Cell Human Leukocyte Research Articles

Abstract 6169: Identification of molecules mediating natural killer cell-pancreatic stellate cell interactions

Abstract Pancreatic stellate cells (PSCs) are the main sources of cancer-associated fibroblasts in pancreatic ductal adenocarcinoma (PDAC), which account for at least 50% of PDAC’s total tumor volume and its dense desmoplastic stroma. In recent years, immune-stromal cell interactions have been the subject of research in a variety of diseases. Interestingly, natural killer (NK) cells are relatively understudied in PDAC and may be exploited to identify new treatment opportunities. We have explored how NK cell-PSC interactions are mediated by known NK cell ligands. Human PSC (hPSC, CRC-811) or PDAC (MIA-PaCa-2, PANC-1) cell lines were co-cultured with DiO stained NK cells (NK-92, donor NK cells) for 4 hours, then target cells were assessed for apoptosis by flow cytometry using AnnexinV and Sytox stains. We found that although NK cells lyse both PSCs and PDAC cells, there was more NK cell-mediated lysis against PSCs. Using flow cytometry, we found that PSCs express the well-known MICA/B ligand, which binds the activating NKG2D receptor on NK cells to initiate its cytotoxic activity. In co-culture, neutralizing MICA/B in vitro did not completely inhibit NK cell-mediated lysis of PSCs, suggesting other interactions need to be explored. To identify other molecular determinants, NK cell ligand and receptor expression was evaluated by quantitative PCR in human PSC, PDAC, NK, and other leukocytic (K562) cell lines. Cell surface validation of highly expressed NK cell ligands and receptors were confirmed by flow cytometry and/or Western blot. NK cell ligands that were both highly expressed and localized to the cell surface were: NKG2D ligands—MICA and ULBP2 (both activating), KIR2DS4 (CD158I) ligand—HLA-A (activating), and NKp44 ligands—NKp44L and PCNA (activating and inhibitory, respectively). Their corresponding receptors were also expressed and localized to the cell surface by NK cells. These results highlight novel NK cell-PSC interactions that may mediate immune suppression in PDAC. These findings warrant further investigation. Citation Format: Zoe X. Malchiodi, Allison Fitzgerald, Elena Galvano, Louis M. Weiner. Identification of molecules mediating natural killer cell-pancreatic stellate cell interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6169.

Synergy between Pelargonium endlicherianum essential oil and conventional antibiotics against Neisseria meningitidis and Haemophilus influenzae

This study investigated the bactericidal effects of the essential oil of Pelargonium endlicherianum in combination with four antibiotics commonly used in the treatment of bacterial meningitis (penicillin, ampicillin, ciprofloxacin, and gentamicin) against the meningitis causative pathogens Neisseria meningitidis and Haemophilus influenzae. The phagocytic effects of these combinations were also tested against human leukocyte cells. The bactericidal effect of P. endlicherianum essential oil (PEO) and antibiotic combinations was dynamically detected by time-kill assay. The function of PEO and antibiotic in permeating outer membrane barriers, when used singly or in combination, was analyzed by UV spectrophotometer.The interactions between antibiotic and essential oil were calculated according to the fractional inhibitory concentration (FIC) index. While a synergistic effect of the ciprofloxacin + PEO combination was determined on N. meningitidis (FIC≤ 0.5), an additive effect was observed on H. influenzae (FIC= 1). Combined use of PEO with gentamicin showed a synergistic effect against N. meningitidis and H. influenzae (FIC ≤ 0.5). The antimicrobial effect of the penicillin + PEO combination was higher than that of penicillin + PEO used alone. The ampicillin + PEO combination had a synergistic effect on N. meningitidis and an additive effect on H. influenzae. The results of our study showed that the essential oil increases membrane permeability activity and also has phagocytic activity in human leukocyte cells. Combining antibiotics with essential oils that target resistant bacteria may open up new options in combating microbial resistance.

Activation of the Constitutive Androstane Receptor Inhibits Leukocyte Adhesiveness to Dysfunctional Endothelium.

