Specific cell targeting to deliver nanoparticles can be achieved by tailored modifications of the material surface with chemical moieties. The selection of the cell targets can be optimized by covering the nanoparticle with molecules, the receptor expression of which is restricted to particular cell subsets. Chemokines perform their biological action through 7-TM Gi-protein-coupled receptors differently expressed in all tissues. We decorated the surface of biocompatible polymer nanoparticles with full-length CCL5, an inflammatory chemokine that attracts leukocytes by binding CCR5, which is highly expressed in blood-circulating monocytes. Our observations showed that CCL5 functionalization does not affect the nanoparticle biocompatibility. Notably, CCL5 NPs delivered to PBMCs are selectively internalized by CCR5+ monocytes but not by CCR5- lymphocytes. The efficacy of PBMC subpopulation targeting by chemokine-decorated nanoparticles establishes an easy-to-use functionalization for specific leukocyte delivery.

Some biochemical events following the binding of prolactin (PRL) to its receptor in normal human leukocytes were investigated. PRL enhanced JAK2 phosphorylation in peripheral blood mononuclear cells (PBMC) but not in granulocytes. PRL also induced phosphorylation of Stat-5 in PBMC and Stat-1 in granulocytes. Subsequent binding of Stat-5- and of Stat-1-like molecules to a GAS responsive element from the beta-casein promoter was detected by EMSA. p38 MAPK (but not p42/p44 MAPK) was activated by PRL in both leukocyte populations. PRL induced iNOS and CIS mRNA expression in granulocytes. Increased expression of IRF-1 and SOCS-2 was observed in granulocytes and of SOCS-3 and iNOS in PBMC. Similar effects were obtained with ovine and human PRL. Antiserum to PRL reduced iNOS and IRF-1 expression induced by PRL in granulocytes and reduced iNOS expression in PBMC. Also, pretreatment of granulocytes with a p38 MAPK inhibitor (SB 203580) prevented in part PRL-induced iNOS and IRF-1 expression. In PBMC, the p38 inhibitor decreased PRL-induced iNOS gene expression. These results indicate that PRL-induced gene regulation in leukocytes requires the activation of at least two different pathways: the Stat and the MAP kinase pathways. Moreover, although PRL activates Stat in both leukocyte types, signal transduction is different in granulocytes and in PBMC. Most importantly, PRL modulates the expression of genes crucial to leukocyte function. The present findings reinforce the concept that PRL has "cytokine-like" activity in human leukocytes.

Dengue virus (DENV) co-circulation in Brazil represents a challenge for treatment and vaccine development. Despite public health impact, the occurrence of coinfections with other viruses is a common event. Increased T cell activation and altered inflammatory response are found during DENV coinfection with Human Immunodeficiency Virus (HIV) impacting HIV-pathogenesis. Even with Antiretroviral therapy (ART), HIV- treated patients had chronic immune activation and lymphocyte apoptosis. However, apoptotic mechanisms have not been investigated during coinfection with DENV. Our attention was attracted to apoptotic cell markers expressions in PBMCs from DENV and DENV/HIV coinfected patients. We found CD4/CD8 ratio inversion in most coinfected patients. CD4 T and CD8 T-cell subsets from DENV and DENV/HIV groups expressed low levels of anti-apoptotic protein Bcl-2. Furthermore, CD8 CD95 double positive cells frequency expressing low levels of Bcl-2 were significantly higher in these patients. Additionally, the density of Bcl-2 on classical monocytes (CD14++CD16-) was significantly lower during DENV infection. Upregulation of pro-apoptotic proteins and anti-apoptotic proteins were found in DENV and DENV/HIV, while catalase, an antioxidant protein, was upregulated mainly in DENV/HIV coinfection. These findings provide evidence of apoptosis triggering during DENV/HIV coinfection, which may contribute to knowledge of immunological response during DENV acute infection in HIV-patients treated with ART.

