Peripheral Blood Mononuclear Cell Populations Research Articles

Abstract 16882: Temporal Changes in the Peripheral Blood Mononuclear Cell Populations After Acute Myocardial Infarction and Prediction of Improvement in Left Ventricular Function in the Cardiovascular Cell Therapy Research Network (CCTRN) TIME and Late-TIME Trials

Background: Despite many cell-mediated attempts to promote myocardial repair after myocardial infarction (MI), no cell treatments are currently available. Instead, cell therapy clinical results remain mixed. We hypothesized that by evaluating changes in circulating cells in improvers and non-improvers in an MI cell therapy study, we could identify cells associated with repair that might be candidates for future cell therapy studies. Methods: In this study, peripheral blood (PB) was evaluated by flow cytometry at baseline and days 1, 30, 90 and 180 after bone marrow mononuclear cell (BMMNC) or placebo therapy from 207 consented patients in the CCTRN TIME (n=120) and Late-TIME (n=87) studies. A linear mixed model was used to determine changes over time in cell frequency. Multiple regression and discriminatory analyses were used to identify if changes in circulating cells predict changes in left ventricular ejection fraction (LVEF). Results: Changes in circulating cells were observed independent of treatment group. In TIME, a progressive decrease of CD11b+ cells started at day 30 (91.69±4.57 vs. 93.23±3.97%, p<0.05) and CD14+ cells began at day 90 (73.53±10.82 vs. 79.17±10.97%, p<0.05) compared to baseline. In Late-TIME, there was an increase in CD11b+ (94.01±3.82 vs. 92.81±3.98%, p<0.05) and CD14+ (81.70±6.64 vs. 77.94±9.29%, p<0.05) cells at day 1 when compared to baseline followed by a progressive decrease in the follow-up visits. A progressive decline in CXCR4+ cells started at day 30 in TIME (35.39 ±14.81 vs. 42.86±13.83%, p<0.05) and at day 1 in Late-TIME (42.81±14.33 vs. 47.74±15.13%, p<0.05). In TIME, a decrease of CXCR4+ cells at day 90 was a negative predictor of changes in LVEF from baseline to six months. Conclusion: Although multiple cell frequencies changed after treatment, only one cell frequency predicted changes in LVEF from baseline to six months. A significant reduction in CXCR4+ circulatory cells at day 90 after treatment is associated with improvement in LVEF. Further studies will be necessary to confirm the changes in circulatory cells and its impact on the regenerative capacity of patients with AMI.

Separation, banking, and quality control of peripheral blood mononuclear cells from whole blood of melanoma patients.

Peripheral blood mononuclear cells (PBMCs) are essential to the study of autoimmune, infectious, parasitic diseases, and cancer. In the rapidly growing field of cancer immunology, cellular phenotyping provides critical information about patient responses to treatments and treatment efficacies. Notably, the evaluation of T cell based therapies relies on the isolation of highly viable CD3+ T cell, CD4+ Helper T cell, and CD8+ Cytotoxic T cell populations before and during patient treatments. Cryopreservation of PBMC populations allows researchers to thaw and characterize clinical samples by flow cytometry, mass cytometry, sequencing, etc. in a high-throughput manner and in batches. Therefore, it is important to separate and bank an abundance of robust circulating immune cells. Here, we report our internal protocols for the high-quality separation, banking, and thawing of clinically relevant PBMC populations. We present quality control data from 11 melanoma patients and characterize their CD3+, CD4+, and CD8+ T cells by 4-color flow cytometry.

Interleukin-6 Rescues Lymphocyte from Apoptosis and Exhaustion Induced by Chronic Hepatitis C Virus Infection.

