Apoptotic Lymphocytes Research Articles

Neither gender nor menstrual cycle phase influences exercise-induced lymphocyte apoptosis in untrained subjects

Lymphocyte apoptosis increases following maximal exercise. Estrogen hormones (E2) have been shown to protect lymphocytes from apoptosis in vitro, but it is unknown whether they can attenuate the apoptotic response to maximal exercise. The purpose of this study was to examine the effect of menstrual cycle variation on exercise-induced lymphocyte apoptosis in humans following exercise. Untrained healthy young men and regularly menstruating women not using hormonal contraceptives volunteered for the study. Women performed a maximal effort treadmill test for VO2 max once in the follicular phase (FOL) and once in the mid-luteal phase (ML) of their cycles. Men completed two VO2 max tests with periods of time between tests matched to those of the female subjects. Blood was collected before (PRE) and immediately after exercise (POST), and analyzed for apoptotic lymphocytes and estradiol. E2 concentrations in women were significantly greater during ML versus during FOL, both PRE and POST (p<0.0001). The percent of exercise-induced lymphocyte apoptosis was similar between women (23.2%+/-1.0%) and men (21.5%+/-0.4%). In women, the apoptotic response to maximal exercise was similar regardless of menstrual cycle phase (FOL=23.7%+/-0.9%, ML=22.7%+/-1.1%). Although elevated female sex hormones in vitro may exert anti-apoptotic effects, these data suggest that in vivo concentrations confer no protection to lymphocytes during exhaustive exercise.

The EGF repeat and discoidin domain protein, SED1/MFG-E8, is required for mammary gland branching morphogenesis

SED1, also known as MFG-E8, is a secreted protein composed of two EGF repeats (the second of which contains an RGD motif) and two discoidin/Factor V/VIII C domains. SED1 is expressed by a wide range of cell types, where it participates in diverse cellular interactions, such as sperm binding to the egg coat and macrophage recognition of apoptotic lymphocytes. Although SED1 was originally identified as a milk protein, its function in the mammary gland remains unclear; suggested functions include inhibition of viral infection and clearance of apoptotic cells during mammary gland involution. We report here that SED1 has an unexpected obligatory role during mammary gland development. Unlike that seen in WT glands, SED1-null glands show severely reduced branching from epithelial ducts and from terminal end buds, which are thin and poorly developed. SED1 is expressed by both luminal and myoepithelial cells in the developing epithelial duct, and binds to alpha(v) integrin receptors on myoepithelial cells leading to MAPK activation and cell proliferation. The absence of SED1 leads to greatly reduced levels of activated MAPK and a concomitant reduction in cell proliferation and branching throughout the epithelial tree. These results suggest that SED1 contributes, at least partly, to the intercellular signaling between luminal and myoepithelial cells that is required for branching morphogenesis.

Exercise‐Induced Muscle Damage and Apoptotic Protein Expression in Immune Cells

We previously reported (Med Sci Sports Exerc 2006) a higher proportion of apoptotic lymphocytes and neutrophils 24h and 48h after a -10% downhill run (DH1) compared to level running (L) or a second downhill run (DH2). We also found greater muscle damage and lower antioxidant status 24h after DH1. PURPOSE: to determine if the elevation in apoptotic leukocytes was mediated by changes in Bcl-2 and Bax protein expression by further analyzing the blood samples. METHODS: 12 moderately trained subjects (mean±SE age=22.7±0.7yr, VO2max=53.1±1.3ml.kg−1.min−1) performed three 40 min treadmill runs at ~70% VO2max: L followed by DH1 and DH2. Blood samples taken at rest (PRE) and immediately, 2h, 24h, and 48h after each run were further analyzed for Bcl-2 and Bax protein expression (ELISA). Data were analyzed using a 2-way repeated measures ANOVA with post hoc Tukey tests. RESULTS: Bax concentration was higher in DH1 than L and DH2 (P<0.01). Bax at 24h was greater than PRE (P<0.05) following DH1. No significant changes were found for Bcl-2. Bax/Bcl-2 ratio was greater in DH1 than L (P<0.01) and DH2 (P<0.05). At 24h post, Bax/Bcl-2 ratio was higher in DH1 than L and DH2 (P<0.01). In DH1, Bax/Bcl-2 ratio at 24h (P<0.01) and 48h post (P<0.05) was elevated above PRE. CONCLUSIONS: Increased oxidative stress following muscle damaging exercise may alter the Bax/Bcl-2 ratio in white cells towards a pro-apoptotic state.