Leukocyte cell recruitment into the vascular subendothelium constitutes an early event in the atherogenic process. As the effect of the constitutive androstane receptor (CAR) on leukocyte recruitment and endothelial dysfunction is poorly understood, this study investigated whether the role of CAR activation can affect this response and the underlying mechanisms involved. Under physiological flow conditions, TNFα-induced endothelial adhesion of human leukocyte cells was concentration-dependently inhibited by preincubation of human umbilical arterial endothelial cells with the selective human CAR ligand CITCO. CAR agonism also prevented TNFα induced VCAM-1 expression, as well as MCP-1/CCL-2 and RANTES/CCL-5 release in endothelial cells. Suppression of CAR expression with a small interfering RNA abrogated the inhibitory effects of CITCO on these responses. Furthermore, CITCO increased interaction of CAR with Retinoid X Receptor (RXR) and reduced TNFα-induced p38-MAPK/NF-κB activation. In vivo, using intravital microscopy in the mouse cremasteric microcirculation treatment with the selective mouse CAR ligand TCPOBOP inhibited TNFα-induced leukocyte rolling flux, adhesion, and emigration and decreased VCAM-1 in endothelium. These results reveal that CAR agonists can inhibit the initial inflammatory response that precedes the atherogenic process by targeting different steps in the leukocyte recruitment cascade. Therefore, CAR agonists may constitute a new therapeutic tool in controlling cardiovascular disease-associated inflammatory processes.

Structural and morphological characterization of Ti6Al4V alloy surface functionalization based on Nb2O5 thin film for biomedical applications

This paper proposes an applied and innovative research for the development of Nb2O5 thin film on the Ti6Al4V alloy surface with great prospects of being used in the biomedical area. Thin films of Nb2O5 have been deposited on the Ti6Al4V alloy surface by using the reactive sputtering technique. The system was characterized morphologically and structurally via SEM/EDX, AFM, FTIR, Raman spectroscopy and XPS. Interfacial tension theory was used for the determination of the surface free energy and showed that the treated surface became more hydrophilic when compared to the bare material. The high-resolution X-ray photoelectron spectra from Nb 3d and O 1s core-levels demonstrate unequivocally the presence of a single-phase Nb2O5. Cytotoxicity and biocompatibility (VERO-CCL-81 cell line) tests as well as analyzes of inflammatory responses with Human blood leukocyte cells were also performed by showing that Nb2O5 coating reduced both Ti6Al4V alloy toxicity and its inflammation induction besides increasing the anti-inflammatory cytokine levels. Since the surface functionalization resulted in both higher biocompatibility and tolerance by immune cells besides low inflammatory levels concerning the base material, we present a superior material with wide spectra of usage in the biomedical area.

Sensitive Immunopeptidomics by Leveraging Available Large-Scale Multi-HLA Spectral Libraries, Data-Independent Acquisition, and MS/MS Prediction

Mass spectrometry (MS) is the state-of-the-art methodology for capturing the breadth and depth of the immunopeptidome across human leukocyte antigen (HLA) allotypes and cell types. The majority of studies in the immunopeptidomics field are discovery driven. Hence, data-dependent tandem MS (MS/MS) acquisition (DDA) is widely used, as it generates high-quality references of peptide fingerprints. However, DDA suffers from the stochastic selection of abundant ions that impairs sensitivity and reproducibility. In contrast, in data-independent acquisition (DIA), the systematic fragmentation and acquisition of all fragment ions within given isolation m/z windows yield a comprehensive map for a given sample. However, many DIA approaches commonly require generating comprehensive DDA-based spectrum libraries, which can become impractical for studying noncanonical and personalized neoantigens. Because the amount of HLA peptides eluted from biological samples such as small tissue biopsies is typically not sufficient for acquiring both meaningful DDA data necessary for generating comprehensive spectral libraries and DIA MS measurements, the implementation of DIA in the immunopeptidomics translational research domain has remained limited. We implemented a DIA immunopeptidomics workflow and assessed its sensitivity and accuracy by matching DIA data against libraries with growing complexity—from sample-specific libraries to libraries combining 2 to 40 different immunopeptidomics samples. Analyzing DIA immunopeptidomics data against a complex multi-HLA spectral library resulted in a two-fold increase in peptide identification compared with sample-specific library and in a three-fold increase compared with DDA measurements, yet with no detrimental effect on the specificity. Furthermore, we demonstrated the implementation of DIA for sensitive personalized neoantigen discovery through the analysis of DIA data with predicted MS/MS spectra of clinically relevant HLA ligands. We conclude that a comprehensive multi-HLA library for DIA approach in combination with MS/MS prediction is highly advantageous for clinical immunopeptidomics, especially when low amounts of biological samples are available.

