PHA-stimulated Lymphocyte Proliferation Research Articles

Additional effect of perioperative, compared with preoperative, immunonutrition after pancreaticoduodenectomy: A randomized, controlled trial

BackgroundWe have reported that perioperative and preoperative immunonutrition reduced infectious complications in patients undergoing pancreaticoduodenectomy; however, it is unclear whether perioperative immunonutrition has additional effects compared with preoperative immunonutrition. The present study evaluated whether perioperative, compared with preoperative, immunonutrition has additional effects on cell-mediated immunity and the infection rate after pancreaticoduodenectomy. Materials and methodsThis was a prospective, randomized clinical trial conducted in our institution. Oral supplementation enriched with arginine, ω-3 fatty acids, and dietary nucleotides was given by enteral infusion to 30 patients before and after surgery (perioperative group); 30 patients received the same enriched formula before surgery and standard enteral nutrition following surgery (preoperative group). The primary endpoint was concanavalin (Con A)- or phytohemagglutinin (PHA)-stimulated lymphocyte proliferation on postoperative day (POD) 7, which is an index of cell-mediated immunity; the secondary endpoint was the postoperative infection rate. ResultsThere were no significant differences in Con A- or PHA-stimulated lymphocyte proliferation on POD 7 between the groups. There was no significant difference in the postoperative infection rate between the two groups. In the post hoc subgroup analysis, with respect to the effect on the infection rate, a significant interaction was found only between a long operative time and perioperative immunonutrition. ConclusionsThere were no additional effects of perioperative, compared with preoperative, immunonutrition on postoperative immunity and infectious complications in patients undergoing pancreaticoduodenectomy.

Dietary nucleotide improves markers of immune response to strenuous exercise under a cold environment

BackgroundStrenuous exercise has been classically associated to immune-suppression and consequently to an increased risk of infections, especially at the upper respiratory tract. The administration of dietary nucleotides has been demonstrated useful to maintain the immune function in situations of stress and thus could be an appropriate strategy to counteract the decline of the immune function associated to strenuous exercise. The aim of the present study was to asses the impact of a specific nucleotide formulation (Inmunactive®) on the markers of immune function of athletes after a heavy exercise bout under cold conditions.MethodsTwenty elite male taekwondo athletes were randomly divided into two groups of 10 subjects that were supplemented with placebo (P) or Inmunactive (I) at 480 mg/day during 30 days. At baseline (day 0) and after 4 wk of supplementation (day 30) each subject undertook an exhaustion exercise test using a cycloergometer. Skin temperature, core temperature, heart rate, lactate concentration and rating of perceived exertion (RPE) were recorded during the test. Blood and saliva samples were obtained before and after each exercise test for determination of blood cell concentrations, PHA-stimulated lymphocyte proliferation (PHA-LP) and salivary immunoglobulin A (SIgA).ResultsExercise tests induced neutrophilia and reduction in lymphocyte blood counts on day 0 and on day 30 in both groups. However, the I group exhibited a faster recovery from the lymphopenic response than the P group, so that lymphocyte levels were higher after 150 min (P < 0.0028). Furthermore, the lymphoproliferative response was modulated by nucleotide supplementation, since it was higher in the I group on day 30 despite an almost significant (P < 0.06) exercise-evoked decrease at baseline.ConclusionsThese findings suggest that supplementation with a nucleotide-based product for 4 weeks could counteract the impairment of immune function after heavy exercise.

Characterization of regulatory T cells in urban newborns

BackgroundIn the United States, asthma prevalence is particularly high among urban children. Although the underlying immune mechanism contributing to asthma has not been identified, having impaired T regulatory (Treg) cells at birth may be a determining factor in urban children. The objective of this study was to compare Treg phenotype and function in cord blood (CB) of newborns to those in peripheral blood (PB) of a subset of participating mothers.MethodsTreg numbers, expression, and suppressive function were quantified in subjects recruited prenatally from neighborhoods where ≥ 20% of families have incomes below the poverty line. Proportion of Treg cells and expression of naïve (CD45RA) or activated (CD45RO, CD69, and HLA-DR) markers in CD4+T cells was measured by flow cytometry. Treg suppressive capacity was determined by quantifying PHA-stimulated lymphocyte proliferation in mononuclear cell samples with and without CD25 depletion.ResultsIn an urban cohort of 119 newborns and 82 mothers, we found that newborns had similar number of cells expressing FOXP3 as compared to the mothers but had reduced numbers of CD4+CD25+bright cells that predominantly expressed the naïve (CD45RA) rather than the activated/memory (CD45RO) phenotype found in the mothers. Additionally, the newborns had reduced mononuclear cell TGF-β production, and reduced Treg suppression of PHA-stimulated lymphocyte proliferation compared to the mothers.ConclusionU.S. urban newborns have Treg cells that express FOXP3, albeit with an immature phenotype and function as compared to the mothers. Longitudinal follow-up is needed to delineate Treg cell maturation and subsequent risk for atopic diseases in this urban birth cohort.

