Exercise-induced changes to in vitro T-lymphocyte mitogen responses using CFSE.

Carboxyfluorescein diacetate succinamidyl ester (CFSE) labeling of lymphocyte populations can provide unique insights into cell function at rest and with exercise, due to its ability to quantify cell division on an individual cell basis. This study aimed to characterize the effect of acute, intense exercise on T-lymphocyte function. Well-trained endurance runners completed 60 min of treadmill running at 95% of individual anaerobic threshold. Blood samples were collected before exercise; after 30 and 60 min of exercise; and after 30, 60, and 90 min of recovery. Isolated peripheral blood mononuclear cells were labeled with CFSE and cultured with or without mitogen (phytohemagglutinin). After culture, cell suspensions were labeled with CD3 (allophycocyanin) and CD8 (phycoerythrin), and expansion rates and cell death rates were calculated for each sample, as well as mitosis rates for each cell generation. Exercise was associated with a 60% decrease in cell expansion in both CD4 and CD8 cell types from before exercise to midexercise (P < 0.05). The significant decrease in expansion rate in the midexercise samples for both cell types was mirrored by a 65% increase in cell death (P < 0.05) in both cell types at that sample point. Exercise had no effect on the mitosis rate of either CD4 or CD8 cells in any cell generation (generations 0-3). This study indicates that 1 h of intense exercise affects in vitro T-lymphocyte function. These data suggest, for the first time, that exercise decreases cell expansion rate via an increase in cell death of both CD4 and CD8 T lymphocytes, rather than a decrease in mitosis.     

Carbohydrate supplementation and exercise-induced changes in T-lymphocyte function.

Carbohydrate (CHO) ingestion during exercise has been shown to reduce perturbations in immune cell numbers and function, possibly through a reduction in the cortisol response to exercise. We have previously observed that exercise decreases T-lymphocyte responses to mitogen via an increase in cell death of both CD4 and CD8 T lymphocytes (Green KJ and Rowbottom DG. J Appl Physiol. 95: 57-63, 2003). This study tested the hypothesis that CHO ingestion rather than placebo (Pl) would result in an attenuation of the cortisol response to exercise and a reduction of the exercise-associated alterations in cell death. Six well-trained cyclists completed two exercise trials consisting of 2.5 h of cycling at 85% of individual ventilatory threshold. In a random order, trials were completed under either CHO (6% CHO solution, 3.2 g CHO/kg body wt total) or Pl conditions. Blood samples were collected before exercise, midexercise (after 60 min of exercise), immediately after exercise, and after 60 min of recovery. T-lymphocyte responses to mitogen were determined by using carboxyfluorescein diacetate succinimidyl ester fluorescent cell division tracking and expansion rates, and cell death rates were calculated for each sample as well as mitosis rates for each cell generation. Cellular expansion of T lymphocytes was decreased after exercise in Pl only. The reduction in cellular expansion was related to an increase in cell death of both CD4 and CD8 cells in culture rather than a decrease in the ability of cells to undergo mitosis. CHO ingestion compared with Pl was associated with no reductions in cellular expansion or increases in cell death. CHO ingestion during exercise acted to reduce the impairment of T-lymphocyte function by decreasing cell death within mitogen-stimulated cell cultures; however, the mechanism of action appears to be independent of cortisol.     

Cell numbers and in vitro responses of leucocytes and lymphocyte subpopulations following maximal exercise and interval training sessions of different intensities.