Synergic potential of Pelargonium endlicherianum Fenzl. Essential oil and antibiotic combinations against Klebsiella pneumoniae

In this study we investigated antimicrobial activity against Klebsiella pneumoniae and the phagocytic functions of human leukocyte cells as revealed in an in vitro experimental model combining cefepime and gentamicin with Pelargonium endlicherianum Fenzl. essential oil treatments. The bactericidal effects of this essential oil and antibiotic combinations were dynamically detected by time-kill assay. To examine the function of this essential oil and antibiotics in permeating outer membrane barriers when used singly or in combination, a UV spectrophotometer was used, and morphologic images were captured by scanning electron microscopy.The antibacterial activity of the essential oil and antibiotics was assessed using broth microdilution and agar well diffusion. The combined effects of the essential oils of P. endlicherianum and gentamicin and cefepime were evaluated by means of the checkerboard method against K. pneumoniae. In the assays, fractional inhibitory concentration (FIC) values were calculated to characterize the interactions between the combinations. In the combinations of essential oil and antibiotics, the sensitivity of the bacteria to antibiotics increased and the antibiotics had a synergistic effect, and the antibacterial effect on the microorganisms increased. The cefepime + essential oil pair tested showed a synergistic effect (FIC ≤ 0.5), but the gentamicin + essential oil pair did not (FIC > 0.5–4.0). Thus, the cefepime + essential oil pair has been found to exhibit a synergistic effect against K. pneumoniae compared to the gentamicin + essential oil pair. According to the results obtained here, the combined use of essential oils with antibiotics can be applied as a treatment strategy to reduce the use of antibiotics, reduce side effects, and possibly reverse antibiotic resistance to these microorganisms in light of the increase in multiple antibiotic resistance.

Biological effects, including oxidative stress and genotoxic damage, of polystyrene nanoparticles in different human hematopoietic cell lines

In recent years the terms "micro-/nanoplastics" (MNPLs) have caught special attention due to the increasing levels by which humans are exposed. Among MNPLs, polystyrene nanoparticles (PSNPs) are one of the most represented MNPLs in the environment. These tiny particles may enter into the human body, translocate through human barriers, interacting with blood and lymphatic immune cells, and reaching secondary organs. By using three different human leukocytic cell lines: Raji-B (B-lymphocytes), TK6 (lymphoblasts) and THP-1 (monocytes), we pursued to determine the effects of these PSNPs on the immune cell population. With this aim, the three cell lines were exposed to spherical PSNPs of about 50 nm of diameter and cytotoxicity, cellular uptake, reactive oxygen species (ROS) production, and genotoxicity were assessed at different time-points. Results show differences in all the measured endpoints, among the selected cell lines. Thus, whilst the monocytic THP-1 cells showed the highest particle internalization, no adverse effects were observed in such cells. On the other side, although Raji-B and TK6 showed lesser PSNPs uptake, mild toxicity, ROS production and genotoxicity were detected. These results highlight the importance of the cell line selection when the biological effects of PSNPs are evaluated.

