Peripheral Blood Mononuclear Cell Suspensions Research Articles

P234 The effect of calcitriol on the expression of interferon signature genes in dendritic cells and macrophages in systemic lupus erythematosus

Abstract Background/Aims A number of studies have demonstrated the inverse association between vitamin D and (i) disease activity and (ii) interferon (IFN) signature gene expression in systemic lupus erythematosus (SLE). The aim of the study was to establish the effect of calcitriol supplementation to cultured macrophages and dendritic cells (DC) from a SLE patient and control, on the expression of 12 IFN signature genes, interferon regulatory factor (IRF)7 and IRF8. Methods Blood samples were collected from a 41-year-old female SLE patient who fulfilled the SLICC classification criteria for SLE and from a 36-year-old healthy female control after obtaining informed consent. Both patients had a serum 25-hydroxyvitamin D level of 25ng/ml. Peripheral blood mononuclear cells (PBMC) were isolated by using histopaque®-1077. CD83 and CD14 expression was measured by flow cytometry and cell count and viability were assessed. The PBMC suspension was cultured in 6-well plates using: (i) ImmunoCultTM DC culture kit (following kit protocol) for DC differentiation, (ii) culture medium for macrophage differentiation containing RPMI 1640 Medium, DMEM, fetal bovine serum and LPS. Following 7 days of incubation, differentiation of cells was confirmed using microscopy and flow cytometry to measure CD83 and CD14 expression. Calcitriol at a concentration of 20nM was added to half of the wells of each sample category. Following 24 and 48 hours, RNA extraction was carried out on treated and untreated samples. QuantiGene Plex technology was used to measure the expression of 12 IFN signature genes, IRF7, IRF8 and four housekeeping genes in the extracted RNA. The normalised median florescence intensity for the 12 IFN signature genes was used to calculate the IFN score. Results A significantly lower IFN score was noted at 24 hours in SLE patient dendritic cell and macrophage cell cultures and in control macrophage cell cultures treated with calcitriol when compared with respective untreated samples (p = 0.014, p = 0.034, p = 0.031 respectively). On comparing IRF8 expression in treated and untreated cell cultures, a significantly lower IRF8 expression was noted in treated macrophage cell cultures obtained from both SLE patient and control (p = 0.034, p = 0.041). No difference in IRF7 expression was noted in treated and untreated cultures for all samples. Conclusion The suppression of the interferon signature genes in dendritic cell and macrophage cell cultures with vitamin D was confirmed in the in vitro experiment carried out. In this experiment a decrease in the expression of IRF8 (known to have a VDR binding site) was also observed in macrophage cultures from both SLE patient and control. Disclosure R. Magro: None. E. Seria: None. G. Grech: None. A.A. Borg: None. C. Scerri: None.

T Cell Immune Profiles of Blood and Tumor in Dogs Diagnosed With Malignant Melanoma.

Investigation of canine T cell immunophenotypes in canine melanomas as prognostic biomarkers for disease progression or predictive biomarkers for targeted immunotherapeutics remains in preliminary stages. We aimed to examine T cell phenotypes and function in peripheral blood mononuclear cells (PBMC) and baseline tumor samples by flow cytometry, and to compare patient (n = 11–20) T cell phenotypes with healthy controls dogs (n = 10–20). CD3, CD4, CD8, CD25, FoxP3, Ki67, granzyme B, and interferon-γ (IFN-γ) were used to classify T cell subsets in resting and mitogen stimulated PBMCs. In a separate patient cohort (n = 11), T cells were classified using CD3, CD4, CD8, FoxP3, and granzyme B in paired PBMC and single cell suspensions of tumor samples. Analysis of flow cytometric data of individual T cell phenotypes in PBMC revealed specific T cell phenotypes including FoxP3+ and CD25+FoxP3- populations that distinguished patients from healthy controls. Frequencies of IFN-γ+ cells after ConA stimulation identified two different patient phenotypic responses, including a normal/exaggerated IFN-γ response and a lower response suggesting dysfunction. Principle component analysis of selected T cell immunophenotypes also distinguished patients and controls for T cell phenotype and revealed a clustering of patients based on metastasis detected at diagnosis. Findings supported the overall hypothesis that canine melanoma patients display a T cell immunophenotype profile that is unique from healthy pet dogs and will guide future studies designed with larger patient cohorts necessary to further characterize prognostic T cell immunophenotypes.

Liquid biopsy of the immune environment: Evaluation of peripheral blood mononuclear cells (PBMCs) with CyTOF and response to trastuzumab (T)-based neoadjuvant chemotherapy (NAC) in HER2+ breast cancer (BC).