In vitroassay for the quantitative measurement of apoptotic lymphocytes phagocytosis by peripheral blood monocytes

Currently used assays for the quantification of apoptotic cells uptake by phagocytes have several methodological problems. Our assay overcomes some of these problems. As a source of apoptotic cells we used peripheral blood lymphocytes obtained from the patients with chronic lymphoblast leukaemia. Apoptosis was induced by incubating cells with cycloheximide for up to 24 h. The assay was performed in suspension of peripheral blood mononuclear cells. For the visualisation of the phagocytes and phagocyted cells and discrimination of phagocyted from bound apoptotic cells we used Acridine orange/Ethidium bromide double staining. Here we offer a simple test which enables reliable measurement and it can show the difference of phagocytic potential between different individuals.

Functional polymorphisms in FAS and FASL contribute to increased apoptosis of tumor infiltration lymphocytes and risk of breast cancer

The FAS-FASL system plays crucial role in counterattack of cancer cell against immune system. This study examined the effects of FAS (-1377G/A and -670A/G) and FASL (-844T/C and 7896G/C) polymorphisms on breast cancer risk and apoptosis of T lymphocytes. The effect on breast cancer risk was determined by case-control analysis of 840 patients and 840 controls. The effects on T-lymphocyte apoptosis were determined by activation-induced cell death (AICD) of T cells ex vivo and by analyzing apoptotic tumor-infiltrating lymphocytes (TILs) in breast cancer tissue. We found moderately increased risk associated with FAS -1377AG [odds ratio (OR), 1.29; 95% confidence interval (CI), 1.05-1.59] and -1377AA (OR, 1.36; 95% CI, 1.01-1.82) genotypes compared with the -1377GG genotype and decreased risk associated with FASL -844CT (OR, 0.76; 95% CI, 0.62-0.94) and -844TT (OR, 0.66; 95% CI, 0.43-1.00) genotypes compared with the -844CC genotype. T lymphocytes with the FASL -844CC genotype had heightened FASL expression that is associated with increased AICD of the T cells stimulated by MCF-7 cells or phytohemagglutinin compared with the FASL -844TT genotype (10.38 +/- 4.09% and 24.29 +/- 1.50% versus 6.03 +/- 0.41% and 17.96 +/- 3.66%; P < 0.05 and 0.001). Breast cancer patients with the FASL -844CC genotype had higher apoptotic TILs in their cancer tissues than those with the FASL -844TT genotype (33.7 +/- 1.2% versus 19.1 +/- 2.0%; P = 0.007). These findings indicate that functional polymorphisms in FAS and FASL contribute to increased apoptosis of tumor infiltration lymphocytes and risk of breast cancer.

A Monoclonal Antibody to Lactadherin Inhibits Sickle Red Blood Cell Adhesion to Vascular Endothelial Cells in a Plasma Milieu.

Adherence of red blood cells to the endothelium initiates vaso-occlusion in sickle cell anemia. The increased adhesiveness of sickle erythrocytes is accompanied by several changes in the lipids of the erythrocyte membrane, including increased expression of phosphatidylserine (PS). One important PS-binding protein is lactadherin (also known as milk fat globule-EGF factor 8), a 45-kDa glycoprotein containing an Arg-Gly-Asp (RGD) sequence. It is secreted by macrophages and is present in normal plasma. Lactadherin promotes phagocytosis of PS-expressing apoptotic lymphocytes and sickle red blood cells by anchoring them to integrins on macrophages. Here, we investigated the role of endogenous lactadherin in adhesion of sickle erythrocytes to the endothelium. We developed a murine monoclonal antibody to human lactadherin, called L688, and investigated its effect on the adhesion of sickle red blood cells to histamine-stimulated human umbilical vein endothelial cells under hydrodynamic flow. In three experiments using washed erythrocytes resuspended in autologous plasma from three different patients with sickle cell anemia, L688 (20 μg/ml) inhibited adhesion by 24–30% (p<0.01). Further evidence for an important role for lactadherin in sickle erythrocyte adhesion to endothelial cells was provided by the observation that exogenous lactadherin enhanced adhesion in a concentration-dependent manner. Lactadherin-mediated adhesion was also inhibited by monoclonal antibody abciximab, (c7E3, 10 μg/ml) which targets the β3 integrin subunit common to both αIIbβ3 and αVβ3. Control antibodies had no effect. Finally, the lactadherin-dependent adhesion of sickle erythrocytes to activated endothelium was inhibited by PS vesicles but not by phosphatidylcholine vesicles, confirming an important role for PS in sickle cell adhesion. Consistent with this, normal erythrocytes can be induced to adhere to stimulated HUVEC in a lactadherin-dependent manner by treatment with N-ethylmaleimide (10 mM) and calcium ionophore A23187 (4 μM) — treatment that exposes PS on the outer leaflet of the red cell membrane. Together, these results indicate that lactadherin mediates sickle cell adhesion to the endothelium by bridging PS on the erythrocytes with αVβ3 integrin on the endothelium. We propose that anemia in sickle cell disease is at least partially due to phagocytosis of lactadherin-coated sickle erythrocytes in the spleen, liver, and lymph nodes. Those erythrocytes that are not ingested immediately by macrophages will become more adhesive for endothelium. Thus, lactadherin appears to be involved both in sickle cell clearance from the circulation and in adhesion to endothelium.