In vitro lymphocyte function and the mobilisation of peripheral blood leucocytes was examined in eight trained subjects who undertook an incremental exercise test to exhaustion and a series of interval training sessions. Venous blood samples were obtained before the incremental test, immediately after, and 30, 60, and 120 min after the test. Interval training sessions were undertaken on separate days and the exercise intensities for each of the different sessions were 30%, 60%, 90% and 120% of their maximal work capacity respectively, as determined from the incremental exercise test. There were 15 exercise periods of 1-min duration separated by recovery intervals of 2 min in each session. Venous blood samples were obtained immediately after each training session. Significant increases in lymphocyte subpopulations (CD3+, CD4+, CD8+, CD20+, and CD56+) occurred following both maximal and supramaximal exercise. This was accompanied by a significant decrease in the response of cultures of peripheral blood lymphocytes to Concanavalin A (ConA), a T-cell mitogen. The state of lymphocyte activation in vivo as measured by CD25+ surface antigen was not, however, affected by acute exercise. The total number of lymphocytes, distribution of lymphocyte subpopulations and in vitro lymphocyte response to ConA had returned to pre-exercise levels within half an hour of termination of exercise but serum cortisol concentrations had not begun to fall at this time. There was a significant decrease in the CD4+:CD8+ cell ratio following exercise; this was more the result of increases in CD3-CD8+ cells (CD8+ natural killer cells) than to CD3+CD8+ cells (CD8+ T-lymphocytes). Decreased responsiveness of T-cells to T-cell mitogens, postexercise, may have been the result of decreases in the percentage of T-cells in postexercise mixed lymphocyte cultures rather than depressed cell function. The cause of this was an increase in the percentage of natural killer cells which did not respond to the T-cell mitogen. The results indicated that while a substantial immediate in vitro "immunomodulation" occurred with acute exercise, this did not reflect an immunosuppression but was rather the result of changes in the proportions of reactive cells in mononuclear cell cultures. We have also demonstrated that the degree of the change in distribution of lymphocyte subpopulation numbers and responsiveness of peripheral blood mononuclear cells in in vitro mitogen reactions increased with increasing exercise intensity. Plasma volume changes may have contributed to some of the changes seen in leucocyte population and subpopulation numbers during and following exercise.     

The Effect of Krill Oil Supplementation on Exercise Performance and Markers of Immune Function

Background

Krill oil is a rich source of the long-chain n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which may alter immune function after exercise. The aim of the study was to determine the effects of krill oil supplementation on post exercise immune function and performance.

Methods

Nineteen males and 18 females (age: 25.8 ± 5.3 years; mean ± S.D.) were randomly assigned to 2 g/day of krill oil (n = 18) or placebo (n = 19) supplementation for 6 weeks. A maximal incremental exercise test and cycling time trial (time to complete set amount of work) were performed pre-supplementation with the time trial repeated post-supplementation. Blood samples collected pre- and post- supplementation at rest, and immediately, 1 and 3h post-exercise. Plasma IL-6 and thiobarbituric acid reactive substances (TBARS) concentrations and, erythrocyte fatty acid composition were measured. Natural killer (NK) cell cytotoxic activity and peripheral blood mononuclear cell (PBMC) IL-2, IL-4, IL-10, IL-17 and IFNγ production were also measured.

Results

No effects of gender were noted for any variable. PBMC IL-2 and NK cell cytotoxic activity were greater (P < 0.05) 3h post exercise in the krill oil compared to the control group. Plasma IL-6 and TBARS, PBMC IL-4, IL-10, IL-17 and IFNγ production, along with performance and physiological measures during exercise, were not different between groups.

Conclusion

Six weeks of krill oil supplementation can increase PBMC IL-2 production and NK cell cytotoxic activity 3h post-exercise in both healthy young males and females. Krill oil does not modify exercise performance.     

Mobilization of NK cells by exercise: downmodulation of adhesion molecules on NK cells by catecholamines

The change of plasma catecholamine concentration correlates with the change of natural killer (NK) activity and NK cell number in peripheral blood mononuclear cells (PBMC) during and after moderate exercise. We studied the causal relation between exercise-induced catecholamine and expression of adhesion molecules on NK cells during and after exercise. The expression of CD44 and CD18 on CD3(-)CD56(+) NK cells was significantly reduced during exercise (P < 0.01). When PBMC were stimulated with 10(-8)M norepinephrine in vitro, the expression of these adhesion molecules on CD3(-)CD56(+) NK cells was downmodulated within 30 min. The binding capacity of NK cells to a CD44 ligand, hyaluronate, was reduced by the stimulation with norepinephrine (P < 0.01). The intravenous injection of norepinephrine in mice decreased the expression of CD44 and CD18 on CD3(-)NK1.1(+) cells (P < 0.01) and increased the number of CD3(-)NK1.1(+) cells in PBMC (P < 0.01). These findings suggest that exercise-induced catecholamines modulate the expression of adhesion molecules on NK cells, resulting in the mobilization of NK cells into the circulation.     