592 Background: Immune responses in the tumor microenvironment have prognostic and predictive value in BC. However, the potential of immune responses observed in peripheral blood as biomarkers in BC remains unclear. We have shown that a higher frequency of circulating monocytes and a lower frequency of antigen-experienced memory CD8+ T cells are associated with response to NAC in triple negative BC (Leon-Ferre et al SABCS 2019). Here, we used cytometry by time-of-flight (CyTOF) to evaluate associations between circulating immune cells, clinical features and response to T-based NAC in HER2+ BC. Methods: PBMC suspensions from 36 pts with stage I-III HER2+ BC were prospectively collected prior to initiation of T-based NAC, stained with 29 metal-tagged antibodies optimized to identify major human immune cell subsets, and acquired in the Helios CyTOF instrument. Differential abundance analysis of immune cells by clinical characteristics and by NAC response was evaluated using Wilcoxon rank sum test. % of immune cell subsets is presented as % of all PBMCs. Results: Most pts presented with ER- tumors (56%), measuring > 5cm (64%) and with nodal metastases (78%). After NAC, 16 pts (44%) achieved pathologic complete response (pCR). Analysis of preNAC PBMCs demonstrated a significantly higher number of B cells (8% vs 5%, p = 0.05) and effector memory CD8+ T cells (CD45RA-/CCR7-, 3 vs 1%, p = 0.02) in pts with pCR compared to those with residual disease. Of the B cell subsets, naïve B cells (CD24-/CD27-) were higher in pts who achieved pCR vs not (7% vs 4%, 0 = 0.04). Regarding clinical characteristics, cN+ pts at presentation exhibited a lower number of peripheral blood T cells compared to cN- pts (47% vs 63%, p = 0.03). Of the T cell subsets, overall CD4+ and naïve CD4+ T cells (CD45RA+/CCR7+) were lower in cN+ vs cN- pts (31% vs 45%, p = 0.05; and 11% vs 24%, p = 0.04). We also observed differences in CD56+/CD16- NK cells by ER status (ER- 1% vs ER+ 3%, p = 0.01), and a moderate negative correlation between age and % circulating CD8+ T cells (rho -0.4669, p = 0.004). Conclusions: Distinct peripheral blood immune cell profiles are observed in HER2+ BC at diagnosis, and are associated with response to T-based NAC and initial clinical characteristics. Notably, pts who later achieved pCR had a relative abundance of B cells and effector memory CD8+ T cells at diagnosis. These data suggest that immune cell phenotyping in peripheral blood may have potential as a biomarker to predict response to NAC in BC.

In vitro assessment of tick-borne encephalitis vaccine: Suitable human cell platforms and potential biomarkers.

Tick-borne encephalitis (TBE) virus causes a severe disease that can lead to permanent neurological complications. The whole inactivated TBE vaccine is highly effective, as proven by high seroconversion rates and near eradication of the disease in countries where vaccination programs have been implemented. TBE vaccine potency testing currently requires the use of in vivo methods that present issues of reproducibility as well as animal discomfort. As an alternative, public and private entities are currently exploring a batch-to-batch consistency approach that would demonstrate conformity of a newly produced vaccine batch with a batch of proven in vivo efficacy with respect to a range of measurable in vitro quality parameters. To identify a suitable cellular platform to be used in a panel of in vitro batch-to-batch assessments for the TBE vaccine, we exposed human cell-based systems, both of primary origin and cell line-derived, to vaccine formulations of high and low quality. Following stimulation, cell responses were evaluated by assessing the expression of selected genes by RT-qPCR. Our findings show that the expression of interferon-stimulated genes differed after treatment with non-adjuvanted vaccine batches of different quality in peripheral blood mononuclear cells (PBMCs) and in monocyte-derived dendritic cells, but not in monocyte-free PBMC suspensions nor in cell line-derived immune cells. These results indicate suitable platforms and potential biomarkers for a cell-based assay that, together with other immu­nochemical analyses, could serve for batch-to-batch assessment of the TBE vaccine, reducing, and eventually replacing, in vivo methods for potency testing.

In vitro assessment of tick-borne encephalitis vaccine: Suitable human cell platforms and potential biomarkers_suppl

Tick-borne encephalitis (TBE) virus causes a severe disease that can lead to permanent neurological complications. The whole inactivated TBE vaccine is highly effective, as proven by high seroconversion rates and near eradication of the disease in countries where vaccination programs have been implemented. TBE vaccine potency testing currently requires the use of in vivo methods that present issues of reproducibility as well as animal discomfort. As an alternative, public and private entities are currently exploring a batch-to-batch consistency approach which would demonstrate conformity of a newly produced vaccine batch with a batch with proven in vivo efficacy with respect to a range of in vitro measurable quality parameters. For the identification of a suitable cellular platform to be used in a panel of in vitro batch-to-batch assessments for the TBE vaccine, we exposed human cell-based systems, both of primary origin and cell line-derived, to vaccine formulations of high and low quality. Following stimulation, cell responses were evaluated by assessing the expression of selected genes by qPCR. Our findings show that the expression of interferon-stimulated genes differed after treatment with non-adjuvanted vaccine batches of different quality in peripheral blood mononuclear cells (PBMCs) and in monocyte-derived dendritic cells, but not in monocyte-free PBMC suspensions nor in cell line-derived immune cells. These results indicate suitable platforms and potential biomarkers for a cell-based assay that, together with other immunochemical analyses, could serve for batch-to-batch assessment of the TBE vaccine, reducing (and eventually replacing) in vivo methods for potency testing.

