Phorbol Myristate Acetate-ionomycin Research Articles

Core Needle Biopsies as an Alternative Source for Ex Vivo Expanded TIL for Adoptive Cell Therapy in Triple-Negative Breast Cancer.

Adoptive transfer of ex vivo expanded tumor-infiltrating lymphocytes (TILs) have produced long-term response in metastatic cancers. TILs have traditionally been expanded from surgically resected specimens. Ultrasound-guided core needle biopsy (CNB) is an alternative method that avoids the morbidity of surgery and have added benefits which may include patients not amenable to surgery as well as the potential to produce TILs from multiple lesions in the same patient. We assessed the ability to produce and expand TILs from primary triple-negative breast cancer tumors from CNB (n=7) and demonstrate comparable expansion, phenotype and cytokine secretion after phorbol myristate acetate-ionomycin stimulation to TILs expanded from surgery (n=6). T cell Receptor clonality and diversity were also comparable between the two cohorts throughout the TIL culture. CNB is a safe and feasible method to obtain tumor tissue for TIL generation in patients with triple-negative breast cancer.

Maternal diet supplementation with high-docosahexaenoic-acid canola oil, along with arachidonic acid, promotes immune system development in allergy-prone BALB/c mouse offspring at 3weeks of age.

To study the effects of feeding docosahexaenoic acid (DHA, derived from novel canola oil), with same amount of arachidonic acid (ARA), supplemented diet to lactating dams on the immune system development of suckled offspring using a T helper type-2 (Th2)-dominant BALB/c mouse. Dams received nutritionally complete control (no ARA or DHA) or DHA + ARA diet (1% DHA and 1% ARA of total fatty acids) from 5days pre-parturition to the end of 3-week suckling period. After euthanization, relevant tissues were collected to study fatty acids, splenocyte phenotype and function (ex vivo cytokines with/without lipopolysaccharide (LPS, bacterial challenge) or phorbol myristate acetate + ionomycin (PMAi) stimulation). Feeding dams a DHA diet significantly increased the mammary gland milk phospholipid concentration of DHA and ARA. This resulted in 60% higher DHA levels in splenocyte phospholipids of the pups although ARA levels showed no difference. In dams fed DHA diet, significantly higher proportion of CD27+ cytotoxic T cell (CTL) and CXCR3+ CCR6- Th (enriched in Th1) were observed than control, but there were no differences in the splenocyte function upon PMAi (non-specific lymphocyte stimulant) stimulation. Pups from DHA-fed dams showed significantly higher IL-1β, IFN-γ and TNF-α (inflammatory cytokines) by LPS-stimulated splenocytes. This may be due to higher proportion of CD86+ macrophages and B cells (all p's < 0.05) in these pups, which may influence T cell polarization. Plant-based source of DHA in maternal diet resulted in higher ex vivo production of inflammatory cytokines by splenocytes due to change in their phenotype, and this can skew T cell towards Th1 response in a Th2-dominant BALB/c mouse.

Effect of High-Fat and Low-Fat Dairy Products on Cardiometabolic Risk Factors and Immune Function in a Low Birthweight Swine Model of Diet-Induced Insulin Resistance.

Although dairy intake has been shown to have a neutral or some beneficial effect on major cardiometabolic risk factors, the impact of dairy, and especially dairy fat, on immune function remains to be investigated. To understand the effect of consuming dairy fat on cardiometabolic risk factors and immune function, we used an established low birthweight (LBW) swine model of diet-induced insulin resistance to compare high-fat and low-fat dairy products to a control high-fat diet (CHF). LBW piglets were randomized to consume one of the 3 experimental HF diets: (1) CHF, (2) CHF diet supplemented with 3 servings/day of high-fat dairy (HFDairy) and (3) CHF diet supplemented with 3 servings/day of low-fat dairy (LFDairy). As comparison groups, normal birthweight (NBW) piglets were fed a CHF (NBW-CHF) or standard pig grower diet (NBW-Chow). A total of 35 pigs completed the study and were fed for a total of 7 weeks, including 1 week of CHF transition diet. At 12 weeks of age, piglets were euthanized. Fasting blood and tissue samples were collected. Ex vivo cytokine production by peripheral blood mononuclear cells (PBMCs) stimulated with pokeweed (PWM), phytohemagglutinin (PHA) and phorbol myristate acetate-ionomycin (PMA-I) were assessed. As expected, LBW-CHF piglets showed early signs of insulin resistance (HOMA-IR, P model = 0.08). Feeding high-fat dairy products improved fasting plasma glucose concentrations more than low-fat dairy compared to LBW-CHF (P < 0.05). Irrespective of fat content, dairy consumption had neutral effect on fasting lipid profile. We have also observed lower production of IL-2 after PWM and PHA stimulation as well as lower production of TNF-α and IFN-γ after PWM stimulation in LBW-CHF than in NBW-Chow (all, P < 0.05), suggesting impaired T cell and antigen presenting cell function. While feeding high-fat dairy had minimal effect on immune function, feeding low-fat dairy significantly improved the production of IL-2, TNF-α and IFN-γ after PWM stimulation, IL-2 and IFN-γ after PHA stimulation as well as TNF-α after PMA-I stimulation compared to LBW-CHF (all, P < 0.05). These data provide novel insights into the role of dairy consumption in counteracting some obesity-related cardiometabolic and immune perturbations.