Apoptosis of lymphocytes in SLE: the level, correlation with prednisolone dosage and lymphocyte phenotypes

Introduction: SLE is a systemic autoimmune disorder. One of the mechanisms by which elimination of autoreactive lymphocytes takes place is apoptosis. Adefect in apoptosis may thus contribute to the development of autoimmune disease. Patients and methods: The study included 34 Russian children patients with SLE in age between 6-17 years old. Lymphocytes were isolated from venous blood by method of gradient centrifugation of all the blood through a Ficoll-pak solution. The quantity apoptotic cells was determined in leukocytes by flow cytometry Epics XL-2 (“Beckman Coulter”, USA). Analysis of lymphocyte subpopulations was carried by using two fluorescent labels: FITC conjugated monoclonal antibodies (CD-25 and CD-20) and phycoerythrin conjugated monoclonal antibody (CD-3, CD-4 and CD-8). Results: After in vitro incubation in CO2 incubator for 36hr; the percentage of apoptotic lymphocytes from patients with SLE was significantly higher than from healthy donors (7.2* ± 4.7 % vs. 5.5 ± 3.2% respectively). This was found to be depending upon the SLE disease activity. Moreover, observed correlations between level apoptosis lymphocyte in SLE and the titer of antibodies to dsDNA (r = 0.5) and dosage prednisolone (r = 0.6). By analyzing lymphocyte phenotypes; there was no significant difference in percentage of CD3+T-cells from SLE compared to normal donors (62.4±7.2 vs. 68.7±9.9.); the same had been observed in CD8+ T-cells (20.9±5.5 vs. 25.3±5.6); and in CD20+ B-cells (9.7±8.9 vs. 13.7±8.0). However, the percentage of CD4+T cell was significantly decreased (P = 0,001) in patients with SLE compared to normal donors (25.5*±7.3 vs. 40.6±8.7). The immunoregulatory index was decreased in SLE compared to normal donors (1.2 vs 1.6 respectively). This phenotypes disbalance was associated with significant increased in the level of activated CD25+ lymphocytes in SLE compared to normal healthy donors. Keywords: SLE, apoptosis, lymphocyte phenotypes, prednisolone, dsDNA autoantibodies, flow cytometry Sudan Journal of Medical Sciences Vol. 1(1) 2006: 14-19

Lymphocyte apoptosis as an immune subversion strategy of microbial pathogens

Apoptosis is a component of cellular death in several immunological reactions. Lymphocyte apoptosis is a feature of negative selection of thymic lymphocytes. Target cells die by apoptosis during their interaction with cytotoxic T cells. Antigens derived from apoptotic cells can be cross-presented by antigen presenting cells (APCs). In these examples, apoptotic death is a beneficial feature for the individual. The apoptosis of cells also occurs during infection with a variety of microorganisms, but this process can be detrimental to the handling of the infection by the host. Here, we aim to highlight some of the recent advances in understanding why apoptosis can be a detrimental event during infection. We will focus on recent research with the intracellular bacterial pathogen Listeria monocytogenes, which demonstrates how apoptosis is induced, some of the host pathways that are exploited and the immunological consequences of cell death. We propose that L. monocytogenes causes lymphocyte death by enhancing the cell-death programs of the host. The presence of apoptotic lymphocytes downregulates early innate immunity, creating a permissive environment for bacterial growth.