Abstract B157: Modulation of natural killer cell dysfunction in human bladder cancer

Abstract T-cells undergo a progressive program of dysfunction termed “exhaustion” during chronic inflammatory pathologies such as cancer. T-cell exhaustion is characterized by diminished effector functionality and inefficient tumor immunosurveillance. However, it is unknown whether an analogous program undermines the contribution of innate lymphocytes like natural killer cells (NK) to tumor-surveillance, and what phenotype might define it. Therefore, we examined expression of inhibitory receptors and the corresponding functional potential of NK and T-cells present in peripheral blood mononuclear cells (PBMC) and tumor tissue of 59 individuals representative of the clinical spectrum of human bladder cancer (BC). PBMC and cell suspensions from freshly dissociated primary tumors were analyzed by flow cytometry to measure changes in the expression of the inhibitory receptors Tim-3, TIGIT, and PD-1, and to determine the composition of hematopoietic lineages in each tissue. NK effector function was assessed via activation with IL-2 or IL-15, followed by co-culture with Class I HLA-deficient targeT-cells to measure cytokine production and degranulation. Additionally, we determined the effect on NK cell function of ex vivo blockade of Tim-3 and TIGIT by the addition of monoclonal antibodies prior to functional assays. We found that NK cells significantly up-regulate expression of Tim-3 and TIGIT, but not PD-1, in both the PBMC and tumors of BC patients. T-cells demonstrate a similar pattern of expression. but with a far lower frequency of positive cells. The magnitude of Tim-3 expression by NK cells is a barometer of tumor invasiveness on cells in both PBMC and tumor tissue, while TIGIT is induced equivalently in BC patients independently of tumor stage. Importantly, both molecules are expressed at similar frequencies on NK cells isolated from blood or tumor, independent of the magnitude of overall expression, yet define NK with different functional potential. NK cells in PBMC from BC patients are functionally comparable to NK cells from healthy donors in their ability to produce IFNγ/TNFα and degranulate in response to targeT-cells, while tumor NK are refractory to both stimuli. NK cells from tumor tissue are not terminally exhausted as effector functions are restored after “resting” ex vivo prior to stimulation. Ex vivo blockade of Tim-3, but not TIGIT, enhances effector function in peripheral NK and T-cells from BC patients, but is ineffective for NK cells in tumor tissue, implicating suppressive factors specific to tumor in mediating NK dysfunction. Tim-3 blockade was most efficient in peripheral NK cells from BC patients that were activated with IL-15 versus IL-2, suggesting that local cytokine milieu can affect responsiveness to subsequent checkpoint inhibition. Overall, our data suggest that both NK and T-cells from patients with BC are enriched for expression of the shared inhibitory receptors Tim-3 and TIGIT, and that expression in the peripheral blood mirrors expression in the tumor. However, tissue-specific cues present in bladder tumors, but not in the periphery, ultimately confer effector dysfunction. Furthermore, because blockade of Tim-3 enhances IFNγ and TNFα production by NK cells present in the peripheral blood of BC patients, it may represent a new strategy to modulate innate, antitumor immunity while simultaneously conferring benefit in the adaptive compartment. Citation Format: Adam M. Farkas, Francois Audenet, Harry Anastos, Matthew Galsky, John P. Sfakianos, Nina Bhardwaj. Modulation of natural killer cell dysfunction in human bladder cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B157.

Dielectric properties of plasma membrane: A signature for dyslipidemia in diabetes mellitus

Dielectric properties of a living biological membrane play crucial role indicating the status of the cell in pathogenic or healthy condition. A distinct variation in membrane capacitance and impedance was observed for peripheral blood mononuclear cell (PBMC) suspensions for diabetic and diabetic-dyslipidemic subjects compared to healthy control. Low frequency region were explicitly considered in electrical analysis to address complex membrane dielectric factors that alter the system capacitance of a PBMC suspension. Such variation was marked in size, morphology and membrane function of PBMCs for control and diseased cases. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) studies reveal significant alteration in surface morphology of PBMCs in diseased condition. Side scatter of flow cytometry reveals complexity of PBMCs in diseased condition. Changes in size between groups were not found by SEM and forward scatter. Functional alteration in PBMCs was manifested by significant changes in cell membrane properties like Na+, K+ ATPase and Ca2+, Mg2+ ATPase activity, reduced plasma membrane fluidity and changes in intracellular Ca2+ content, which bear significant correlation in diabetic and diabetic dyslipidemic subjects. Therefore, dielectric parameters of PBMCs in diabetic-dyslipidemic challenges may led to interesting correlation opening the possibility of identifying crucial signature biomarkers.

Mutated Regions of Nucleophosmin 1 Elicit CD8+ T-Cell Responses in Patients with Acute Myeloid Leukemia

