Phytohemagglutinin-stimulated Lymphocyte Proliferation Research Articles

Immunomodulatory activity of Tityus serrulatus scorpion venom on human T lymphocytes.

BackgroundTityus serrulatus scorpion venom (TsV) contains toxins that act on K+ and Na+ channels and account for the venom’s toxic effects. TsV can activate murine peritoneal macrophages, but its effects on human lymphocytes have been poorly investigated. Considering that lymphocytes may play an important role in envenomation, we assessed whether TsV affects the expression of phenotypic (CD3, CD4, and CD8) and activation (CD69, CD25, and HLA-DR) markers, cell proliferation, and cytokine production in peripheral blood mononuclear cells.MethodsCytotoxicity of TsV was evaluated via the MTT assay. Cell proliferation, expression of phenotypic and activation markers, and release of cytokines were assessed using flow cytometry, after treatment with non-cytotoxic concentrations of TsV. The combined use of carboxyfluorescein diacetate succinimidyl ester and monoclonal antibodies against phenotypic and activation markers enabled us to simultaneously assess cell proliferation extent and cell activation status, and to discriminate among cell subpopulations.ResultsTsV at concentrations of 25 to 100 μg/mL were not cytotoxic towards peripheral blood mononuclear cells. TsV did not induce significant changes in lymphocyte subpopulations or in the expression of activation markers on CD4+ and CD8+ T cells. TsV inhibited the phytohemagglutinin-stimulated lymphocyte proliferation, particularly in the CD8+ CD25+ T lymphocyte subset. TsV alone, at 50 and 100 μg/mL, did not induce peripheral blood mononuclear cell proliferation, but elicited the production and release of IL-6, a proinflammatory cytokine that plays an important role in innate and adaptive immune responses.ConclusionsTsV is a potential source of molecules with immunomodulatory action on human T lymphocytes.Electronic supplementary materialThe online version of this article (doi:10.1186/s40409-015-0046-3) contains supplementary material, which is available to authorized users.

Phytochemical screening of the dichloromethane⿿ethanolic extract of Eriosema campestre var. macrophylum roots and its antiproliferative effect on human peripheral blood lymphocytes

Eriosema campestre var. macrophylum (Grear) Fortunato, Fabaceae, is a native plant of the Brazilian Cerrado and the decoction of its roots has been used by folk medicine for the therapy of inflammatory diseases. In this study we aimed to investigate the effect of the dichloromethane⿿ethanolic extract of E. campestre roots on the proliferative response of lymphocytes and to examine the profile of IL-2 production. The effect of dichloromethane⿿ethanolic extract of E. campestre on the proliferation of phytohemagglutinin-stimulated lymphocytes was evaluated by using flow cytometry and the cell supernatants were assayed for IL-2 concentrations by using an enzyme-linked immunosorbent assay. The phytochemical screening of E. campestre roots was performed to determine the main secondary metabolites through chromogenic and precipitation reactions and by using HPLC-PAD. In addition to the presence of subclasses of flavonoids (flavones and flavonols) in dichloromethane⿿ethanolic extract of E. campestre, we observed that the extract induced a concentration-dependent decrease in IL-2 levels on the supernatant of the cell cultures as well as an antiproliferative effect on T lymphocytes, including CD4+ and CD8+ cells. The anti-inflammatory effects attributed to E. campestre by folk medicine may partly be explained by its antiproliferative action on T lymphocytes.

Relationship between Immunological Parameters and the Severity of Neutropenia and Effect of Enteral Nutrition on Immune Status during Neoadjuvant Chemotherapy on Patients with Advanced Esophageal Cancer