Chronic hepatitis C virus (HCV) infection appears to trigger the onset of immune exhaustion and apoptosis to potentially assist viral persistence inside the host, eventually leading to exacerbated conditions of inflammation and hepatocarcinogenesis. Growing evidence suggests that spontaneous apoptosis of peripheral blood mononuclear cells (PBMCs) could be one of the potential immune impairment mechanisms in chronic viral infection. Interleukin-6 (IL-6) is a pleiotropic cytokine that plays an essential role in regulating immune and inflammatory responses. Owing to its known role in priming T cell growth, differentiation, and inhibition of lymphocyte apoptosis, we investigated the protective effect of IL-6 in rescuing lymphocytes from apoptosis and functional exhaustion in chronic HCV infection. The expression pattern of antiapoptotic (Mcl-1 and Bcl-2), proapoptotic (caspase-3 and Bim) genes along with interferon gamma (IFN-γ) and T cell inhibitory receptor (TIM-3) was analyzed before and after in vitro IL-6 treatment of patient-derived PBMCs. It was observed that the expression of antiapoptotic genes, Mcl-1 and Bcl-2 increased (threefolds and twofolds, respectively) and there was a considerable downregulation in T cell inhibitory receptor (TIM-3) and caspase-3. Similarly, the capacity of PBMCs to produce IFN-γ was also significantly increased (p < 0.001) depicting the promising nature of IL-6 in enhancing lymphocyte effector function. Summing it up, the study supports the positive role of IL-6 in rescuing PBMC population; however, the cytokine alone is not sufficient to sustain the adaptive immunity. It could be used as a potential candidate for combinational therapy along with other regulatory factors for ex vivo enhancement of lymphocyte and may help in moving one step toward adoptive T cell therapy in chronic HCV infection.

Ibrutinib induces rapid down-regulation of inflammatory markers and altered transcription of chronic lymphocytic leukaemia-related genes in blood and lymph nodes.

In chronic lymphocytic leukaemia (CLL) patients, treatment with the Bruton tyrosine kinase inhibitor ibrutinib induces a rapid shift of tumour cells from lymph nodes (LN) to peripheral blood (PB). Here, we characterized in depth the dynamics of ibrutinib-induced inflammatory, transcriptional and cellular changes in different compartments immediately after treatment initiation in seven relapsed/refractory CLL patients. Serial PB and LN samples were taken before start and during the first 29days of treatment. Changes in plasma inflammation-related biomarkers, CLL cell RNA expression, B-cell activation and migration markers expression, and PB mononuclear cell populations were assessed. A significant reduction of 10 plasma inflammation markers, the majority of which were chemokines and not CLL-derived, was observed within hours, and was paralleled by very early increase of CD19+ circulating cells. At the RNA level, significant and continuous changes in transcription factors and signalling molecules linked to B-cell receptor signalling and CLL biology was observed in both PB and LN CLL cells already after 2days of treatment. In conclusion, ibrutinib seems to instantly shut off an ongoing inflammatory response and interfere with diverse sensitive pathways in the LN.

Adaptive metabolic pattern biomarker for disease monitoring and staging of lung cancer with liquid biopsy

In this manuscript, we demonstrate the applicability of a metabolic liquid biopsy for the monitoring and staging of patients with lung cancer. This method provides an unbiased detection strategy to establish a more precise correlation between CTC quantification and the actual burden of disease, therefore improving the accuracy of staging based on current imaging techniques. Also, by applying statistical analysis techniques and probabilistic models to the metabolic status and distribution of peripheral blood mononuclear cell (PBMC) populations “perturbed” by the presence of CTCs, a new category of adaptive metabolic pattern biomarker (AMPB) is described and unambiguously correlated to the different clinical stages of the patients. In fact, this strategy allows for classification of different categories of disease within a single stage (stage IV) before computed tomography (CT) and positron emission tomography (PET) scans and with lower uncertainty.

Critical issues in genotoxicity assessment of TiO2 nanoparticles by human peripheral blood mononuclear cells.

In the last years, a number of in vitro studies have been performed to assess the genotoxic activity of titanium dioxide (TiO2 ). To resolve the contradictory results, in this study, we investigated the genotoxic activity of commercial TiO2 nanoparticles (NPs) and microparticles of different forms (anatase, rutile and mix of both). We evaluated micronucleus formation in stimulated lymphocytes, as well as DNA strand breaks and 8-oxo-7,8-dihydro-2'-deoxyguanosine in peripheral blood mononuclear cells (PBMCs), a mixed population of lymphocytes and monocytes. Different responses to TiO2 exposure were obtained depending on the assay. Both TiO2 NPs and microparticles and all the crystalline forms elicited a significant increase in 8-oxo-7,8-dihydro-2'-deoxyguanosine and DNA strand breaks in the whole PBMC population, without a concurrent increase of micronuclei in proliferating lymphocytes. The distribution of DNA damage in PBMCs, detected by the comet assay, that measures DNA damage at level of single cells, indicated the presence of a more susceptible cell subpopulation. The measurement of side scatter signals by flow cytometry highlighted the preferential physical interaction of TiO2 particles with monocytes that also displayed higher reactive oxygen species generation, providing a mechanistic explanation for the different responses observed in genotoxicity assays with PBMCs and lymphocytes. This study confirmed the suitability of human PBMCs as multi-cell model to investigate NP-induced DNA damage, but suggested some caution in the use of stimulated lymphocytes for the assessment of NP clastogenicity.

