Effect of cage population density on plasma corticosterone and peripheral lymphocyte populations of laboratory mice

The effect of different population densities of mice per cage on plasma corticosterone, peripheral lymphocytes and specific lymphocyte subpopulations was investigated. The animals were housed in groups of 2, 4 or 8 mice per cage and the blood samples were taken from each animal of these groups on days one, 7 and 14. A significant elevation (P less than 0.05) in plasma corticosterone concentration was observed in the group of 8 mice per cage on days one and 7 as compared with those of 2 or 4 mice per cage. The number of peripheral lymphocytes was significantly decreased in the groups of 2 (P less than 0.01) and 8 (P less than 0.05) mice per cage as compared with the group of 4 mice per cage on day one. A significantly decreased number of lymphocytes (P less than 0.01) in the group of 8 mice per cage continued to day 7. There were no significant differences in specific lymphocyte subpopulations observed among these groups. The results of this study suggest that a population density of 4 mice per cage induced minimal stress compared to that induced by the population densities of 2 or 8 mice per cage. Since stress is known to induce alteration in a variety of biological functions, the population density of mice per cage should be considered in the interpretation of research data.     

Mechanism of suppression of human peripheral blood lymphocyte proliferation by platelet activation factor

The influence of the phospholipid platelet activation factor (PAF) was studied on PHA-stimulated proliferation of peripheral blood lymphocytes in patients with bronchial asthma and normal subjects. It was found that influencing on the whole population of lymphocytes PAF suppressed proliferation mainly of T-cells. Besides, the influence of PAF on lymphocyte proliferation seemed to be mediated by monocytes since removal of monocytes from the whole population of mononuclear cells abolished the suppression of lymphocyte proliferation induced by PHA. Pretreatment of lymphocytes with PAF antagonist--BL 8705 almost completely blocked the suppression of lymphocyte proliferation induced by PHA.     

The expression profile of Fc receptor-like Y in B lymphocytes with hepatitis B virus induced diseases

The Fc Receptor-like Y (FcRY) molecule is preferentially expressed by B lymphocytes and has recently been considered as a potential therapeutic target for B cell malignancies. In this study, we investigated the correlation between FcRY expression profile, B lymphocytes population and different HBV infection disease status. The FcRY expression level on B lymphocytes and the number of B lymphocytes population from peripheral blood in 27 healthy controls (HC) and 65 patients with HBV-induced diseases, including chronic hepatitis B (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC), were analyzed using quantitative real-time PCR and flow cytometry. The results showed the level of FcRY expression and frequency of germinal center (GC) B lymphocytes from peripheral blood were significantly correlated with the HBV-related disease status, which was highest in HCC and LC patients, lowest in healthy donors, and in the middle in patients with CHB. Our study indicates that there is a significant correlation between FcRY expression profile, B lymphocytes population and HBV-induced diseases. However, the roles of FcRY and B lymphocytes in HBV-induced diseases are unclear and need further investigation.     

Rosette-forming ability of virus-treated human leukocytes (V-rosettes)

Rosette-formation with auto- and allogeneic red blood cells was applied to detection of human leucocyte subpopulations interacting with Sendai virus (V-rosettes). It was shown that the majority of V-rosette-forming cells appeared to be monocytes. T lymphocytes did not take part in V-rosette-formation since selective elimination of T cells from the mononuclear cells population did not lead to reduction of but increased the number of V-rosettes. Enrichment of cell suspension with B lymphocytes was followed by a rise in the number of V-rosettes thereby allowing the attribution of B lymphocytes along with monocytes to the cell population interacting with virus. The results suggest that ability of virus-exposed immunocompetent cells to react with their own red blood cells may lie at the basis of the development of autoimmune hemolytic anemia and other autoimmune diseases.     