Objectives: Chemotherapy may cause various toxicities as well as impair immunological function. However, little is known about the relationship between toxicities and immunological parameters or the effect of enteral nutrition (EN) on immunological status during chemotherapy. Methods: 91 patients who received neoadjuvant chemotherapy (NACT) for esophageal cancer were randomly assigned to receive either EN or parenteral nutrition (PN). Immunological parameters, including total lymphocyte count (TLC), type 1 and type 2 CD4-positive T cells (Th1/Th2) balance, human leukocyte antigen (HLA)-DR expression on monocytes, natural killer cell activity, and phytohemagglutinin-stimulated lymphocyte proliferation were measured at baseline and day 14 of the first chemotherapy cycle. Results: In the PN group, patients with grade 3–4 neutropenia showed significantly lower TLC, HLA-DR expression, and Th1/Th2 balance at day 14 compared to those with grade 0–2 neutropenia. Among pretherapeutic factors, Th1/Th2 balance was the only factor significantly associated with the severity of neutropenia. Concerning the comparison of immunological parameters between the EN and PN groups, HLA-DR expression at day 14 was significantly higher in the EN group. Conclusions: Baseline Th1/Th2 balance predicted the severity of neutropenia, and EN significantly reduced the decline of monocyte HLA-DR expression in patients with esophageal cancer receiving NACT.

4SC-101, A Novel Small Molecule Dihydroorotate Dehydrogenase Inhibitor, Suppresses Systemic Lupus Erythematosus in MRL-(Fas)lpr Mice

Immunosuppressive treatments of systemic lupus (SLE) remain associated with significant toxicities; hence, compounds with better toxicity profiles are needed. Dihydroorotate dehydrogenase (DHODH) inhibition with leflunomide has proven to be effective in autoimmune diseases including SLE, but leflunomide can cause a variety of side effects. We hypothesized that 4SC-101, a novel DHODH inhibitor with a more favorable toxicity profile, would be as effective as high-dose cyclophosphamide (CYC) in controlling experimental SLE of female MRL(Fas)lpr mice. Daily oral gavage of 30, 100, and 300 mg/kg 4SC-101 from 12 to 22 weeks of age was compared with either vehicle or CYC treatment (30 mg/kg/week, i.p.) in terms of efficacy and toxicity. Three hundred milligrams per kilogram 4SC-101 was as effective as CYC in depleting spleen autoreactive T cells, B cells, and plasma cells as well as the respective DNA and RNA serum autoantibodies. This was associated with a comparable amelioration of the renal, dermal, and pulmonary SLE manifestations of MRL(Fas)lpr mice. However, even the highest dose of 4SC-101 had no effect on bone marrow neutrophil counts, which were significantly reduced in CYC-treated mice. Together, the novel DHODH inhibitor 4SC-101 is as effective as high dose CYC in controlling SLE without causing myelosuppression. Hence, DHODH inhibition with 4SC-101 might be suitable to treat active SLE with fewer side effects than CYC.

The Uleine-Rich Fraction ofHimatanthus lancifoliusBlocks Proliferative Responses of Human Lymphoid Cells

The purpose of the present work is to report the effects shown by the uleine-rich fraction extracted from the barks of Himatanthus lancifolius (Muell. Arg.) Woodson (Apocynaceae) on human peripheral blood mononuclear cells in which we were able to inhibit the proliferation of phytohemagglutinin-stimulated lymphocytes by blocking their transformation into blast-dividing cells. Furthermore, it inhibited the proliferation of Daudi and Reh cells, two leukemic cell lines of lymphoid origin. The present study widens the biological applications of H. lancifolius' alkaloids to include its possible use as a modulator of the immune system.

β-Glucan, Immune Function, and Upper Respiratory Tract Infections in Athletes

This study investigated the effects of oat beta-glucan (BG) supplementation on chronic resting immunity, exercise-induced changes in immune function, and self-reported upper respiratory tract infection (URTI) incidence in human endurance athletes. Trained male cyclists were randomized to BG (N = 19) or placebo (P; N = 17) groups and under double-blind procedures received BG (5.6 g x d(-1)) or P beverage supplements for 2 wk before, during, and 1 d after a 3-d period in which subjects cycled for 3 h x d(-1) at approximately 57% maximal watts. URTI symptoms were monitored during BG supplementation and for 2 wk afterward. Blood samples were collected before and after 2 wk of supplementation (both samples, 8:00 a.m.), immediately after the 3-h exercise bout on day 3 (6:00 p.m.), and 14 h after exercise (8:00 a.m.) and were assayed for natural killer cell activity (NKCA), polymorphonuclear respiratory burst activity (PMN-RBA), phytohemagglutinin-stimulated lymphocyte proliferation (PHA-LP), plasma interleukin 6 (IL-6), IL-10, IL-1 receptor agonist (IL-1ra), and IL-8, and blood leukocyte IL-10, IL-8, and IL-1ra mRNA expression. 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 during the 2-wk postexercise period did not differ significantly between groups. An 18-d period of BG versus P ingestion did not alter chronic resting or exercise-induced changes in immune function or URTI incidence in cyclists during the 2-wk period after an intensified exercise.

