Effect of olive oil-based emulsion on human lymphocyte and neutrophil death

Background The incorporation of lipid emulsions in parenteral diets is a requirement for energy and essential fatty acid supply to critically ill patients. The most frequently used IV lipid emulsions (LE) are composed with long-chain triacylglycerols rich in omega-6 polyunsaturated fatty acids (PUFA) from soybean oil, but these LE promote lymphocyte and neutrophil death. A new emulsion containing 20% soybean oil and 80% olive oil rich in (omega-9 monounsaturated fatty acids (MUFA) has been hypothesized not to cause impairment of immune function. In this study, the toxicity of an olive oil-based emulsion (OOE) on lymphocytes and neutrophils from healthy volunteers was investigated. Methods: Twenty volunteers were recruited and blood was. collected before a 6-hour infusion of an OOE, immediately after infusion, and again 18 hours postinfusion. Lymphocytes and neutrophils were isolated by gradient density. The cells were studied immediately after isolation and after 24 hours or 48 hours in culture. The following determinations were carried out: triacylglycerol levels and fatty acid composition and levels in plasma, lymphocyte proliferation, production of reactive oxygen species, and parameters of lymphocyte and neutrophil death (viability, DNA fragmentation, phosphatidylserine externalization, mitochondrial depolarization, and neutral lipid accumulation). Results: OOE decreased lymphocyte proliferation, provoked lymphocyte necrosis, and had no effect on the proportion of viable neutrophils. The mechanism of cell death induced by OOE involved neutral lipid accumulation but had no effect on mitochondrial membrane depolarization. Conclusions: The OOE given as a single dose of 500 mL induced low toxicity to lymphocytes from healthy volunteers, probably by necrosis.

The effect of an adventure race on lymphocyte and neutrophil death

The effect of an adventure race (Ecomotion Pr), which lasted for 4-5 days, on neutrophil and lymphocyte death from elite athletes was investigated. Blood was collected from 11 athletes at rest and after the adventure race. The following parameters of cell death were measured in neutrophils and lymphocytes: cell membrane integrity, DNA fragmentation, mitochondrial transmembrane depolarization and reactive oxygen species (ROS) production. Phagocytosis capacity was also evaluated in neutrophils. The adventure race raised the proportion of cells with the loss of membrane integrity; lymphocytes by 14% and neutrophils by 16.4%. The proportion of lymphocytes with DNA fragmentation (2.9-fold) and mitochondrial transmembrane depolarization (1.5-fold) increased. However, these parameters did not change in neutrophils. ROS production remained unchanged in lymphocytes, whereas an increase by 2.2-fold was found in neutrophils due to the race. Despite these changes, the phagocytosis capacity did not change in neutrophils after the race. In conclusion, the Ecomotion Pr race-induced neutrophil death by necrosis (as indicated by the loss of membrane integrity) and led to lymphocyte death by apoptosis (as indicated by increase DNA fragmentation and depolarization of mitochondrial membrane).

Induction of Lymphocyte Death by Short- and Long-Duration Triathlon Competitions

LEVADA-PIRES, A. C., M. F. CURY-BOAVENTURA, R. GORJAO, S. M. HIRABARA. E. F. PUGGINA, I. L. PELLEGRINOTTI, L. A. DOMINGUES FILHO, R. CURI, and T. C. PITHON-CURI. Induction of Lymphocyte Death by Short- and Long-Duration Triathlon Competitions. Med. Sci. Sporty Exerc., Vol. 4 1, No. 10, pp. 1896-1901, 2009. Purpose: The effect of triathlon competitions on death of lymphocytes from elite athletes was investigated. Material and Methods: Blood was collected from sedentary volunteers and triathletes at rest and after a short-duration triathlon (SDT) and after a long-duration triathlon (LDT-half Ironman) competitions. Results: The athletes had lowered lymphocyte proliferation capacity compared with sedentary volunteers either at rest or after the competitions. There was no difference in the parameters associated with lymphocyte death when sedentary volunteers were compared with triathletes at rest. Lymphocytes from triathletes after SDT competition showed an increase in DNA fragmentation, phosphatidylserine externalization, and mitochondrial transmembrane depolarization and did not alter membrane integrity when compared with cells from athletes at rest. In contrast, the LDT competition raised the proportion of lymphocytes with loss of membrane integrity when compared with cells from athletes at rest and did not change the apoptotic parameters. The LDT competition induced an increase of reactive oxygen species (ROS) production by lymphocytes compared with triathletes at rest. The SDT competition did not alter ROS production by lymphocytes when compared with cells from triathletes at rest. ROS production by lymphocytes after LDT competition was 60% higher than in SDT. Conclusions: Evidence is presented herein that an LDT competition caused lymphocyte death by necrosis, whereas an SDT induced lymphocyte apoptosis. The mechanism for lymphocyte death induced by the triathlon competitions may involve an increase in ROS production at different extents.