Elevated binding activity of CD8+ cells with phytohaemagglutinin by asbestos fibrein vitro

Asbestos fibres are known to depress the mitogenic stimuli of phytohaemagglutinin (PHA) to lymphocytes. We examined effects of asbestos (chrysotile) fibre on the proliferation of PHA-stimulated lymphocytes and the PHA binding activity of lymphocytes in vitro. The incorporation of 3H-thymidine and the expression of interleukin-2 receptor were depressed when the cells were exposed to 50 micrograms/ml of chrysotile fibre. The PHA binding activity of lymphocytes was significantly enhanced after chrysotile fibre exposure, as compared with non-exposed group. These results indicate that the depression of PHA stimuli with chrysotile fibre was not due to blocking of PHA binding to lymphocytes. The enhancement of PHA binding activity by chrysotile fibre was observed on CD8+ but not on CD4+ cells. It is possible, therefore, that treatment of peripheral blood mononuclear cells with chrysotile fibre induces increased reactivity of CD8+ cells with PHA, and that intensely activated CD8+ cells suppress proliferation of lymphocytes.

Modulation of human lymphocyte proliferation by amino acids

The amino acids required for phytohaemagglutinin (PHA) induced lymphocyte proliferation were determined by the 3H-thymidine incorporation in amino acid-deficient media. Results indicate that the PHA-stimulated lymphocytes require alanine and serine in addition to 13 other amino acids present in Eagle's minimal essential medium (arginine, cysteine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine). The omission of any one of the 13 amino acids would stop almost completely the proliferation of PHA-stimulated lymphocytes. The omission of serine from RPMI 1640 medium caused a mean reduction of 64% of cell proliferation, while the addition of alanine to PRMI 1640 culture medium caused a mean increment of 52%. The lymphocyte proliferation appears to be modulated by amino acids in the culture medium, and for optimal growth of lymphocytes, all these 15 amino acids are essential.

Effects Of β-Glucan On Immune Function And Upper Respiratory Tract Infections In Endurance Athletes

Animal data indicate that oat β-glucan (BG) supplements offset the increased risk of upper respiratory tract infection (URTI) associated with exercise stress through augmentation of neutrophil and macrophage function. PURPOSE: This study investigated the effects of BG supplementation on chronic resting and exercise-induced changes in immune function and URTI incidence in human endurance athletes. METHODS: Trained male cyclists were randomized to BG (N=19) or placebo (P) (N=17) groups and under double blind procedures received BG (5.6 grams/day) or P for two weeks prior to and during a 3-d period in which subjects cycled for 3 h/day at ~57% Wattsmax. URTI symptoms were monitored for two weeks before and two weeks after the 3-day period of intensified exercise. Blood samples were collected before and after two weeks supplementation (both samples, 8:00 am), immediately after the 3 h exercise bout on day 3 (6:00 pm), and 14 h post-exercise (8:00 am), and assayed for natural killer cell activity (NKCA), polymorphonuclear respiratory burst activity (PMN-RBA), PHA-stimulated lymphocyte proliferation (PHA-LP), plasma IL-6, IL-10, IL-1ra, and IL-8, and blood leukocyte IL-10, IL-8, and IL-1ra mRNA expression. RESULTS: Chronic resting levels and exercise-induced changes in NKCA, PMN- RBA, PHA-LP, plasma cytokines, and blood leukocyte cytokine mRNA did not differ significantly between BG and P groups. URTI incidence rates during the two week period following intensified exercise did not differ significantly between groups (9/19 or 47% for BG and 6/17 or 35% for P, Chi-square = 0.16, P=0.693). CONCLUSION: A 17-day period of BG vs. P ingestion did not alter chronic resting or exercise-induced changes in immune function, or URTI incidence in cyclists during the 2-week period following intensified exercise. Funded by a grant from the Gatorade Sports Science Institute.