Diverse production system and social status effects on pig immunity and behavior

Pig performance, immunity, and behavior may be influenced by production system and social status. A conventional indoor housing system was compared with an outdoor system to determine the effects of diverse production systems on pig performance, behavior, and immunity. At 21 days of age, weight matched pigs reared in a conventional indoor or an outdoor production system were weaned and randomly selected for use in the present study. Six replicate pens were evaluated per treatment, with two littermate female pigs in each pen. Social status was determined at 7 weeks of age by a feed competition test. Pig behavior was recorded for 24 h using a scan sample technique. At 9 weeks of age, blood samples were taken from indoor- and outdoor-reared pigs and immune assays performed. Both indoor- and outdoor-reared pigs were given a sheep red blood cell (SRBC) challenge at 6 and 8 weeks of age, and antibody titers to SRBC were measured at 6, 7, and 9 weeks of age. Performance data were recorded throughout the study period. Body weight and average daily gain were not influenced by the production system in which the pig was reared. The production system effected standing, lying, drinking, and oral/nasal/facial behaviors. The production system influenced the expression of certain behaviors depending on the time of day. Immune and blood measures were affected by production system. The percentage of phagocytosis was greater (P ≤0.05) and antibody titers to SRBC challenge tended to be greater (P=0.066) among outdoor-reared pigs compared with indoor-reared pigs. Outdoor reared pigs had higher hemoglobin concentrations (P<0.005), percentage of hematocrit (P<0.005), mean corpuscular volume (P<0.005), and mean corpuscular hemoglobin (P<0.005) compared with indoor-reared pigs. Furthermore, dominance order influenced the immune system. Dominant pigs had greater phytohemagglutinin stimulated lymphocyte proliferation (P<0.01) and baseline antibody titers to sheep red blood cells (P<0.05) compared with submissive pigs. In conclusion, in the present study body weight and average daily gain were similar among diverse production systems. However, pig behavior and immune system were significantly influenced by the production system. Dominance order influenced the immune system regardless of the production system. © 2007 Elsevier B.V. All rights reserved.

The Post-Operative Level of Serum Monocyte Chemoattractant Protein-1 and Its Correlation With the Severity of Surgical Stress

Several basic studies have demonstrated that monocyte chemoattractant protein-1 (MCP-1) influences type 2 cytokine production and cell-mediated immunity. However, there have been few reports on the changes in serum MCP-1 levels in patients undergoing major thoracoabdominal surgery. In this study, we examined the kinetics of serum MCP-1 post-operatively and clarified its significance regarding the extent of surgical stress. Seventeen patients who underwent surgical operations were included in this study. All of them were fed by total parenteral nutrition to deliver the same amount of calories and nitrogen. Of the severely stressed group, nine patients underwent esophagectomy with thoracotomy. Of the group of moderately stressed group, eight patients underwent gastric or colorectal surgery. Serum MCP-1 and interleukin-6 (IL-6) were measured after operation. Cell-mediated immunity was measured by concanavalin A- or phytohemagglutinin-stimulated lymphocyte proliferation on the seventh post-operative day. Serum MCP-1 and IL-6 were increased immediately after surgery in both groups. The level of MCP-1 was significantly higher in the group of severely stressed patients than in the moderately stressed group. Concanavalin A or phytohemagglutinin-stimulated lymphocyte proliferation was significantly lower in the severely stressed group than in the moderately stressed group. These results indicate that serum MCP-1 levels are directly correlated, and cell-mediated immunity inversely correlated, with the severity of surgical stress.