BOTH GRAM-NEGATIVE AND GRAM-POSITIVE EXPERIMENTAL PNEUMONIA INDUCE PROFOUND LYMPHOCYTE BUT NOT RESPIRATORY EPITHELIAL CELL APOPTOSIS

To determine whether and by which pathway (via the death receptor or mitochondrial mediated pathway) lymphocyte apoptosis occurs in pneumonia and to determine if increased bronchial epithelial cell apoptosis occurs in pneumonia. Prospective randomized study in a university research laboratory. Male C57BL/6 mice (n = 30). Animals received an intratracheal injection of Streptococcus pneumoniae or Pseudomonas aeruginosa to induce gram-positive or gram-negative pneumonia, respectively and were killed 24, 30, or 48 h later. Presence of pneumonia was confirmed via gross visual examination of lungs and by histology. Lymphocyte apoptosis in spleen and thymus was analyzed by flow cytometry for active caspases 3, 8, and 9 and by immunohistochemical (IHC) staining for active caspase 3 and DNA strand breaks. Respiratory epithelial cell apoptosis was assessed by IHC. Histologically, pneumonia was present in all bacteria-treated animals but none in sham-treated mice. Extensive lymphocyte apoptosis in spleen and thymus was documented by characteristic morphological changes on hematoxylin and eosin staining and by IHC staining in both S. pneumonia and P. aeruginosa infection. Flow cytometry confirmed IHC and showed apoptotic lymphocytes positive for active caspases 3, 8, and 9 in both thymi and spleens in both infections. In contrast to the extensive lymphocyte apoptosis, only rare scattered apoptotic changes were seen in respiratory epithelial or endothelial cells in pneumonia due to either organism. Increased lymphocyte but not bronchial cell apoptosis occurs in both gram-positive and gram-negative pneumonia and probably involves both the extrinsic and intrinsic pathway.

Prolonged exercise does not cause lymphocyte DNA damage or increased apoptosis in well-trained endurance athletes

Recent research has demonstrated that lymphocyte apoptosis sensitivity appears to be related to training status and exercise intensity. This work investigated the effect of prolonged, submaximal treadmill running on percentage (%) apoptosis, % necrosis and DNA strand breaks in lymphocytes and related these to changes in total lymphocyte and blood cortisol concentrations in well-trained runners. Venous blood samples (n = 14) were taken immediately before (PRE), immediately after (IPE) and 3 h after (3PE) 2.5 h of treadmill running at 75% of VO2 max from eight well-trained male endurance athletes (age 34.2 +/- 2.44 years) and analysed for cellular content and serum cortisol concentrations. Lymphocytes were isolated from whole blood and % apoptotic and necrotic cell were detected by flow cytometry using Annexin V-FITC and propidium iodide uptake. DNA strand breaks were measured by single-cell gel electrophoresis. Despite a significant (P < 0.001) exercise-induced increase in mean serum cortisol concentrations and reduction in lymphocyte counts, the mean % Annexin-V positive cells (13.3 +/- 6.78 in PRE, 11.3 +/- 5.51 in IPE and 12.8 +/- 6.75 in 3PE samples) were not significantly different at the three time-points (P > 0.05). Mean DNA strand breaks in the lymphocytes also did not change significantly (P > 0.05) rising from 25.7 +/- 2.16 to 26.9 +/- 1.89 and 27.1 +/- 1.38 microm in IPE and 3PE samples, respectively. The exercise-induced changes in total blood lymphocyte counts and cortisol concentrations did not result in a significant change in % apoptotic lymphocytes or DNA strand breaks in the endurance-trained athletes during this prolonged, submaximal exercise.