Salix alba (white willow) medicinal plant presents genotoxic effects in human cultured leukocytes

ABSTRACTSalix alba (SA), commonly known as white willow, is a plant used in folk medicine for the treatment of chronic and acute inflammation, infection, pain, and fever. The phytochemical characterization of the bark extract of this plant indicated that its main component is salicin, a precursor of the anti-inflammatory agent acetylsalicylic acid. Considering the lack of studies evaluating the genetic toxicity and cytotoxic action of SA bark extract on human cells, as well as the chemical characterization of its major phenolic compounds, the present study was designed to (1) investigate the cytotoxic and genotoxic potential of SA bark extract on human peripheral leukocyte cells and human hepatoma cell line HepG2, and (2) characterize its major phenolic constituents. The phenolic compounds found were salicylic acid, salicin, salidroside, saligenin, tremulodin, salicoylsalicin, salicortin, and tremulacin. The results using trypan blue staining test showed viability decreases (viability less than 70%) for concentrations of SA extract equal and higher to 200 µg/ml. Low genotoxic activity (comet assay) was exhibited for 50 and 100 µg/ml SA extract in human leukocytes. SA did not exert a marked clastogenic/aneugenic effect on leukocytes and HepG2 human cells. Data suggest that the genotoxic effects of SA bark extract occur when it is not metabolized by liver enzymes.

Human Leukocyte Antigen and Red Blood Cells Impact Umbilical Cord Blood CD34+ Cell Viability after Thawing.

Hematopoietic progenitor cell (HPC) transplantation is a treatment option for malignant and nonmalignant diseases. Umbilical cord blood (UCB) is an important HPC source, mainly for pediatric patients. It has been demonstrated that human leukocyte antigen (HLA) matching and cell dose are the most important features impacting clinical outcomes. However, UCB matching is performed using low resolution HLA typing and it has been demonstrated that the unnoticed mismatches negatively impact the transplant. Since we found differences in CD34+ viability after thawing of UCB units matched for two different patients (p = 0.05), we presumed a possible association between CD34+ cell viability and HLA. We performed a multivariate linear model (n = 67), comprising pre-cryopreservation variables and high resolution HLA genotypes separately. We found that pre-cryopreservation red blood cells (RBC), granulocytes, and viable CD34+ cell count significantly impacted CD34+ viability after thawing, along with HLA-B or -C (R2 = 0.95, p = 0.01; R2 = 0.56, p = 0.007, respectively). Although HLA-B*40:02 may have a negative impact on CD34+ cell viability, RBC depletion significantly improves it.

Design, synthesis, and evaluation of novel 2-substituted 1,4-benzenediol library as antimicrobial agents against clinically relevant pathogens

Development of new antimicrobial agents, capable of combating resistant and multidrug-resistant fungal and bacterial clinical strains, is necessary. This study presents the synthesis and antimicrobial screening of 42 2-substituted-1,4-benzenediols, being 10 novel compounds. In total, 23 compounds showed activity against fungi and/or bacteria. Benzenediol compounds 2, 5, 6, 8, 11, and 12 demonstrated broad spectrum antimicrobial actions, including resistant and multidrug-resistant species of dermatophytes (Trichophyton mentagrophytes), Candida spp. and the ESKAPE panel of bacteria. Minimum inhibitory concentrations of these compounds for fungi and bacterial strains ranged from 25 to 50 µg/ml and 8–128 µg/ml, respectively. The antifungal mechanism of action is related to the fungal cell wall of dermatophytes and membrane disruption to dermatophytes and yeasts, in the presence of compound 8. Specific structural changes, such as widespread thinning along the hyphae and yeast lysis, were observed by scanning electron microscopy. The effects of compound 8 on cell viability are dose-dependent; however they did not cause genotoxicity and mutagenicity in human leukocyte cells nor haemolysis. Moreover, the compounds were identified as nonirritant by the ex-vivo Hen’s egg test-chorioallantoic membrane (HET-CAM). The furan-1,4-benzenediol compound 5 showed in vivo efficacy to combat S. aureus infection using embryonated chicken eggs. Therefore, the compounds 8, and 5 are promising as hits for the development of new antimicrobial drugs with reduced toxicity.