Quercetin Reduces Illness but Not Immune Perturbations after Intensive Exercise

To investigate the effects of quercetin supplementation on incidence of upper respiratory tract infections (URTI) and exercise-induced changes in immune function. Trained male cyclists (N=40) were randomized to quercetin (N=20) or placebo (N=20) groups and, under double-blind procedures, received 3 wk quercetin (1000 mg.d(-1)) or placebo before, during, and for 2 wk after a 3-d period in which subjects cycled for 3 h.d(-1) at approximately 57% Wmax. Blood and saliva samples were collected before and after each of the three exercise sessions and assayed for natural killer cell activity (NKCA), PHA-stimulated lymphocyte proliferation (PHA-LP), polymorphonuclear oxidative-burst activity (POBA), and salivary IgA output (sIgA). Pre- to postexercise changes in NKCA, PHA-LP, POBA, and sIgA did not differ significantly between quercetin and placebo groups. URTI incidence during the 2-wk postexercise period differed significantly between groups (quercetin=1/20 vs placebo=9/20, Kaplan-Meier analysis statistic=8.31, P=0.004). Quercetin versus placebo ingestion did not alter exercise-induced changes in several measures of immune function, but it significantly reduced URTI incidence in cyclists during the 2-wk period after intensified exercise.

Effect of dietary lactoferrin on the immune functions and serum iron level of weanling piglets1

A total of 90 weanling female pigs (Duroc x Landrace x Yorkshire) were used in a 30-d growth experiment to investigate the effect of lactoferrin (LF) on growth performance, immune function, and serum iron concentrations. The pigs were allocated on the basis of BW and litter to 3 dietary treatments in a randomized complete block design. The dietary treatments were: control group (basal diet), antibiotics group (basal diet + 20 mg/kg of flavomycin + 110 mg/kg of aureomycin), and LF group (basal diet + 1.0 g/kg of LF). There were 3 replicate pens per treatment, and pigs were grouped with 10 pigs per pen. Six pigs, randomly selected from each treatment (2 pigs/pen), were slaughtered for serum and spleen samples on d 15 and 30. Supplementation with LF improved the phytohemagglutinin (PHA)-stimulated peripheral lymphocyte proliferation by 36% (P < 0.01), increased concanavalin A (ConA)- and PHA-induced spleen lymphocyte proliferation by 332% (P < 0.01) and 258% (P < 0.01), enhanced serum IgG by 20% (P < 0.05), IgA by 13% (P < 0.05), IgM by 15% (P < 0.05), complement 4 (C4) by 29% (P < 0.05), IL-2 by 12% (P < 0.01), and serum iron values by 22% (P < 0.05) on d 15 compared with the control. Lactoferrin supplementation increased PHA-stimulated lymphocyte proliferation (P < 0.01), serum IgG by 16% (P < 0.05), IgA by 17% (P < 0.05), C4 by 11% (P < 0.05), IL-2 by 14% (P < 0.05), and serum iron values by 23% (P < 0.01), and decreased the diarrhea ratio (P < 0.05) relative to the control on d 30. Compared with the controls, supplementation with antibiotic increased ConA- and PHA-induced spleen lymphocyte proliferation (P < 0.05) on d 15, decreased the diarrhea ratio (P < 0.05), and increased the PHA-induced spleen lymphocyte proliferation (P < 0.05) and serum iron values (P < 0.01) on d 30. These results support the possible use LF as an immunostimulant to improve immune functions and strengthen host defenses and would seem to be a good method for defending weanling piglets from infections and weanling stress.