Supplementing Docosahexaenoic Acid and Arachidonic Acid During Suckling Period Is Beneficial for Developing Tolerance to Egg Protein in Allergy Prone Brown Norway Rats

ObjectivesWe aimed to study the effect of supplementing arachidonic acid (ARA) and docosahexaenoic acid (DHA) during suckling (through Suckling Diet, SD) and weaning (through weaning diet, WD) on oral tolerance (OT) development in allergy susceptible Brown Norway rats. MethodsDams were assigned to consume either ARA+DHA (0.4% ARA, 0.8% DHA w/w total fat) SD or control SD (0% ARA, DHA) for 21d during the pups’ suckling period. At 21d, pups from each SD group were randomized to receive ARA + DHA WD (0.5% ARA, 0.5% DHA w/w total fat) or control WD (0% ARA, DHA) until 8wk. Between 21–25 d, pups from each of the 4 diet groups were randomized to receive ovalbumin (Ova, 3 mg) or a placebo (8% sucrose) for OT induction. At 7 wk, pups received an Ova + alum injection IP to induce systemic immunization. In necropsy, plasma was collected and splenocytes were isolated to determine immune cell types and ex-vivo cytokine production to phorbol myristate acetate + ionomycin (PMAi). ResultsAlthough higher DHA levels in dams’ breastmilk were seen with ARA+DHA SD, this SD effect was not seen in the 8wk pups. At 8wks, ARA+DHA WD resulted in significantly higher DHA level in (1.5x) plasma phospholipids (PL) and (2x) splenocytes. ARA composition in pups’ plasma and splenocytes PL was not different among SD and WD groups. OT development was confirmed by a significantly lower plasma level of Ova-IgG1 and Ova-IgE in Ova OT groups. Further, SD ARA+DHA supplementation also had 30% lower plasma Ova-IgG1 than control SD (P = 0.02) but the SD had no effect on production of IL2, IL4, IL10, IFNg, TNFα and IL13 by PMAi stimulated splenocytes. However, pups from Ova group that received ARA+DHA SD and control WD produced more TGFβ than control for SD and WD (P = 0.05). Pups from sucrose OT groups, when supplemented with ARA+DHA SD had 50% higher T regulatory cells (CD3+CD4+CD25+FoxP3+, Tregs) than controls SD in spleen. In pups that received control SD groups, the Ova group had lower activated T helper (CD3+CD4+CD28+) than sucrose, however, when pups received ARA+DHA SD, OT effect was absent. ConclusionsARA and DHA in WD increased DHA status without affecting ARA status in plasma. ARA and DHA provided in SD was beneficial for OT development shown by lowered ova-IgG1, which may be due to higher Tregs and TGFβ produced by splenocytes in brown Norway offspring. Funding SourcesNSERC, AGES-ALES (Patel).

Splice-site variant in ACSL5: a marker promoting opposing effect on cell viability and protein expression