Histological chorioamnionitis (HCA) is an infection of fetal membranes and complicates 5.2% to 28.5% of all live births. HCA is associated with increased mortality and morbidity in both premature and term neonates. Exposure to HCA may have long-term consequences, including an increased risk for allergic disorders and asthma later in childhood, the mechanism(s) of which are still not yet well understood. The objective of this study was to determine the mRNA transcriptome of cord blood mononuclear leukocytes from term neonates to identify key genes and pathways involved in HCA. We found 366 differentially expressed probe IDs with exposure to HCA (198 upregulated, 168 downregulated). These transcriptomes included novel genes and pathways associated with exposure to HCA. The differential gene expression included key genes regulating inflammatory, immune, respiratory and neurological pathways, which may contribute to disorders in those pathways in neonates exposed to HCA. Our data may lead to understanding of the role of key genes and pathways identified on the long-term sequelae related to exposure to HCA, as well as to identifying potential markers and therapies to prevent HCA-associated complications.

Background: Histological chorioamnionitis (HCA) is an infection/inflammation of fetal membranes and complicates 5.2-28.5% of all live births. Exposure to HCA can have long-term consequences including abnormal neurodevelopment and an increased risk for allergic disorders and asthma later in childhood. HCA may incite epigenetic changes, which have the potential to modulate both the immune and neurological systems as well as increase the risk of related disorders later in life. However, there is limited data on the impact of HCA on epigenetics, in particular DNA methylation, and changes to immune and neurological systems in full-term human neonates. Objective: To determine differential DNA methylation in cord blood mononuclear leukocytes from neonates exposed to HCA. Methods: Cord blood was collected from 10 term neonates (5 with HCA and 5 controls without HCA) and mononuclear leukocytes were isolated. Genome-wide DNA methylation screening was performed on Genomic DNA extracted from mononuclear leukocytes. Results: Mononuclear leukocytes from cord blood of HCA-exposed neonates showed differential DNA methylation of 68 probe sets compared to the control group (44 hypermethylated, 24 hypomethylated) with a p ≤ 0.0001. Several genes involved in immune modulation and nervous system development were found to be differentially methylated. Important canonical pathways as revealed by Ingenuity Pathway Analysis (IPA) were CREB Signaling in Neurons, FcγRIIB Signaling in B Lymphocytes, Cell Cycle: G1/S Checkpoint Regulation, Interleukin-1, 2, 3, 6, 8, 10, 17, and 17A signaling, p53 signaling, dopamine degradation, and serotonin degradation. The diseases and disorders picked up by IPA were nervous system development and function, neurological disease, respiratory disease, immune cell trafficking, inflammatory response, and immunological disease. Conclusions: HCA induces differential DNA methylation in cord blood mononuclear leukocytes. The differentially methylated genes may contribute to inflammatory, immunological and neurodevelopmental disorders in neonates exposed to HCA.

The principal active component of isoforskolin (ISOF) is from the plant Coleus forskohlii, native to China, which has attracted much attention for its biological effects. We hypothesize that ISOF and forskolin (FSK) pretreatment attenuates inflammation induced by lipopolysaccharide (LPS) related to toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor kappa B (NF-κB) signaling. Mononuclear leukocytes (MLs) from healthy donors' blood samples were separated by using density gradient centrifugation. Protein levels of TLR4, MyD88, and NF-κB were detected using western blot and inflammatory cytokines interleukin (IL) 1β, IL-2, IL-6, IL-21, IL-23, tumor necrosis factor (TNF) α, and TNF-β were tested by enzyme-linked immunosorbent assay and Quantibody array in MLs. Our results showed that LPS augmented the protein levels of TLR4, MyD88, and NF-κB in MLs and the production of IL-1β, IL-2, IL-6, IL-21, IL-23, TNF-α, and TNF-β in supernatants of MLs. Despite treatment with ISOF and FSK prior to LPS, the protein levels of TLR4, MyD88, NF-κB, IL-1β, IL-2, IL-6, IL-21, IL-23, TNF-α, and TNF-β in MLs were apparently decreased. roflumilast (RF) and dexamethasone (DM) had a similar effect on MLs with ISOF and FSK. Our results, for the first time, have shown that ISOF and FSK attenuate inflammation in MLs induced by LPS through down-regulating protein levels of IL-1β and TNF-α, in which TLR4/MyD88/NF-κB signal pathway could be involved.