The effect of transportation on the immune status of Bos indicus steers1

This study investigated the effect of 72 h of road transport on the immune status of Bos indicus steers (n = 10; age = 15 to 18 mo). Total and differential leukocyte numbers and lymphocyte function were determined at 2 d before transport (-48 h), immediately after 72 h of transport (72 h), and 6 d after transport (216 h). Phytohemagglutinin (PHA)-stimulated lymphocyte proliferation, interferon-gamma production, and tetanus-toxoid specific antibody levels were determined. Total leukocyte and eosinophil numbers showed a transient decrease at 72 h (immediately after transport; P < 0.05) and returned to baseline values by 6 d after transport. Lymphocyte numbers and antibody titers were unaffected by transportation. The PHA-stimulated lymphocyte proliferation decreased (P < 0.05) at 72 h and returned to baseline levels 6 d after transport. This study demonstrated that transportation of mature Bos indicus steers caused transient decreases in leukocyte numbers and lymphocyte function, although all measures recovered by 6 d after transport. Therefore, Bos indicus cattle may be vulnerable to infection during this period.

Ferulate inhibits mixed lymphocyte reaction stimulated cell proliferation and inhibits IL-2 and IL-2 receptor expression

Introduction. We have previously shown that curcumin (di-feruloyl methane) has immunosuppressive properties in addition to its anti-oxidant, anti-inflammation, and anti-mutagenic properties. Ferulic acid is a metabolic product of phenylalanine and tyrosine and also a degradation product of curcumin. In this experiment we investigated whether ferulic acid retained the immunosuppressive properties of curcumin. We used ethyl--ferulic acid (FE, from Sigma) in all our experiments due to its higher water solubility than free ferulic acid and therefore more bioavailable. Methods. Lymphocytes were isolated from human spleen. Mixed lymphocyte reaction (MLR), exogenous IL-2, and PHA-stimulated human lymphocyte proliferation were assayed by 3H-thymidine incorporation. Cell-secreted IL-2 in the culture medium was assayed by ELISA, and IL-2 Receptor (IL-2R) expression was assayed by flow-cytometry. Trypan Blue exclusion was used to rule out direct cytotoxic effects of FE. Microsoft Excel T-test program was used for statistics. P values less than 0.05 were considered statistically significant. Results. MLR stimulated cell proliferation > 8 times over the control response cells. FE at 10, 20, 40 and 80 μM inhibited MLR stimulated 3H-thymidine uptake (CPM/well) from 5349 ± 178 to 4635 ± 119, 4294 ± 125, 3450 ± 79, and 1756 ± 62, respectively ( P < 0.01, n = 6). MLR stimulated IL-2 secretion in the culture medium from control (pg/ml) 24 ± 5.2 to 214.8 ± 5.5. FE at 40 and 80 μM significantly inhibited IL-2 level from 214.8 ± 5.5 to 161.8 ± 6.6 and 138.5 ± 9.6, respectively ( P < 0.04, n = 3). Exogenous IL-2 (0.5 nM) stimulated IL-2 receptor (IL-2R) expression in human lymphocytes from control of 10.52 to 19.95%. FE at 80 μM inhibited exogenous IL-2-stimulated IL-2R expression to 10.95%. Mitogen PHA (5 μg/ml) stimulated IL-2R expression in the lymphocytes from control 10.52 to 58.34%, FE at 80 μM inhibited IL-2R expression to 25.5%. (This experiment has been done once.) Conclusion. FE has immunosuppressive properties like its precursor curcumin but possesses the additional advantage of being bioavailable. These effects warrant further investigation.

Mesenchymal stem cells inhibit the expression of CD25 (interleukin-2 receptor) and CD38 on phytohaemagglutinin-activated lymphocytes.

Mesenchymal stem cells (MSC) are immunomodulatory and inhibit lymphocyte proliferation. We studied surface expression of lymphocyte activation markers and secreted cytokines, when lymphocytes were activated in the presence of MSC. MSC suppressed the proliferation of phytohaemagglutinin (PHA)-stimulated CD3+, CD4+ and CD8+ lymphocytes. MSC significantly reduced the expression of activation markers CD25, CD38 and CD69 on PHA-stimulated lymphocytes. Mixed lymphocyte culture (MLC) supernatants containing MSC suppressed proliferation of MLC and PHA-stimulated lymphocytes dose-dependently. MSC secrete osteoprotegerin (OPG), but not hepatocyte growth factor (HGF) or transforming growth factor-beta (TGF-beta). Stromal-cell-derived factor-1 (SDF-1) is not expressed on the cell surface. A recent report suggested that T-cell suppression by MSC is mediated by HGF and TGF-beta. MSC suppression was not restored by the addition of neutralizing antibodies against SDF-1, OPG, HGF or TGF-beta, alone or in combination. Addition of guanosine to PHA-stimulated lymphocyte cultures containing MSC did not affect lymphocyte proliferation. The immunosuppressive effects of cyclosporine and MSC did not interfere, when present in the cultures of PHA-activated lymphocytes. In summary, human MSC suppress proliferation of both CD4+ and CD8+ lymphocyte and decrease the expression of activation markers.

