Chemotactic Properties Research Articles

LL-37: Cathelicidin-related antimicrobial peptide with pleiotropic activity.

Antimicrobial peptides (AMPs) is a large family of compounds serving as natural antibiotics, widely distributed across the organism, mainly in mucus layers. They are designed to prevent pathogens from colonization. Among them, defensins and cathelicidins could be found. LL-37, the sole human cathelicidin draws particular attention because of its outstanding abilities. In addition to being a broad spectrum antibiotic, LL-37 has potent chemotactic and immunomodulatory properties. In this review, we discussed the potency of LL-37 as a therapeutic agent in four systems: immunological, respiratory, gastrointestinal and in the skin. We analyzed the main molecular pathways dependent on human cathelicidin and related them to specific diseases. We conclude that LL-37 shows a great potential to be further investigated and developed as a drug with clinical use.

Differential lipid metabolism in monocytes and macrophages: influence of cholesterol loading

The influence of the hypercholesterolemia associated with atherosclerosis on monocytes is poorly understood. Monocytes are exposed to high concentrations of lipids, particularly cholesterol and lysophosphatidylcholine (lyso-PC). Indeed, in line with recent reports, we found that monocytes accumulate cholesteryl esters (CEs) in hypercholesterolemic mice, demonstrating the need for studies that analyze the effects of lipid accumulation on monocytes. Here we analyze the effects of cholesterol and lyso-PC loading in human monocytes and macrophages. We found that cholesterol acyltransferase and CE hydrolase activities are lower in monocytes. Monocytes also showed a different expression profile of cholesterol influx and efflux genes in response to lipid loading and a different pattern of lyso-PC metabolism. In monocytes, increased levels of CE slowed the conversion of lyso-PC into PC. Interestingly, although macrophages accumulated glycerophosphocholine, phosphocholine was the main water-soluble choline metabolite being generated in monocytes, suggesting a role for mono- and diacylglycerol in the chemoattractability of these cells. In summary, monocytes and macrophages show significant differences in lipid metabolism and gene expression profiles in response to lipid loading. These findings provide new insights into the mechanisms of atherosclerosis and suggest potentials for targeting monocyte chemotactic properties not only in atherosclerosis but also in other diseases.

Antimicrobial peptides from mussel Mytilus galloprovincialis: The myticins

Antimicrobial peptides (AMPs) have been found in different bivalve species but a remarkable abundance of these small peptides has been reported for the Mediterranean mussel, Mytillus galloprovincialis. In particular, the myticin C (Myt C) is the most abundantly expressed gene in cDNA libraries and also in Next generation sequencing projects after immune stimulation. This AMP presents a high genetic variability but until now the mechanism to generate it is unknown. Myt C is mainly present in hemocytes and also in serum where it is constitutively found in proteomic studies. Also, Myt C modulates the immune system because its overexpression is able to alter the expression of other mussel immune-related genes. Our results indicate that Myt C has antibacterial, antiviral and chemotactic properties being active across species due to its strong antiviral activity against a fish rhabdovirus. All these results suggest that this molecule should be considered not only as an AMP but also as the first chemokine/cytokine-like molecule identified in bivalves and one of the few examples in all of the invertebrates.

(7R,8S)-9-Acetyl-dehydrodiconiferyl alcohol inhibits inflammation and migration in lipopolysaccharide-stimulated macrophages