Effects of phospholipid platelet activation factor on proliferation of B-lymphocytes of the peripheral blood of healthy donors and patients with bronchial asthma

The influence of the phospholipid platelet activation factor (PAF) was studied on pokeweed mitogen (PWM)-stimulated proliferation of peripheral blood lymphocytes in patients with bronchial asthma and normal subjects. It was found that PAF exerts dose-dependent activation with following suppression mainly on B-cells after its action on the whole population of lymphocytes activated by PWM. Besides, the influence of PAF on lymphocyte proliferation seemed to be mediated by monocytes since removal of monocytes from the whole population of mononuclear cells abolished the lymphocyte activation induced by PWM. Indomethacin inhibits lymphocyte proliferation activation induced by PAF. The results indicate that PAF has an effect on the IgE and IgG synthesis by blood B-lymphocytes in patients with bronchial asthma.     

Isolation and fractionation of porcine peripheral lymphocytes

A method is described for the separation and purification of porcine peripheral blood lymphocytes and their subsequent sub-cellular fractionation. The isolated cell population consisted of 99% lymphocytes with a viability of 100% and an overall recovery of 30%. The lymphocyte : thrombocyte ratio was 56 : 1 and the population contained no detectable erythrocytes. The lymphocytes proved resistant to mechanical breakage but a method of homogenisation in a hypotonic medium was developed which resulted in complete cell rupture with minimal damage to sub-cellular components.     

Cytotoxic activity against maedi-visna virus-infected macrophages.

The cell type predominantly infected by maedi-visna virus (MVV) is the macrophage, and we have looked at the ability of MVV-infected macrophages to interact with cytotoxic T lymphocytes (CTL), important effector cells against virus infections. MVV-specific CTL precursors were detected, after in vitro culture with MVV antigen and recombinant human interleukin-2, in peripheral blood lymphocytes of all MVV-infected sheep. MVV-infected monocyte-derived macrophages and alveolar macrophages were able to stimulate CTL activity in vitro and were targets for these activated CTL. The major effector cell population using MVV-infected macrophage targets was CD8+ lymphocytes, although another population, lymphokine-activated killer cells, may also have been involved. There was no direct cytotoxic activity found in alveolar lymphocytes from MVV-infected sheep without lung lesions.     

Stimulation and inhibition of human T-lymphocyte colony cell proliferation by hemopoietic cell factors.

Human lymphocytes, isolated from peripheral blood and stimulated with phytohemagglutinin M (PHA) prior to being seeded on a two-layer medium of soft agar which contained the mitogen, developed into colonies 3–4 days after seeding in the culture system. The cloning potential of PHA-treated lymphocytes is significantly enhanced by adding, to the soft agar culture, culture fluid (CF) obtained from mitogen-treated lymphocytes or a feeder layer (FL) prepared either from lymphocytes isolated from peripheral blood or from T-cell enriched populations. PHA seems to stimulate the release of lymphocyte colony enhancing factor (LCEF) from the T-sensitized lymphocytes. The addition of CF or FL to the culture medium appears to increase the amount of LCEF, resulting in enhancement of the number and size of lymphocyte colonies. When CF derived from spleen cells or from the peripheral blood adherent-cell population was added to the lower layer of the soft agar culture, the growth and development of lymphocyte colonies was inhibited. This suggests that monocyte-macrophages release a lymphocyte colony inhibiting factor (LCIF) into the CF. The extent of inhibition or stimulation of colony formation is a function of the number and type of cells used to prepare the CF or FL and the concentration of CF in the culture medium. The presence of FL or CF derived from spleen non-adherent cells, white blood cells, bone marrow cells, or a B-cell enriched population had no effect on colonies growing in the culture. This may possibly be due to the paucity of T lymphocytes and monocyte-macrophages present in these materials. A control system in which LCIF, produced by monocyte-macrophages, and LCEF, produced by T lymphocytes, participate in the regulation of lymphocyte production is postulated.     