Influence of skinfold sum and peak VO(2) on immune function in children.

To measure the relationship of skinfold sum and peak VO(2) power with immune function in children. Cross-sectional, with all children tested twice during a 2 month period for peak VO(2), sum of two skinfolds, and immune function, with data from the two measures averaged and then correlated (alpha level, < or = 0.01). Immune measures included leukocyte and lymphocyte subset counts, delayed-typed hypersensitivity (DTH), global IgG antibody response over 4 weeks to pneumococcal vaccination (pIgG), salivary IgA concentration (sIgA), PHA-stimulated lymphocyte proliferation (PHA-SLP), natural killer cell activity (NKCA), and granulocyte and monocyte phagocytosis and oxidative burst activity. Seventy-three children (n=42 males, n=31 females) ranging in age from 7 to 13 y (mean+/-s.d. age, 9.9+/-1.7 y). The mean skinfold sum was 28.9+/-17.1 mm, and peak VO(2) 45.8+/-8.1 ml/kg/min. Peak VO(2), skinfold sum, and immune measures did not differ significantly by age or gender. Therefore, correlations were made on combined indices for all subjects. Peak VO(2) and the skinfold sum were not significantly correlated with NKCA, oxidative burst activity, plgG or DTH. Peak VO(2) was negatively correlated with monocyte phagocytosis (r=-0.30, P=0.012) and positively correlated with PHA-SLP (6.25 microg/ml; r=0.35, P=0.004). The skinfold sum was positively correlated with the total leukocyte count (r=0.39, P<0.001), granulocyte count (r=0.36, P=0.002), monocyte count (r=0.38, P=0.001), monocyte phagocytosis (r=0.41, P<0.001), granulocyte phagocytosis (r=0.35, P=0.003), and sIgA (r=0.32, P=0.006), and negatively correlated with PHA-SLP (6.25 microg/ml; r=-0.39, P=0.001). Data from this study indicate that a high skinfold sum is related to elevated leukocyte subset counts and monocyte/granulocyte phagocytosis, and low PHA-SLP in children.

Lymphocyte proliferation modulated by glutamine: involved in the endogenous redox reaction.

Decreased glutamine concentrations are found during catabolic stress and are related to susceptibility to infections. However, little is known about the mechanism of glutamine modulation of lymphocyte functions. Glutamine is not only an important energy source in mitochondria, but is also a precursor of glutamate, which is used for cellular glutathione (GSH) biosynthesis in lymphocytes. In this study, we investigated the effects of glutamine on the redox reaction during lymphocyte proliferation. Peripheral blood mononuclear cells, obtained from healthy adult volunteers, were cultured and stimulated by phytohaemagglutinin (PHA) in the presence of different glutamine concentrations. Cells were harvested and prepared for analysis of lymphocyte proliferation, cell cycle propagation, intracellular glutathione levels and reactive oxygen species (ROS) production. We found that glutamine supplementation significantly enhanced PHA-stimulated lymphocyte proliferation and propagation of the cell cycle from the G1 to S and G2/M phases. Glutamine also enhanced production of both intracellular ROS and GSH levels in PHA-stimulated lymphocytes. Flow cytometric analysis by the mercury orange staining method showed that glutamine significantly enhanced intracellular non-protein thiols in PHA-stimulated CD4+, but not CD8+ lymphocyte subsets. Furthermore, intracellular GSH detected by monochlorobimane dye probe showed that glutamine enhanced GSH both in PHA-stimulated CD4+ and CD8+ lymphocyte subsets. Inadequate glutamine supplementation resulted in decreased lymphocyte proliferation in association with decreased levels of intracellular GSH. Addition of exogenous GSH significantly enhanced lymphocyte proliferation, whereas blockade of GSH synthesis enhanced ROS production and suppressed lymphocyte proliferation. These results suggest that the modulation of PHA-stimulated lymphocyte proliferation by glutamine is closely related to the maintenance of appropriate intracellular redox status.