Endurance training modulates lymphocyte function in rats with post-MI CHF

Purpose: Exercise training restores innate immune system cell function in post-myocardial infarction (post-MI) rats. However, studies of the involvement of lymphocyte (Ly) in the setting of the congestive heart failure (CHF) are few. To address this issue, we investigated the function of Ly obtained from cervical lymph nodes from post-MI CHF rats submitted to treadmill running training. Methods: Twenty-five male Wistar rats were randomly assigned to the following groups: rats submitted to ligation of the left coronary artery, which were sedentary (MI-S, N= 7, only limited activity) or trained (MI-T, N= 6, on a treadmill (0% grade at 13-20 m.m(-1)) for 60 min.d(-1), 5 d.wk(-1), for 8-10 wk); or sham-operated rats, which were sedentary (sham-S, N = 6) or trained (sham-T, N = 6). The incorporation of [2-C-14]-thymidine by Ly cultivated in the presence of concanavalin A (Con A) and lipopolysaccharide (LPS), cytokine production by Ly cultivated in the presence of phytohemagglutinin (PHA), and plasma concentration of glutamine were assessed in all groups, 48 h after the last exercise session. Results: Proliferative capacity was increased, following incubation with Con-A in the MI groups, when compared with the sham counterparts. When incubated in the presence of PHA, MI-S produced more IL-4 (96%) than sham-S (P < 0.001). The training protocol induced a 2.2-fold increase in the production of interleukin-2 (P < 0.001) of the cells obtained from the cervical lymph nodes of MI-T, compared with MI-S. Conclusion: The moderate endurance training protocol caused an increase in IL-2 production, and a trend toward the reversion of the Th-1/Th-2 imbalance associated with IL-4 production increased in the post-MI CHF animal model.

Endothelin A receptor antagonist modulates lymphocyte and eosinophil infiltration, hyperreactivity and mucus in murine asthma

Levels of endothelins are particularly high in the lung, and there is evidence that these peptides are involved in asthma. Asthma is a chronic inflammatory disease associated with lymphocyte infiltration. In the present study, we used a murine model of asthma to investigate the role of endothelins in lymphocyte and eosinophil infiltration into the airway hyperreactivity and mucus secretion. Sensitized C57B1/6 mice were treated with endothelin ET(A) receptor antagonist (BQ123) or endothelin ET(B) receptor antagonist (BQ788) 30 min before an antigen aerosol challenge. After 24 h, dose response curves to methacholine were performed in isolated lungs, FACS analysis of lymphocytes and eosinophil counts were performed in bronchoalveolar lavage fluid and mucus index was determined by histopathology. In sensitized and antigen-challenged mice there is a marked increase in the T CD(4)(+), T CD(8)(+), B220(+), T gamma delta(+) and NK1.1(+) lymphocyte subsets. Treatment with BQ123 further increased these cell populations. The number of eosinophils, airway hyperreactivity and mucus were all reduced by BQ123 treatment. The BQ788 had no significant effect on the parameters analyzed. Treatment with BQ123 reduced the endothelin concentration in lung homogenates, suggesting that endothelins exert a positive feedback on their synthesis. We show here that in murine asthma the ET(A) receptor antagonist up-regulates lymphocyte infiltration and reduces eosinophils, hyperreactivity and mucus. (C) 2008 Elsevier B.V. All rights reserved.