The Influence of Immunosuppressive Drugs on T- and B-cell Apoptosis via p53-Mediated Pathway In Vitro and In Vivo

This study was designed to assess the effects of calcineurin and inosine-5-monophosphate-dehydrogenase inhibitors on p53-mediated-apoptosis in T- and B-cells in vitro and in human heart-transplanted recipients (HTx-R). For in vitro experiments, peripheral blood from healthy volunteers was collected and treated either with 1 microM cyclosporin A (CsA; n = 6), 10 microM mycophenolic acid (MPA; n = 6) or 100 nM tacrolimus (TRL; n = 6). For the second part, peripheral blood was collected from HTx-R undergoing CsA-MPA (n = 11) or TRL-MPA (n = 11) therapy before (0 hr) and after (2 hr) acute drug application and from healthy volunteers (n = 11) without drug therapy. Whole blood (part 1+2) was stimulated (24 hr) with eight different concentrations of actinomycin-D (0-800 nM), an apoptosis inductor acting via p53-pathway. Apoptotic lymphocytes were measured by TUNEL and expression of Annexin-V using FACS. Drug effects were calculated by taking the effects of actinomycin-D as baseline values. In vitro drug treatment with CsA, MPA, and TRL significantly (P < 0.05) decreased the apoptotic effect of actinomycin-D in T-cells in a noncompetitive manner. In HTx-R undergoing drug therapy, there was a similar antiapoptotic effect observed in both T- and B-cells (P < 0.05). Differences between 0 hr and 2 hr after acute drug application did not exist. Apoptosis induced by actinomycin-D can be completely blocked by caspase-inhibitor zVAD-FMK. Our results suggest that, in vitro and in HTx-R, an inhibition of calcineurin and inosine-5-monophosphate-dehydrogenase by CsA, TRL, or MPA lead to an inhibition of T-and B-cell apoptosis via p53-pathway. This assay may be helpful to provide insights into mechanisms of immunosuppressive drugs in regulation of apoptosis in lymphocytes.

Apoptosis of Peripheral Blood Lymphocytes in Primary Nephrotic Syndrome

ABSTRACT: Background: Apoptosis is a highly programmed form of cell death that has significant role in such physiological processes as normal cell turnover, as well as many pathological conditions. Several studies implicates altered lumphocyte function in the mechanisms underlying idiopathic nephrotic syndrome. An increased apoptosis rate may also contribute to the functional abnormalities of T-cells already found in these patients. Objectives: The objectives of the present work were to investigate the phenotypic characteristic of T-lymphocyte subsets in newly diagnosed nephrotic children and after induction of remission. This study also aimed at detecting the percentage of apoptotic lymphocytes in the peripheral blood of the same group of the patients. Methods: The study was carried out on 30 selected children. Twenty children with first attack nephrotic Syndrome suggestive of minimal change type and ten healthy children of comparable age as a control group. All children were subjected to history taking, clinical examination and laboratory investigations including complete blood picture, blood urea, serum creatinine, total serum proteins, serum cholesterol, serum C3, complete urine analysis and 24 hours urine for proteins. All children were also further subjected to specific laboratory tests to study peripheral blood lymphocytes including detection of lymphocytes subsets by three color flow analysis using monoclonal antibodies CD3 and CD4 and also determination of apoptotic lymphocytes in the peripheral blood by flow cytometry using annexin-v-flourecein isothiocynate (FITC) and propidium iodide (PI). Results: There was no statically significant difference between nephrotic patients at presentation and control group as regards percentage of CD3 and CD4 lymphocytes in peripheral blood (p=0.89 and 0.30 respectively). Also, the percentage of apoptotic lymphocytes in peripheral blood was significantly higher in the patient group at presentation of nephrotic disease than after induction of remission and than the control group (p=0.000 and 0.000 respectively). Finally, the percentage of apoptotic lymphocytes was significantly higher at remission of the disease than the control group (p=0.001). Conclusions: In the present study increase in the apopotosis rate of CD3+Lymphocytes was found. This could be a crucial point in the pathogenesis of the nephrotic syndrome but clearly more prospective studies of large groups are needed with the analysis of the apoptosis rate within different subsets of lymphocytes. It is very difficult at the moment to explain why the lymphocyte apoptosis is increased in nephrotic syndrome. This might result from impaired production of and dysregulation in the synthesis of different cytokines, which are essential for the normal life span of lymphocytes such as interleukin (IL)-2 and TNF- α. Another explanation for the abnormalities found in our study may be reduced anti-oxidant defense in patients with nephrotic syndrome which contribute to an increase in the apoptosis rate of circulating lymphocytes.