ObjectiveStimulating lymphocytes from healthy donors by DCs loaded with NPM1mut peptides in vitro in order to induce specific cellular immune responses against the peptides. And detecting the NPM1mut peptide specific T-cell in peripheral blood of patients with NPM1mut positive acute myeloid leukemia (AML), to provide a theoretical basis for immune therapy of AML.Methods1. NPM1 mutation detection: newly diagnosed patients of AML underwent gene mutations screening routinely.2. Samples collection: peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood collected from healthy donors with HLA-A* 0201 or A*1101 and NPM1mut positive AML patients in complete remission.3. Inducing differentiation of PBMCs into DCs: non-adherent cells were removed from PBMCs suspension, and then adherent cells were subsequently cultured for 5 days with GM-CSF and IL-4 to induce differentiation. Then cells were cultured in medium containing GM-CSF, IL-4, IL-6, IL-1β, TNF-α, and PGE2 for another 2 days to induce maturation.4. Generation of NPM1-specific cytotoxic T cells: DCs were pulsed with different NPM1 peptides followed by irradiation with 40 GY. The non-peptide pulsed DCs group was set as negative control. The above mentioned DCs were then co-incubated with their own PBMCs, maintained by IL-2 and IL-7. On day 7 of co-incubation, the same treated DCs as mentioned above were added again to restimulate lymphocytes.5. ELISPOT analysis and intracellular staining: peptide-stimulated T cells were tested by ELISPOT and intracellular staining method on day 7 and 14 . These two methods both detected the secretion of IFN-γ of cytotoxic T cells. T2 cells were used as target cells, and in terms of HLA-A*1101 positive donors, their own DCs were used as targets.Results1. In the Han Chinese population, the most common alleles of HLA-A loci were A* 1101, A*2402, and A*0201 allele.2. Through prediction of the aforementioned software, HLA-A*0201 restricted wild type NPM1 amino acid sequences (DLWQWRKSL), mutated NPM1-A/D amino acid sequences (AIQDLCLAV), and HLA-A*1101 restricted mutated NPM1-A/D amino acid sequences (AVEEVSLRK) were synthesized. There were no proper epitopes for either HLA-A*1101 restricted wild type NPM1 protein or HLA-A*2402 restricted wild type and mutated NPM1 protein.3. Expression of surface antigens of DCs on day 7 were as follows: CD14(2.6%), CD80(43.4%), CD83(7.8%), CD86(99.9%), HLA-DR(67.1%), which was consistent with DCs phenotype.4. About 10 days after DCs were co-incubated with their own PBMCs, the number of lymphocytes increased significantly, especially in the NPM1mut peptide pulsed group. On day 7 of co-incubation, ELISPOT analysis results of all samples were negative. However, 3 cases’ ELISPOT results were positive for the mutated peptide holes on day 14, in contrast, the results of the wild type peptide holes were negative. The NPM1mut peptide-specific T lymphocytes positive rate [=(average number of spots in mutated peptide holes-the average number of spots in negative control holes)/total lymphocytes per hole] was about 1/2500. Intracellular staining showed that in the aforementioned 3 cases, the proportion of CD8+IFN-γ+cells of the mutant peptide group was higher than either the wild type peptide group or negative control group, but there was no difference between the later two groups.5. 6 peripheral blood samples of patients with NPM1mut +AML were performed ELISPOT analysis, with only 1 case (16.7%) showing a approaching positive result.ConclusionsNPM1mut peptide pulsed DCs can stimulate their own PBMCs from healthy donors in vitro to produce mutated NPM1 specific T lymphocytes, and it is expected to be used as immune therapy of AML. The mutated NPM1 specific CTL in NPM1mut+AML patients are almost undetectable that indicates immune system have been comprised because of chemotherapy and disease, and can not responses to antigens efficiently. Perhaps AML patients can accept CTL transfusion from their relatives. Our study provide an experimental basis for cellular immunotherapy of AML. DisclosuresNo relevant conflicts of interest to declare.

Synergistic protein secretion by mesenchymal stromal cells seeded in 3D scaffolds and circulating leukocytes in physiological flow

Mesenchymal stromal cells (MSC) play an important role in natural wound healing via paracrine and juxtacrine signaling to immune cells. The aim of this study was to identify the signaling factors secreted by preseeded cells in a biomaterial and their interaction with circulating leukocytes, in the presence of physiological biomechanical stimuli exerted by the hemodynamic environment (i.e. strain and shear flow). Electrospun poly(ε-caprolactone)-based scaffolds were seeded with human peripheral blood mononuclear cells (PBMC) or MSC. Protein secretion was analyzed under static conditions and cyclic strain. Subsequently, the cross-talk between preseeded cells and circulating leukocytes was addressed by exposing the scaffolds to a suspension of PBMC in static transwells and in pulsatile flow. Our results revealed that PBMC exposed to the scaffold consistently secreted a cocktail of immunomodulatory proteins under all conditions tested. Preseeded MSC, on the other hand, secreted the trophic factors MCP-1, VEGF and bFGF. Furthermore, we observed a synergistic upregulation of CXCL12 gene expression and a synergistic increase in bFGF protein production by preseeded MSC exposed to PBMC in pulsatile flow. These findings identify CXCL12 and bFGF as valuable targets for the development of safe and effective acellular instructive grafts for application in in situ cardiovascular regenerative therapies.

Cryopreservation of human monocytes for pharmacopeial monocyte activation test

EU Directive 2010/63/EU regarding the protection of experimental animals came into force in November 2010 with an obligation for EU member states to incorporate its requirements into their respective national legislations by 1st of January 2013. The directive stipulates the application of in vitro methods to replace animal experiments whenever such an in vitro method exits and is recognized by EU legislation. The monocyte activation test (MAT) for the detection and quantification of pyrogenic contamination in medicines is recognized by the European Directorate for the Quality of Medicines & Health Care (EDQM) and was published in the European Pharmacopeia (Pharm. Eur.) in April 2010. The methodology described here facilitates the use of the MAT by making monocytes available, in the form of cryopreserved human peripheral blood mononuclear cells (PBMCs). We have developed and qualified a procedure to prepare functional monocytes in the form of PBMCs from the leukocyte filters that are used for the separation of blood in blood donation centers. Once used, these filters are normally treated as biological waste. Here we describe the procedures that are critical for the successful cryopreservation of PBMCs, demonstrate protection of PBMC functionality using various ligands for the toll-like receptors (TLRs) that mediate pyrogenic responses, report validation of the methodology for linearity, precision and robustness and show examples of the practical application of cryopreserved in MATs with samples of drugs and vaccines. Another application of cryopreserved PBMCs, only mentioned here, is to serve as an alternative to freshly isolated PBMCs in tests for unwanted intrinsic pro-inflammatory activities of new biological therapeutics. Such tests use PBMCs or PBMCs over a layer of endothelial cells to detect (unwanted) cytokine release, PBMCs being more suited to this purpose than tests using whole blood.