Differential expression of new LTA splice variants upon lymphocyte activation

Lymphotoxin alpha (LTA) is a member of the TNF cytokine superfamily, produced principally by lymphocytes. It plays an important role in immune and inflammatory responses. Many TNF superfamily members have functionally important isoforms generated by alternative splicing but alternative splicing of LTA has never been studied. The known LTA protein is encoded by a transcript containing four exons. Here we report seven new LTA splice variants, three of them evolutionary conserved. We demonstrate their presence in cytoplasmic RNA suggesting that they could be translated into new LTA isoforms. We observed that their expression is differentially regulated upon activation of peripheral blood mononuclear cells and lymphocyte subpopulations (CD4+, CD8+, and CD19+). Our data suggest that the new LTA splice variants might play a role in the regulation of the immune response. (C) 2007 Elsevier Ltd. All rights reserved.

Hypoallergenic variants of the major latex allergen Hev b 6.01 retaining human T lymphocyte reactivity

Hev b 6.01 is a major allergen of natural rubber latex with sensitization of 70–86% of latex glove-allergic subjects. Recently, we mapped the immunodominant T cell sites of Hev b 6.01 to the highly IgE-reactive hevein (Hev b 6.02) domain. Hev b 6.01 contains 14 cysteine residues with multiple disulphide bridges stabilizing tertiary conformation. With the goal of a standardized specific immunotherapy we developed hypoallergenic Hev b 6.01 mutants by site-directed mutagenesis of selected cysteine residues (3, 12, 17, and 41) within the Hev b 6.02 domain. Peptides corresponding to the Hev b 6.02 domain of two of the mutants were also synthesized. These mutants and peptide variants showed markedly decreased or ablated latex-allergic patient serum IgE binding by immunoblotting and ELISA. Basophil activation testing confirmed markedly decreased activation with successive cysteine substitutions of the mutants and complete abrogation with the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide. Retention of T cell reactivity is crucial for effective specific immunotherapy and all mutants and peptide variants maintained their latex-specific T cell reactivity. The ablated allergenicity but retained T cell reactivity of the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide suggests this peptide is a suitable candidate for inclusion in a latex immunotherapy preparation.

Studies of the human lymphocyte P2Z receptor and its activation of phospholipase D