Comparative studies on in vitro reactivity of fresh and cryopreserved pig lymphocytes.

The effect of cryopreservation on in vitro reactivity of pig lymphocytes was studied. Peripheral blood mononuclear cells (PBMC) were frozen by controlled-rate freezing and stored in liquid nitrogen (LN2) between 4 and 36 days. Following thawing 74.7 +/- 2.6% of cells were recovered of which 94.5 +/- 0.9% were viable as determined by trypan blue exclusion. Functional parameters measured included the concentration of free intracellular Ca2+ ([Ca2+]i) in resting and mitogen-stimulated PBMC, mitogen and alloantigen-induced blastogenesis, as well as cell-mediated cytotoxicity. Irrespective of storage time and cell donor, [Ca2+]i in frozen-thawed PBMC (67.7 +/- 4.3 nM) was significantly lower (P less than 0.001) when compared to fresh cells (96.2 +/- 4.5 nM). In addition, cryopreserved PBMC only weakly responded with an increase of [Ca2+]i after stimulation by various concentrations of phytohemagglutinin (PHA). Following activation by PHA (2 micrograms/ml) for 4 days fresh lymphocytes (84,047 +/- 5475 cpm) incorporated significantly more (P less than 0.005) [3H]thymidine than frozen PBMC (66,001 +/- 4117 cpm). A similar difference in proliferation rates (P less than 0.05) between fresh (10,046 +/- 1915 cpm) and frozen-thawed PBMC (5852 +/- 1304 cpm) was observed in one-way mixed lymphocyte cultures (MLC), while the spontaneous incorporation of radiolabel was unchanged in frozen stored cells. By using MLC-derived cytotoxic effector cells (E) and [3H]thymidine-labeled concanavalin A blasts as targets (T), cryopreserved PBMC displayed a severe deficiency of cytotoxic effector functions at all tested E:T ratios. These results indicate that pig PBMC are very sensitive to LN2 storage although some immunological functions are more affected by cryopreservation than others.     

Gamma-irradiated peripheral blood mononuclear cells can express LAK activity

Peripheral blood mononuclear cells (PBMC) irradiated with high dose gamma-radiation (1000-5000 rad) are commonly used as feeder cells during the cloning of T lymphocytes, natural killer (NK) and lymphokine activated killer (LAK) cells. We report here that such gamma-irradiated PBMC can be stimulated with interleukin 2 (IL-2) to express the ability to lyse a variety of tumor cell targets. The non-major histocompatibility complex (MHC) restricted cytotoxicity demonstrated by irradiated PBMC is, however, lower than that expressed by their non-irradiated counterparts. The numbers of viable, gamma-irradiated LAK cells are significantly increased by the addition of the mitogen, phytohemagglutinin (PHA). Purification of the gamma-irradiated cells expressing cytotoxic activity by flow cytometry determined that the effector cells were predominantly CD3- cells, although some CD3+ cells also expressed moderate LAK activity. The ability of gamma-irradiated cells to proliferate in the presence of PHA alone, or with IL-2 + PHA, was maximal at day 4-5; but proliferation, as detected by 3H-thymidine uptake, was not detectable beyond 12-15 days of in vitro culture. Because many of the LAK, T cell and NK cell cloning procedures require the presence of feeder layers, growth factors (usually IL-2) and mitogens, the presence of residual feeder cells expressing cytotoxic activity may affect the specificity of such clones. Thus, efforts should be made to ensure that such gamma-radiation-resistant cells capable of expressing cytotoxic activity are completely eliminated before the cloned cells are used for further experiments.     

Anti HLA class I monoclonal antibody effect on PKC kinetics in PHA activated human peripheral blood mononuclear and E+ cells

Cytoplasmic protein kinase C (PKC) has been studied in phytohemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMC) and macrophage depleted E+ cell culture. Within 10' after contemporanous addition of PHA and anti HLA class I monoclonal antibody 01.65 (MoAb) PKC is depleted in both cell types. Enzyme activity recovers in the following hours however at 72 hours is at control values in E+ cultures while in PBMC cultures it is still depleted at 68% of the control. Anti HLA class I MoAb induced tritiated lymidine (3H-TdR) incorporation inhibition appears to be related to low levels of PKC activity.     