MALL LYMPHOCYTE POPULATIONS IN THE MOUSE BONE MARROW

Autoradiography and scintillation counting have been used for evaluation of lymphocyte turnover and life span in the bone marrow, peripheral blood and thoracic duct lymph of BALB/C mice. It was shown that the bone marrow contained two populations of small lymphocytes. One population was labelled 100% after 3–4 days of intensive injections of ³H-thymidine and constituted about 75% of the lymphocytes. The remaining 25% of the lymphocytes turned over at a much slower rate comparable to the rate of increase in labelled small lymphocytes of the thoracic duct. More than 10% of the small lymphocytes of the bone marrow were found to be unlabelled after 10 days of intensive injections of ³H-thymidine. Nine weeks after giving ³H-thymidine for 30 consecutive days, 8·6% of the small lymphocytes in the bone marrow remained labelled. The mean grain counts of cells in this population were comparable to those of thoracic duct lymphocytes at corresponding times. About 90% of the peripheral blood lymphocytes were found to have a slow turnover and a long life span.     

Cytogenetic results of irradiation of Southern Ural residents

The paper summarizes the results of studies of 85 individuals exposed in the Southern Ural region. The spontaneous frequency of the cells with micronuclei (MN) in a population of human blood lymphocytes after PHA stimulation and cytokinetic block with cytochalasin B has been determined. The sensitivity of lymphocytes to the irradiation at the dose of 1.0 Gy and the adaptive response (AR) after the irradiation at the low adaptive dose of 0.05 Gy, and the challenge dose of 1.0 Gy 5 h later have been studied too. It was shown that the peculiarity of the Urals population consists in a higher individual variability of the frequency of cells with MN in all groups have been investigated (spontaneous, after acute irradiation in the dose 1.0 Gy) in comparison with Moscow people. The proportion of persons with a significant AR in the Urals groups was considerably lower than that identified among Moscow residents, and the number of persons with enhanced radiosensitivity increased following low-dose irradiation. We can suppose that prolonged action of low level radiation with another ecological factors, living in the contaminated regions result in the enhancement of the sensitivity to the genotoxic agents in the separate individuals.     

The migration of lymphocytes across specialized vascular endothelium. IV. Prednisolone acts at several points on the recirculation pathways of lymphocytes

Radioactively labelled thoracic duct lymphocytes from syngeneic rat donors were injected iv into recipients which had been given a continuous iv infusion of prednisolone at 1 mg/hr for 15–18 hr previously. The tissue distribution and recirculation into lymph of the labelled lymphocytes were compared quantitatively in the prednisolone-treated and control recipients by scintillation counting and autoradiography. The most prominent effect of prednisolone was to retard recirculating lymphocytes within the tissues to which they are normally distributed by the blood, namely the bone marrow, the spleen, and the lymph nodes. Although lymphocyte traffic was almost completely frozen by prednisolone, recirculating lymphocytes were not killed. A second effect of prednisolone was to impair the influx of lymphocytes from the blood into lymph nodes. Different groups of lymph nodes varied in the extent to which prednisolone inhibited the entry of lymphocytes, and previous antigenic stimulation completely exempted lymph nodes from this inhibition. Lymphocytes took a longer time to cross the walls of high endothelial venules in the lymph nodes of prednisolone-treated rats. A third effect of prednisolone was to increase the rate at which lymphocytes entered the bone marrow from the blood by crossing sinusoidal endothelium.     

The in vivo micronucleus test in human capillary blood lymphocytes: methodological studies and effect of ageing.

The in vivo micronucleus test in lymphocytes of human capillary blood collected by skin puncture is described. This method needs only 1-2 drops of finger blood. The normal value of micronucleus frequencies in lymphocytes from 250 healthy persons aged 6-88 years was determined. The upper limits of normal values were estimated by means of percentile. The 95th percentiles were 1/1000 and 1.5/1000 for the 6-45-year age group and the 46-88-year age group, respectively. There was no significant difference in micronucleus frequency between men and women. On the basis of micronucleus assays in more than 3000 cases, we consider that the micronucleus test in human capillary blood lymphocytes is a rapid, convenient and sensitive procedure for monitoring a human population exposed to environmental and occupational mutagens and carcinogens.     

Mechanism of lymphocyte activation by periodic acids. Blastogenesis and induction of immunomodulator populations]

Mild periodate treatment is mitogenic for T lymphocytes. With murine spleen cells periodate oxidation is effective between C8 and C9 on sialyl acid residues. With human blood lymphocytes this oxidation occurs between C7 and C8 of these residues. In vitro immune response is inhibited by periodate treatment. Activation of an immunosuppressive T lymphocyte population is obtained. Similar results are performed with human blood lymphocytes.     