Background(7R, 8S)-9-Acetyl-dehydrodiconiferyl alcohol (ADDA), a novel lignan compound isolated from Clematis armandii Franch (Ranunculaceae) stems, has been found to exert potential anti-inflammatory activities in vitro. PurposeTo investigate the pharmacological effects and molecular mechanisms of ADDA on lipopolysaccharide (LPS)-induced activation and migration of macrophages. Study design/methodsMacrophages were stimulated with LPS in the presence or absence of ADDA. Expression of inflammatory mediators, including cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nitric oxide (NO) were measured by Western blot and commercial NO detection kit. Cellular viability and chemotactic properties of macrophages were investigated using MTT and transwell migration assays. The activation and expression of mitogen activated protein kinases, nuclear factor-κB (NF-κB), protein kinase B (Akt), Src, and focal adhesion kinase (FAK) were analyzed by Western blot. ResultsNon-toxic concentrations (12.5–50µM) of ADDA concentration-dependently inhibited expression/release of inflammatory mediators (COX-2, iNOS, and NO), suppressed Akt and c-jun N-terminal kinase 1/2 (JNK) phosphorylation, and NF-κB activation in LPS-stimulated macrophages. In addition, ADDA blocked LPS-mediated macrophage migration and this was associated with inhibition of LPS-induced Src and FAK phosphorylation as well as Src expression in a concentration dependent manner. Notably, the inhibitory effects of ADDA on iNOS, NO, and Src could be mimicked by a Src inhibitor PP2 or an iNOS inhibitor l-NMMA. ConclusionOur results suggested that ADDA attenuated LPS-induced inflammatory responses in macrophages and cell migration, at least in part, through inhibition of NF-κB activation and modulation of iNOS/Src/FAK axis.

Liquid and Gel Platelet Rich Plasma as Skin Healing Adjuvant

Background: In recent decades, many researches have been conducted on processes involved in tissue repairing, mainly in the development of resources and technology designed to improve the wound healing progress. Platelet rich plasma (PRP) derived from autologous blood is defined as a plasma volume with platelet concentration higher than physiological level. It is an autogenous and low cost source of growth factors, which are essential for tissue regeneration due to their angiogenic, mitogenic, and chemotactic properties. The aim of this study was evaluate two forms of PRP- liquid and gel - regarding their capacity to influence quality and repair time of standardized skin injuries.Materials, Methods & Results: New Zealand healthy rabbits were distributed in three groups (n = 6): control group (CG), liquid platelet rich plasma group (LIQPRP), and gel platelet rich plasma group (GELPRP). Acute skin lesions were inducted in two areas approximately 2 cm close to scapular edge and depth including epidermis, dermis, and hypodermis to external muscular fascia. Animals received treatment according to each group. Injuries were measured with digital pachymeter in two directions: longer length (l) and longer width (w), every two days. Areas and healing rates were calculated. Microscopic analysis samples were collected on days seven and 14 and evaluated through hematoxylin and eosin staining (HE) for global tissue examination, and through Masson’s trichrome (MT) to collagen fibers present within the interstice. These analyzes considered: angiogenesis, inflammation infiltrated and collagen fibers quantity. Immunohistochemistry with anti-Ki-67 antibody was utilized for proliferative profile assessment. Kruskal-Wallis’ non-parametric tests of independent samples was performed for comparison of values obtained through platelet count, referring to evaluation of platelet increase on treatments. Scar contraction rate (CR) was evaluated through Shapiro-Wilk’s normality test, and then submitted to mixed models test. Results obtained by histopathological and immunohistochemistry were also evaluated by Shapiro-Wilk’s normality test (for all tests a 5% level of significance was considered). Platelet concentration achieved with liquid PRP was 8.64 and gel PRP reached 5.62 times higher than physiological values. Platelet increase mean for both groups was 7.95. No statistical significance was observed between groups. No side-effects or adverse reactions related to PRP usage were observed while study was conducted.Discussion: In the present study, there was a need to raise platelet poor plasma volume in order to obtain autogenous thrombin required for gel PRP. After this modification, a stable and reasonable platelet concentration gel was produced. However, this form of PRP application requires more time for sample preparation, increasing the production cost. Furthermore, injection of liquid PRP directly in the wound site activates platelets by generated substances due to needle perforation, and mainly due to tissue trauma generated at the lesion site. Relating to the therapies administered, gel PRP was considered more manageable, since 3D structure could easily adapt to wound site after simply deposition of it. Liquid PRP was administered with needle and syringe, which required the surgeon to be more careful and perform a slow injection in order to avoid any spill and loss of material. Furthermore, histopathological analysis did not point any clot traces formed by gel PRP dehydration, although it is not possible to ensure that the clot was eliminated, reabsorbed, or even removed by the animal. By this protocol, a stable and reasonable platelet concentration gel was produced. Further studies are encouraged as well as employment of alternative diagnostic tools, in order to better understand found results.