Analysis of DNA Damage Biomarkers in Human Leukocytes by PAHs Exposure

Aims: To study the potential genotoxicity of two polynuclear aromatic hydrocarbons (PAHs), exposed cultured human leukocytes in vitro using two types of biomarkers genotoxicity: DNA strand breaks (DNA-SB) and adduct formation (DNA-PAHs).
 Study Design: Human leukocytes were exposed to toxic cultures with different concentrations of anthracene (ANT), phenanthrene (PHE) and benzo(a)pyrene (B(a)P) for 24 hours. Four toxic test groups, PAHs, control group, analytic blank group and standard fluorescence group were considered. 
 Place and Duration of Study: Laboratorio de Citopatología Ambiental, of Departamento de Morfología at Escuela Nacional de Ciencias Biológicas, IPN; between february 2016 and july 2018.
 Methodology: Human leukocytes cells were isolated and cell viability was previously verified by Trypan dye exclusion test. Firstly, the lethal concentrations with neutral red (NR50) assay for each one PAHs was obtained. Then sublethal concentrations range of these toxics for both biomarkers were used. In case of DNA fragmentation, a fluorochrome was used to mark DNA strandbreaks and isolation with alkaline solution finally determined with fluorescence spectroscopy. To test the formation of DNA-PAHs adducts, first of all they were isolated with a solvent system with polarity gradient and finally determined with fluorescence spectroscopy.
 Results: PAHs with 3 aromatic rings showed lethal cytotoxicity, lower in case of the B(a)P with 5 aromatic rings. These results are in contrast with previously reported observations, in which non-adduct with DNA and DNA strandbreaks formation were detected with Anthracene and Phenanthrene while DNA adduction formation and DNA strandbreaks were produced in case of B(a)P.
 Conclusion: Biomarkers may be used as suitable discriminants of genotoxic agents as well as of environmental pollutants with genotoxic potential and for application in studies of environmental risk assessment and in hazardous waste evaluation. The application of both genotoxic biomarkers, DNA strand breaks and production of adducts DNA-PAHs, as genotoxicity assays are quickly and accurate techniques for determining the carcinogenic potential of environmental samples.

Molecular Pathways Associated with Sperm Biofunction Are Not Affected by the Presence of Round Cell and Leukocyte Proteins in Human Sperm Proteome.

In human sperm proteomic experiments, leukocyte and round cell proteins may contaminate the sperm proteome and affect the bioinformatic results. The main objective of this study was to identify the possible interference of these proteins, especially from leukocytes, in identification of sperm functional pathways through proteomic and bioinformatic tools. We have evaluated the sperm proteome by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in four groups: (1) neat semen with round cells and leukocytes ≥1 × 106/mL; (2) samples with round cells and leukocytes ≥1 × 106/mL processed by 65% density gradient centrifugation; (3) neat semen with round cells <1 × 106/mL; and (4) samples with round cells <1 × 106/mL processed by 65% density gradient centrifugation. Pure leukocyte culture was used as a control group. The difference in the conserved DEPs (common to both sperm and leukocytes) between the sperm samples with leukocytes ≥1 × 106/mL and round cells <1 × 106/mL was negligible. Comparative analysis between groups 1, 2, 3, and 4 with the control group revealed that the presence of leukocyte proteins does not significantly alter the activation z-score of the identified canonical pathways or biological functions in sperm proteome. Our experimental results demonstrate that the presence of round cell and leukocyte proteins do not affect the identification of the molecular pathways associated with human spermatozoa protein function. Hence, the use of neat frozen semen samples for proteomic studies showed no significant impact on the downstream bioinformatic analysis.