The aim was to investigate if hydrogen sulfide (H2S) induces the formation of the NLRP3 inflammasome and subsequent IL-1β and IL-18 secretion in human peripheral blood mononuclear cells (PBMCs) and in the human monocyte cell line THP1. Bacterial production of H2S has been suggested to participate in the inflammatory host response in periodontitis pathogenesis. H2S is a toxic gas with pro-inflammatory properties. It is produced by bacterial degradation of sulfur-containing amino acids, for example, cysteine. We hypothesize that H2S affects the inflammatory host response by inducing formation of the NLRP3 inflammasome and thereby causes the secretion of IL-1ß and IL-18. PBMCs from eight healthy blood donors, the human monocyte cell line THP1 Null, and two variants of the THP1 cell line unable to form the NLRP3 inflammasome were cultured in the presence or absence of 1 mM sodium hydrosulfide (NaHS) in 24-well plates at 37°C for 24 hr. Supernatants were collected and the IL-1β and IL-18 concentrations were measured with DuoSet ELISA Development kit. PBMCs exposed to NaHS produced more IL-1ß and IL-18 than unexposed control cells (p = .023 and p = .008, respectively). An increase of extracellular potassium ions (K+) inhibited the secretion of IL-1ß and IL-18 (p = .008). Further, NaHS triggered the secretion of IL-1ß and IL-18 in human THP1-Null monocytes (p = .0006 and p = .002, respectively), while the NaHS-dependent secretion was reduced in the monocyte cell lines unable to form the NLRP3 inflammasome. Hence, the results suggest that NaHS induces the formation of the NLRP3 inflammasome and thus the secretion of IL-1ß and IL-18. Enhanced NLRP3 inflammasome-dependent secretion of IL-1β and IL-18 in human mononuclear leukocytes exposed to NaHS in vitro is reported. This may be a mode for H2S to contribute to the inflammatory host response and pathogenesis of periodontal disease.

Dietary fish oil, rich in n-3 fatty acids (n-3 FAs), e.g. docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), regulate inflammatory reactions by various mechanisms, e.g. gene activation. However, the effects of long-term treatment with DHA and EPA in humans, using genome wide techniques, are poorly described. Hence, our aim was to determine the effects of 6 mo of dietary supplementation with an n-3 FA preparation rich in DHA on global gene expression in peripheral blood mononuclear cells.

Nanocomposition comprised of interleukin-2 in suboptimal noneffective concentration and β-cyclodextrin was studied in vitro. This preparation as well as interleukin-2 in optimal concentration was shown to increase natural killer activity to K-562 cells and cytotoxicity of activated peripheral blood mononuclear cells (PBMCs) against PC-3 and DU 145 cells. At the same time β-cyclodextrin or interleukin-2 in equimolar concentrations did not influence the spontaneous killer activity of PBMC. This combination of cyclodextrin + interleukin-2 led to the decrease of interleukin-2 effective concentration by an order. This phenomenon could be explained by cyclodextrins ability to promote the formation of nanoparticles with drugs, which results in enhancing their water solubility and bioavailability. Besides, interleukine-2/β-cyclodextrin nanocomposition as opposed to interleukin-2 alone led to increasing the number of not only lymphocytes, but also macrophages contained in activated PBMC population. Application of low concentration of interleukin-2 allowing for good clinical efficiency may significantly mitigate the side effects of the drug and enable to develop adoption of immunotherapy for patients with androgen-resistant prostate cancer.

DNA methylation changes may predispose becoming IgE-sensitized to allergens. We analyzed whether DNA methylation in peripheral blood mononuclear cells (PBMC) is associated with IgE sensitization at 5 years of age (5Y). DNA methylation was measured in 288 PBMC samples from 74 mother/child pairs from the birth cohort ALADDIN (Assessment of Lifestyle and Allergic Disease During INfancy) using the HumanMethylation450BeadChip (Illumina). PBMCs were obtained from the mothers during pregnancy and from their children in cord blood, at 2 years and 5Y. DNA methylation levels at each time point were compared between children with and without IgE sensitization to allergens at 5Y. For replication, CpG sites associated with IgE sensitization in ALADDIN were evaluated in whole blood DNA of 256 children, 4 years old, from the BAMSE (Swedish abbreviation for Children, Allergy, Milieu, Stockholm, Epidemiology) cohort. We found 34 differentially methylated regions (DMRs) associated with IgE sensitization to airborne allergens and 38 DMRs associated with sensitization to food allergens in children at 5Y (Sidak p ≤ 0.05). Genes associated with airborne sensitization were enriched in the pathway of endocytosis, while genes associated with food sensitization were enriched in focal adhesion, the bacterial invasion of epithelial cells, and leukocyte migration. Furthermore, 25 DMRs in maternal PBMCs were associated with IgE sensitization to airborne allergens in their children at 5Y, which were functionally annotated to the mTOR (mammalian Target of Rapamycin) signaling pathway. This study supports that DNA methylation is associated with IgE sensitization early in life and revealed new candidate genes for atopy. Moreover, our study provides evidence that maternal DNA methylation levels are associated with IgE sensitization in the child supporting early in utero effects on atopy predisposition.