Controlling keratinocyte activity by LL-37 conjugated nanoparticles

Controlling keratinocyte activity by LL-37 conjugated nanoparticles

Effects of Aggregatibacter actinomycetemcomitans leukotoxin on neutrophil migration and extracellular trap formation

BackgroundAggressive periodontitis is associated with the presence of Aggregatibacter actinomycetemcomitans, a leukotoxin (Ltx)-producing periodontal pathogen. Ltx has the ability to lyse white blood cells including neutrophils.ObjectivesThis study was aimed at investigating the interactions between neutrophils and Ltx with regard to the chemotactic properties of Ltx and the release of neutrophil extracellular traps (NETs).MethodsNeutrophils from healthy blood donors were isolated and incubated for 30 min and 3 h with increasing concentrations of Ltx (1, 10, and 100 ng/mL) as well as with A. actinomycetemcomitans strains (NCTC 9710 and HK 1651) producing different levels of Ltx. Formation of NETs and cell lysis were assessed by microscopy, fluorescence-based assays, and measurement of released lactate dehydrogenase. Neutrophil migration in response to different Ltx gradients was monitored by real-time video microscopy, and image analysis was performed using ImageJ software.ResultsAlthough Ltx (10 and 100 ng/mL) and the leukotoxic A. actinomycetemcomitans strain HK 1651 lysed some neutrophils, other cells were still capable of performing NETosis in a concentration-dependent manner. Low doses of Ltx and the weakly leukotoxic strain NCTC 9710 did not lead to neutrophil lysis, but did induce some NETosis. Furthermore, all three concentrations of Ltx enhanced random neutrophil movement; however, low directional accuracy was observed compared with the positive control (fMLP).ConclusionsThe results indicate that Ltx acts both as a neutrophil activator and also causes cell death. In addition, Ltx directly induces NETosis in neutrophils prior to cell lysis. In future studies, the underlying pathways involved in Ltx-meditated neutrophil activation and NETosis need to be investigated further.

Chemotactic dynamics of catalytic dimer nanomotors.

Synthetic chemically powered nanomotors possessing the ability of chemotaxis are desirable for target cargo delivery and self-assembly. The chemotactic properties of a sphere dimer motor, composed of linked catalytic and inactive monomers, are studied in a gradient field of fuel. Particle-based simulation is carried out by means of hybrid molecular dynamics/multiparticle collision dynamics. The detailed tracking and motion analysis describing the running and tumbling of the sphere dimer motor in the process of chemotaxis are investigated. Physical factors affecting chemotactic velocity are discussed, and quantitative relations are presented. The influence of the geometry of sphere dimer motors on the chemotactic dynamics is explored, which is beneficial for the design of motors with high sensitivity for detecting the surrounding environment.

Nanotechnology for mesenchymal stem cell therapies

Mesenchymal stem cells (MSC) display great proliferative, differentiative, chemotactic, and immune-modulatory properties required to promote tissue repair. Several clinical trials based on the use of MSC are currently underway for therapeutic purposes. The aim of this article is to examine the current trends and potential impact of nanotechnology in MSC-driven regenerative medicine. Nanoparticle-based approaches are used as powerful carrier systems for the targeted delivery of bioactive molecules to ensure MSC long-term maintenance in vitro and to enhance their regenerative potential. Nanostructured materials have been developed to recapitulate the stem cell niche within a tissue and to instruct MSC toward the creation of regeneration-permissive environment. Finally, the capability of MSC to migrate toward the site of injury/inflammation has allowed for the development of diagnostic imaging systems able to monitor transplanted stem cell bio-distribution, toxicity, and therapeutic effectiveness.