Characterisation of peripheral blood mononuclear cell populations in periparturient dairy cows that develop metritis

Bacterial contamination of the uterus following calving is ubiquitous in the dairy cow, 40% of cows develop postpartum uterine infection, including metritis. While predisposing factors like twinning and dystocia are associated with metritis, it is unclear why some cows remain healthy following calving and others develop uterine infection, negatively impacting animal health, milk production and economic return. Here, we profiled peripheral blood mononuclear cells of cows before calving and during postpartum metritis. We hypothesized that peripheral blood mononuclear cell function and proportions would be altered during the prepartum period in cows that develop postpartum metritis. Using flow cytometry we observed reduced proportions of peripheral CD3+/CD4+, CD4+/CD62L+, FOXP3+ and CD21+ populations from −10 to 40 days relative to calving associated with metritis, while the proportion of peripheral CD3+/CD4+ lymphocytes were specifically reduced in the prepartum period before the onset of metritis. Peripheral blood mononuclear cells from cows with metritis had a perturbed capacity to secrete IL-1β or IFNγ in response to in vitro stimulus; cells collected during the prepartum period from cows that would go on to develop metritis failed to increase IL-1β secretion in response to stimulation, while IFNγ secretion was altered at calving and postpartum in cows with metritis compared to healthy herd mates. No effect of metritis was observed in the capacity of cows to mount a humoral immune response to antigen administered on the day of calving. The studies discussed here suggest that while minor changes to the prepartum immune system are observed in cows that develop metritis, changes observed in the postpartum period are more prevalent and likely a consequences of disease and not causative. Future studies to modulate the prepartum immune system may help to limit postpartum metritis.

Functional Studies of T Regulatory Lymphocytes in Human Schistosomiasis in Western Kenya.

.Immunoregulation is considered a common feature of Schistosoma mansoni infections, and elevated levels of T regulatory (Treg) lymphocytes have been reported during chronic human schistosomiasis. We now report that the removal of Treg (CD4+/CD25hi/CD127low lymphocytes) from peripheral blood mononuclear cells (PBMCs) of S. mansoni–infected individuals leads to increased levels of phytohemagglutinin (PHA)-stimulated interferon gamma (IFNγ) production and decreased interleukin-10 (IL-10) responses. Exposure to schistosome antigens did not result in measurable IFNγ by either PBMC or Treg-depleted populations. Interleukin-10 responses to soluble egg antigens (SEA) by PBMC were unchanged by Treg depletion, but the depletion of Treg greatly decreased IL-10 production to soluble worm antigenic preparation (SWAP). Proliferative responses to PHA increased upon Treg removal, but responses to SEA or SWAP did not, unless only initially low responders were evaluated. Addition of anti-IL-10 increased PBMC proliferative responses to either SEA or SWAP, but did not alter responses by Treg-depleted cells. Blockade by anti-transforming growth factor-beta (TGF-β) increased SEA but not SWAP proliferative responses by PBMC, whereas anti-TGF-β increased both SEA- and SWAP-stimulated responses by Treg-depleted cultures. Addition of both anti-IL-10 and anti-TGF-β to PBMC or Treg-depleted populations increased proliferation of both populations to either SEA or SWAP. These studies demonstrate that Treg appear to produce much of the antigen-stimulated IL-10, but other cell types or subsets of Treg may produce much of the TGF-β. The elevated levels of Treg seen in chronic schistosomiasis appear functional and involve IL-10 and TGF-β in antigen-specific immunoregulation perhaps leading to regulation of immunopathology and/or possibly decreased immunoprotective responses.

Identification of Anti-Trypanosoma cruzi Lead Compounds with Putative Immunomodulatory Activity.