Melatonin inhibits PHA-stimulated chicken lymphocyte proliferation in vitro.

Many studies have shown that melatonin plays a fundamental role in neuroimmunomodulation. There are known differences between mammals and birds in immunomodulatory function of melatonin exerted in vivo. In present study the effect of exogenous melatonin on chicken lymphoid cell proliferation in vitro was examined. Melatonin alone (10(-10)M-10(-5)M) did not exert any effect on the proliferation of the chicken thymocytes, splenocytes and lymphocytes from the bursa of Fabricius. On the other hand, melatonin addition strongly inhibited the proliferation of PHA-activated thymocytes and splenocytes from young chickens. The effect of melatonin was hormone- and mitogen-(PHA) dose-dependent; the most pronounced effect was obtained at low PHA and melatonin concentrations i.e., 1.6-3.125 microg/100 microl of PHA and 10(-10) M-10(-7) M melatonin, respectively. Prior immunization of chickens with sheep red blood cells (SRBC) inhibited the proliferative response of splenocytes to the same dose of PHA and, consequently, the effect of melatonin addition was not observed. The results reported demonstrate a direct inhibitory effect of melatonin on in vitro activated chicken lymphocytes.

Mitotic Index and Cell Proliferation Kinetics as Additional Variables for Assessment of Genotoxic Effect of The Herbicide Modown

·ivikova K. , J . Dianovsk : Mitotic Index and Cell Proliferation Kinetics as Additional Variables for Assessment of Genotoxic Effect of The Herbicide Modown. Acta Vet. Brno 2000, 69: 45–50. The in vitro effect of the herbicide Modown (with active component bifenox) was tested for the ability to influence cell proliferation of PHA-stimulated bovine peripheral lymphocytes. Mitotic (MI) and proliferation (PI) indices were determined as an alternative for the screening of the cytostatic activity. The herbicide Modown exerted a clear effect on the inhibition of MI and PI over a concentration-tested range of 25 μg/ml to 1000 μg/ml. An expressive proliferation delays was found after the treatment with herbicide at a dose of 250 μg/ml (P < 0.001), while the higher doses of 500 and 1000 μg/ml had caused nearly complete mitotic inhibition in each donor (P < 0.05 and P < 0.001, respectively). A correlation between the PI and MI inhibition refers rather to cytostatic than cytotoxic effects of the herbicide. The results support the possibility of immunosuppression by herbicide exposure. Herbicide Modown, bovine peripheral lymphocytes, cell proliferation, cytostatic effect Nitrodiphenylether herbicides are persistent and lipophilic environmental contaminants may contribute to the impairment of animal health and production. The presence of pesticide residues has been demonstrated in raw bovine milk and in different domestic animal tissues (Lioi et al. 1998). All nitrodiphenylether herbicides exert their phytotoxic activity in chlorophyll of plants by the reduction itself to radical species, which initiate destructive reactions in membrane lipids, leading to the cell leakage (Corbet t et al. 1984). Members, as acifluorfen and oxyfluorfen, e.g. are extremely potent inhibitors of protoporphyrinogen oxidase, a membrane–bound enzyme involved in the heme and chlorophyll biosynthesis pathways (Camadro et al. 1995). The later was studied for the induction of haematological diseases in human erythroid progenitor cells but a cytotoxic effect had been seen only at very high concentrations (Rio et al. 1997). Bifenox is a selective herbicide used in control of annual broad-leaved weeds and some grasses, e. g. in cereals, maize, soya beans, rice and other crops. J inno et al. (1999) described the cytotoxic and porphyrogenic effects of bifenox in rat hepatocytes. They found the maximum porphyrin accumulation at 0.25 mM of the herbicide and an inhibion of protoporphyrogen oxidase, resulting in the accumulation of protoporphyrin IX. Francis et al. (1999) evaluated maternal and developmental toxicity of ten diphenylether herbicides (including bifenox) in mice. They found a correlation among the position of chlorine substituents and the potential for inducing prenatal and postnatal syndroms. But no prenatal and postnatal embryotoxicity was shown. ACTA VET. BRNO 2000, 69: 45–50 Address for correspondence: RNDr. Katarina ·ivikova, CSc. Department of Veterinary Genetics, University of Veterinary Medicine Komenskeho 73, 041 81 Ko‰ice, Slovak Republic Phone: +421 95 63 321 11, (ext. 482) Fax: +421 95 63 236 66 E-mail: sivikova@uvm.sk http://www.vfu.cz/acta-vet/actavet.htm

Effect of glutamine on Th1 and Th2 cytokine responses of human peripheral blood mononuclear cells.