Extracellular adenosine 5′-triphosphate (ATP) is an agonist for the P2Z receptor of human leukaemic lymphocytes and opens a Ca ²⁺-selective ion channel, which also conducts Ba²⁺, Sr²⁺ and the small fluorescent dye, ethidium⁺. A wide range of receptor agonists, many of which raise cytosolic [Ca²⁺] activate phospholipase D (PLD). In the present study, it was shown that both ATP and 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) stimulated PLD activity in a concentration-dependent manner, and the inhibitory effects of suramin, oxidised ATP, extracellular Na⁺ and Mg²⁺ suggested that the effect of these agonists is mediated by P2Z receptors. The role of divalent cations in ATP-stimulated PLD activity was investigated. Several agonists (eg ATP, thapsigargin, ionomycin) stimulated a rise in cytosolic [Ca²⁺] in human lymphocytes, but only ATP and ionomycin stimulated PLD activity. When Ca²⁺ influx was prevented by EGTA, the majority of ATP-stimulated and all of ionomycin-stimulated PLD activity was inhibited. Preloading cells with the Ca²⁺ chelator, BAPTA, reduced cytosolic [Ca²⁺] and, paradoxically, ATP-stimulated PLD activity was potentiated. ATP-stimulated PLD activity was supported by both Ba²⁺ and Sr²⁺ when they were substituted for extracellular Ca²⁺. Furthermore, both ATP-stimulated PLD activity and ATP-stimulated ¹³³Ba²⁺ influx showed a linear dependence on extracellular [Ba²⁺]. Thus it was concluded that ATP stimulated PLD activity in direct proportion to the influx of divalent cations through the P2Z ion channel and this PLD activity was insensitive to changes in bulk cytosolic [Ca²⁺]. The calmodulin (Ca²⁺/CaM) inhibitor, trifluoperazine (TFP) inhibited ionomycin- and ATP-stimulated PLD activity and ATP-stimulated apoptosis, but had no effect on PLD activity already activated by ATP. However, TFP inhibited ATP-stimulated Ca²⁺, Ba²⁺ and ethidium⁺ fluxes, at concentrations below those which inhibit Ca²⁺/CaM, suggesting that TFP inhibits the P2Z receptor. Similarly, the isoquinolinesulphonamide, KN-62, a selective inhibitor of Ca²⁺/CaM-dependent protein kinase II (CaMKII), also prevented ATP-stimulated apoptosis, but had no effect on pre-activated PLD. In addition, KN-62, and an analogue, KN-04, which has no effect on CaMKII, potently inhibited ATP-stimulated Ba²⁺ influx (IC₅₀ 12.7 ± 1.5 and 17.3 ± 2.7 nM, respectively), ATP-stimulated ethidium⁺ uptake (IC₅₀ 13.1 ± 2.6 and 37.2 ± 8.9 nM, respectively), ATP-stimulated phospholipase D activity (50% inhibition 5.9 ± 1.2 and 9.7 ± 2.8 nM, respectively) and ATP-induced shedding of the surface adhesion molecule, L-selectin (IC₅₀ 31.5 ± 4.5 and 78.7 ± 10.8 nM, respectively). They did not inhibit phorbol ester- or ionomycin-stimulated PLD activity or phorbol ester-induced L-selectin shedding. Neither KN-62 nor KN-04 (both 500 nM) have any effect on UTP-stimulated Ca²⁺ transients in fura-2-loaded human neutrophils, a response which is mediated by the P2Y₂ receptor, neither did they inhibit ATP-stimulated contractile responses mediated by the P2X₁ receptor of guinea pig urinary bladder. Thus, KN-62 and KN-04 are almost equipotent as P2Z inhibitors with IC₅₀s in the nanomolar, indicating that their actions cannot be due to CaMKII inhibition, but rather that they are potent and direct inhibitors of the P2Z receptor. Extracellular ATP-induced shedding of L-selectin from lymphocytes into the medium is a Ca²⁺-independent response. L-selectin is either cleaved by a metalloproteinase or a PLD with specificity for glycosylphosphatidylinositol (GPI). The novel hydroxamic acid-based zinc chelator, Ro-31-9790 blocks ATP-induced L-selectin shedding, but was without effect on ATP-induced Ba²⁺ influx or ATP-stimulated PLD activity. Furthermore, another zinc chelator, 1,10-phenanthroline, an inhibitor of a GPI-PLD, potentiated rather than inhibited ATP-stimulated PLD activity, suggesting that ATP-induced L-selectin shedding and ATP-stimulated PLD activity are independent of each other. Although extracellular ATP is the natural ligand for the lymphocyte P2Z receptor, it is less potent than BzATP in stimulating Ba²⁺ influx. Concentration-response curves for BzATP- and ATP-stimulated ethidium⁺ influx gave EC₅₀s 15.4 ± 1.4 µM and 85.6 ± 8.8 µM, respectively. The maximal response to ATP was only 69.8 ± 1.9% of that for BzATP. Hill coefficients were 3.17 ± 0.24 and 2.09 ± 0.45 for BzATP and ATP respectively, suggesting greater positive cooperativity for BzATP than for ATP in opening the P2Z-operated ion channel. A rank order of agonist potency of BzATP > ATP = 2MeSATP > ATPγS was observed for agonist-stimulated ethidium⁺ influx, while maximal influxes followed a rank order of BzATP > ATP > 2MeSATP > ATPγS. When ATP (300 -1000 µM) was added simultaneously with 30 µM BzATP (EC₉₀), it reduced both ethidium⁺ and Ba²⁺ fluxes by 30 - 40% relative to values observed with BzATP alone. KN-62, previously shown to be a specific inhibitor of the lymphocyte P2Z receptor, was a less potent antagonist of BzATP-induced fluxes than ATP, when maximal concentrations of both agonists (50 and 500 µM respectively) were used. However, when BzATP (18 µM) was used at a concentration equiactive with a maximally effective ATP concentration, KN-62 showed the same inhibitory potency for both agonists. The ecto-ATPase antagonist, ARL-67156, inhibited both ATP- and BzATP-stimulated Ba²⁺ influx, suggesting that the lower efficacy of ATP compared with BzATP was not due to preferential hydrolysis of ATP. Thus, the natural ligand, ATP, is a partial agonist for the P2Z receptor while BzATP is a full agonist. Moreover the competitive studies show that only a single class of P2-receptor (P2Z class) is expressed on human leukaemic lymphocytes. Both ATP- and BzATP-stimulated PLD activity were significantly inhibited (P < 0.05) when cells were suspended in iso-osmotic choline Cl medium. Choline⁺ was found to be a permeant for the P2Z ion channel, since ATP induced a large uptake of [¹⁴C]choline⁺ (60 to 150 µmol/ml intracellular water) during a 5 min incubation, which remained in the cells for several hours, and ATP was used to load cells with these levels of choline⁺. Intracellular choline⁺ inhibited ATP-, BzATP-, PMA- and ionomycin-stimulated PLD activity. Brief exposure of lymphocytes to ATP increased the subsequent basal rate of ethidium⁺ uptake, and this was prevented by intracellular choline⁺. It is proposed that P2Z-mediated Ca²⁺ influx in lymphocytes activates PLD leading to significantly changes of the phospholipid composition of the plasma membrane, which subsequently produces a permeability lesion, which in turn contributes to cell death.