Quantifying oxygen in paper-based cell cultures with luminescent thin film sensors.

Paper-based scaffolds are an attractive material for generating 3D tissue-like cultures because paper is readily available and does not require specialized equipment to pattern, cut, or use. By controlling the exchange of fresh culture medium with the paper-based scaffolds, we can engineer diffusion-dominated environments similar to those found in spheroids or solid tumors. Oxygen tension directly regulates cellular phenotype and invasiveness through hypoxia-inducible transcription factors and also has chemotactic properties. To date, gradients of oxygen generated in the paper-based cultures have relied on cellular response-based readouts. In this work, we prepared a luminescent thin film capable of quantifying oxygen tensions in apposed cell-containing paper-based scaffolds. The oxygen sensors, which are polystyrene films containing a Pd(II) tetrakis(pentafluorophenyl)porphyrin dye, are photostable, stable in culture conditions, and not cytotoxic. They have a linear response for oxygen tensions ranging from 0 to 160 mmHg O2, and a Stern-Volmer constant (K sv) of 0.239 ± 0.003 mmHg O2 (-1). We used these oxygen-sensing films to measure the spatial and temporal changes in oxygen tension for paper-based cultures containing a breast cancer line that was engineered to constitutively express a fluorescent protein. By acquiring images of the oxygen-sensing film and the fluorescently labeled cells, we were able to approximate the oxygen consumption rates of the cells in our cultures.

Neutrophil Recruitment Is Regulated By Adamts-13 in a Murine Model of Invasive Aspergillosis

▪Introduction: During inflammation von Willebrand factor (VWF) multimers are secreted as an acute phase protein whereupon the size and the prothrombotic activity play an essential role. The size of VWF multimers is regulated by the specific proteolytic activity of ADAMTS-13 (a disintegrin and metalloprotease with ThromboSpondin type 1 repeats-13) which is diminished under several pathological conditions. Employing a murine model of invasive pulmonary aspergillosis (IPA) we aimed to determine the relevance of this regulatory pathway for innate inflammatory responses and polymorphonuclear neutrophil (PMN) recruitment which is crucial for fungal clearance and survival.Methods: IPA was induced by intratracheal application of Aspergillus fumigatus (A. fumigatus) conidia in wildtype (129/Sv/Pas) or Adamts13 deficient (Adamts13-/-) mice. 24 h after infection some mice were sacrificed for analysis of fungal load and histology. To assess fungal load as CFU lung homogenates were plated and cultured on Sabourough agar plates. Paraffin sections of the lungs were prepared and stained with mouse complement component C3d antibody for histological analysis. In addition, degranulation, oxidative burst activity and CD62L shedding were assessed in PMN of wildtype and Adamts13-/- mice. Chemotactic properties of A. fumigatus -activated and control serum from wildtype and knock-out mice was evaluated by migration of purified human PMN, isolated by dextran sedimentation and Histopaque® centrifugation, in a transwell assay (Corning® HTS Transwell®-96 well permeable support; 3µm).Results: Adamts13 deficiency in mice was accompanied by a worse outcome of IPA infections compared to wildtype controls. Adamts13-/- mice displayed more severe signs of disease and a lethal course was observed in about 24% of animals. Compared to Adamts13-/- mice all neutropenic mice developed lethal IPA after infection, but all wild-type mice survived the disease. Besides, examination of the lungs revealed a higher fungal burden along with increased signs of acute lung injury, complement deposition and levels of pro-inflammatory cytokines in Adamts13-/- mice. Furthermore a more pronounced perivascular leukocyte infiltration was observed in histological sections indicating a dysregulated inflammatory response in Adamts13-/- mice. Importantly, the activation of neutrophil effector functions in response to TLR agonist or in PMN-mediated fungal killing of conidia or hyphae revealed no general defect in vitro. Despite enhanced complement deposition in the lungs in Adamts13-/- mice, PMN migration towards complement-activated serum revealed unaltered chemotactic properties comparing A. fumigatus -activated serum of wildtype and knock-out mice.Conclusion: In acute inflammatory processes such as fungal pneumonia the proteolytic regulation of VWF by ADAMTS-13 is an important mechanism to control PMN recruitment and outcome of infections. DisclosuresNo relevant conflicts of interest to declare.