Активатор ППАР-гама піоглітазон знижує експресію транскрипційного фактора NF-kB та апоптоз в мононуклеарних клітинах СD64+ крові in vitro

In order to examine the impact of PPAR-gamma activator on the expression of kB nuclear transcription factor and apoptosis, the peripheral blood mononuclear cells (PBMC) were incubated with pioglitazone at concentrations 10, 30 and 100 MM. Expression of NF-kB in CD64+ cells was evaluated by flow cytometry using monoclonal antibodies. The development of apoptosis was evaluated using annexin V and propidium iodide. Activation of PPAR-y resulted in a reduction of the number of CD64+ cells and expression of the maximum levels of these receptors. We also obesrved a dose-dependent decrease of NF-kB concentration in PBMC suspension and in CD64+ cells. While analyzing the apoptosis, the reduction of apoptotic cells (reduction of AnV+PI+ cells) has been observed. The results obtained allow to prove that PPAR-gamma activation participates in the PBMC apoptosis regulation and in the expression of transcriptional factor NF-kB. Our results indicate for the involvement of PPAR-gamma in the regulation of NF-kB activity which plays one of the most significant roles in the inflammation realization, oncogenesis, and so on.

33 IN VITRO IMMUNOGENICITY OF SOMATIC CELL NUCLEAR TRANSFER-DERIVED TRANSGENIC CLONED DOGS

Histocompatible tissue has been generated by somatic cell nuclear transfer (SCNT) and the resultant tissues were not rejected by the immune system of the nucleus donors. In addition, many transgenic animals combined with SCNT have been produced. However, in vitro immunogenicity of transgenic cloned animals originated from the same donor cell with nontransgenic cloned animals has not been assessed until now. The objective of this study was to evaluate the in vitro immunogenicity of cloned dogs with each other, between cloned dogs and transgenic cloned dogs and between transgenic cloned dogs with each other by mixed lymphocyte reaction. In this study, we used cloned beagles (BG1, 2) derived from SCNT using fetal fibroblasts (BF3). Serially, 4 transgenic cloned beagles (Ruppy 1–3, 5) were also genetically engineered from the same donor cell, BF3, with red fluorescent protein (RFP) gene inserted into their genome. We used 2 age-matched healthy female beagle dogs as control dogs. They have different 3 DLA types with all cloned dogs. Peripheral blood mononuclear cells (PBMC) of 2 cloned beagles and 4 transgenic cloned beagles were isolated from whole bloods using Ficoll gradient solution. PBMC from each dog were mixed to auto PBMC, other transgenic cloned dogs and non-related control dogs under the experimental designs. All the mixtures were incubated at 37°C for 4 days, adding BrdU labeling reagent and re-incubated for 24 h. Results are expressed in absorbance mean value ± standard deviation of 450-nm wavelength read by microplate reader. Each cell combination was assayed in 8 replicates. In Experiment 1, PBMC of cloned beagles were combined with equal concentrations of another cloned beagle's PBMC. In Experiment 2, PBMC suspension of Ruppy 1–3, 5 were mixed with equal concentrations of another transgenic cloned beagle's PBMC suspension. In Experiment 3, PBMC suspensions of cloned beagles were mixed with PBMC suspensions of transgenic cloned beagles and reverse reaction was performed. Statistical analysis was performed by using Mann-Whitney U test. In Experiment 1, whereas the absorbance value of mixture of cloned dogs and control dogs shows apparent proliferation, auto mixture of each dog and allo-mixture of BG1 and BG2 show no proliferation (Table 1), indicating immunological factors exposed to PBMC in 2 cloned dogs were compatible. In Experiment 2 among transgenic cloned dogs, no evidence of proliferations in mixed allo-PBMC was shown (Table 1), suggesting in vitro immunogenicity between transgenic cloned dogs was also not shown. In Experiment 3 among cloned dogs and transgenic cloned dogs, no significant difference was found (Table 1). In conclusion, cloned dogs derived from SCNT shared immunological phenotype. Next, immunogenicity among transgenic cloned beagle dogs was not shown despite random insertion of a foreign gene. Lastly, cloned beagles and transgenic cloned beagles show lymphocyte antigen compatibility irrespective of having a foreign gene or not. Table 1.The absorbance values of mixed lymphocytes of 4 transgenic cloned dogs and 2 cloned dogs This study was supported by RNL BIO (#0468-20110001), IPET, MKE (#10033839-2011-13) and Natural Balance Korea.