Exercise and blood lymphocyte subset responses: intensity, duration, and subject fitness effects

This study examined the effect of exercise intensity and duration on the percent blood lymphocytes in men of low [LF; maximal O2 uptake (VO2max) less than 50 ml.kg-1.min-1 and sedentary], moderate (MF; VO2max = 50-60 ml.kg-1.min-1 and recreationally active), and high (HF; VO2max greater than 60 ml.kg-1.min-1 and recent training history) fitness. Thirty healthy adult men (aged 20-31 yr) participated in four randomly ordered cycle ergometer rides: ride 1 (65% VO2max, 30 min), ride 2 (30% VO2max, 60 min), ride 3 (75% VO2max, 60 min), and ride 4 (65% VO2max, 120 min). Blood samples were drawn at various times before and after the exercise sessions. Lymphocyte subsets were determined by flow cytometry using monoclonal antibodies for total T (CD3+), T-helper (CD4+), and T-suppressor (CD8+) lymphocytes and for a subset of cells expressing a natural killer (NK) cell antigen (Leu7+). Plasma catecholamines were assayed to determine exercise stress. There were sharp reductions (P less than 0.01) in the percentage of pan-T and T-helper lymphocytes immediately after exercise across all fitness levels; the magnitude of this reduction was greatest after the highest intensity (ride 3) or longest duration (ride 4) work. In contrast, the absolute number of T and T-helper cells tended to increase after exercise and significantly so in the HF subjects (P less than 0.005). There was no significant effect of exercise or subject fitness category on the percentage of T-suppressor lymphocytes, although the absolute numbers of this subset increased significantly after exercise in LF subjects. Marked increases (P less than 0.01) in the percentage of NK cells occurred immediately after exercise at all intensities and durations tested; numerical increases in total NK cells were significant in all fitness groups after the highest intensity work (ride 3; P less than 0.005). Irrespective of whether the changes were expressed as percentage or total numbers, recovery to base line occurred at 30 min after exercise. The results suggest that the exercise effect on blood lymphocyte subset percentages in men is transient and occurs across all fitness levels. Concomitant changes in plasma catecholamine concentrations are only weakly associated with these lymphocyte subset percentage responses to exercise. Furthermore, this study shows that the exercise-induced changes in lymphocyte percentages do not consistently reflect changes in the absolute numbers of cells.     

Biosynthesis of the third component of complement in rat liver epithelial cell lines and its stimulation by effector molecules from cultured human mononuclear cells

Summary Rat liver epithelial cell lines, growing in a serum-supplemented medium, synthesize and secrete into the culture medium the third component of complement (C₃). We studied the regulation of C₃ production in this system. We found that human peripheral blood mononuclear leukocytes in culture released one or more soluble factors which stimulated rat liver epithelial cells to produce increased quantitites of C₃. This stimulting effect was strongly enhanced when the mononuclear cell cultures were treated with phytohemagglutinin, a T-lymphocyte mitogen. The factor(s) failed to enhance C₃ biosynthesis by rat dermal fibroblasts, which are known to produce this protein. This reveals a tissue-specific differential response between the fibroblasts and the liver epithelial cells. The physical and chemical characteristics, such as heat sensitivity, 2.8M ammonium sulphate precipitation, and lower activity after digestion by proteases unambiguously indicate that the effector molecules are proteins. When the crude supernatant of mononuclear leukocytes was fractionated by gel filtration, the stimulating factor(s) eluted as two peaks with apparent molecular weight of 25 to 60 and 15 to 20 kdalton, respectively. As to the cellular origin of the C₃-stimulating factor(s), several observations were made: (a) in separate cultures containing either T-cells or monocyte-enriched populations from the same sample of blood mononuclear cells, no activity was detected in the presence or absence of phytohemagglutinin, (b) conditioned media from each of these cultures could not substitute for the corresponding intact cell populations, and (c) the addition of purified T-cells to the monocyte-enriched population in the presence of phytohemagglutinin restored the production of the stimulating activity by the mixed culture. Finally, experiments were carried out to verify whether monokine interleukin 1 affects the hepatic C₃ biosynthesis. It was demonstrated that interleukin 1 enhanced this biosynthesis, but could not completely substitute for conditioned medium from stimulated mononuclear cells.     