Long-chain Acyl-CoA synthetases (ACSLs) activate fatty acids (FAs) by thioesterification with Coenzyme A (CoA), generating FA-CoAs. These products are essential for lipid metabolism and carcinogenesis. In previous study, we identified an intronic variant rs2256368:A>G, whose G allele promotes exon 20 skipping in up to 43% of ACSL5 transcripts but its functional relevance is unclear. Here, we compared the expression of splice (Spl) and nonsplice (NSpl) ACSL5 variants and the effect on cell viability under culture conditions that force cells to metabolize fatty acids. We found that lymphoblastoid cell lines from 1000 Genomes Project, bearing Spl genotypes, showed a reduced expression of total ACSL5 protein due to an inefficient translation of the Spl RNA. These cells impaired growth in cultures with phorbol myristate acetate-ionomycin (PMA-Io) or medium deprived of glucose, while production of reactive oxygen species increased in PMA-Io. Specific ACSL5-isoform transfection in HEK239T (kidney), U87 (astroglioma), and HOG (oligodendrocyte) cells showed the Spl protein to be the causal factor of cell-growth inhibition, despite its reduced protein expression. Our findings indicate that the variant rs2256368:A>G can predict a growth inhibitory activity, caused by the Spl isoform of ACSL5 protein, opposed to the activity of the NSpl. Deep understanding of its functioning might have application in metabolic diseases and cancer.

PMA/IONO affects diffuse large B-cell lymphoma cell growth through upregulation of A20 expression.

Diffuse large B-cell lymphoma (DLBCL) is a common non-Hodgkin lymphoma. A20 and mucosa-associated lymphoid tissue lymphoma translocation gene1 (MALT1) are known to be related to DLBCL pathogenesis and progression. This study aimed to assess the effects of phorbol myristate acetate/ionomycin (PMA/IONO) on the growth and apoptosis of the DLBCL cell line OCI-LY1, and their associations with A20, MALT1 and survivin levels. Cell viability was assessed by MTT assay. Cell cycle distribution and apoptosis were evaluated using flow cytometry after incubation with AnnexinV-FITC/propidium iodide (PI) and RNase/PI, respectively. Gene and protein expression levels were determined by quantitative real-time PCR and western blotting, respectively. To further determine the role of A20, this gene was silenced in the OCI-LY1 cell line by specific siRNA transfection. A20 protein levels were higher in the OCI-LY1 cells treated with PMA/IONO compared with the controls, and were positively correlated with the concentration and treatment time of IONO, but not with changes of PMA and MALT1. Meanwhile, survivin expression was reduced in the OCI-LY1 cells after PMA/IONO treatment. In addition, OCI-LY1 proliferation was markedly inhibited, with a negative correlation between cell viability and IONO concentration. In concordance, apoptosis rates were higher in the OCI-LY1 cells after PMA+IONO treatment. Cell cycle distribution differed between the OCI-LY1 cells with and without PMA/IONO treatment only at 24h, with increased cells in the G0/G1 stage after PMA/IONO treatment. These findings indicate that PMA/IONO promotes the apoptosis and inhibits the growth of DLBCL cells, in association with A20 upregulation. Thus, A20 may be a potential therapeutic target for DLBCL.

SAT0371 Ustekinumab Treatment in Ankylosing Spondylits (AS) Patients Does not Modulate Effector CD4+ T Cell Frequencies Including TH17 Cells

BackgroundUstekinumab blocks the action of IL-12 and IL-23 by interfering with binding of the p40 subunit to the IL-12Rβ1 subunit used by IL-12 and IL-23 receptors. IL-12 and IL-23 are...

Evaluation of bovine-derived lacteal complex supplementation on gene expression in BALB/c mice

Evaluation of bovine-derived lacteal complex supplementation on gene expression in BALB/c mice Mario Clerici1,2, Emmanuel Pauze3, Arienne de Jong3, Mara Biasin1, Larry E Miller41Department of Biomedical Sciences and Technology, University of Milan, Milan, Italy; 2Don C Gnocchi Foundation, IRCCS, Milan, Italy; 3Sprim Advanced Life Sciences, Milan, Italy; 4Sprim USA, San Francisco, CA, USAAbstract: We conducted an evaluation of gene expression associated with innate and adaptive immunity in a double-blind ex vivo mouse study using a bovine-derived dietary ingredient (Ai/E10&reg;, Health Technology Resources, Inc., Scottsdale, AZ, USA). BALB/c female mice (5&ndash;6 weeks of age) were fed chewy granola bars supplemented with (Test) or without (Control) Ai/E10 for 10 days. After the feeding period, the animals were sacrificed and spleen cells were isolated and incubated with lipopolysaccharide and phorbol myristate acetate-ionomycin. RNA was extracted and mRNA expression of 84 genes involved in innate and acquired immunity was determined with real-time PCR arrays. Numerous genes associated with innate and adaptive immunity were upregulated in the Test group when stimulated with mitogens. Significant upregulation was observed in 30% (25 of 84) of genes upon lipopolysaccharide stimulation and in 14% (12 of 84) of genes upon phorbol myristate acetate + ionomycin stimulation in the Test group relative to Controls. This study illustrates that Ai/E10 supplementation results in significant and specific upregulation of genes associated with innate and adaptive immunity in mice. Notably, this effect was observed only in stimulated cultures.Keywords: dietary supplementation, immunomodulation, mice