Improving the timeframe between blood collection and interferon gamma release assay using T-Cell Xtend ®

Vacutainer CPT tubes require blood samples for TSPOT.TB to be processed within 8 h. In this study we evaluated the ability of T-Cell Xtend to maintain the number and function of lymphocytes after 24 and 48 h of blood storage, giving similar test results as in freshly isolated specimens. Whole blood specimens from 59 individuals were collected in Vacutainer CPT tubes (CPT) and lithium heparin (LH) tubes. CPT tubes were processed within 8 h. T-Cell Xtend was added to LH tubes after 24 or 48 h. We also left LH tubes untreated for 48 h. Total number of white blood cells (WBC) and proportions of lymphocytes and granulocytes were determined in the isolated Peripheral Blood Mononuclear Cells (PBMC). We also evaluated the performance of T-Cell Xtend in the TSPOT.TB assay. PBMC yields from T-Cell Xtend treated LH samples did not differ from PBMC yields from CPT tubes, but T-Cell Xtend had a pronounced effect on the proportions of lymphocytes and granulocytes. The mean lymphocyte percentage in PBMCs isolated from fresh CPT blood was 84.31 ± 1.14% (at t = 48 h), but was decreased to 52.72 ± 3.34% (p < 0.05) in untreated LH blood (at t = 48 h). This effect was neutralized by T-Cell Xtend (85.44 ± 0.74%). We observed a similar but opposite effect on granulocytes: The mean proportion in untreated LH blood was increased to 40.9 ± 3.67% (p < 0.001) compared to CPT blood (8.26 ± 0.89%). Treatment of LH samples with T-Cell Xtend (48 h) restored the proportion of granulocytes to 8.47 ± 0.61%. Enumeration of spots in the TSPOT.TB assay demonstrated good agreement between CPT and T-Cell Xtend results, even after 48 h. T-Cell Xtend efficiently removes granulocytes from PBMC suspensions and increases the proportion of lymphocytes. TSPOT.TB results from T-Cell Xtend treated blood samples are at least comparable to the results obtained from the current CPT method. Use of standard lithium heparin blood combined with T-Cell Xtend allows up to 48 h storage of blood samples for batched processing and may further decrease the rate of indeterminate TSPOT.TB results.

Long-term effects of low doses of ionising radiation on Chernobyl clean-up workers from Latvia

Around 6000 inhabitants (20-49 years old in 1986) of Latvia took part in clean-up work in Chernobyl from 1986 till 1991. Most of them were officially documented as recipients of ionising radiation exposure (1-50 cGy). ABM (a 3-aminobenzanthrone derivative developed at the Riga Technical University, Riga, Latvia) has been previously shown to be a potential probe for determining the immune state of patients with different pathologies. The first study (using ABM) of Peripheral Blood Mononuclear Cell (PBMC) membranes of Chernobyl clean-up workers (n-97) from Latvia was conducted in 1996-1997. In 2006-2007 we examined the same (n = 54) individuals. Lipid peroxidation, ABM and ANS spectral parameters in PBMC suspension, fluorescence anisotropy and blood plasma albumin characteristics were recorded. In 1997 screening showed five different patterns of fluorescence spectra, but in 2007 we obtained only four. These patterns of spectra had never been seen previously in healthy individuals or patients with tuberculosis, multiple sclerosis, rheumatoid arthritis, etc., examined by us. The patterns of ABM fluorescence spectra were associated with membrane anisotropy and conformational changes of blood plasma albumin. It is necessary to note that all investigated parameters significantly differed in the observed groups of patients. These findings reinforce our understanding that the cell membrane is a significant biological target of radiation. These studies reveals a progressive trend towards certain resemblances to PBMC membranes of chronic B-cell lymphoid leukaemia and protein coformational alterations.

Fc receptor engagement mediates differentiation of cardiac fibroblast precursor cells

We previously described a critical role for a fibroblast precursor population in the development of a murine fibrotic cardiomyopathy model (I/RC). These precursors arose from circulating bone marrow-derived cells of monocytic origin. Administration of serum amyloid P (SAP) prevented the presence of this cell population in the heart and the cardiomyopathy. Because SAP binds to Fc receptors (FcRs) expressed on monocytes, we investigated the involvement of FcR signaling. We chose mice lacking the FcRgamma chain protein (FcRgamma(-/-)), a common membrane-signaling component of activating FcRs. Like wild-type mice, FcRgamma(-/-) mice developed fibrosis and cardiac dysfunction when subjected to I/RC. However, unlike wild-type mice, SAP in FcRgamma(-/-) mice failed to inhibit the development of fibrosis and cardiac dysfunction and did not diminish the numbers of alpha-smooth muscle actin(+) and CD34(+), CD45(+) fibroblasts that were typical for I/RC. To further examine the role of SAP in monocyte-to-fibroblast transition, we performed in vitro assays in which human peripheral blood mononuclear cells (PBMCs) migrated through human umbilical vein endothelial cells (HUVECs). We found that MCP-1-dependent transendothelial migration of monocytes markedly accelerated their differentiation into fibroblasts. This monocyte differentiation to fibroblasts was eliminated when SAP was added to the PBMC suspension before endothelial transmigration. Adding SAP to cells after successful migration did not inhibit fibroblast maturation. These data indicate that SAP inhibits the differentiation of a blood-borne, myeloid cell population into fibroblasts by signaling through activating FcRs before transendothelial migration has occurred. We suggest that FcR activation of circulating precursor cells may represent a new treatment target for adverse remodeling and cardiac fibrosis.