Trypanosoma cruzi suppresses the ability of activated human lymphocytes to enter the cell cycle

Using an in vitro system in which human peripheral blood mononuclear cells stimulated with the T-lymphocyte mitogen phytohemagglutinin were cocultured with Trypanosoma cruzi trypomastigotes, we demonstrated a marked accumulation of cells in G0/G1a during the 72-hr observation period whereas mitogen-stimulated cells in parallel cultures lacking the parasite readily entered the G1b, S, and G2/M phases. These results suggest that the immunological alterations that occur in acute Chagas' disease may result from an early cell cycle blockade induced by the parasite.     

Lymphocyte subset responses to repeated submaximal exercise in men

The effects of repeated bouts of submaximal cycle ergometry exercise on changes in the percentage of peripheral blood T-lymphocytes, the T-helper/inducer and T-cytotoxic/suppressor subsets, and natural killer (NK) cells were studied in 18 healthy young men who had no history of regular exercise training. Subjects were matched on the basis of maximal O2 uptake and assigned randomly to exercise or control groups, with controls resting quietly during the exercise sessions. The percentage of peripheral blood mononuclear leukocytes that reacted with monoclonal antibodies specific for T-lymphocytes (CD3+ cells), the helper/inducer subset (CD4+ cells) and cytotoxic/suppressor subset (CD8+ cells) of T-lymphocytes, and cells with NK activity (Leu7+ cells) were enumerated by fluorescence-activated flow cytometry for samples obtained immediately before and after exercise on days 1, 3, and 5 of a 5-day exercise regimen. The results of this study were mixed with decreases in the percentage of T-lymphocytes before vs. after exercise on days 1 and 3 (P less than 0.001), a decrease in the percentage of T-helper/inducer cells before vs. after exercise on day 3 (P less than 0.05), no effect of exercise on the percentage of T-cytotoxic/suppressor cells, and a marked increase in the percentage of NK cells after exercise on days 1 (P less than 0.05) and 3 (P less than 0.01). The total number of recovered NK cells in the mononuclear leukocyte fraction of blood also increased significantly after exercise on days 1 (P less than 0.05) and 3 (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation.

Production of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) after stimulation by lipopolysaccharide (LPS) and phytohemagglutinin (PHA) was studied in 1/10 diluted whole blood (WB) culture and in peripheral blood mononuclear cell (PBMC) culture. Cytokines IL-1 beta, TNF-alpha and IL-6 are preferentially stimulated by LPS whereas IL-2, IFN-gamma and GM-CSF are stimulated by PHA. Combination of 5 micrograms/ml PHA and 25 micrograms/ml LPS gave the most reliable production of the six cytokines studied. IL-1 beta, TNF-alpha and IL-6 represent a homogeneous group of early-produced cytokines positively correlated among themselves and with the number of monocytes in the culture (LeuM3). Furthermore, IL-1 beta was negatively correlated with the number of T8 lymphocytes. IL-2, IFN-gamma and GM-CSF represent a group of late-produced cytokines. Kinetics and production levels of IL-6 and GM-CSF are similar in WB and PBMC cultures. In contrast, production levels of TNF-alpha and IFN-gamma are higher in WB than in PBMC whereas production levels of IL-6 and IL-2 are lower in WB than in PBMC. Individual variation in responses to PHA + LPS was always higher in PBMC cultures than in WB cultures. The capacity of cytokine production in relation to the number of mononuclear cells is higher in WB, or in PBMC having the same mononuclear cell concentration as WB, than in conventional cultures of concentrated PBMC (10(6)/ml). Because it mimics the natural environment, diluted WB culture may be the most appropriate milieu in which to study cytokine production in vitro.     

The effects of a single bout pilates exercise on mRNA expression of bone metabolic cytokines in osteopenia women

[Purpose]

The purpose of this study was to examine the effect of a single bout pilates exercise on mRNA expression of bone metabolic cytokines in elderly osteopenia women.