Trivalent live attenuated influenza-simian immunodeficiency virus vaccines: efficacy and evolution of cytotoxic T lymphocyte escape in macaques.

There is an urgent need for a human immunodeficiency virus (HIV) vaccine that induces robust mucosal immunity. CD8+ cytotoxic T lymphocytes (CTLs) apply substantial antiviral pressure, but CTLs to individual epitopes select for immune escape variants in both HIV in humans and SIV in macaques. Inducing multiple simian immunodeficiency virus (SIV)-specific CTLs may assist in controlling viremia. We vaccinated 10 Mane-A1*08401+ female pigtail macaques with recombinant influenza viruses expressing three Mane-A1*08401-restricted SIV-specific CTL epitopes and subsequently challenged the animals, along with five controls, intravaginally with SIVmac251. Seroconversion to the influenza virus vector resulted and small, but detectable, SIV-specific CTL responses were induced. There was a boost in CTL responses after challenge but no protection from high-level viremia or CD4 depletion was observed. All three CTL epitopes underwent a coordinated pattern of immune escape during early SIV infection. CTL escape was more rapid in the vaccinees than in the controls at the more dominant CTL epitopes. Although CTL escape can incur a &quot;fitness&quot; cost to the virus, a putative compensatory mutation 20 amino acids upstream from an immunodominant Gag CTL epitope also evolved soon after the primary CTL escape mutation. We conclude that vaccines based only on CTL epitopes will likely be undermined by rapid evolution of both CTL escape and compensatory mutations. More potent and possibly broader immune responses may be required to protect pigtail macaques from SIV.

T lymphocyte subsets of the umbilical cord blood of dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Old beagle dogs have lower faecal concentrations of some fermentation products and lower peripheral lymphocyte counts than young adult beagles

The effects of age on microbiota composition, gut fermentation end-product formation and peripheral lymphocyte numbers were compared between old and young adult Beagle dogs fed four kibble diets differing in yeast cell wall contents. The experiment had a double 4 x 4 Latin square design, one with four mature dogs (4 years old) and the other with four old dogs (10 years old), with four replicates (diets) per dog. In each period a 15d adaptation period preceded a 5d total collection of faeces for the digestibility trial. on day 21, fresh faecal samples were collected for the determination of bacterial enumeration, pH, biogenic amine and short-chain fatty acid. Flow cytometry was used for immunophenotypic evaluation. Dogs were fed four kibble diets with similar composition with 0, 0.15, 0.30 and 0.45% of yeast cell wall (as-fed), respectively. Data were evaluated using general linear models of Statistical Analysis Systems statistical software (P<0.05). No evidence of a difference in faecal bacteria counts between ages was found (total aerobes, total anaerobes, Bifidobacterium, Lactobacillus, Clostridium and Escherichia coli: P. 0.15). Faecal concentrations of butyrate, histamine, agmatine and spermine were lower (P <= 0.05) and faecal pH was higher (P=0.03) in older dogs than in mature adult dogs, suggesting an alteration in bacterial metabolic activity, or in the rate of intestinal absorption of these compounds. Concentrations of T-lymphocytes, T-cytotoxic lymphocytes and B-lymphocytes were also lower (P <= 0.01) in older dogs than in mature adult dogs. The study confirmed alterations in peripheral lymphocytes and revealed a reduced concentration of some fermentation end products in the colon of old dogs.

