measured by 3H-thymidine incorporation over a further 24 hours. Inhibition of PBMC proliferation was assessed over 10 FK concentrations ranging from 1 x 10(-8) to 10 micrograms/ml using both volunteer controls (n = 51) and dialysis patients (n = 23). A wide range of interindividual variability of FK inhibition occurred throughout the range of FK concentrations tested, becoming less variable at the higher concentrations. The interactions of FK with CyA, MP, 6ME and MPA were assessed using PBMCs from 12 volunteer controls. For FK at 1 x 10(-8) micrograms/ml; CyA, MP, 6ME and MPA were used at 0.01 microgram/ml. For FK at 1 x 10(-7) micrograms/ml; CyA, MP, 6ME and MPA were used at 0.1 microgram/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclosporine↗