In seeking substitutions for the current Chagas disease treatment, which has several relevant side effects, new therapeutic candidates have been extensively investigated. In this context, a balanced interaction between mediators of the host immune response seems to be a key element for therapeutic success, as a proinflammatory microenvironment modulated by interleukin-10 (IL-10) is shown to be relevant to potentiate anti-Trypanosoma cruzi drug activity. This study aimed to identify the potential immunomodulatory activities of the anti-T. cruzi K777, pyronaridine (PYR), and furazolidone (FUR) compounds in peripheral blood mononuclear cells (PBMC) from noninfected (NI) subjects and chronic Chagas disease (CD) patients. Our results showed low cytotoxicity to PBMC populations, with 50% cytotoxic concentrations (CC50) of 71.0 μM (K777), 9.0 μM (PYR), and greater than 20 μM (FUR). In addition, K777 showed no impact on the exposure index (EI) of phytohemagglutinin-stimulated leukocytes (PHA), while PYR and FUR treatments induced increased EI of monocytes and T lymphocytes at late stages of apoptosis in NI subjects. Moreover, K777 induced a more prominent proinflammatory response (tumor necrosis factor alpha-positive [TNF-α+] CD8+/CD4+, gamma interferon-positive [IFN-γ+] CD4+/CD8+ modulated by interleukin-10-positive [IL-10+] CD4+ T/CD8+ T) than did PYR (TNF-α+ CD8+, IL-10+ CD8+) and FUR (TNF-α+ CD8+, IL-10+ CD8+). Signature analysis of intracytoplasmic cytokines corroborated the proinflammatory/modulated (K777) and proinflammatory (PYR and FUR) profiles previously found. In conclusion, the lead compound K777 may induce beneficial changes in the immunological profile of patients presenting the chronic phase of Chagas disease and may contribute to a more effective therapy against the disease.

Aberrant STAT phosphorylation signaling in peripheral blood mononuclear cells from multiple sclerosis patients

BackgroundMultiple sclerosis (MS) is characterized by increased activation of peripheral blood mononuclear cells (PBMCs), linked to perturbations in the phosphorylation of signaling proteins.MethodsWe developed a phosphoflow cytometry protocol to assess the levels of 11 phosphorylated nuclear proteins at baseline conditions and after cell activation in distinct PBMC populations from 41 treatment-naïve relapsing-remitting (RR) MS subjects and 37 healthy controls, and in a second cohort of 9 untreated RRMS patients and 10 secondary progressive (SP) MS patients. Levels of HLA-ABC, HLA-E, and HLA-DR were also assessed. Phosphorylation levels of selected proteins were also assessed in mouse splenocytes isolated from myelin oligodendrocyte glycoprotein (MOG)35–55-induced experimental autoimmune encephalomyelitis (EAE).ResultsModest differences were observed at baseline between patients and controls, with general lower phosphorylation levels in cells from affected individuals. Conversely, a dramatic increase in phosphorylated p38MAPK and STAT proteins was observed across all cell types in MS patients compared to controls after in vitro activation. A similar phosphorylation profile was observed in mouse lymphocytes primed in vivo with MOG. Furthermore, levels of all p-STAT proteins were found directly correlated with HLA expression in monocytes. Levels of phosphorylated proteins did not differ between relapsing-remitting and secondary progressive MS patients either in baseline conditions or after stimulation. Lastly, phosphorylation levels appear to be independent of the genotype.ConclusionThe response to IFN-α through STAT proteins signaling is strongly dysregulated in MS patients irrespective of disease stage. These findings suggest that the aberrant activation of this pathway could lead to changes in the expression of HLA molecules in antigen presenting cells, which are known to play important roles in the regulation of the immune response in health and disease.

ARV7/AR ratio and neutrophil-to-lymphocyte ratio (NLR) as predictors of docetaxel benefit in metastatic castration-resistant prostate cancer patients (mCRPC).

254 Background: The constitutively active androgen receptor (AR) variant 7 (ARV7) has been associated to AR inhibitors resistance, but its role in taxane-benefit has not been stablished. Moreover, the neutrophil-to-lymphocyte ratio (NLR) has been associated with poor outcome in prostate cancer.The PBMC population may contain circulating tumor cells (CTCs) but is mainly constituted by lymphocytes and monocytes, that also express the AR. Here we studied the expression of ARV7 and AR in peripheral blood mononuclear cells (PBMC) and its correlation with NLR and clinical outcome in mCRPC treated with docetaxel. Methods: ARV7 and AR mRNAs were tested by quantitative reverse-transcription PCR. Baseline neutrophil and lymphocyte values and PBMC samples were prospectively collected before treatment initiation. Patients were stratified according to NLR ratio (low NLR: ≤3; high NLR:&gt;3) and to ARV7/ARFL ratio expression (Maxstat Cutoff 1.99). Correlation between NLR and PSA progression-free (PSA-PFS) and overall survival (OS), were measured from the start of docetaxel treatment and calculated by log rank test. Results: Fifty patients were included:25 (50%) had low NLR. Thirty-eight patients (76%) expressed high ARV7/ARFL. No significant correlation between NLR with ARV7, AR and ARV7/AR was observed. High ARV7/ARF ratio correlated to a better PSA-PFS (median PSA-PFS 7.47; HR 0.38, 95%CI 0.17-0.85; P=0.0179) , but not OS. High NLR was associated to a lower OS [Median OS was 9.2 months versus 30.17 months in those with low NLR (HR 2.19, 95% CI 1.17-4.09, p= 0.01)]. When both parameters were combined, high NLR and low ARV7/ARFL ratio were associated to a worse PSA-PFS in comparison with low NLR and high ARV7/ARFL ratiogroup(median PSA-PFS: 4.9 vs. 7.5; p=0.008 and HR 5.08, 95% CI 1.33-19.41, p= 0.08) and OS (median OS: 7.4 vs. 30.5; p=0.001 and HR 22.79, 95% CI 4.29-121.17, p= 0.00). Conclusions: The combination of low ARV7/ARFL high NLR and ratio evaluated in PBMC prior to docetaxel treatment has a predictive impact on response and survival in mCRPC patients.