Cytokine Imbalance after Measles Virus Infection Has No Correlation with Immune Suppression

Measles virus infection leads to immune suppression. A potential mechanism is the reduction of interleukin 12 (IL-12) secretion during acute measles, resulting in a TH2 response. Studies in humans have reported conflicting results, detecting either a TH2 or a TH1 response. We have investigated the correlation between a TH2 response and immune suppression in specific-pathogen-free inbred cotton rats which were infected with measles vaccine and wild-type viruses. After infection of bone marrow-derived macrophages with wild-type virus, IL-12 secretion was reduced in contrast to the level for vaccine virus infection. In bronchoalveolar lavage cells, IL-12 secretion was suppressed after infection with both wild-type and vaccine virus on days 2, 4, and 6 and was detectable on days 8 and 10. After stimulation of mediastinal lymph node and spleen cells with UV-inactivated measles virus at various time points after infection, gamma interferon but no IL-4 was found. After stimulation with phorbol myristate acetate-ionomycin, high gamma interferon and low IL-4 levels were detected. To investigate whether the secretion of IL-4 contributes to immune suppression, a recombinant vaccine virus was created which secretes cotton rat IL-4. After infection with this recombinant virus, IL-4 secretion was enhanced. However, neither inhibition of concanavalin A-stimulated spleen cells nor keyhole limpet hemocyanin-specific proliferation of spleen cells was altered after infection with the recombinant virus in comparison to the levels with the parental virus. Our data indicate that measles virus infection leads to a decrease in IL-12 secretion and an increase in IL-4 secretion, but this does not seem to correlate with immune suppression.

Alteration of peripheral blood T-reg cells and cytokines production in angiography personnel exposed to scattered X-rays.

Angiocardiography is an X-ray examination of the blood vessels or chambers of the heart. Cardiologists and staff members applying this procedure are exposed to high levels of scattered radiation. In our previous study the incidence of unstable chromosomal aberrations and cytokinesis-blocked micronuclei were found to be significantly higher in exposed individuals than the age and sex matched controls. In the present study we assessed cytokine production by peripheral blood mononuclear cells of the above cases and the percentage of Treg cells. According to film dosimeter analysis, personnels received 0.25-15 mSv during the previous year (average of 3 mSv/y). Isolated PBMCs from the test and control groups were stimulated with Phorbol Myristate Acetate/ Ionomycin (PMA/I). Cytokine production was measured in the supernatants of cultured lymphocytes. The percentage of Treg cells was studied by flow cytometry. The production of IL-10 and IL-5 was significantly down-regulated in the test group compared to the control group. In contrast, IL-12 was up-regulated. Yet, no statistically significant difference was found for IFN- gamma between two groups. In addition, we found higher percentage of CD4+CD25+(bright) Treg cells in the study group compared to the controls. Taken together, it was shown that low doses of scattered X-rays could skew cytokine profile of peripheral blood mononuclear cells in favour of inflammatory response causing the increase of Treg cells.

Cytokine dysregulation in activated cystic fibrosis (CF) peripheral lymphocytes.

Recent studies demonstrate in vivo and in vitro cytokine dysregulation in CF epithelial cells. To see if these abnormalities may be generalized to other cells expressing cystic fibrosis transmembrane conductance regulator (CFTR) but not directly exposed to local inflammation, we studied mRNA transcription, intracellular protein production and extracellular secretion of IL-2, IL-4, IL-5, IL-10 and interferon-gamma (IFN-gamma) from freshly isolated blood mononuclear and CD4+ T cells from CF patients and controls. Cells were activated by phorbol myristate acetate (PMA) and anti-CD3, PMA-ionomycin, or lipopolysaccharide (LPS) and assessed for cytokine mRNA transcription by semiquantitative reverse transcriptase-polymerase chain reaction, intracellular protein production by flow cytometry, and secretion by supernatant ELISA. Cytokine expression was highly stimulus-dependent. CF cells showed higher IL-10 transcription than control cells after maximal activation by LPS (P = 0.01); despite this, cytokine production and secretion were equivalent to controls. CF cells showed lower cellular IL-10 production after PMA-anti-CD3 activation (P = 0.002). CF cells secreted less IFN-gamma than control cells after maximal activation by PMA-anti-CD3 (1836 +/- 273 pg/ml versus 9635 +/- 3437 pg/ml, P = 0.04). IL-2, IL-4 and IL-5 regulation was similar to controls. We conclude that CF mononuclear cells show selective cytokine dysregulation after maximal activation, namely reduced IFN-gamma secretion and increased IL-10 mRNA without increased production or secretion. These findings extend defects described in respiratory epithelial cells to circulating immunoregulatory cells, suggesting a link between CF genotype and cytokine dysregulation.