Decreased glutamine concentrations are found in patients with catabolic stress and are related to susceptibility to infections. In this study, we evaluated the role of glutamine in Th1/Th2 cytokine responses. Peripheral blood mononuclear cells were stimulated with phytohemagglutinin (PHA), live attenuated bacillus Calmette-Guérin (BCG), or measles virus in the presence of different glutamine concentrations. We found that glutamine at an optimal concentration (0.6 mM) significantly enhanced PHA-stimulated lymphocyte proliferation as well as Th1 [interferon-gamma (IFN-gamma) and interleukin-2 (IL-2)] and Th2 cytokine (IL-4 and IL-10) production. In the absence of glutamine, BCG and measles virus elicited minimal lymphocyte proliferation, whereas BCG enhanced Th1 cytokine response and measles virus promoted Th2 cytokine response. Interestingly, addition of glutamine promoted the BCG-elicited Th1 cytokine response (IFN-gamma), but suppressed the measles-induced Th2 cytokine response (IL-10). These results suggest that appropriate glutamine levels may influence host responses to different antigens and microorganisms. Furthermore, predominately Th1, but not Th2, cytokine responses required the presence of optimal concentrations of glutamine.

Changes in immune function following surgery for esophageal carcinoma.

Changes in immune function due to surgical injury have been well-documented. Immunosuppression is one of the causes of infectious complications leading to organ dysfunction in critical illness. It is not known what kind of surgery in the daily clinical practice causes immunosuppression. Stress response and immune function following surgery for esophageal carcinoma, assuming a highly-stressed operation, were studied and then compared with the stress response and immune function following gastric surgery, a moderately-stressed procedure. Forty patients who underwent esophagectomy and 39 patients receiving gastric operation were studied. The concentrations of serum interleukin-6 (IL-6) were measured preoperatively, at 1, 2, and 6 h, and at 1, 3, and 10 d after operation. Total protein, serum albumin, rapid turnover protein, serum CRP, and cortisol were measured before operation and at 1, 3, 7, and 21 d after operation. ConA- and PHA-stimulated lymphocyte proliferation, IgA, IgG, and IgM were also measured preoperatively, and on 7 and 21 d following surgery. The patients were fed exclusively by total parenteral nutrition (TPN). A striking rise of IL-6 was observed, with a peak in both groups at 1 to 6 h following operation. The peak values were 419+/-30 pg/mL, which was approximately twice as high in the esophagectomy patients as in the gastrectomy patients (195+/-40 pg/mL). CRP and cortisol also increased after operation, and these increases were also significantly greater in the esophagectomy patients. ConA- and PHA-stimulated lymphocyte proliferation decreased significantly 7 d after esophagectomy (P<0.05), but was unchanged in the patients receiving gastrectomy. Suppression of cellular immunity correlated significantly with serum cortisol, and was preceded by a rise in serum IL-6. The IgA, IgG, and IgM levels, however, remained unchanged from their preoperative values throughout the study in both groups. Nutritional status in terms of serum protein, albumin, and rapid turnover protein, decreased postoperatively, but there was no difference between the two groups. It is, therefore, concluded that cell-mediated immunosuppression, preceded by a hyperinflammatory response, is an observable reaction in patients following esophageal surgery, but not in patients undergoing gastric surgery.

TGF-beta is not the principal immunosuppressive component in coagulation factor concentrates.