Autoreactive MZ and B-1 B-cell activation by Faslpr is coincident with an increased frequency of apoptotic lymphocytes and a defect in macrophage clearance

AbstractMurine autoreactive anti-Smith (Sm) B cells are negatively regulated by anergy and developmental arrest, but are also positively selected into the marginal zone (MZ) and B-1 B-cell populations. Despite positive selection, anti-Sm production occurs only in autoimmune-prone mice. To investigate autoreactive B-cell activation, an anti-Sm transgene was combined with the lpr mutation, a mutation of the proapoptotic gene Fas (Faslpr), on both autoimmune (MRL) and nonautoimmune backgrounds. Faslpr induces a progressive and autoantigen-specific loss of anti-Sm MZ and B-1 B cells in young adult Faslpr and MRL/Faslpr mice that does not require that Faslpr be B-cell intrinsic. This loss is accompanied by a bypass of the early pre–plasma cell (PC) tolerance checkpoint. Although the MRL bkg does not lead to a progressive loss of anti-Sm MZ or B-1 B cells, it induces a robust bypass of the early pre-PC tolerance checkpoint. Faslpr mice have a high frequency of apoptotic lymphocytes in secondary lymphoid tissues and a macrophage defect in apoptotic cell phagocytosis. Since Sm is exposed on the surface of apoptotic cells, we propose that anti-Sm MZ and B-1 B-cell activation is the result of a Faslpr-induced defect in apoptotic cell clearance.

Role of HMGB1 in apoptosis-mediated sepsis lethality

Severe sepsis, a lethal syndrome after infection or injury, is the third leading cause of mortality in the United States. The pathogenesis of severe sepsis is characterized by organ damage and accumulation of apoptotic lymphocytes in the spleen, thymus, and other organs. To examine the potential causal relationships of apoptosis to organ damage, we administered Z-VAD-FMK, a broad-spectrum caspase inhibitor, to mice with sepsis. We found that Z-VAD-FMK–treated septic mice had decreased levels of high mobility group box 1 (HMGB1), a critical cytokine mediator of organ damage in severe sepsis, and suppressed apoptosis in the spleen and thymus. In vitro, apoptotic cells activate macrophages to release HMGB1. Monoclonal antibodies against HMGB1 conferred protection against organ damage but did not prevent the accumulation of apoptotic cells in the spleen. Thus, our data indicate that HMGB1 production is downstream of apoptosis on the final common pathway to organ damage in severe sepsis.

Choline deficiency increases lymphocyte apoptosis and DNA damage in humans

Whereas deficiency of the essential nutrient choline is associated with DNA damage and apoptosis in cell and rodent models, it has not been shown in humans. The objective was to ascertain whether lymphocytes from choline-deficient humans had greater DNA damage and apoptosis than did those from choline-sufficient humans. Fifty-one men and women aged 18-70 y were fed a diet containing the recommended adequate intake of choline (control) for 10 d. They then were fed a choline-deficient diet for up to 42 d before repletion with 138-550 mg choline/d. Blood was collected at the end of each phase, and peripheral lymphocytes were isolated. DNA damage and apoptosis were then assessed by activation of caspase-3, terminal deoxynucleotide transferase-mediated dUTP nick end-labeling, and single-cell gel electrophoresis (COMET) assays. All subjects fed the choline-deficient diet had lymphocyte DNA damage, as assessed by COMET assay, twice that found when they were fed the control diet. The subjects who developed organ dysfunction (liver or muscle) when fed the choline-deficient diet had significantly more apoptotic lymphocytes, as assessed by the activated caspase-3 assay, than when fed the control diet. A choline-deficient diet increased DNA damage in humans. Subjects in whom these diets induced liver or muscle dysfunction also had higher rates of apoptosis in their peripheral lymphocytes than did subjects who did not develop organ dysfunction. Assessment of DNA damage and apoptosis in lymphocytes appears to be a clinically useful measure in humans (such as those receiving parenteral nutrition) in whom choline deficiency is suspected.

Apoptotic lymphocytes and CD34+ cells in cryopreserved cord blood detected by the fluorescent vital dye SYTO 16 and correlation with loss of L-selectin (CD62L) expression