It is Time for Action: Consuming Evidence: Critical Appraisal: Part II

Dengue is a major public health problem in tropical regions around the world. Viral and immune host factors determine the clinical courses of the infection. We analyzed the dynamics of viremia (by real-time polymerase chain reactions), antigenemia (through detection of the viral non-structural protein [NS]-1 by enzyme-linked immunosorbent assays) and the frequency of virus-infected peripheral blood mononuclear cells (PBMCs) (by multiparametric flow cytometry) in children with primary or secondary dengue virus (DENV) infection in mild to severe cases. Additionally, we evaluated the association of these factors with clinical severity and laboratory parameters. The levels of viremia and antigenemia peaked during the early days of illness and these viral parameters were correlated (rho = 0.37, P = 0.003). Circulating monocytes were the most naturally infected subset within the PBMCs population, with kinetics similar to those of viremia and antigenemia. The levels of viremia and antigenemia were higher in children with primary infections than in those with secondary infections (P ≤ 0.04). Although there were no associations between the three evaluated factors and clinical severity, the levels of plasma NS1 and the frequency of dengue virus-infected monocytes correlated with prolonged coagulation times. In short, the viremia, antigenemia and infected monocytes were detected early and were not related to clinical severity. The magnitude of antigenemia and infected circulating monocytes was associated with coagulation disorders.

Response to Trypanosoma cruzi by Human Blood Cells Enriched with Dentritic Cells Is Controlled by Cyclooxygenase-2 Pathway.

Chagas disease (Cd) or American human trypanosomiasis is caused by Trypanosoma cruzi and affects ~7 million people, mostly in Latin America. The infective trypomastigote forms of the parasite can invade several human blood cell populations, including monocytes and dendritic cells (DC). Although these cells display a wide functional diversity, their interactions with T. cruzi via cyclooxygenase (COX) and cyclic adenosine monophosphate (cAMP) dependent pathways have not been analyzed. To exploiting this mechanism, DC-enriched peripheral human blood mononuclear cell populations (DC-PBMC) were used as our model. Our results showed that the treatment of these cell populations with celecoxib (CEL), a cyclooxygenase-2 selective inhibitor or SQ 22,536, an adenilate cyclase inhibitor, significantly caused marked inhibition of T. cruzi infection. In contrast, aspirin (ASA, a non-selective COX-1 and COX-2 inhibitor) treatment did not inhibit the infection of the cells by the parasite and was independent of nitric oxide (NO) production. The expression of co-stimulatory molecules CD80 and CD86 were similar on cells treated or not with both COX-inhibitors. The infection stimulated the release of TNF-α, IL-1β, IL-6, IL-8, and IL-10 production by infected cells. Treatment with ASA or CEL did not affect TNF-α, IL-6, IL-8, IL-10, and NO production by infected cells, but increased IL-1β production by them. Our results suggest a key role of COX-2 and cAMP pathways in T. cruzi invasion process of human blood cells and these pathways may represent targets of new therapeutic options for Cd.

Naltrexone Inhibits IL-6 and TNFα Production in Human Immune Cell Subsets following Stimulation with Ligands for Intracellular Toll-Like Receptors.