Coagulation factor concentrates are known to inhibit a variety of immune reactions when assessed in vitro. This study assessed the immunomodulatory activity of a wide range of coagulation factor concentrates by measuring their inhibition of PHA-stimulated lymphocyte proliferation and reduction in IL-2 secretion. The hypothesis that TGF-beta is responsible for most of these effects was tested by measuring biologically active TGF-beta and immunoreactive TGF-beta1 in the concentrates and comparing the levels recorded with immunosuppressive activity. In addition, the coagulation factors were compared directly with a standard preparation of TGF-beta in a TGF-beta-specific bioassay and in lymphocyte proliferation assays. Although there was a broad correlation between levels of total or active TGF-beta and immunosuppressive activity across all of the coagulation factors tested, individual data sets showed clear discrepancies. Implying that TGF-beta probably serves as a surrogate marker for other immunomodulatory contaminants and that neither TGF-beta nor any other single substance could account for all of the immunosuppressive activity observed. Furthermore, there was a difference of more than 100-fold in the relative potencies of coagulation factors and pure TGF-beta, when compared in immunosuppression assays, indicating that the different assays did not measure the same substance. Whereas anti-TGF-beta antibody almost completely blocked the activity of coagulation factor concentrates (TGF-beta-specific bioassay) and abrogated the effect of authentic TGF-beta (immunosuppression assays) at high concentrations it achieved <50% reversal of the immunosuppressive effects of coagulation factors in immunosuppression assays. These findings indicated that TGF-beta accounted for only a minor proportion of the immunosuppressive activity in most coagulation factor concentrates.

Effects of soybean oil emulsion and eicosapentaenoic acid on stress response and immune function after a severely stressful operation.

To investigate the effects of soybean oil emulsion and oral or enteral administration of eicosapentaenoic acid (EPA) on stress response, cytokine production, protein metabolism, and immune function after surgery for esophageal cancer. It has been reported that safflower oil, rich in n-6 polyunsaturated fatty acid (n-6 PUFA), affects the survival rate of septic animals and decreases the immune function. It has also been reported that the administration of fish oil, in contrast, reduces these stress responses and stress-induced immunosuppression. In humans, the effects of soybean oil emulsion and the administration of EPA on stress response and immune function after surgery have not been established. Patients who underwent esophagectomy with thoracotomy were divided into three groups. Seven patients were fed by total parenteral nutrition (TPN) with soybean oil emulsion, which accounted for 20% of total calories. Seven patients were given oral or enteral administration of 1.8 g/day EPA, in addition to TPN with soybean oil emulsion. Nine patients served as the control group; these patients received fat-free TPN. Serum interleukin-6 (IL-6), C-reactive protein, concanavalin A (con A)- or phytohemagglutinin (PHA)-stimulated lymphocyte proliferation, natural killer cell activity, and stress hormones were measured. The postoperative level of serum IL-6 was significantly higher in the group receiving soybean oil emulsion than in the fat-free group. Oral or enteral supplementation of EPA with soybean oil emulsion significantly reduced the level of serum IL-6 compared with the patients receiving soybean oil emulsion. Con A- or PHA-stimulated lymphocyte proliferation decreased significantly on postoperative day 7 in all groups of patients. The supplementation of EPA with soybean oil emulsion significantly improved the lymphocyte proliferation and natural killer cell activity on postoperative day 21 compared with the group receiving soybean oil emulsion. Soybean oil emulsion amplifies, and the supplementation of EPA reduces, the stress response and stress-induced immunosuppression.

Synergistic interaction between dehydroepiandrosterone and prednisolone in the inhibition of rat lymphocyte proliferation.

The interaction between dehydroepiandrosterone (DHEA) and prednisolone (PD) in the inhibition of rat lymphocyte proliferation was evaluated. The in vitro proliferative response of splenocytes from male Sprague-Dawley rats stimulated with phytohemagglutinin (PHA) was measured by the incorporation of 3H-thymidine into the DNA. DHEA and PD were added at the initiation of cultures individually and in various molar ratio combinations. The search for synergy, additivity or antagonism between the two compounds was performed by using the median-effect method of Chou and Talalay and Drewinko's statistical analysis. Both compounds individually inhibit the proliferation of PHA-stimulated rat lymphocytes. DHEA with an IC50 value of 13.4 microM was a thousand times less potent than PD which had an IC50 value of 5.4 nM. Synergy was observed between DHEA and PD. The intensity of the interaction appeared to be function of the molar ratio of the two drugs. The association of DHEA and PD could produce enhanced steroid effects in anti-inflammatory therapy.