Discrimination between live and apoptotic cells is important for accurate determination of viable CD34(+) cells in hematopoietic stem cell transplant products. SYTO16 is a sensitive fluorescent dye for discriminating live from apoptotic leukocytes. The incidence of apoptotic leukocytes in paired samples of fresh and cryopreserved-thawed cord blood (CB) was determined by the SYTO16/7-AAD flow cytometric assay. Cell migration and expression of the cell homing molecule L-selectin (CD62L) was determined in relation to SYTO16 staining. SYTO16 detected significant proportions of apoptotic lymphocytes and CD34(+) cells in fresh and thawed CB buffy-coat samples that were not detected by 7-AAD. Compared to fresh CB, the proportion of apoptotic lymphocytes and CD34(+) cells significantly increased following thawing. Significantly higher proportions of live SYTO16(bright) lymphocytes and CD34(+) cells were found in the migrated cell population compared to the non-migrated population. Significantly fewer lymphocytes and CD34(+) cells expressed CD62L following thawing. Absence of CD62L expression was strongly correlated with apoptotic/SYTO16(dim) lymphocytes and CD34(+) cells. Cryopreserved-thawed CB contains significant proportions of apoptotic lymphocytes and CD34(+) cells that are not detected by 7-AAD. SYTO16 offers a sensitive method for discrimination of live from apoptotic leukocytes and assists in accurate assessment of CB quality and suitability for use in clinical transplantation.

Freewheel Running and Submandibular Lymph Node Lymphocyte Response to Exercise Stress in Older Mice

The submandibular lymph nodes (SLN) are the primary sites for drainage of and sensitization to pathogens from the anterior eye; intraocular tumor antigens also drain to the SLN and result in activation of cytotoxic T lymphocytes. Thus, changes in lymphocyte distribution and viability in SLN with training and acute exercise may directly impact ocular health. PURPOSE: To examine the impact of acute exercise on SLN lymphocytes after long-term freewheel (voluntary) running in mice. METHODS: Five week old female C57BL/6 mice were assigned to wheel running (WR, n=20) or non-wheel running groups (NR, n=21) for 8 months and then randomized to treadmill running and sacrifice immediately (EX+Imm) or sacrifice 24h (EX+24h) after cessation of exercise, or a control group sacrificed immediately after exposure to treadmill noise and vibrations without running (NonEX). Lymphocytes from bilateral SLN were collected, enumerated and analyzed for CD3, CD4, CD8 and CD19 surface markers and phosphatidylserine externalization by flow cytometry, intracellular glutathione by spectrofluorometry and plasma corticosterone by radioimmuno assay. RESUlTS: Monthly running distances differed significantly over time (p<0.01), with an average of 136±11.8 km•month−1 during months 1–6 and thereafter declined (months 7 and 8). Plasma corticosterone was elevated in NR mice immediately following treadmill exercise (NR EX+IMM: 73.5±7.6 ng•mL−1 vs. NR NonEX: 28.2±9.5ng•mL−1; p<0.05), but did not differ between WR Ex+IMM and WR NonEX. WR mice had a higher %T cells in SLN (WR: 71.7±1.7%vs. NR: 62.3±2.3%; p<0.05) and lower % B cells compared to NR mice (WR: 12.5±0.9%vs. NR: 27.7±2.2%; p<0.001). Despite a higher % apoptotic SLN lymphocytes in NR compared to WR mice (NR: 20.9±1 .4% vs.WR: 12.4 ±2.7%; p<0.01), intracellular glutathione concentration and total SLN lymphocyte numbers were not altered by training. However, the percent apoptotic lymphocytes was decreased in the SLN immediately, and further 24h after exercise in WR (p<0.001) but not in NR mice. CONCLUSION: Eight months of freewheel running results in a higher T cell proportion and less acute exercise-induced apoptosis of mouse SLN lymphocytes. These effects may be beneficial for ocular health in athletes and older adults. Supported by NSERC of Canada.