Expression of YKL-40 and MIP-1a proteins in exudates and transudates: biomarkers for differential diagnosis of pleural effusions? A pilot study.

BackgroundYKL-40 is an extracellular matrix glycoprotein with a significant role in tissue inflammation and remodeling. MIP-1a has chemotactic and pro-inflammatory properties, and is induced by YKL-40 in several lung disorders. The aim of this study was to determine the levels of YKL-40 and MIP-1a in blood serum and pleural fluids of various pulmonary diseases, and to evaluate their potential role as differential diagnosis biomarkers.MethodsWe recruited 60 patients (age: 62.5 ± 20.6 years) with pleural effusions: 49 exudates and 11 transudates (T). Exudates were further classified based on the underlying disease: ten with tuberculosis (TB), 13 with lung cancer (LCa), 15 with metastatic cancer (MCa) of non-lung origin and 11 with parapneumonic (PN) effusions. YKL-40 and MIP-1a levels were measured by ELISA.ResultsPleural YKL-40 levels (ng/ml) were similar among all patient groups (TB: 399 ± 36, LCa: 401 ± 112, MCa: 416 ± 34, PN: 401 ± 50, T: 399 ± 42, p = 0.92). On the contrary, YKL-40 was significantly lower in the serum of TB patients (TB: 58 ± 22, LCa: 212 ± 106, MCa: 254 ± 140, PN: 265 ± 140, T: 229 ± 123, p < 0.001). Pleural MIP-1a protein levels (ng/ml) were statistically lower only in patients with LCa (TB: 25.0 ± 20.2, LCa: 7.3 ± 6.0, MCa: 16.1 ± 14.9, PN: 25.4 ± 27.9, T: 18.5 ± 7.9, p = 0.012), a finding also observed in serum MIP-1a levels (TB: 17.1 ± 7.6, LCa: 9.4 ± 7.0, MCa: 28.7 ± 28.7, PN: 33.3 ± 24.0, T: 22.9 ± 8.7, p = 0.003).ConclusionsOur data suggest that both YKL-40 and MIP-1a, particularly in serum, could prove useful for the differentiation of pleural effusions in clinical practice, especially of TB or LCa origin. However, large-scale studies are needed to validate these findings.

APD3: the antimicrobial peptide database as a tool for research and education.

The antimicrobial peptide database (APD, http://aps.unmc.edu/AP/) is an original database initially online in 2003. The APD2 (2009 version) has been regularly updated and further expanded into the APD3. This database currently focuses on natural antimicrobial peptides (AMPs) with defined sequence and activity. It includes a total of 2619 AMPs with 261 bacteriocins from bacteria, 4 AMPs from archaea, 7 from protists, 13 from fungi, 321 from plants and 1972 animal host defense peptides. The APD3 contains 2169 antibacterial, 172 antiviral, 105 anti-HIV, 959 antifungal, 80 antiparasitic and 185 anticancer peptides. Newly annotated are AMPs with antibiofilm, antimalarial, anti-protist, insecticidal, spermicidal, chemotactic, wound healing, antioxidant and protease inhibiting properties. We also describe other searchable annotations, including target pathogens, molecule-binding partners, post-translational modifications and animal models. Amino acid profiles or signatures of natural AMPs are important for peptide classification, prediction and design. Finally, we summarize various database applications in research and education.