Influences of soybean oil emulsion on stress response and cell-mediated immune function in moderately or severely stressed patients

OBJECTIVES: We previously reported that ω-6 fat emulsion increases cytokine production in burned rats. Effects of soybean oil emulsion on surgical stress responses and lymphocyte function according to the surgical severity have not been studied in detail. We investigated the effects of soybean oil emulsion, which contains 50% ω-6 fatty acid, on postoperative stress responses and cell-mediated immune function according to the severity of surgical stress. METHODS: Eight patients who underwent gastric or colorectal surgery and nine who underwent esophagectomy were fed fat-free total parenteral nutrition. Ten patients who underwent gastric or colorectal surgery and seven who underwent esophagectomy were fed total parenteral nutrition with soybean oil emulsion. Total parenteral nutrition provided 1.5 g of protein and 40 kcal per kilogram every day from 7 d before surgery to postoperative day 14. Soybean oil emulsion (Intralipid) accounted for 20% of the total calories. Serum interleukin-6, C-reactive protein, glucagon, and concanavalin A-N or phytohemagglutinin-stimulated lymphocyte proliferation were determined. RESULTS: In the group of moderately stressed patients, soybean oil emulsion did not amplify the measured levels. In the group of severely stressed patients, soybean oil emulsion amplified the level of serum interleukin-6 and decreased concanavalin A- or phytohemagglutinin-stimulated lymphocyte proliferation. CONCLUSIONS: Soybean oil emulsion amplifies the stress responses and possibly suppresses cell-mediated immune function induced by surgical stress in severely stressed patients, but not in moderately stressed patients.

Interactions of aspirin and salicylic acid with prednisolone for inhibition of lymphocyte proliferation

The anti-inflammatory properties of salicylates and prednisolone were investigated using inhibition of in vitro phytohemagglutinin-stimulated lymphocyte proliferation in human whole blood from healthy male and female subjects. Steroids and salicylates are used in conjunction for the treatment of inflammatory disorders such as rheumatoid arthritis and inhibit the proinflammatory transcription factor, NFκB, by different mechanisms. Data generated using various combinations of these drugs were analyzed using isobolograms and the universal surface response approach based on the Loewe additivity principle. The interaction between aspirin and prednisolone was mildly antagonistic while that between salicylic acid and prednisolone was modestly synergistic at therapeutic concentrations. Further, aspirin was slightly more potent in males, but the nature of the steroid–salicylate interaction was similar across genders. This study helps rationalize part of the beneficial effects of steroids and salicylates in treatment of inflammatory disorders.

Responses of bovine lymphocytes to heat shock as modified by breed and antioxidant status.

We tested whether resistance of lymphocytes to heat stress is modified by breed, intracellular glutathione content, and extracellular antioxidants. In the first experiment, lymphocytes from Angus (Bos taurus, non-heat-tolerant), Brahman (B. indicus, heat-tolerant), and Senepol (B. taurus, heat-tolerant) heifers (12 heifers per breed) were cultured at 45 degrees C for 3 h to evaluate thermal killing, at 42 degrees C for 12 h in a 60-h phytohemagglutinin-induced proliferation test, and at 42 degrees C for 1 h to measure induction of heat shock protein 70 (HSP70). Killing at 45 degrees C was affected by breed x temperature (P < .01); the decrease in viability caused by a temperature of 45 degrees C was greater for Angus than for Brahman or Senepol. For phytohemagglutinin-stimulated lymphocytes, heating to 42 degrees C reduced [3H]thymidine incorporation equally for all breeds. Viability at the end of culture was affected (P < .001) by a breed x temperature interaction because the decrease in viability caused by culture at 42 degrees C was greatest for lymphocytes from Angus heifers. Heat shock for 1 h at 42 degrees C caused a two- to threefold increase in intracellular concentrations of HSP70, but there was no interaction of temperature with breed. In another experiment (with lymphocytes harvested from three Holstein cows), buthionine sulfoximine, a glutathione synthesis inhibitor, inhibited (P < .01) proliferation of phytohemagglutinin-stimulated lymphocytes at 38.5 and 42 degrees C. Addition of the antioxidants glutathione or thioredoxin to culture did not reduce the effects of heating to 42 degrees C on proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of mitogen-induced lymphocyte proliferation in sheep.