[Methods]

We selected 11 people of elderly osteopenia women and loaded a single bout pilates exercise about RPE 10-14 level. The blood samples were collected before, immediately after and 60 minute after pilates exercise, then examined calcium metabolic markers in serum and extracted peripheral blood mononuclear cell (PBMC) from whole blood and confirmed mRNA expression of bone metabolic cytokines from PBMC. To clarify the changes during exercise, we designed repeated measure ANOVA as the control group to perform blood sampling without exercise.

[Results]

As a result, serum P showed significant interaction effect between group and time (p<.001), the pilates exercise group decreased about 9% at immediately after exercise and 13% during recovery after exercise (p<.05), while the control group showed a tendency to increase. Serum CK also showed a significant interaction between group and time (p<.05), the pilates group significantly increased at immediately after exercise and during recovery after exercise (p<.05) but the control group didn’t have changes. TNF-α and IL-6 mRNA expression in PBMC was significantly increased in the pilates group (p<.01, p<.05), although INF-γ mRNA expression didn’t show statistically significant difference, it tended to increase in the pilates group (NS).

[Conclusion]

These results suggested that a single bout pilates exercise of elderly osteopenia women cause hypophosphatemia with temporary muscle damage, and it leading high turnover bone metabolic state with to activate both of bone formation and bone resorption.     

Autologous tumor cell killing activity of tumor-associated lymphocytes in patients with head and neck carcinomas

In order to select the most cytotoxic effector cells for adoptive immunotherapy, lymphokine activated killer (LAK) cells, tumor infiltrating lymphocytes (TILs) and autologous mixed lymphocyte tumor cell culture (MLTC) cells derived from peripheral blood mononuclear cells (PBMC) in the same subject with head and neck carcinomas were prepared. The autologous tumor cell killing activity and cell surface phenotypes of each of the three effector cells were studied. MLTC cells cultured with interleukin-2 (IL-2) showed the strongest cytotoxic activity among these three different effector cells. Although TILs had suppressed killing activity immediately after isolation, after successive cultivations with IL-2, a cytotoxic activity against autologous tumor cells stronger than that of LAK cells appeared. Both IL-2 stimulated MLTC cells and TILs showed an enrichment of CD8 positive and CDU negative cells in a CD3 positive subpopulation.Abbreviations CD cluster differentiation - IL-2 interleukin-2 - LA lymphokine activated - LAK lymphokine activated killer - MLTC mixed lymphocyte tumor cell culture - NK natural killer - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes     

Release of lymphokines after infection with Epstein Barr virus in vitro. II. A monocyte-dependent inhibitor of interleukin 1 downregulates the production of interleukin 2 and interferon-gamma in rheumatoid arthritis

Epstein Barr virus (EBV)-infection of normal peripheral blood mononuclear cells (PBMC) in vitro induces IFN-alpha secretion from B cell and natural killer (NK) cell populations, and IFN-gamma secretion from T cells. IFN-gamma depends on prior elaboration of IL 2 and IL 1 that originates from monocytes and NK cells. PBMC from rheumatoid arthritis (RA) patients released moderately elevated levels of IFN-alpha (236 +/- 62 U/ml vs 168 +/- 34 in normals). In contrast, IFN-gamma was significantly lower in RA (88 +/- 34 U/ml vs 209 +/- 32) with an associated deficit in IL 2. A monocyte-dependent factor was shown to be responsible for this deficit, since monocyte depletion of RA cultures normalized the levels of IL 2 and IFN-gamma. Significantly lower levels of IL 1 activity were present in the supernatants of RA PBMC cultures as compared with normal cultures, and this was shown to be associated with presence of a nondialyzable IL 1 inhibitor. This inhibitor was capable of preventing the IL 1-dependent synthesis of IL 2 and IFN-gamma by normal PBMC. Exogenous IL 1 or IL 2 restored the deficient IFN-gamma secretion in RA PBMC. Thus, the deficient ability of RA lymphocytes to control EBV infection may be secondary to impairment of a monocyte-T cell interaction at the level of IL 1.     

Synthesis of Gm allotypes by human tonsillar lymphocytes in culture: concordance with serum phenotype.