Defining the function of a novel TNF receptor superfamily member

Background: &#x0D; Insulin Growth Factor Like 1 Receptor (IGFLR1) is a putative TNF receptor superfamily member of unknown function. Transcripts for IGFLR1 are most abundant in T cells in mice and several leukocyte subsets in humans. A major limitation to understanding IGFLR1 function is the lack of biochemical data regarding IGFLR1 expression and processing in leukocytes. &#x0D; Experimental Design: &#x0D; A panel of human leukocyte and keratinocyte cell lines was screened for IGFLR1 transcript expression by quantitative PCR. Immunoblotting was then used to detect IGFLR1 protein and characterize which isoform(s) were expressed. Chimeric IGFLR1 construct that could be used to mimic IGFLR1 activation was also generated. This chimeric protein, hNGFR-DmrB2- hIGFLR, contains two DmrB domains that allow for multimerization in the presence of AP20187. &#x0D; Results: &#x0D; IGFLR1 transcript expression varied between hematopoietic cell lines with relatively low expression in MOLT4 cells (T cell line), intermediate expression in IM9 and ARH77 cells (B cell lines) and high expression in MKB cells (T cell line). Immunoblotting in these cell lines revealed a similar protein signal expression pattern. Immunoblotting also suggested that the most abundant isoform of IGFLR1 was likely isoform c, which lacks ligand-binding and transmembrane domains. With respect to the hNGFR-DmrB2-hIGFLR1 construct, the chimeric protein was detected in CHO cells by immunoblotting and biochemical analyses confirmed that addition of AP20187 induced multimerization of this protein. These findings confirm the functionality of hNGFR-DmrB2-hIGFLR1. &#x0D; Potential Impact: &#x0D; Biochemical studies of endogenously expressed IGFLR1 in these cell lines suggest differential mRNA splicing may play an important role in the production and function of IGFLR1 isoforms. Future studies using the hNGFR-DmrB2-hIGFLR1 construct in the aforementioned human cell lines can be performed to study IGFLR1 signaling.

Novel Models to Study Stromal Cell-Leukocyte Interactions in Health and Disease.

To study human immunology in general and stromal immunology in particular, it is highly motivated to move from monolayers to 3D cultures, such as organotypic models, that better mimic the function of living tissue. These models can potentially contain most if not all cell types present in tissues, in combination with different extracellular matrix components that can critically affect cell phenotype. Besides their well-established use in studies of tissue-specific cells, such as epithelial cells, endothelial cells and stromal fibroblasts in combination with extracellular components, these models have also been shown to be valuable to study how tissue participates in the regulation of leukocyte differentiation and function. Organotypic models with leukocytes represent novel powerful tools to study human stromal immunology and mechanisms involved in the regulation of leukocyte functions and inflammatory processes in human health and disease. In particular, these models are robust, long-lived and reproducible and allow monitoring of disease progression in real time, as well as the mixing of cellular constituents from healthy and pathological tissues. These models are also easy to manipulate, either genetically or by adding external stimulants, such as cytokines and pathogens, to mimic pathological conditions. It is thus not surprising that these models are proposed to be useful in toxicology screening assays, evaluating therapeutic efficacy of drugs and antibiotics, as well as in personalized medicine. Within this chapter, the most recent developments in creating organotypic models for the purpose of study of human leukocyte and stromal cell interactions, in health and disease, will be discussed, in particular focusing on live imaging. Special emphasis will be given on an organotypic model resembling human lung and its usefulness in studying the fine control of physiological and pathological processes in human health and disease. Using these models in studies on human stromal cell and leukocyte interactions will likely help identifying novel disease traits and may point out new potential targets to monitor and treat human diseases.

&lt;b&gt;Cd, Cu, and Mn from Uruguay River Basin in Uruguaiana, RS, Brazil, and their toxicological potential for human leukocyte

This study assessed the limnology from the Medium Uruguay River Basin in Uruguaiana, Brazil, with a focus on the concentration of heavy metals (Cd, Cu, and Mn), to assess the toxicological potential (cytotoxicity and genotoxicity) for humans using as biological matrix of study human leukocyte cells. The conductivity, resistivity, and dissolved O 2 levels exceeded the limits recommended by the National Environmental Council ( Conselho Nacional do Meio Ambiente - CONAMA). The percentage of non-viable human leukocyte cells exposed to water samples was approximately 20% higher than that of the negative control (<3%), but similar to the positive control. The DNA damage index was high for all heavy metal concentrations assayed when compared to the negative control 12±2.96, p < 0.0001, with a range of 155.66±23.89 to 194.33±23.23, but similar to the positive control (210.62±27.48). Moreover, the leukocyte degeneration index was higher in all samples containing heavy metals than in the negative control (4%), which demonstrates to be due the presence of Cu (11.8-12.5%), Cd (13-15.6%), and Mn (15.6-22.5%). Taken together, our results show that the quality from water samples analyzed is below than recommended by CONAMA and offers risk of contamination by heavy metals for the general population.