: The main objective of this study was to assess PAG2 mRNA expression in maternal blood cells at the peri-implantation period in water buffalo; moreover, we wanted to evaluate the earliest time in which PAG-2 could be detected in maternal blood. Thirty-two lactating buffaloes artificially inseminated (AI) were utilized. Blood was collected at Days 0, 14, 18, 28, 40 after AI (AI = day 0). Pregnancy was diagnosed by ultrasound at Days 28 and 40 post AI. Out of 32 buffaloes, 14 were pregnant (P group) and 18 were not pregnant (NP group). The plasma PAG-2 threshold of 1.0 ng/mL in the P group was reached at day 40 post AI. PAG2 mRNA expression differed between the P and NP groups, and was either evaluated in Peripheral Blood Mononuclear Cells (PBMC) or Polymorphonuclear Leukocytes (PMN), starting from day 14. However, both the estimated marginal means and multiple comparisons showed that PAG2 mRNA expression was higher in PMN than PBMC. In the present study, PAG-2 appeared in the blood (40 Days post AI), and an early expression of PAG2 mRNA at Day 14 post AI was also observed. Although further research is undoubtedly required, PAG2 mRNA in peripheral blood leukocytes could be using to better understand the role that PAGs play during pregnancy in buffalo.

The present study investigates the antioxidant, anticancer, anticholinesterase, anti-genotoxic activities and phenolic contents of flower, fruit and seed methanol extracts of Hypericum retusum AUCHER. The amounts of protocatechuic acid, catechin, caffeic acid and syringic acid in methanol extracts were determined by HPLC. Total phenolic content of H. retusum seed extract was found more than fruit and flower extracts. The DPPH free radical scavenging activity of flower and seed methanol extracts showed close activity versus BHT as control. Among three extracts of H. retusum only flower methanol extract was exhibited considerable cytotoxic activities against to HeLa and NRK-52E cell lines. Moreover, seed methanol extract showed both acetyl and butyrl-cholinesterase inhibitory activity. The highest anti-genotoxic effects were seen 25 and 50 μg/mL concentrations. In this study, the extracts showed a strong antioxidant and anti-genotoxic effect. The seed extract was more efficient- than extracts of fruit and flowers. Our results suggest that the antioxidant and anti-genotoxic effects of extracts depend on their phenolic contents. Further studies should evaluate the in-vitro and in-vivo the benefits of H. retusum seed methanol extracts.

Prostasomes are membranous vesicles (150-200 nm diameter) present in human semen. They are secreted by the prostate gland and contain large amounts of cholesterol, sphingomyelin and Ca2+. In addition, some of their proteins are enzymes. Prostasomes enhance the motility of ejaculated sperm and are involved in a number of biological functions. In a previous work, we found that prostasome can fuse to spermatozoa at slightly acidic pH values, as demonstrated by the transfer of the lipophilic octadecylrhodamine probe. In this paper, we study the interactions of two leukocyte populations (polymorphonuclear and mononuclear) with prostasomes and find a pH-dependent adhesion (revealed by microscopic observation), but no fusion. These phenomena may be relevant for the functions of leukocytes in human reproduction.