Desloratadine citrate disodium injection, a potent histamine H1 receptor antagonist, inhibits chemokine production in ovalbumin-induced allergic rhinitis guinea pig model and histamine-induced human nasal epithelial cells via inhibiting the ERK1/2 and NF-kappa B signal cascades

Chemokines have chemotactic properties on leukocyte subsets whose modulation plays a pivotal role in allergic inflammatory processes. Our present study was designed to investigate the anti-allergic and anti-inflammatory properties of desloratadine citrate disodium injection (DLC) and elucidate the molecular mechanisms of its anti-inflammatory properties. The anti-allergic effects of DLC were evaluated based on allergic symptoms, serological marker production and histological changes of the nasal mucosa in guinea pigs model of allergic rhinitis. The anti-inflammatory properties and molecular mechanisms of DLC were explored by studying the regulation of a set of chemokines and extracellular signal-regulated kinase (ERK)1/2 and nuclear factor-kappa B (NF-κB) pathways, after DLC treatment in guinea pigs model of allergic rhinitis in vivo and histamine-activated human nasal epithelial cells (HNECs) in vitro. In vivo model in guinea pigs, DLC alleviated the rhinitis symptoms, inhibited inflammatory cells infiltration in nasal lavage fluid (NLF) and histamine, monocyte chemotactic protein (MCP)-1, regulated on activation normal T cell expressed, and presumably secreted (RANTEs) and interleukin (IL)-8 release in sera and P-ERK1/2 and NF-κB activation in nasal mucosa. In vitro, DLC markedly inhibited histamine-induced production of MCP-1, RANTEs and IL-8 and suppressed c-Raf, mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) and ERK1/2 activation in HNECs. These results provide evidence that DLC possesses potent anti-allergic and anti-inflammatory properties. The mechanism of action underlying DLC in allergic inflammation appears to be inhibition of the phosphorylation of ERK1/2, in addition to blocking of the NF-κB pathway.

Group A Streptococcal M1 Protein Sequesters Cathelicidin to Evade Innate Immune Killing.

The antimicrobial peptide LL-37 is generated upon proteolytic cleavage of cathelicidin and limits invading pathogens by directly targeting microbial membranes as well as stimulating innate immune cell function. However, some microbes evade LL-37-mediated defense. Notably, group A Streptococcus (GAS) strains belonging to the hypervirulent M1T1 serogroup are more resistant to human LL-37 than other GAS serogroups. We show that the GAS surface-associated M1 protein sequesters and neutralizes LL-37 antimicrobial activity through its N-terminal domain. M1 protein also binds the cathelicidin precursor hCAP-18, preventing its proteolytic maturation into antimicrobial forms. Exogenous M1 protein rescues M1-deficient GAS from killing by neutrophils and within neutrophil extracellular traps and neutralizes LL-37 chemotactic properties. M1 also binds murine cathelicidin, and its virulence contribution in a murine model of necrotizing skin infection is largely driven by its ability to neutralize this host defense peptide. Thus, cathelicidin resistance is essential for the pathogenesis of hyperinvasive M1T1 GAS.

Olive oil compounds inhibit the paracrine regulation of TNF-α-induced endothelial cell migration through reduced glioblastoma cell cyclooxygenase-2 expression