Conditions were established to optimize the lymphocyte proliferation test in sheep, a species where proliferation under standard conditions is lower than for other species. Optimum proliferation of phytohemagglutinin-stimulated lymphocytes was achieved in TCM-199 supplemented with 5% (v/v) horse serum, 0.2 mM sodium pyruvate and 5 x 10(-5) M beta-mercaptoethanol. Proliferative responses in this medium were 2.5-fold higher or more than when using a standard medium (RPMI-1640 + 10% heat-inactivated fetal bovine serum). Lymphocytes cultured in medium containing horse serum also responded to concanavalin A as well as or better than lymphocytes cultured in medium with bovine serum. For both PHA and Con A, optimal incorporation of radioactivity was achieved when cells were pulsed with [3H-methyl]-thymidine 72 h after addition of mitogen. The medium based on TCM-199 + horse serum could support interleukin-2-induced proliferation. Furthermore, known inhibitors of lymphocyte proliferation in sheep were effective in inhibiting lymphocytes cultured in this medium.

Can daily dietary arginine supplement affect the function and subpopulation of lymphocytes in patients with advanced gastric cancer?

Since arginine can stimulate lymphocyte proliferation in the healthy human, its effect on lymphocyte proliferation in vitro was studied in 7 patients with far advanced gastric cancer. These patients with normal nourishment were ambulatory and could consume a regular diet. A daily dietary supplement of 30 g arginine for 7 days did not alter the total lymphocyte counts or the T/B cell ratio in the peripheral blood. Enhancement of lymphocyte proliferation in response to mitogen stimulation was not observed. Furthermore, an in vitro study on the effect of arginine on phytohemagglutinin-stimulated lymphocyte proliferation showed that lymphocytes from gastric cancer patients had poorer responses than those obtained from normal subjects, despite the supplement in the culture medium with normal serum, patient serum, or fetal bovine serum. Arginine ingestion did not impair liver function and had no detectable side effects except transient nausea in 1 patient. These results indicate that dietary arginine supplement appears safe but does not stimulate lymphocyte function in far advanced gastric cancer patients. The suppressed immune function in gastric cancer patients may be the result of their intrinsic lymphocyte defect.

The Effects of Acute Moderate Exercise on Lymphocyte Function and Serum Immunoglobulin Levels

The extent and duration of changes on lymphocyte function and serum immunoglobulin (Ig) levels were examined in 12 women who walked 45 min at 60% VO2 max in a laboratory setting. A 2-factor, 2 x 6 design with repeated measures on both factors was utilized. The first factor was condition (exercise and rest), and the second factor was time (six times of measurement over a 24-h period), with treatment order counterbalanced. The 45-min walk, in comparison to rest in a seated position, was not associated with significant changes in circulating numbers of interleukin-2-activated T cells (CD5 and CD25) or on spontaneous or concanavalin-A-stimulated lymphocyte proliferation. A trend for decreased phytohemagglutinin-stimulated lymphocyte proliferation in comparison to the rest condition, however, was seen 1.5 h following the exercise bout (p = 0.047). The patterns of change for serum IgG, IgA, and IgM were significantly different (p = 0.001, p less than 0.001, p = 0.010, respectively) between conditions. IgG rose 7.2% immediately following exercise, and then returned to baseline 1.5 h later, which contrasted significantly with changes in the rest condition. These same patterns of change occurred also with IgA and IgM, but increases immediately following exercise were not significant, although a trend was seen for IgA (p = 0.03). The 45-min walk had no effect on plasma cortisol and epinephrine levels relative to the rest condition, but was associated with a significant 89% increase in norepinephrine.(ABSTRACT TRUNCATED AT 250 WORDS)

The nevoid basal cell carcinoma syndrome: Sensitivity to ultraviolet and x-ray irradiation