Lymphocyte transformation assay for C neoformans antigen is not reliable for detecting cellular impairment in patients with Neurocryptococcosis

Background: Cryptococcus neoformans causes meningitis and disseminated infection in healthy individuals, but more commonly in hosts with defective immune responses. Cell-mediated immunity is an important component of the immune response to a great variety of infections, including yeast infections. We aimed to evaluate a specific lymphocyte transformation assay to Cryptococcus neoformans in order to identify immunodeficiency associated to neurocryptococcosis (NCC) as primary cause of the mycosis.Methods: Healthy volunteers, poultry growers, and HIV-seronegative patients with neurocryptococcosis were tested for cellular immune response. Cryptococcal meningitis was diagnosed by India ink staining of cerebrospinal fluid and cryptococcal antigen test (Immunomycol-Inc, SP, Brazil). Isolated peripheral blood mononuclear cells were stimulated with C. neoformans antigen, C. albicans antigen, and pokeweed mitogen. The amount of H-3-thymidine incorporated was assessed, and the results were expressed as stimulation index (SI) and log SI, sensitivity, specificity, and cut-off value (receiver operating characteristics curve). We applied unpaired Student t tests to compare data and considered significant differences for p<0.05.Results: The lymphotoxin alpha showed a low capacity with all the stimuli for classifying patients as responders and non-responders. Lymphotoxin alpha stimulated by heated-killed antigen from patients with neurocryptococcosis was not affected by TCD4+ cell count, and the intensity of response did not correlate with the clinical evolution of neurocryptococcosis.Conclusion: Response to lymphocyte transformation assay should be analyzed based on a normal range and using more than one stimulator. The use of a cut-off value to classify patients with neurocryptococcosis is inadequate. Statistical analysis should be based on the log transformation of SI. A more purified antigen for evaluating specific response to C. neoformans is needed.

Lymphoproliferative response and T lymphocyte subsets in a medium-term multi-organ bioassay for carcinogenesis in Wistar rats

The lymphoproliferative response and T lymphocyte subsets were evaluated at different stages of carcinogenesis in male Wistar, rats sequentially initiated with N-diethylnitrosamine (DEN), N-butyl-N-4(hydroxybutyl)nitrosamine (BBN), N-methyl-N-nitrosourea (MNU), dihydroxy-di-N-propylnitrosamine (DHPN) and N,N'-dimethylhydrazine (DMH) (DMBDD initiation). One group was evaluated at the 4th week and other initiated group at the 30th week. Two initiated groups were also exposed through diet to 7-acetylaminofluorene (2-AAF) or phenobarbital (PB), from the 6th until the 30th week. Two groups received only 2-AAF or PB until the 30th week. Five groups were studied to evaluate the effects of each initiator. The lymphoproliferative response was induced in vitro by concanavalin A and the percentage of T lymphocyte subsets was determined by flow cytometry, All groups submitted to initiation only, initiation plus promotion, or promotion only, developed significantly more preneoplastic: lesions than the untreated control group. The main target organs for tumor development were the liver, colon, urinary bladder, kidneys and Zymbal glands, mainly in the group treated with DMBDD + 2-AAF, There were no alterations of the lymphoproliferative response and of the T lymphocyte subsets percentage in the DMBDD-treated group at the 4th and 30th weeks. At the 30th week, the T lymphocyte subsets percentage was also not affected in the initiated groups after treatments with 2-AAF or PB. The lymphoproliferative response, however, was decreased in the DMBDD + 2-AAF group and in the groups treated only with 2-AAF or PB, the present results indicate that the initiating chemicals used in the DMBDD initiation protocol do not exert any influence on the immune system. The alteration of lymphoproliferative response induced at the advanced stage of carcinogenesis without alteration of T lymphocyte subsets may indicate that the influence of 2-AAF and PB on the immune system is functional and not toxic. (C) 2000 Elsevier B.V. Ireland Ltd. All rights reserved.