The mechanisms of hematogenous leukocyte trafficking at the human blood-nerve barrier (BNB) are largely unknown. Intercellular adhesion molecule-1 (ICAM-1) has been implicated in the pathogenesis of Guillain-Barré syndrome (GBS). We developed a cytokine-activated human in vitro BNB model using primary endoneurial endothelial cells. Endothelial treatment with 10 U/ml tissue necrosis factor-α and 20 U/ml interferon-γ resulted in de novo expression of pro-inflammatory chemokines CCL2, CXCL9, CXCL11, and CCL20, with increased expression of CXCL2-3, CXCL8, and CXCL10 relative to basal levels. Cytokine treatment induced/enhanced ICAM-1, E- and P-selectin, vascular cell adhesion molecule-1 and the alternatively spliced pro-adhesive fibronectin variant, fibronectin connecting segment-1 expression in a time-dependent manner, without alterations in junctional adhesion molecule-A expression. Lymphocytes and monocytes from untreated GBS patients express ICAM-1 counterligands, α(M)- and α(L)-integrin, with differential regulation of α(M) -integrin expression compared to healthy controls. Under flow conditions that mimic capillary hemodynamics in vivo, there was a >3-fold increase in total GBS patient and healthy control mononuclear leukocyte adhesion/migration at the BNB following cytokine treatment relative to the untreated state. Function neutralizing monoclonal antibodies against human α(M)-integrin (CD11b) and ICAM-1 reduced untreated GBS patient mononuclear leukocyte trafficking at the BNB by 59% and 64.2%, respectively. Monoclonal antibodies against α(L)-integrin (CD11a) and human intravenous immunoglobulin reduced total leukocyte adhesion/migration by 22.8% and 17.6%, respectively. This study demonstrates differential regulation of α(M)-integrin on circulating mononuclear cells in GBS, as well as an important role for α(M)-integrin-ICAM-1 interactions in pathogenic GBS patient leukocyte trafficking at the human BNB in vitro.

Interferon (IFN)-alpha and IFN-gamma positive cells were revealed by flow cytometry in peripheral blood mononuclear cells (PBMC) of Specific Pathogen Free (SPF) pigs. A low prevalence of IFN-gamma positive cells was also detected in PBMC of some Porcine Reproductive and Respiratory Syndrome virus-infected pigs and uninfected, control pigs. IFN-alpha positive cells showed phenotypes of both monocytes and plasmacytoid dendritic cells. The presence of IFN-alpha in PBMC was also confirmed by Western blotting. By immunoprecipitation, IFN-alpha was detected as 32 and 55-57 kDa bands in PBMC of healthy SPF piglets. These samples were also IFN-gamma positive; the cytokine was revealed as 24, 37 and 54 kDa bands. The unusual molecular mass values of intracellular interferons were probably due to oligomerization, as previously described for human IFN-alpha. Swine intracellular IFN-alpha displayed the expected antiviral activity on bovine MDBK cells. The results indicate that interferons are constitutively expressed in swine leukocytes with peculiar molecular features.

In life sciences, antibodies are among the most commonly used tools for identifying, tracking, quantifying and isolating molecules, mainly proteins. However, it has recently become clear that antibodies often fall short with respect to specificity and selectivity and in many cases target proteins are not even known. When commercial availability of antibodies is scarce, e.g. for targeting proteins from farm animals, researchers face additional challenges: they often have to rely on cross-reactive antibodies, which are poorly characterized for their exact target, their actual cross-reactivity and the desired application. In this study, we aimed at identifying the true target of mouse monoclonal antibody 8F2, which was generated against chicken PBMC and used for decades in research, while it's actual target molecule remained unknown. We used 8F2 antibody for immunoprecipitation in chicken PBMC and subsequently identified its true target as CD11d, which was never described in chicken lymphocytes before, by quantitative LC-MSMS. The most abundant interactor of CD11d was identified as integrin beta 2. The existence of this alpha integrin was therefore clearly proven on protein level and provides a first basis to further assess the role of CD11d in chickens in future studies. Data are available via ProteomeXchange with identifier PXD017248. SIGNIFICANCE: Our studies determined CD11d as the true target of a previously uncharacterized mouse monoclonal antibody 8F2, generated against chicken peripheral blood derived mononuclear cells (PBMC). This is therefore now first member of alpha integrins in chickens, that existence was now clearly identified on protein level. The additional identification of CD11d interactors provides information on integrin-dependent regulation of signaling networks, allowing further functional studies.