Urinary antimony and leukocyte telomere length: An analysis of NHANES 1999–2002

Telomeres are repetitive DNA sequences (TTAGGG) at the end of chromosomes. Cells with critically short telomeres enter replicative senescence and apoptosis. Several in vitro studies report that antimony causes cell apoptosis in human leukocyte cell lines. The goal of this analysis was to investigate whether there is an association between antimony exposure and leukocyte telomere length (LTL) among US adults aged 20 and older based on the National Health and Nutrition Examination Survey (NHANES) 1999–2002. We used multivariate linear regression to analyze the association of urinary antimony with LTL. LTL was log-natural transformed and the results were re-transformed and presented as percent differences. After adjustment for potential confounders, individuals in the 3rd and 4th quartiles of urinary antimony had statistically significantly shorter LTL (−4.78%, 95% CI: −8.42,−0.90; and −6.11%, 95% CI: −11.04,−1.00, respectively) compared to the lowest referent quartile, with evidence of a dose-response relationship (p-value for trend =0.03). Shorter LTL with antimony was driven by middle aged (40–59 years) and older (60–85 years) adult groups. The association may be biologically plausible because of reported oxidative stress and apoptosis effects of antimony on blood cells, effects known to shorten telomere length.

The role of sepiolite and palygorskite on the migration of leukocyte cells to an inflammation site

Sepiolite and palygorskite have shown beneficial health effects but understanding human cell-clay interactions has yet to become unveiled. This paper reports on the effects of sepiolite (Vallecas, Spain) and palygorskite (Torrejon El Rubio, Spain) on the infiltration of human blood leukocytes to an infiltration site. Quantification of human blood leukocyte cells under pro- and anti-inflammatory conditions was conducted, and cells visualized in an Axioscope (Carl Zeiss; Oberkochen, Germany). Images were recorded with an Axiocam Mrm monochromatic camera and ZEN Pro software (Carl Zeiss). The distribution of human blood leukocyte cells at the inflammation site varied before and after adding the clay. The relative proportion of PMN-to-monocytes(MN) (PMN/MN) exposed to the inflammatory activity by 12-O-tetradecanoylphorbol-13-acetate (TPA) changed in the presence of sepiolite (TPA+sepiolite) or palygorskite (TPA+palygorskite) either after 4 or 24h, namely, 0.60, 2.5, and 2.33; and 4.33, 1.53, and 2.8, respectively. PMN/MN values compared in the presence of TPA or TPA and palygorskite, however decreased sharply in the presence of TPA and sepiolite. Proposedly, decreases in PMN/MN values caused by adding sepiolite may alter PMN and MN immunological functions, by lessening the destruction extent of invasive bacteria via phagocytosis and the conversion of MN to macrophages. Proposedly, limiting a conversion of MN to macrophages impedes resolving inflammation because of an incomplete digestion of aged cells. Evidently, shifting from pro- to anti-inflammatory conditions due to the addition of the clay altered the mechanism of infiltration of different leukocyte cells to an inflammation site. Finally, the presence of few macrophages at the inflammation site was attributed to resolution of inflammation, whereby macrophages participated in anti-inflammatory mechanisms leading to the return to homeostasis in tissues.

Imidazolium salts with antifungal potential against multidrug-resistant dermatophytes

