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A simple, reproducible method is described for in vitro culture of mouse lymphocytes purified by differential centrifugation through Ficoll-Hypaque gradient. Peripheral blood, spleen and thymus purified lymphocytes responded well to in vitro PHA stimulation as measured by blast transformation and tritiated thymidine incorporation; the lowest values were observed with thymus lymphocytes.
The purpose of the present study was to detail the spinal cord (SC) trajectories and arborization patterns of vestibulospinal axons descending from the lateral vestibular nucleus (LVN). An anterograde neural tracer, Phaseolus vulgaris-leucoagglutinin (PHA-L), was focally injected into the right-side LVN in 8 cats. Their subsequent survival times varied from 4 days to 12 weeks. The labeled axons were found mainly in the brainstem after 4-5 days and in successively more caudal spinal segments after longer survival times: i.e., in C1-T2 after 2-3 weeks, in C3-T11 after 6-7 weeks, and in T7-S1 after 10-12 weeks. The trajectories of 28 single, thick (diameter >/=2.4 microm) lateral vestibulospinal tract (LVST) axons were traced from serial transverse sections of the SC from C1-8 (n = 10), T1-9 (n = 11), and T11-L7 (n = 7). In the cervical segments, the LVST axons gave off collateral fibers, which terminated mainly in Rexed's laminae VII-VIII. The terminal-field patterns of these collaterals differed from one stem axon to another. In the thoracic segments, the terminal-field patterns from a given LVST axon were similar at each segmental level, i.e., a few main branches with or without short side branches. At the L3-5 midlumbar level, the collaterals usually arborized more extensively, such that their terminal fields occupied a much greater region of laminae VII-VIII. In contrast, at the L6-7 lower lumbar level, collaterals arising from thin axons (diameter <1.0 microm) tended to innervate, with even more extensive arborization, the medial part of the lamina VIII. These results revealed common and segment-specific collateral distribution patterns of LVST axons along the full extent of the spinal neuraxis.
Thymocytes of C3H/He mice were poorly triggered to induce DNA synthesis by phytohemagglutinin but fairly well triggered when they were mixed with small number oble product(s) of these cells. Peritoneal exudate cells were mainly harvested at 3 hr, largely consisting of macrophages, after intraperitoneal injection of 0.2% sodium caseinate. Addition of 2(2) to 2(6) x 10(3) of 3-hr exudate cells, 2(2) to 2(4) x 10(3) of 48-hr exudate cells, or 2(2) to 2(4) x 10(3) of 96-hr exudate cells to syngeneic thymocyte culture (3 x 10(6) resulted in marked enhancement of DNA synthesis by thymocytes stimulated with 1 mul PHA. Similar enhancement of DNA synthesis was also observed in the presence of 200 mul of cellfree supernatant fluid of peritoneal exudates of 3 hr or 9 hr after casein stimulation. Conversely, two hundred microliters of 0-hr, 48-hr, or 96-hr peritoneal fluid showed a slight suppressing effect on thymocyte response and 50 to 100 mul of these exudates showed no enhancing effect. Furthermore, 50 to 800 mul of culture supernatants from 1.6 x 10(6) cells/4 ml of 3-hr exudate cells, 96-hr exudate cells, or purified macrophages also caused a similar enhancement of DNA synthesis by thymocytes. On the other hand, excess numbers of exudate cells (2(7) to 2(8) x 10(3) of 3-hr exudate cells, or 2(5) to 2(9) of 96-hr exudate cells) or excess amounts of culture supernatants (800 mul from 6.4 to 12.8 x 10(6)/4 ml of 3-hr cells, or 200 mul from 12.8 x 10(6)/4 ml, 400 mul from 6.4 x 10(6)/4ml, or 800 mul from 3.2 x 10(6)/4 ml of 96-hr cells) diminished the enhancing potency on thymocytes. The cooperative function by polymorphonuclear leukocytes or by macrophages on the lymphocyte response was thus postulated.
Beryllium in various physical forms was studied for its ability to induce increased 14C-thymidine incorporation by allergised guinea pig lymphocytes. No increase was observed. It was further shown that beryllium had an inhibitory effect on the response of normal lymphocytes to PHA and on the response of allergised lymphocytes to antigen. These results support the finding in other systems that beryllium can suppress DNA synthesis.