The established causal relationship between the chronic inflammatory microenvironment, tumor development and cancer recurrence has provided leads for developing novel preventive strategies. Accumulating experimental, clinical and epidemiological data has provided support for the chemopreventive properties of olive oil compounds traditionally found within the Mediterranean diet. In this study, we investigated whether tyrosol (Tyr), hydroxytyrosol, oleuropein and oleic acid (OA), four compounds contained in extra virgin olive oil, can prevent tumor necrosis factor (TNF)-α-induced expression of cyclooxygenase (COX)-2 (an inflammation biomarker) in a human glioblastoma cell (U-87 MG) model. We found that Tyr and OA significantly inhibited TNF-α-induced COX-2 gene and protein expression, as well as PGE2 secretion. Both compounds also inhibited TNF-α-induced JNK and ERK phosphorylation, whereas only Tyr inhibited TNF-α-induced NF-κB phosphorylation. Paracrine-regulated migration of human brain microvascular endothelial cells (HBMECs) was assessed using growth factor-enriched conditioned media (CM) isolated from U-87 MG cells. We found that while PGE2 triggered HBMEC migration, the CM isolated from U-87 MG cells, where either COX-2 or NF-κB had been silenced or had been treated with Tyr or OA, exhibited decreased chemotactic properties. These observations demonstrate that olive oil compounds inhibit the effect of the chronic inflammatory microenvironment on glioblastoma progression through TNF-α actions and may be useful in cancer chemoprevention.

Abstract 5468: Tirapazamine as a strategy to prevent cell dissemination and overcome drug resistance

Abstract Introduction: Multiple myeloma (MM) and Waldenström's macroglobulinemia (WM) are lymphoplasmacytic malignancies which are characterized by continuous spread through cell trafficking in and out of the BM. We have recently shown that the spread process in these diseases is mediated by hypoxia. Moreover, despite the introduction of novel therapies, cancer patients relapse due to the development of a drug-resistant stem cell-like subpopulation, which we recently found to be, at least in part, promoted by hypoxia. Therefore, in this study we aimed to overcome spread and drug resistance in MM and WM by inhibition of the hypoxic responses in these cells. Tirapazamine (TPZ) is a hypoxia-activated pro-drug causing cell apoptosis, which has been shown to improve cancer patient outcome when combined with radiotherapy. We use TPZ for the first time in hematologic malignancies to target the drug-resistant cancer cells and sensitize them to therapy. Procedures: Cell survival was tested on MM (MM1.s, H929, OPM1, RPMI8226) and WM (BCWM.1, MWCL.1) cell lines exposed to normoxia (21% O2) or hypoxia (1% O2) for 24hrs in the presence of bortezomib and carfilzomib, different concentrations of TPZ (in order to obtain an IC50), or combination of those drugs using MTT assay. For adhesion assay, cancer cells were pre-labeled with calcein-AM, treated with TPZ, applied to unlabeled endothelial cells, non-adherent cells were washed after 1hr, and adherent cells were analyzed by a fluorescent reader. Chemotactic properties of cells were tested using a transwell chamber and the migrated cells were counted by flow cytometry. For in vivo study, 5 × 106 MM1s-Luc-GFP cells were injected i.v. into 6 SCID mice and tumor progression was monitored for 3 weeks by bioluminescent imaging. All mice were treated with (1) high-dose bortezomib (1.5mg/kg) only (n = 3), or (2) bortezomib and TPZ (40mg/kg; n = 3), all administered i.p. sequentially twice a week. Bone marrow and peripheral blood were collected and analyzed for the presence of GFP+ cells by flow cytometry. Summary of data: We found that hypoxia induced drug resistance to bortezomib and carfilzomib of MM and WM cells, and that TPZ was active in a dose-dependent manner only in hypoxic conditions. Combination of TPZ with bortezomib and carfilzomib, resensitized cancer cells to death in hypoxia. Addition of TPZ increased adhesion of cancer cells to endothelial cells, and decreased chemotactic properties of cancer cells in hypoxia in vitro. Moreover, post-treatment residual cancer cells were further decreased using TPZ, preventing recurrence of MM in vivo. Conclusion: We report that TPZ prevents hypoxia-induced metastasis and overcomes hypoxia-induced drug resistance in MM and WM. This is the first study to show the efficacy of TPZ in hematologic malignancies in general and in MM and WM in particular. This data provide a preclinical basis for future clinical trials testing efficacy of TPZ in MM and WM. Citation Format: Barbara Muz, Pilar de la Puente, Feda Azab, Micah Luderer, Abdel Kareem Azab. Tirapazamine as a strategy to prevent cell dissemination and overcome drug resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5468. doi:10.1158/1538-7445.AM2015-5468

The calcium-activated potassium channel KCa3.1 plays a central role in the chemotactic response of mammalian neutrophils.