Bordetella bronchiseptica dermonecrotizing toxin suppresses in vivo antibody responses in mice

The effects of Bordetella bronchiseptica dermonecrotic toxin (DNT) on the in vivo antibody response of mice were investigated. Intravenous injection of DNT at doses of 0.5 and 2.0 ng resulted in a significant suppression of the antibody response both to sheep red blood cells and to Escherichia coli lipopolysaccharide as measured by plaque-forming cell and hemagglutination assays. Spleen weights of mice given the same doses of DNT were significantly reduced, while the weights of thymuses and mesenteric lymph nodes were not. Numbers of Thy-1,2+ T lymphocytes, L3T4+ T lymphocytes, Lyt-2+ T lymphocytes and surface-immunoglobulin-positive lymphocytes decreased in spleens of the DNT-treated mice. Since the ratio of each lymphocyte population to the total number of splenic lymphocytes was not significantly different between the DNT-treated and non-treated mice, it is unlikely that DNT has a cytotoxic activity or a mitogen activity to some specific population of lymphocytes. Thus, we considered that the immunosuppression was attributable to a dysfunction of the spleen atrophied by the DNT.     

Binding of acridine orange by chromatin in human lymphocytes with different capabilities for adhesion]

A population of morphologically homogenous peripheral blood lymphocytes separated on a column with glass beads was studied microfluorimetrically. It was found that high adhesive lymphocytes bound acridine orange in a greater extent than did low adhesive cells.     

Accumulation of mu-type opiate receptor ligands during the culture of phytohemagglutinin-stimulated lymphocytes

Substances displacing labelled ligands from opiate receptors of the rat brain membrane fractions were found in the extracts of human blood lymphocytes. Pronase treatment has shown that at least part of the opiate receptor ligands present in lymphocyte extracts are of peptide nature. Opiate mu-receptor ligands content was identical in the extracts of the total population, T-lymphocyte and B-cell-enriched population. However, opiate delta-receptor ligands concentration in T-cell population is 15 times lower than in B-cell-enriched population. Opiate receptor ligands content is not changed during cultivation of nonstimulated lymphocytes. However, phytohemagglutinin activation results in a threefold increase of opiate mu-receptor ligands content in lymphocytes. The results obtained suggest that synthesis and processing of opiate mu-receptor ligands precursor occur in stimulated T-lymphocytes.     

Comparison of lymphocyte subpopulations in arterial and venous blood of the rat

Lymphocyte subpopulation as well as other hematological properties were compared between arterial and venous bloods of Wistar-Imamichi rats. Lymphocyte subsets were defined with four monoclonal antibodies which were specific to the respective cell surface glycoproteins. Using these monoclonal antibodies, subsets of B lymphocytes, T lymphocytes, helper T lymphocytes, and suppressor and cytotoxic T lymphocytes in the peripheral lymphocytes were identified. The blood samples were taken from aorta abdominalis and venae cava caudalis. The population of these subsets were enumerated by a laser flow-cytometry system. The result showed that there was no significant difference in hematological properties between the arterial and venous blood except in leukocyte count and hemoglobin concentration. The difference in leukocyte counts was thought to depend mainly on the fluctuation of the lymphocyte counts. However, no significant difference was recognized in the proportion of positive cells to each monoclonal antibody. It was concluded that the difference in leukocyte counts found between the arterial and venous bloods of the Wistar-Imamichi rat did not produce any effects on the proportion of the subpopulation in the peripheral lymphocytes, and the lymphocyte subpopulations in both arterial and venous bloods were substantially equivalent to each other.     