Demographic studies in patients with skin cancer have demonstrated the importance of exposure to ultraviolet and x-ray irradiation. This paper describes in vitro studies in peripheral lymphocytes from three patients with the nevoid basal cell carcinoma syndrome. Particular stress was placed on the following factors: (1) the distribution of the lymphocyte subsets, (2) the frequency of spontaneous sister chromatid exchange, (3) the effect of ultraviolet C (UVC) (254 nm) on deoxyribonucleic acid (DNA) synthesis, (4) the effect of UVC on the phytohemagglutinin-stimulated lymphocyte proliferation, and (5) the capacity to repair x-ray-induced DNA damage. Our data indicate that the distribution of the peripheral lymphocytes was normal, while the frequency of spontaneous sister chromatid exchange was high. The capacity of the lymphocytes to repair x-ray-induced DNA damage was low in all three patients. In two patients the UVC-induced DNA synthesis was reduced, while an increased UVC-induced inhibition of lymphocyte proliferation was observed. These cellular responses in vitro to ultraviolet and x-ray irradiation correspond to the clinical features of the nevoid basal cell carcinoma syndrome. A clearly defective in vitro cellular response to x-ray irradiation, reflecting the clinically evident x-ray sensitivity in the nevoid basal cell carcinoma syndrome, has not been reported previously.

Quinolones affect thymidine incorporation into the DNA of human lymphocytes.

The incorporation of [3H]thymidine into DNA was increased in phytohemagglutinin-stimulated human lymphocytes exposed to four of the new quinolone derivatives (ciprofloxacin, norfloxacin, ofloxacin, and A 56620) at concentrations achievable in clinical situations. However, proliferation of phytohemagglutinin-stimulated lymphocytes was not influenced by ciprofloxacin at concentrations of 0.5 to 10 micrograms/ml.

485 ROLE OP SOMATOMEDIN-C (SMC) IN LYMPHOCYTE PROLIFERATION

As lymphocytes have receptors for SMC, we studied the role of SMC in lymphocyte proliferation. Peripheral lymphocytes from 27 controls and 17 children with growth hormone deficiency (GHD) were cultured in either autologous serum (AS) or pooled serum from normal adults (NS) with and without phytohemagglutinin(PHA). The mitogenic response (MR) was defined as thymidine incorporated in AS ÷ thymidine incorporated in NS. Control MR = 1.01 ± 0.05 (X ± SEM). Two different MRS were observed in the untreated GHD children. Most GHD sera had a normal MR (1.09 ± 0.06), but 4 had a very low MR (0.05 ± 0.02). MR did not correlate with SMC or growth rate. The two groups of children were similar in all other parameters that we measured. After 1 week of growth hormone treatment (0.1u/kg/d), MR was normal in all (0.97 ± 0.06). Partially purified SMC (10–500 ng/ml) did not increase MR in the presence of varying (0–5%) amounts of AS. Although PHA stimulated lymphocyte proliferation, culture medium SMC was not increased and was proportional to the SMC content of the serum used (r = 0.8). Conclusions: SMC does not have a direct role in lymphocyte proliferation since the MR in GHD serum was not proportional to SMC; exogenous SMC did not increase MR nor was SMC synthesized by lymphocytes in vitro. Serum from some GHD children lacks another factor(s) that supports lymphocyte proliferation in vitro.

Abnormal lymphocyte response to u.v. radiation in multiple skin cancer.

The lymphocyte response to u.v. radiation (254 nm) was investigated by two different methods in 29 unselected patients with multiple epidermal cancer. The u.v.-induced DNA synthesis was determined as the increase in incorporation of [3H]thymidine in irradiated cells compared with nonirradiated cells after incubation for 2 h. The u.v. tolerance was measured as the u.v. dose necessary for 50% reduction in phytohemagglutinin-stimulated lymphocyte proliferation. Patients with both squamous cell differentiated tumors and basal cell carcinomas had very high u.v.-induced DNA synthesis values (p less than 0.01, when compared with patients with basal cell carcinoma only and p less than 0.005, when compared with controls). The u.v. tolerance in patient lymphocytes was considerably lower than in control lymphocytes (p less than 0.001), with the lowest values occurring in patients with clinical sun intolerance (p = 0.05, when compared with the remaining patients). These investigations may be of predictive value in skin carcinogenesis.