The primary abnormality in Down syndrome (DS), trisomy 21, is well known; but how this chromosomal gain produces the complex DS phenotype, including immune system defects, is not well understood. We profiled DNA methylation in total peripheral blood leukocytes (PBL) and T-lymphocytes from adults with DS and normal controls and found gene-specific abnormalities of CpG methylation in DS, with many of the differentially methylated genes having known or predicted roles in lymphocyte development and function. Validation of the microarray data by bisulfite sequencing and methylation-sensitive Pyrosequencing (MS-Pyroseq) confirmed strong differences in methylation (p<0.0001) for each of 8 genes tested: TMEM131, TCF7, CD3Z/CD247, SH3BP2, EIF4E, PLD6, SUMO3, and CPT1B, in DS versus control PBL. In addition, we validated differential methylation of NOD2/CARD15 by bisulfite sequencing in DS versus control T-cells. The differentially methylated genes were found on various autosomes, with no enrichment on chromosome 21. Differences in methylation were generally stable in a given individual, remained significant after adjusting for age, and were not due to altered cell counts. Some but not all of the differentially methylated genes showed different mean mRNA expression in DS versus control PBL; and the altered expression of 5 of these genes, TMEM131, TCF7, CD3Z, NOD2, and NPDC1, was recapitulated by exposing normal lymphocytes to the demethylating drug 5-aza-2'deoxycytidine (5aza-dC) plus mitogens. We conclude that altered gene-specific DNA methylation is a recurrent and functionally relevant downstream response to trisomy 21 in human cells.

Using third harmonic generation (THG) microscopy, we demonstrate that granularity differences of leukocytes can be revealed without a label. Excited by a 1230 nm femtosecond laser, THG signals were generated at a significantly higher level in neutrophils than other mononuclear cells, whereas signals in agranular lymphocytes were one order of magnitude smaller. Interestingly, the characteristic THG features can also be observed in vivo to track the newly recruited leukocytes following lipopolysaccharide (LPS) challenge. These results suggest that label-free THG imaging may provide timely tracking of leukocyte movement without disturbing the normal cellular or physiological status.

The differential analysis of human leukocytes has many important biological and medical applications. In this work, dielectrophoretic field-flow-fractionation (DEP-FFF), a cell-separation technique that exploits the differences in the density and dielectric properties of cells, was used to separate the mixtures of the major human leukocyte subpopulations (T- and B-lymphocytes, monocytes, and granulocytes). The separation was conducted in a thin chamber equipped with an array of microfabricated interdigitated electrodes on the bottom surface, and the separation performance was characterized by on-line flow cytometry. To investigate optimal separation conditions for different leukocyte mixtures, elution fractograms at various DEP field frequencies were obtained for each leukocyte subtype. With appropriately chosen conditions, high separation performance was achieved in separating T- (or B-) lymphocytes from monocytes, T- (or B-) lymphocytes from granulocytes, and monocytes from granulocytes. DEP-FFF does not involve cell-labeling or cell-modification step, and provides a new approach to hematological analysis.

Acute myelogenous leukemia (AML) is one of the major hematological malignancies. In the human genome, several have been found to originate from retroviruses, and some of which are involved in the progression of various cancers. Hence, to investigate whether retroviral-like genes are associated with AML development, we conducted a transcriptome sequencing analysis of 12 retroviral-like genes of 150 AML patients and 32 healthy donor samples, of which RNA sequencing data were obtained from public databases. We found high expression of ERV3-1, an envelope gene of endogenous retrovirus group 3 member 1, in all AML patients examined in this study. In particular, blood and bone marrow cells of the myeloid lineage in AML patients, exhibited higher expression of ERV3-1 than those of the monocytic AML lineage. We also examined the protein expression of ERV3-1 by immunohistochemical analysis and found expression of the ERV3-1 protein in all 12 myeloid-phenotype patients and 7 out of 12 monocytic-phenotype patients, with a particular concentration observed at the membrane of some leukemic cells. Transcriptome analysis further suggested that upregulated ERV3-1 expression may be associated with chromosome 8 trisomy as anomaly was found to be more common among the high expression group than the low expression group. However, this finding was not corroborated by the immunohistochemical data. This discrepancy may have been caused, in part, by the small number of samples analyzed in this study. Although the precise associated molecular mechanisms remain unclear, our results suggest that ERV3-1 may be involved in AML development.