The opioid antagonist naltrexone hydrochloride has been suggested to be a potential therapy at low dosage for multiple inflammatory conditions and cancers. Little is known about the immune-modulating effects of naltrexone, but an effect on the activity of toll-like receptor 4 (TLR4) has been reported. We analyzed the effects of naltrexone hydrochloride on IL-6 secretion by peripheral blood mononuclear cells (PBMC) in vitro following stimulation with ligands for TLR4 and for the intracellular receptors TLR7, TLR8, and TLR9. Naltrexone did not affect cell viability or induce apoptosis of PBMC. Intracellular staining demonstrated that naltrexone inhibited production of IL-6 and TNFα by monocyte and plasmacytoid dendritic cell subsets within the PBMC population following treatment with ligands for TLR7/8 and TLR9, respectively. No effect of cytokine production by PBMC following stimulation of TLR4 was observed. Additionally, naltrexone inhibited IL-6 production in isolated monocytes and B cells after TLR7/8 and TLR9 stimulation, respectively, but no effect on IL-6 production in isolated monocytes after TLR4 stimulation was observed. These findings indicate that naltrexone has the potential to modulate the secretion of inflammatory cytokines in response to intracellular TLR activity, supporting the hypothesis that it may have potential for use as an immunomodulator.

Reduction of T-Helper Cell Responses to Recall Antigen Mediated by Codelivery with Peptidoglycan via the Intestinal Nanomineral–Antigen Pathway

Naturally occurring intestinal nanomineral particles constituently form in the mammalian gut and trap luminal protein and microbial components. These cargo loaded nanominerals are actively scavenged by M cells of intestinal immune follicles, such as Peyer’s patches and are passed to antigen-presenting cells. Using peripheral blood mononuclear cell populations as an in vitro model of nanomineral uptake and antigen presentation, we show that monocytes avidly phagocytose nanomineral particles bearing antigen and peptidoglycan (PGN), and that the presence of PGN within particles downregulates their cell surface MHC class II and upregulates programmed death receptor ligand 1. Nanomineral delivery of antigen suppresses antigen-specific CD4+ T cell responses, an effect that is enhanced in the presence of PGN. Blocking the interleukin-10 receptor restores CD4+ T cell responses to antigen codelivered with PGN in nanomineral form. Using human intestinal specimens, we have shown that the in vivo nanomineral pathway operates in an interleukin-10 rich environment. Consequently, the delivery of a dual antigen–PGN cargo by endogenous nanomineral in vivo is likely to be important in the establishment of intestinal tolerance, while their synthetic mimetics present a potential delivery system for therapeutic applications targeting the modulation of Peyer’s patch T cell responses.

Diagnostic and prognostic value of CD163 for infection-associated hemophagocytic syndrome in children

Objective To investigate the diagnostic and prognostic value of serum soluble CD163 (sCD163) and the positive rate of membrane-bound CD163 (mCD163) in peripheral blood mononuclear cells (PBMC) in children with infection-associated hemophagocytic syndrome (IAHS). Methods Between July 2012 and June 2016, 26 pediatric patients with IAHS (IAHS group) and 28 pediatric patients with sepsis(sepsis group)admitted to Children′s Hospital Affiliated to Shanghai Jiaotong University were selected, and 20 healthy children were taken as healthy control group.Sandwich enzyme linked immunosorbent assay was used to detect serum sCD163.The population of circulating mCD163 positive monocytes was determined by using flow cytometry.Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic and prognostic values of sCD163 and mCD163 in children with IAHS compared with the diagnostic and prognostic values of plasma ferritin, and so on. Results The serum levels of sCD163 in patients of IAHS group, sepsis group and healthy control group were (1 264±538) mg/L, (862±332) mg/L, (610±316) mg/L, respectively.And the population of mCD163-positive PBMC in patients of IAHS group, sepsis group and healthy control group was (88.3±9.7)%, (68.5±18.3)%, (28.9±5.2)%, respectively.Both serum sCD163 and the population of mCD163-positive PBMC were significantly higher in IAHS group compared with those of sepsis group (t=2.031, P=0.048; t=3.191, P=0.002, respectively). The serum sCD163 and population of mCD163-positive PBMC in sepsis group were higher than controls (t=3.848, P=0.002; t=4.049, P=0.000, respectively). Moreover, the areas under the ROC curve (AUC) for the mCD163, sCD163, were 0.853(P=0.013), 0.762(P=0.004), 0.755(P=0.049), respectively.mCD163 at a cutoff of 83.7% had a high diagnosis sensitivity (81.8%) and specificity (72.4%). The optimal cutoff values of sCD163 and ferritin for predicting IAHS was 888 mg/L (sensitivity 66.7% and specificity 63.3%) and 2 880 μg/L (sensitivity 80.0% and specificity 54.5%). In addition, the serum level of sCD163 and the population of mCD163-positive PBMCs were significantly increased in acute phase and decreased in recovery phase[(1 553±542) mg/L vs. (866±92) mg/L, (91.0±6.4)% vs.(79.0±4.6)%, t=2.450, χ2=3.419, P=0.036, 0.007] in IAHS group.Furthermore, subgroup analysis indicated that the serum level of sCD163 and the population of mCD163-positive PBMCs were significantly higher in dead patients than those in survived patients [(1 748.91±518.17) mg/L vs.(909.69±171.35) mg/L, t=3.070, P=0.011; (93.50±8.42)% vs.(77.30±3.28)%, χ2=3.005, P=0.024, respectively]. Conclusion Serum sCD163 and the population of mCD163-positive PMSCs are specific and validity biomarkers for early diagnosis of IAHS, which also are associated with treatment response assessment and prognostic analysis in IAHS. Key words: Soluble CD163; Membrane-bound CD163; Infection-associated hemophagocytic syndrome; Sepsis; Child