To investigate the antidermatophytic action of a complementary set imidazolium salts (IMS), determining structure-activity relationships and characterizing the IMS toxicological profiles. The susceptibility evaluation of 45 dermatophytic clinical isolates, treated in vitro with eleven different IMS (ionic compounds) and commercial antifungals (nonionic compounds), was performed by broth microdilution, following the standard norm of CLSI M38-A2. All dermatophytes were inhibited by IMS, where the lowest minimum inhibitory concentration (MIC) values were observed for salts with n-hexadecyl segment in the cation side chain, containing either the chloride or methanesulfonate anion. 1-n-Hexadecyl-3-methylimidazolium chloride (C16 MImCl) and 1-n-hexadecyl-3-methylimidazolium methanesulfonate (C16 MImMeS) acted as fungicides, even in extremely low concentrations, wherein C16 MImMeS exerted this effect on 100% of the tested dermatophytes. Some of these IMS provoked evident alterations on the fungi cell morphology, causing a total cell damage of ≥ 70%. Importantly, none of the screened IMS were cytotoxic, mutagenic or genotoxic to human leucocyte cells. This report demonstrates for the first time the strong antifungal potential of IMS against multidrug-resistant dermatophytes, without presenting toxicity to human leucocyte cells at MIC. The expressive antifungal activity of IMS, combined with the in vitro nontoxicity, makes them promising compounds for the safe and effective treatment of dermatophytoses, mainly when this skin mycosis is unresponsive to conventional drugs.

&lt;b&gt;&lt;em&gt;In vitro&lt;/em&gt; ZnCl&lt;sub&gt;2&lt;/sub&gt; cytotoxicity and genotoxicity in human leukocytes: Zero-order kinetic cellular zinc influx

Zinc (Zn) is an essential trace element for cellular viability, but concentrations above physiologic level may lead to cellular damage. The purpose of the present study was to evaluate the in vitro ZnCl 2 genotoxicity and cytotoxicity in human leukocyte cells. This was assessed in an unprecedented way that correlated the level of intracellular Zn after cell exposition with the cellular damage. The exposure to increased Zn concentrations (2.5-20 µg mL -1 ), showed significantly reduced cellular leukocyte viability. However, significant DNA damages were observed only when the Zn exposure concentrations were from 10-20 µg mL -1 . The Zn intracellular levels found in leukocytes was from 72.25-268.9 ρg cell -1 , starting to induce cytotoxicity and genotoxicity at concentrations of 95.68 and 126.2 ρg cell -1 , respectively. The relationship between the exposure concentration and intracellular levels of Zn suggests that the influx of Zn, in the form of ZnCl 2 , occurs in human leukocytes under zero-order kinetics.

A Human-Specific α7-Nicotinic Acetylcholine Receptor Gene in Human Leukocytes: Identification, Regulation and the Consequences of CHRFAM7A Expression.

The human genome contains a variant form of the α7-nicotinic acetylcholine receptor (α7nAChR) gene that is uniquely human. This CHRFAM7A gene arose during human speciation and recent data suggests that its expression alters ligand tropism of the normally homopentameric human α7-AChR ligand-gated cell surface ion channel that is found on the surface of many different cell types. To understand its possible significance in regulating inflammation in humans, we investigated its expression in normal human leukocytes and leukocyte cell lines, compared CHRFAM7A expression to that of the CHRNA7 gene, mapped its promoter and characterized the effects of stable CHRFAM7A overexpression. We report here that CHRFAM7A is highly expressed in human leukocytes but that the levels of both CHRFAM7A and CHRNA7 mRNAs were independent and varied widely. To this end, mapping of the CHRFAM7A promoter in its 5'-untranslated region (UTR) identified a unique 1-kb sequence that independently regulates CHRFAM7A gene expression. Because overexpression of CHRFAM7A in THP1 cells altered the cell phenotype and modified the expression of genes associated with focal adhesion (for example, FAK, P13K, Akt, rho, GEF, Elk1, CycD), leukocyte transepithelial migration (Nox, ITG, MMPs, PKC) and cancer (kit, kitL, ras, cFos cyclinD1, Frizzled and GPCR), we conclude that CHRFAM7A is biologically active. Most surprisingly however, stable CHRFAM7A overexpression in THP1 cells upregulated CHRNA7, which, in turn, led to increased binding of the specific α7nAChR ligand, bungarotoxin, on the THP1 cell surface. Taken together, these data confirm the close association between CHRFAM7A and CHRNA7 expression, establish a biological consequence to CHRFAM7A expression in human leukocytes and support the possibility that this human-specific gene might contribute to, and/or gauge, a human-specific response to inflammation.