Isolated human tonsillar lymphocytes were cultured with pokeweed mitogen, phytohemagglutinin, and without mitogen for 9 to 28 days. IgK, Gm(a) and Gm(f) were then quantitated in the cell suspensions. In cultures of cells derived from persons whose blood was heterozygous for IgGl allotype antigens Gm(a+f+), approximately equal amounts of Gm(a) and Gm(f) were found. In cultures of cells of Gma or Gmf homozygotes, there was complete concordance between the Gm allotype antigens produced by the cultures and the donor's serum phenotype-with no instance, either at zero time or at culture termination in which a Gm antigen was detected which was absent from the donor's serum. It was concluded that in vitro genetic allotype synthesis in tonsillar lymphocytes during short-term culture mirrored accurately in vivo Gm expression. IgK and Gm antigen synthesis was highest in the flasks containing pokeweed mitogen although both phytohemagglutinin and no-mitogen control flasks showed, in certain experiments, proliferation and an increase in the Ig per viable cell. It was observed that no-mitogen flasks contained twice as much allotype antigen as did phytohemagglutinin flasks suggesting an inhibition of Ig synthesis associated with the mitogen. The tonsillar lymphocytes, under the experimental conditions employed, were shown by a radio-incorporation and immunoprecipitation technique to be synthesizing polyclonal Ig de novo, at the termination of the cultures.     

A single bout of exercise influences natural killer cells in elderly women, especially those who are habitually active

The purpose of our study was to examine the effects of a single exercise bout on the natural killer (NK) cell count and activity in physically active elderly people, sedentary elderly people, and sedentary young people. Eight elderly women who trained by walking (age, 64 +/- 1 years; Vo(2peak), 32.2 +/- 1.1 ml.kg(-1).min(-1)), 8 age-matched untrained women (63 +/- 1 years, 28.8 +/- 1.0 ml.kg(-1).min(-1)), and 8 young untrained women (25 +/- 1 years, 37.6 +/- 1.6 ml.kg(-1).min(-1)) were studied. Blood samples were taken before, immediately after, and 2 hours after a 30-minute bout of exercise at an intensity equivalent to 70-75% of Vo(2peak). Peripheral blood mononuclear cells were isolated and the NK cell count and activity were analyzed. The NK cell count of the elderly sedentary group immediately after exercise was significantly higher than those of the elderly women who walked and young sedentary women, whereas no significant interaction was detected in NK cell activity and NK cell activity per cell number among the 3 groups. Consequently, an intrinsic defect in the cytotoxic ability of NK cells appeared in sedentary elderly people but not in active elderly people who perform habitual exercise.     

HCG-Activated Human Peripheral Blood Mononuclear Cells (PBMC) Promote Trophoblast Cell Invasion

Successful embryo implantation and placentation depend on appropriate trophoblast invasion into the maternal endometrial stroma. Human chorionic gonadotropin (hCG) is one of the earliest embryo-derived secreted signals in the peripheral blood mononuclear cells (PBMC) that abundantly expresses hCG receptors. The aims of this study were to estimate the effect of human embryo–secreted hCG on PBMC function and investigate the role and underlying mechanisms of activated PBMC in trophoblast invasion. Blood samples were collected from women undergoing benign gynecological surgery during the mid-secretory phase. PBMC were isolated and stimulated with or without hCG for 0 or 24 h. Interleukin-1β (IL-1β) and leukemia inhibitory factor (LIF) expressions in PBMC were detected by enzyme-linked immunosorbent assay and real-time polymerase chain reaction (PCR). The JAR cell line served as a model for trophoblast cells and was divided into four groups: control, hCG only, PBMC only, and PBMC with hCG. JAR cell invasive and proliferative abilities were detected by trans-well and CCK8 assays and matrix metalloproteinase (MMP)-2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 expressions in JAR cells were detected by western blotting and real-time PCR analysis. We found that hCG can remarkably promote IL-1β and LIF promotion in PBMC after 24-h culture. PBMC activated by hCG significantly increased the number of invasive JAR cells in an invasion assay without affecting proliferation, and hCG-activated PBMC significantly increased MMP-2, MMP-9, and VEGF and decreased TIMP-1 and TIMP-2 expressions in JAR cells in a dose-dependent manner. This study demonstrated that hCG stimulates cytokine secretion in human PBMC and could stimulate trophoblast invasion.