Fumaric acid monoethylester (FAME) inhibits the incorporation of 14C-Thymidin, 14C-Uridin, 14C-Alanin and 14C-Leucin into acid-insoluble biopolymers of cultivated PHA-stimulated human lymphocytes. At high concentrations of FAME (500 mug/ml culture medium) the inhibition of nucleic acid synthesis is 6 times higher on the average than the inhibition of protein synthesis. However, the application of the cis-isomer, maleic acid monoethylester (MAME), results in an increase of the incorporation rate of the labelled precursors into the RNA and DNA. This is 3.5--9.3 times higher than after application of FAME. The results demonstrate the specific inhibition by FAME. The rate of labelling of nucleic acids is decreased above 10 mug FAME/ml culture medium and in the case of MAME above 50 mug/ml medium. As an explanation of the specific action of FAME its influence on the enzymes of the nucleic acid synthesis, the citric acid cycle or a faulty synthesis of enzymes are discussed.
The effect of adriamycin on PHA-stimulated lymphocytes is dose-dependent. An adriamycin concentration of 0.2 mug/ml reduces blast transformation and RNA synthesis as well as cell proliferation (DNA-synthesis). The lower concnetration of 0,01 mug/ml, which can be found inpatients for several hours after i.v. application, results only in a transitional inhibition of uridine nucleolar labelling. This inhibition of uridine nucleolar labelling appears more rapidly after adriamycin addition than that of thymidine labelling. Lymphocytes preincubated with adriamycin are effected similarly to lymphocyte cultured in the continuous presence of adriamycin.
Treatment of human perypheral blood lymphocytes with phytohaemagglutinin (PHA), for 30 min and for 72 h, increases their ability to form E-rosettes. Possible interpretations of the phenomenon are discussed.
We studied the cytotoxic effects of recombinant TNF-alpha and supernate of phytohemagglutinin stimulated peripheral blood mononuclear cells individually and in combination against a cholangiocarcinoma cell line. Levels of cyclins D1, E and A in the cell line were detected by immunoblotting, and the cell cycle stage was assayed by propidium iodide staining followed by flow cytometry analysis. Viable and apoptotic cells were assessed by trypan blue dye exclusion, DAPI staining, agarose DNA laddering and propidium iodide staining. At the beginning of each experiment, the majority of cholangiocarcinoma cells expressed cyclin A and were in S phase as determined by propidium iodide staining. Treatment of such cells with recombinant TNF-alpha resulted in cytotoxic effects clearly evident at 36 hours post exposure. There was a synergistic killing effect when recombinant TNF-alpha was combined with PHA supernate and this effect could be partly neutralized by monoclonal anti TNF-alpha, interleukin (IL)-2, IL-12 and IFN-gamma.
The author examined the enzymic content of lymphocytes and the changes, which occurred after stimulation with phytohemaglutinins (PHA). There was a difference in the reaction of lymphocytes in healthy persons, in patients with virus diseases and in persons with malignant lympholeucosis.
The study group consisted of 13 patients, including 5 in complete remission and 8 in incomplete hematologic remission. Six patients had a pause in therapy, and the remaining ones were continuing maintenance therapy. In all cases production of IgG, IgA and IgM immunoglobulins was maintained. Response of lymphocytes to PHA in vitro was normal in 7 patients, mainly in those who had a pause in treatment. The test results were uncorrelated with hematologic status. Both tests showed preserved humoral immunity and delyed hypersensitivity in the early period of remission and had no prognositc value.
The production of interferon-tau (IFN-tau), interleukin-2 (IL-2) and prostaglandin-E2 (PGE2) in peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin (PHA) and the repercussion which these cytokines have on the growth and activity of the 2', 3' and cyclonucleotide-3'-phosphohydrolase (CNP) of cultures of rat oligodendrocytes were analyzed. The study was carried out in a group of patients (20 with active disease, 23 in remission) with defined multiple sclerosis (MS), a group of subjects with other central nervous system diseases (OCNSD) and a group of 20 healthy (HS). Increases in PGE2 were found in PBMC cultures of patients with MS (p < 0.0005, ANOVA test) and the production of PGE2 in patients with active MS correlated positively (R2 = 0.743, p = 0.02) with CNP activity in the oligodendroglial cultures. These results suggest that PGE2 may influence in the re-myelinization process.
Phytohaemagglutinin (PHA)-responsive lymphocytes from human peripheral blood samples, either irradiated or un-irradiated, showed increased frequency of first division metaphase cells (detected by fluorescence plus Giemsa (FPG) staining) as a function of duration of storage. Irradiated and subsequently stored samples showed small but significant increase for the yield of dicentrics. The yield of aberrant metaphases and deletions (excess acentrics) remained unchanged. Increasing Bromodeoxyuridine (BrdU) concentrations slowed down the cell cycle progression but did not influence the yield of aberrations including that of dicentrics.