Anti-HIV Activity Mediated by Natural Killer and CD8+ Cells after Toll-Like Receptor 7/8 Triggering

We previously found that triggering TLR7/8 either by single stranded HIV RNA or synthetic compounds induced changes in the lymphoid microenvironment unfavorable to HIV. In this study, we used selective TLR7 and 8 agonists to dissect their contribution to the anti-HIV effects. While triggering TLR7 inhibited efficiently HIV replication in lymphoid suspension cells from tonsillar origin, its effect was inconsistent in peripheral blood mononuclear cells (PBMC). In contrast, triggering TLR8 showed a very prominent and overall very consistent effect in PBMC and tonsillar lymphoid suspension cells. Depletion of dendritic cells (DC), Natural killer cells (NK) and CD8+ T-cells from PBMC resulted in the reversal of TLR8 induced anti-HIV effects. Especially noteworthy, depletion of either NK or CD8+ T-cells alone was only partially effective. We interpret these findings that DC are the initiator of complex changes in the microenvironment that culminates in the anti-HIV active NK and CD8+ effector cells. The near lack of NK and the low number of CD8+ T-cells in tonsillar lymphoid suspension cells may explain the lower TLR8 agonist's anti-HIV effects in that tissue. However, additional cell-type specific differences must exist since the TLR7 agonists had a very strong inhibitory effect in tonsillar lymphoid suspension cells. Separation of effector from the CD4+ target cells did not abolish the anti-HIV effects pointing to the critical role of soluble factors. Triggering TLR7 or 8 were accompanied by major changes in the cytokine milieu; however, it appeared that not a single soluble factor could be assigned for the potent effects.These results delineate the complex effects of triggering TLR7/8 for an efficient antiviral defense. While the ultimate mechanism(s) remains unknown, the potent effects described may have therapeutic value for treating chronic viral diseases. Notably, HIV replication is blocked by TLR triggering before HIV integrates into the host chromosome which would prevent the establishment or maintenance of the latent reservoir.

Effects of glutamine on the cytokine expression of peripheral blood mononuclear cells

To investigate the effects of glutamine (Gln) on the cytokine expression of the peripheral blood mononuclear cells (PBMCs) stimulated by lipopolysaccharide (LPS). PBMCs were extracted from healthy volunteer by density gradient centrifugation. Suspension of PBMCs was pretreated with Gln of the concentrations of 1 mmol/L, 8 mmol/L, 10 mmol/L, and 15 mmol/L for 0.5 h and 2 h respectively. Suspension of PBMCs without Gln pretreatment but stimulated by LPS was used as positive control group, and suspension without LPS stimulation was used as negative control group. Then LPS was added ELISA was used to examine the levels of the mediators of inflammation, TNF-alpha, IL-1beta, IL-4, IL-6, IL-8, and IL-10, in the supernatant, and Gene Array Protocol was used to examine the mRNA expression of these cytokines. After LPS stimulation, the expression levels of all the cytokines were obviously enhanced. The expression levels of the proinflammatory mediator, except for IL-6, of the Gln 8 mmoL/L 0.5 h group were significantly lower than those of the positive control group; and the expression levels of all the cytokines of the Gln 15 mmol/L 0.5 h group were significantly lower than those of the positive control group, however, all still significantly higher than those of the negative control group (P <0.01 or P <0.05). The expression levels of the proinflammatory mediator except for IL-8 of the Gln 1 mmol/L 0.5 h group did not significantly changed in comparison with the positive control group; and the level of IL-8 was significantly decreased (P <0.01). The expression levels of all the cytokines of the high concentrations Gln groups were all significantly lower than those of the positive control group. The levels of TNF-alpha, IL-6, and IL-8 of the high concentration Gln 2 h groups were all significantly higher then those of the concentration Gln 0.5 h groups (all P <0.01), however, there was not significant difference in the level of IL-1beta between the high concentration 0.5 h and 2 h Gln groups. The level of the anti-inflammatory mediator IL-10 of the high concentration Gln 0.5 h and 2 h groups, especially that of the latter group, were significantly lower than that of the positive control group (both P <0.01), however, the IL-10 level of the high concentration Gln 0.5 h group was still significantly higher than that of the negative control group (P < 0.05). Gene chip analysis confirmed the above results. Glutamine dose- and time-dependently downregulates the expression of the mediators of inflammation, and lessen the excessive expression of those cytokines in cells stimulated by LPS.

Structural Changes in Lymphocytes Membrane of Chernobyl Clean-up Workers from Latvia

ABM (3-aminobenzanthrrone derivative) developed at the Riga Technical University, Riga, Latvia) has been previously shown as a potential probe for determination of the immune state of patients with different pathologies . The fist study (using probe ABM) of peripheral blood mononuclear cells (PBMC) membranes of 97 Chernobyl clean-up workers from Latvia was conducted in 1997. Now we repeatedly examine the same (n = 54) individuals in dynamics. ABM spectral parameters in PBMC suspension, fluorescence anisotropy and blood plasma albumin characteristics were recorded. In 1997 screening showed 5 different patterns of fluorescence spectra, from which in 2007 we obtained only two. These patterns of spectra had never been previously seen in healthy individuals or patients with tuberculosis, multiple sclerosis, rheumatoid arthritis, etc., examined by us. Patterns of ABM fluorescence spectra are associated with membrane anisotropy and conformational changes of blood plasma albumin. We observed that in dynamics 1997-2007 the lipid compartment of the membrane became more fluid while the lipid-protein interface became more rigid. The use of probe ANS and albumin auto-fluorescence allowed show conformational alterations in Chernobyl clean-up workers blood plasma. It is necessary to note that all investigated parameters significantly differ in observed groups of patients. These findings reinforce our understanding that that the cell membrane is a significant biological target of radiation. The role of the membrane in the expression and course of cell damage after radiation exposure must be considered. So ten years dynamic of PBMC membrane characteristics by ABM (spectral shift and anisotropy indexes) in Chernobyl clean-up workers reveal progressive trend toward certain resemblance with those of chronic B-cell lymphoid leukemia.