Peripheral cellular and humoral responses to infestation with the cattle tick Rhipicephalus microplus in Santa Gertrudis cattle.

Resistance to cattle tick infestation in single-host ticks is primarily manifested against the larval stage and results in the immature tick failing to attach successfully and obtain a meal. This study was conducted to identify immune responses that characterize the tick-resistant phenotype in cattle. Thirty-five tick-naïve Santa Gertrudis heifers were used in this study, thirty of which were artificially infested for thirteen weeks with tick larvae while five animals remained at a tick-free quarantine property to serve as a control group. Following thirteen weeks of tick infestation, the animals in this trial exhibited highly divergent tick-resistant phenotypes. Blood samples collected throughout the trial were used to measure peripheral immune parameters: haematology, the percentage of cellular subsets comprising the peripheral blood mononuclear cell (PBMC) population, tick-specific IgG1 and IgG2 antibody titres, IgG1 avidity for tick antigens and the ability of PBMC to recognize and proliferate in response to stimulation with tick antigens in vitro. The tick-susceptible cattle developed significantly higher tick-specific IgG1 antibody titres compared to the tick-resistant animals. These results suggest that the heightened antibody response either does not play a role in resistance or might contribute to increased susceptibility to infestation.

Magnitude of viremia, antigenemia and infection of circulating monocytes in children with mild and severe dengue

Dengue is a major public health problem in tropical regions around the world. Viral and immune host factors determine the clinical courses of the infection. We analyzed the dynamics of viremia (by real-time polymerase chain reactions), antigenemia (through detection of the viral non-structural protein [NS]-1 by enzyme-linked immunosorbent assays) and the frequency of virus-infected peripheral blood mononuclear cells (PBMCs) (by multiparametric flow cytometry) in children with primary or secondary dengue virus (DENV) infection in mild to severe cases. Additionally, we evaluated the association of these factors with clinical severity and laboratory parameters. The levels of viremia and antigenemia peaked during the early days of illness and these viral parameters were correlated (rho=0.37, P=0.003). Circulating monocytes were the most naturally infected subset within the PBMCs population, with kinetics similar to those of viremia and antigenemia. The levels of viremia and antigenemia were higher in children with primary infections than in those with secondary infections (P≤0.04). Although there were no associations between the three evaluated factors and clinical severity, the levels of plasma NS1 and the frequency of dengue virus-infected monocytes correlated with prolonged coagulation times. In short, the viremia, antigenemia and infected monocytes were detected early and were not related to clinical severity. The magnitude of antigenemia and infected circulating monocytes was associated with coagulation disorders.

Immune modulation associated with vascular endothelial growth factor (VEGF) blockade in patients with glioblastoma.

Vascular endothelial growth factor (VEGF), in addition to being pro-angiogenic, is an immunomodulatory cytokine systemically and in the tumor microenvironment. We previously reported the immunomodulatory effects of radiation and temozolomide (TMZ) in newly diagnosed glioblastoma. This study aimed to assess changes in peripheral blood mononuclear cell (PBMC) populations, plasma cytokines, and growth factor concentrations following treatment with radiation, TMZ, and bevacizumab(BEV). Eleven patients with newly diagnosed glioblastoma were treated with radiation,TMZ, and BEV, following surgery. We measured immune-related PBMC subsets using multi-parameter flow cytometry and plasma cytokine and growth factor concentrations using electrochemiluminescence-based multiplex analysis at baseline and after 6weeks of treatment. The absolute number of peripheral blood regulatory T cells (Tregs) decreased significantly following treatment. The lower number of peripheral Tregs was associated with a CD4+ lymphopenia, and thus, the ratio of Tregs to PBMCs was unchanged. The addition of bevacizumab to standard radiation and temozolomide led to the decrease in the number of circulating Tregs when compared with our prior study. There was a significant decrease in CD8+ cytotoxic and CD4+ recent thymic emigrant T cells, but no change in the number of myeloid-derived suppressor cells. Significant increases in plasma VEGF and placental growth factor (PlGF) concentrations were observed. Treatment with radiation, TMZ, and BEV decreased the number but not the proportion of peripheral Tregs and increased the concentration of circulating VEGF. This shift in the peripheral immune cell profile may modulate the tumor environment and have implications for combining immunotherapy with anti-angiogenic therapy.