The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes

We examined the antigenic and functional characteristics of human peripheral blood lymphocytes that differentially express the CD16 (Leu-11) and Leu-19 (NKH-1) antigens. Leu-19 is a approximately 220,000 daltons protein expressed on approximately 15% of freshly isolated peripheral blood lymphocytes. Within the Leu-19+ subset, three distinct populations were identified: CD3-,CD16+,Leu-19+ cells; CD3+,CD16-,Leu-19+ cells; and CD3-,CD16-,Leu-19bright+ cells. Both the CD3+,CD16-,Leu-19+ and CD3-,CD16+,Leu-19+ populations mediated non-major histocompatibility complex (MHC)-restricted cytotoxicity against the NK-sensitive tumor cell K562 and were large granular lymphocytes. CD3-,CD16+,Leu-19+ NK cells were the most abundant (comprising approximately 10% of peripheral blood lymphocytes) and the most efficient cytotoxic effectors. The finding that CD3+,Leu 19+ lymphocytes mediated cytotoxicity against K562 unequivocally demonstrates that a unique subset of non-MHC-restricted cytotoxic CD3+ T lymphocytes are present in the peripheral blood of unprimed, normal individuals. However, CD3+,CD16-,Leu-19+ cells comprised less than 5% of peripheral blood lymphocytes, and the cytotoxic activity of this subset was significantly less than CD3-,CD16+,Leu-19+ NK cells. Most CD3+,Leu-19+ T cells co-expressed the CD2, CD8, and CD5 differentiation antigens. The antigenic and functional phenotype of peripheral blood CD3+,Leu-19+ cytotoxic T lymphocytes corresponds to the interleukin 2-dependent CD3+ cell lines that mediate non-MHC-restricted cytotoxicity against NK-sensitive tumor cell targets. A small population of Leu-19bright+ lymphocytes lacking both CD3 and CD16 was also observed. This population (comprising less than 2% of peripheral blood lymphocytes) contained both large agranular lymphocytes and large granular lymphocytes. CD3-,CD16-,Leu-19bright+ lymphocytes also mediate non-MHC-restricted cytotoxicity. The relationship of these CD3-CD16-,Leu-19bright+ lymphocytes to CD3+ T cells or CD16+ NK cells is unknown.     

Changes in the composition of canine respiratory cells obtained by bronchial lavage following irradiation or drug immunosuppression.

Canine respiratory cells, obtained by bronchial lavage, and blood leukocytes were monitored to observe cellular changes following acute and chronic immunosuppression. Irradiation (350 R) produced bone marrow suppression and prompt peripheral blood leukopenia, but did not affect recovery of pulmonary alveolar macrophages or lymphocytes for 12 days after. Treatment for 6 weeks with daily methylprednisolone (1 mg/kg) caused a progressive decrease in the number of recoverable respiratory lymphocytes, whereas alternate day methylprednisolone (2 mg/kg) had less effect. Cyclophosphamide in combination with steroids generally augmented the progressive loss of blood and respiratiory lymphocytes. Recovery of alveolar macrophages was not changed appreciably. Thus, the population of lung macrophages, sampled by pulmonary lavage, withstood acute and chronic forms of immunosuppression very well. In contrast, canine lymphocytes seem more susceptible to injury, especially to drug regimens containing steroids.     

Mosaicism of peripheral blood lymphocyte populations in females heterozygous for the Lesch-Nyhan mutation

Phytohemagglutinin-stimulated thymidine-H³ incorporation into peripheral blood lymphocytes in vitro was inhibited by 8-azaguanine (AG) in cells from normal individuals, but not in cells from boys with the Lesch-Nyhan (L-N) syndrome. Artificial cell mixtures in which the AG-resistant minority L-N population comprised more than 1.0% of the total were clearly differentiated from normals by this technique. The method was used to search for blood cell mosaicism in females heterozygous for the X-chromosomal L-N mutation. In three adolescent girls, a minority population of AG-resistant lymphocytes comprising 5–10% of the total was found, while no such mosaicism was detected in a heterozygous woman of 40. Although blood cell mosaicism had not previously been found in female carriers of the L-N mutation, the sensitivity of the present method exceeds that of previous studies. Our findings suggest that in vivo selection against lymphocytes with the L-N mutation does occur but is not absolute and may be age related. The present method has applications in studying this phenomenon, and, with modification, may provide a direct assay system to detect the in vivo frequencies of AG-resistant lymphocytes.