Evaluation of the modified ELISPOT assay for gamma interferon production in cancer patients receiving antitumor vaccines.

Frequencies of vaccine-responsive T-lymphocyte precursors in peripheral blood mononuclear cells (PBMC) prior to and after administration of peptide-based vaccines in patients with cancer can be measured by limiting-dilution assays (LDA) or by ELISPOT assays. We have used a modified version of the ELISPOT assay to monitor changes in the frequency of gamma interferon (IFN-gamma)-producing T cells in a population of lymphocytes responding to a relevant peptide or a nonspecific stimulator, such as phorbol myristate acetate-ionomycin. Prior to its use for monitoring of patient samples, the assay was validated and found to be comparable to the LDA performed in parallel, using tumor-reactive cytolytic T-lymphocyte (CTL) lines. The sensitivity of the ELISPOT assay was found to be 1/100,000 cells, with an interassay coefficient of variation of 15%, indicating that it could be reliably used for monitoring of changes in the frequency of IFN-gamma-secreting responder cells in noncultured or cultured lymphocyte populations. To establish that the assay is able to detect the T-cell precursor cells responsive to the vaccine, we used CD8(+) T-cell populations positively selected from PBMC of HLA-A2(+) patients with metastatic melanoma, who were treated with dendritic cell-based vaccines containing gp100, MELAN-A/MART-1, tyrosinase, and influenza virus matrix peptides. The frequency of peptide-specific responder T cells ranged from 0 to 1/2,600 before vaccination and increased by at least 1 log unit after vaccination in two patients, one of whom had a clinical response to the vaccine. However, no increases in the frequency of peptide-responsive T cells were observed in noncultured PBMC or PBMC cultured in the presence of the relevant peptides after the melanoma patients enrolled in another trial were treated with the intramuscular peptide vaccine plus MF59 adjuvant. Thus, while the ELISPOT assay was found to be readily applicable to assessments of frequencies of CTL precursors of established CTL lines and ex vivo-amplified PBMC, its usefulness for monitoring of fresh PBMC in patients with cancer was limited. In many of these patients antitumor effector T cells are present at frequencies of lower than 1/100,000 in the peripheral circulation. Serial monitoring of such patients may require prior ex vivo amplification of specific precursor cells.

Fas ligand-mediated lysis of self bystander targets by human papillomavirus-specific CD8+ cytotoxic T lymphocytes.

Mouse cytotoxic T lymphocytes (CTL) reactive with a H-2Db-presented 9-mer peptide of the human papillomavirus type 16 protein E7(49-57) (RAHYNIVTF) were generated from the spleen cells of wild-type C57BL/6 (B6) or B6 perforin-deficient (B6.P0) mice. CD8(+) B6 CTL displayed peptide-specific perforin- and Fas-mediated lysis of E7-transfected mouse RMA lymphoma cells (RMA-E7), while CD8(+) CTL from B6.P0 mice lysed RMA-E7 cells via Fas ligand (FasL) exclusively. Rapid and efficient lysis of syngeneic bystander B6 blasts or RMA cells by either B6 or B6.P0 Ag-activated CTL was mediated by a FasL-Fas mechanism. Fas-resistant bystanders were not lysed, nor were allogeneic Fas-sensitive C3H/HeJ (H-2(k)) or BALB/c (H-2(d)) bystander blasts. Interestingly, however, phorbol myristate acetate-ionomycin preactivation of B6.P0 effectors enabled lysis of allogeneic H-2(k) and H-2(d) bystanders even in the absence of antigenic stimulation. Lysis of syngeneic bystander cells was always FasL-Fas dependent and required effector-bystander contact and, in particular, an interaction between CTL LFA-1 and bystander ICAM-1. Thus, in the context of major histocompatibility complex class I molecule-peptide ligation of the T-cell receptors of CD8(+) CTL, neighboring bystander cells that are syngeneic and Fas sensitive and express the adhesion molecule ICAM-1 are potential targets of CTL attack.