Exercise-Induced Muscle Damage and Immune Cell Apoptosis

Muscle damage is evident following an acute bout of downhill running (i.e., eccentric ally-biased exercise) at moderate intensity whereas it does not occur following running on a level grade or after a second bout of downhill running. Muscle damage increases intracellular free radicals due to the phagocytic processes of white cells. This increase in reactive oxygen species reduces oxygen radical absorbance capacity (ORAC) which may elevate the number of apoptotic cells. PURPOSE: to examine the effect of muscle damaging exercise on immune cell apoptosis. METHODS: Twelve moderately trained subjects (mean±SE age=22.7±0.7yr, VO2max= 53.1±1.3ml·kg−1·min−1) performed three 40 min treadmill exercises at ∼70% VO2max: a level running trial (L) and two downhill (−10%) running trials (DH1 and DH2). Blood samples were taken at rest and immediately (POST), 2h, 24h, and 48h following each run, and analyzed for creatine kinase (CK) activity, ORAC and apoptotic cells (%) using morphological identification. Data were analyzed using a 2-way repeated measures ANOVA with post hoc Tukey tests. RESULTS: CK activity at 24h following exercise was significantly higher in DH1 than L and DH2 (p < 0.01). ORAC was significantly lower at 24h in DH1 as compared to L and DH2 (p < 0.01). Lymphocyte number peaked at POST and was significantly higher in DH1 than in L and DH2. The number of neutrophils was highest 2h following exercise and also significantly higher in DH1 vs L and DH2. Lymphocyte apoptosis was significantly greater at 24h and 48h following DH1 than L and DH2 (p < 0.01). Neutrophil apoptosis in DH1 was significantly higher at 2h, 24h and 48h than in L and significantly greater at 24h and 48h than in DH2 (p < 0.01). CONCLUSION: An initial bout of downhill running for 40 min at a moderate intensity, which likely induced muscle damage, significantly increased oxidative stress and the proportion of apoptotic lymphocytes and neutrophils in the blood compared to level running or a second downhill run. This suggests that increased oxidative stress following exercise-induced muscle damage may affect the amount of apoptosis in white cells, and perhaps play a role in immune suppression.

Preoperative steroid pulse therapy for invasive thymoma

Glucocorticoid was used in thymomas. The purpose of the study was to evaluate the efficacy of intravenous high-dose glucocorticoid (steroid pulse) therapy in patients with previously untreated advanced thymoma. Causes were also sought for a possible underlying mechanism of the effect of steroid on thymoma. Seventeen patients with invasive thymoma who had not received previous chemotherapy or radiation therapy were enrolled in the study. All cases were treated with 2 courses of glucocorticoid therapy before surgery. Tumor response was assessed by computed tomography (CT) scan 1 week after the steroid pulse therapy. Lymphocytes associated with thymoma were analyzed for their CD4/CD8 phenotype and glucocorticoid receptor (GR). TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining was used to analyze the apoptotic lymphocytes and epithelial cells. The overall response rate to the steroid pulse therapy was 47.1% (8 of 17). The reduction in tumor size was most prominent in type B1 thymomas; there were significant differences between type AB and type B1 thymomas (P = .0234) and type B1 and type B3 thymomas (P = .0068). The reduction in tumor size was accompanied with a marked reduction in the CD4+8+ double-positive immature thymocytes that expressed higher levels of glucocorticoid receptor. Apoptotic changes were observed in both neoplastic epithelial cell and lymphocyte components after glucocorticoid therapy. The efficiency of preoperative steroid pulse therapy in type B1 thymoma was most prominent, which is probably related to the specific effect on GR-rich CD4+8+ double-positive immature lymphocytes, which are abundant in this type of thymoma.

The down-regulation of IL1alpha and IL6, in monocytes exposed to extracorporeal photopheresis (ECP)-treated lymphocytes, is not dependent on lymphocyte phosphatidylserine externalization

Extracorporeal photopheresis (ECP) has been successfully used to treat some inflammatory conditions. Following ECP, lymphocytes become apoptotic and untreated monocytes, exposed to post-ECP lymphocytes, reduce proinflammatory cytokine secretion. This study attempted to establish if this monocyte immunosuppression was linked to phosphatidylserine externalization (detected using Annexin V) on the apoptotic lymphocytes. Using density gradient and magnetic separation, lymphocytes were isolated from three cutaneous T-cell lymphoma and nine chronic graft versus host disease (cGvHD) patients pre-ECP and prior to re-infusion (post-ECP). The collected lymphocytes were cultured overnight and Annexin V levels determined. Peripheral blood was taken from the same patient 20 h later and the monocytes were isolated. The 'fresh' monocytes were introduced to each 20 h pre- and post-ECP lymphocyte culture, stimulated with lipopolysaccharide (LPS) and Brefeldin A and subsequently tested for intracellular tumour necrosis factor alpha, interleukin 1 alpha (IL1alpha), IL1beta, IL6 and IL8. For cGvHD patients, the relative levels of IL1alpha and IL6 were reduced in the untreated, LPS-stimulated monocytes exposed to post-ECP lymphocytes. However, the down-regulation of IL1alpha and IL6 did not correlate to levels of Annexin V-positive lymphocytes. ECP-treated lymphocytes can reduce the ability of LPS-stimulated monocytes to produce some proinflammatory cytokines; however, this effect is not dependent on phosphatidylserine externalization.