Proliferation kinetics and spontaneous yield of chromosomal aberrations phytohemagglutinin (PHA)-responsive peripheral blood lymphocytes were studied from blood samples collected from 45 individuals in 4 different synthetic media. Except for a significant difference for Eagle's MEM and RPMI 1640, the other media did not show difference for the yield of chromatid or chromosome type of aberrations. Differences were however noticed in the proliferation kinetics (mitotic and proliferative rate indices) of cells among the media used. The study indicated that (i) the intrinsic properties of media which influence proliferation rate and yield of chromosomal aberrations are independent of each other as higher proportion of first division cells do not correspond with higher frequency of chromosomal aberrations, (ii) the amount of free-radical scavengers present in the medium, apart from the genetic make-up of the individuals, may contribute to the spontaneous yield of chromosomal aberrations and (iii) RPMI 1640 medium, which showed higher transformation and faster cycling rate for the lymphocytes, may be considered as medium of choice for analysing two main cytogenetic end-points, chromosomal aberrations and sister chromatid exchanges (SCEs).
Lymphocytes were highly purified from synovial fluid and peripheral blood of 10 rheumatoid arthritis patients and assessed for responsiveness to PHA-P and Con-A. In all cases, both synovial and blood lymphocytes showed a marked reduction in response to these mitogens compared with normal blood lymphocytes. The factors responsible for this low T cell responsiveness are discussed.
To describe the development, components, initial results and lessons learned from Thailand's National Access to Antiretroviral Program for People living with HIV/AIDS (NAPHA), a historical review was conducted and program monitoring was analyzed. The national antiretroviral therapy program at different levels of the public health system was implemented with all major program components; ARV protocol development, health care professional training, drug supply chain management, laboratory network formation, monitoring and evaluation, and multi-sector and PHA involvement since 2001, which was based on elements of research, pilot projects, training, national guideline development, experiences and policy making. A national monitoring system was developed to monitor the progress of the program. From February 2001 to December 2004, the monitoring reports received from implementing hospitals showed that 58,133 cases had received antiretroviral therapy (ART), and 85% (49,477) of them were continuing to take ARV drugs. In conclusion, the NAPHA was implemented nationwide with comprehensive systems. The reports indicate achievement of expansion of the ART program. Lessons learned from the program initiation and scaling up show local leadership, comprehensive training, adherence, and coordination are essential to program effectiveness and sustainability.
Serum alphafetoprotein (AFP) is actively taken up, through receptor-mediated endocytosis, by many embryo-fetal cells during ontogenic development but also by neoplastic cells, as well as by normal peripheral T lymphocytes after mitogenic activation. We have previously shown that the ability to internalize AFP is impaired in mitogen-activated T cells from several groups of (HIV+)-seropositive individuals and that this expression roughly correlates with the progression of the disease. It is not clear whether this impaired AFP-endocytosis results from an HIV-mediated inhibition of AFP-receptor expression or if it is the consequence of a target signal transduction impairment due to HIV-infection. In the present work we have explored both possibilities by studying AFP-endocytosis in peripheral blood mononuclear cells (PBMC) from seropositive (HIV+)-asymptomatic heterosexual individuals and in in vitro HIV-infected PBMC from healthy donors, either quiescent or stimulated with phytohemagglutin (PHA). Our results suggest that this novel abnormality of T-cells associated with HIV-infection reflects an unusual proliferative response of PBMC to mitogenic stimuli.
The effect of red blood cells (RBC), fresh plasma (FP), fresh frozen plasma (FFP) and different storage solutions (e.g. CPDA-1, CPD, PAGGS-M, SAG-M, Adsol) on the immune response was investigated in human lymphocyte cultures. The inhibitory activity was measured by determining the effect of different components on the PHA-induced T-cell proliferation. Our results indicate that the inhibitory effect is caused by an as yet unidentified plasma factor (e.g. anti-idiotypic antibodies, soluble HLA-class I or II antigens). In contrast, pure RBC or blood storage solutions revealed only minimal effects on the T-cell response.
Concanavalin-A (Con-A) gave rise to a moderate aggregation in untreated (control) Tetrahymena pyriformis GL populations. The cells pretreated (imprinted) with insulin or lectins underwent aggregation earlier and to a greater degree than the untreated control cells. Lens lectin, although of the same sugar specificity as Con-A, was considerably less active than the latter. Helix lectin and phytohaemagglutinin (PHA), although of similar sugar specificity, accounted for a dissimilar extent of cell aggregation, in that Helix lectin was comparable in activity to Con-A. Pretreatment (imprinting) with insulin enhanced the aggregation inducing effect of those lectins which were capable of overlapping on the insulin receptor owing to similar specificity.