Changes in Killer-Cell Immunoglobulin-like Receptor (KIR) Expression on NK Cells Following DNA Demethylation By Azacitidine in Relapsed and Refractory Lymphomas

IntroductionWe conducted a study to see the activity of hypomethylating agent (HMA) in relapsed refractory lymphoma. In parallel, we studied KIR expression and promoter methylation before and after treatment with HMA. Allele-specific stochastic KIR expression is maintained in mature natural killer (NK) cells by a combination of DNA methylation and histone modification, with DNA methylation being dominant. The aim of this part of the study is to understand how KIR gene expression changes in response to epigenetic therapy, so that we then can manipulate NK cells to optimize the effect of chemo-immunotherapy of neoplastic diseases.MethodsBlood samples were collected from patients enrolled in an open-labeled phase I trial of the combination of azacitidine with cyclophosphamide, vincristine and rituximab in relapsed/refractory lymphoma.Regimen: Azacitidine on days one through five (starting at 25mg/m2), followed by oral cyclophosphamide at 300 mg/m2 on days six through nine, vincristine 1.4 mg/m2 day eight, and rituximab 375 mg/m2 day eight. Each cycle was to be repeated every 21 days, up to eight cycles if participants continued to benefit from therapy.Sampling: Between 10-20uL whole blood was collected from eligible patients before commencement of therapy and on Cycle 1 Day 5, Cycle 2 Day 1 and Cycle 3 Day 1. Peripheral blood mononuclear cells (PBMC) were isolated immediately ex vivo using Lymphocyte Separation Media (Lonza), and were divided into two aliquots: one for flowcytometry and the other for pyrosequencing (Epigendx, Hopkinton, MA).Samples from each time point were stained with fluorescently-labelled antibodies to CD3, CD16, CD56, CD14, CD20 and four different KIRs. For calculating the number of KIRs per NK cell, we used a gating strategy in which CD56dim NK cells are identified, and then the number of KIR expression is counted on those by sequential gating. The percentages of monocytes and lymphocytes among the PBMC population were also determined using CD14 and CD20 antibodies. Monocytes were further characterized according to their CD16 expression into Classical (CD14highCD16negative), Intermediate (CD14highCD16low), and Non-classical (CD14lowCD16high).ResultsPBMC from 10 patients were available for flow cytometric analysis and pyrosequencing. There were statistically significant differences (p=0.0089) in percent of DNA methylation determined by PBMC pyrosequencing, with significant decrease from Cycle 1 Day 1 to Cycle 1 Day 5 and then an increase from Cycle 1 day 5 to Cycle 2 Day 1. The decline in the percent of DNA methylation in PBMC at day 5 of cycle 1 of the chemotherapy was also found to be statistically significant (Figure 1; p=0.0202).There was a large variation in the types and numbers of KIR molecules expressed at baseline (Table 1) on CD56dim NK cells among the patients in the cohort studied, with no significant differences between the various time points and response to treatment. Despite an apparent trend of increasing percentages of non-classical and intermediate monocytes (at the expense of classical monocytes), the data was not statistically significant (Figure 2; p=0.06). We also noticed a trend of decreasing percentage of monocytes and increasing percentage of lymphocytes in the PBMC population with increasing treatment cycles. However, this was not statistically significant either.ConclusionsKIR expression may play role in HMA induced tumor cell killing. In our study, no significant changes were seen in the number of KIR receptors on NK cells post-treatment with hypomethylating agent despite observing a reduction in global DNA methylation in PBMC. This could be due to a number of reasons, including the small size of the study, the rapid turnover of NK cells, or that the reduced methylation was not sufficient to induce changes in KIR expression. The significance of the increase in the number of non-classical and intermediate monocytes following treatment with HMA is not well understood and needs further investigation.(The study was supported by Celgene) [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.