CpG-C ISS-ODN activation of blood-derived B cells from healthy and chronic immunodeficiency virus-infected macaques

Cytosine-phosphate-guanine class C (CpG-C) immunostimulatory sequence oligodeoxynucleotides (ISS-ODNs) activate human B cells and dendritic cells (DCs), properties that suggest potential use as a novel adjuvant to enhance vaccine efficacy. After demonstrating that the CpG-C ISS-ODN C274 activates macaque DCs, we examined in vitro activation of macaque B cells by C274 as a prelude to evaluation of this molecule as an adjuvant in the testing of candidate human immunodeficiency virus vaccines in the rhesus macaque-simian immunodeficiency virus (SIV) model. C274 induced macaque CD20(+) B cells to proliferate more strongly than CD40 ligand or CpG-B ISS-ODN. C274 enhanced B cell survival; increased viability was most evident after 3-7 days of culture. Increased expression of CD40, CD80, and CD86 by B cells was apparent within 24 h of exposure to C274 and persisted for up to 1 week. C274-stimulated, B cell-enriched and peripheral blood mononuclear cell suspensions from naïve and immunodeficiency virus-infected monkeys secreted several cytokines [e.g., interleukin (IL)-3, IL-6, IL-12, interferon-alpha] and chemokines [e.g., monocyte chemoattractant protein-1/CC chemokine ligand 2 (CCL2), macrophage-inflammatory protein-1alpha/CCL3, IL-8/CXC chemokine ligand 8]. In comparison, exposure of macaque B cells to SIV had minimal impact on surface phenotype, despite inducing cytokine and chemokine production in cells from infected and uninfected animals. These observations emphasize the need to identify strategies to optimally boost immune function, as immunodeficiency viruses themselves only partially activate B cells and DCs. The ability of C274 to stimulate B cells and DCs in healthy and infected monkeys suggests its possible use as a broad-acting adjuvant to be applied in the rhesus macaque model for the development of preventative and therapeutic vaccines.

Characterization of B- and T-cell responses and HLA-DR4 binding motifs of the latex allergen Hev b 6.01 (prohevein) and its post-transcriptionally formed proteins Hev b 6.02 and Hev b 6.03.

Multiple immunoglobulin E (IgE)-binding proteins in natural rubber latex extracts have been identified. In the case of Hev b 6 a differentiation was made between the precursor protein prohevein (Hev b 6.01) and its two post-transcriptionally formed proteins, the N-terminal hevein (Hev b 6.02) and the C-terminal domain (Hev b 6.03). All three components act as independent allergens. The aim of this study was a detailed analysis of the T-cell responses and the IgE-binding capacity of Hev b 6.01, Hev b 6.02 and Hev b 6.03 by using these allergens as recombinant maltose-binding fusion (MBP) proteins and the usage of synthetic modified hevein peptides. Latex-allergic health care workers (HCWs) suffering from rhinitis, conjunctivitis, contact urticaria and/or asthma with increased specific IgE-antibodies to latex were tested for their IgE-binding capacity and T-cell reactivity (by proliferation response) to the recombinant MBP-rHev b 6.01, MBP-rHev b 6.02, MBP-rHev b 6.03, to native Hev b 6.02, to modified hevein peptides and wheat germ agglutinin (WGA). For testing of the human leucocyte antigen (HLA) class II restriction of MBP-rHev b 6.01 induced peripheral blood mononuclear cell (PBMC) responses, monoclonal antibodies against HLA-DR, HLA-DP or HLA-DQ were added. Seventeen of 18 (94%) serum samples from latex-allergic HCWs had increased levels of specific IgE to MBP-rHev b 6.01, 16 (89%) to MBP-rHev b 6.02 and 13 (72%) to MBP-rHev b 6.03. A significant difference existed between the specific IgE-values of MBP-rHev b 6.02 and MBP-rHev b 6.03 (P < 0.01). Proliferation responses of PBMC of the same 18 latex-allergic patients were positive for MBP-rHev b 6.01 and MBP-rHev b 6.03 in 83 and 67% of the tested PBMC suspension, whereas the proliferation responses induced with MBP-rHev b 6.02 or native Hev b 6.02 were very low (5.6 and 22.2%). Sera from nine additional latex-allergic patients showed specific IgE binding to the native Hev b 6.02, but none of these sera showed specific IgE binding to the modified Hev b 6.02-peptides [whereby all eight cysteine residues were substituted by serine (C --> S) or by alanine (C --> A)]. Proliferation responses induced by the modified Hev b 6.02 peptides were not significantly different from that induced by Hev b 6.02. Potential HLA-DR4Dw4(DRB1*0401)-restricted T-cell epitopes of Hev b 6.01 predicted by two computer algorithms were only found in the Hev b 6.03-part of Hev b 6.01. In the Hev b 6.01 precursor the regions responsible for IgE binding and those for inducing the T-cell proliferation responses are settled in different parts of the protein. The Hev b 6.02 domain is responsible for IgE binding and carries discontinuous B-cell epitopes whereas Hev b 6.03 is a better inducer of a proliferation response and contains HLA-DR4-binding motifs.