Neutrophils are the first cells to arrive at sites of injury. Nevertheless, many inflammatory diseases are characterized by an uncontrolled infiltration and action of these cells. Cell migration depends on volume changes that are governed by ion channel activity, but potassium channels in neutrophil have not been clearly identified. We aim to test whether KCa3.1 participates in neutrophil migration and other relevant functions of the cell. Cytometer and confocal measurements to determine changes in cell volume were used. Cells isolated from human, mouse and horse were tested for KCa3.1-dependent chemotaxis. Chemokinetics, calcium handling and release of reactive oxygen species were measured to determine the role of KCa3.1 in those processes. A mouse model was used to test for neutrophil recruitment after acute lung injury in vivo. We show for the first time that KCa3.1 is expressed in mammalian neutrophils. When the channel is inhibited by a pharmacological blocker or by genetic silencing, it profoundly affects cell volume regulation, and chemotactic and chemokinetic properties of the cells. We also demonstrated that pharmacological inhibition of KCa3.1 did not affect calcium entry or reactive oxygen species production in neutrophils. Using a mouse model of acute lung injury, we observed that Kca3.1(-/-) mice are significantly less effective at recruiting neutrophils into the site of inflammation. These results demonstrate that KCa3.1 channels are key actors in the migration capacity of neutrophils, and its inhibition did not affect other relevant cellular functions.

Chemokines and their receptors in Atherosclerosis.

Atherosclerosis, a chronic inflammatory disease of the medium- and large-sized arteries, is the main underlying cause of cardiovascular diseases (CVDs) most often leading to a myocardial infarction or stroke. However, atherosclerosis can also develop without this clinical manifestation. The pathophysiology of atherosclerosis is very complex and consists of many cells and molecules interacting with each other. Over the last years, chemokines (small 8–12 kDa cytokines with chemotactic properties) have been identified as key players in atherogenesis. However, this remains a very active and dynamic field of research. Here, we will give an overview of the current knowledge about the involvement of chemokines in all phases of atherosclerotic lesion development. Furthermore, we will focus on two chemokines that recently have been associated with atherogenesis, CXCL12, and macrophage migration inhibitory factor (MIF). Both chemokines play a crucial role in leukocyte recruitment and arrest, a critical step in atherosclerosis development. MIF has shown to be a more pro-inflammatory and thus pro-atherogenic chemokine, instead CXCL12 seems to have a more protective function. However, results about this protective role are still quite debatable. Future research will further elucidate the precise role of these chemokines in atherosclerosis and determine the potential of chemokine-based therapies.

Different maturation cocktails provide dendritic cells with different chemoattractive properties.

BackgroundDendritic cells (DC) are currently implemented as immunotherapeutic strategy for the treatment of tumor patients based on their central role in the immune system. Despite good results were obtained in vitro and in animal models, their clinical use has provided limited success suggesting the requirement to optimise the protocol for their production.MethodsA cDNA array was performed on FastDC obtained from the differentiation of human peripheral blood monocytes stimulated with the clinical gold standard or with two alternative maturation cocktails combining interferon (IFN)γ and ligands for different toll like receptors (TLR).ResultsA stronger modulation of the DC transcriptome with respect to immature DC was found in alternatively stimulated DC when compared to DC stimulated with the clinical gold standard. A major class of molecules differentially expressed using distinct DC stimulation protocols were chemokines. Validation of their differential expression pattern at the mRNA and protein level confirmed the secretion of inflammatory chemokines by the alternative DC. Functional analyses of the chemotactic properties of DC “wash out” supernatants highlighted the ability of alternative, but not of gold standard DC to efficiently recruit immune cells with a prevalence of monocytes. Effector cells belonging to the innate as well as adaptive immunity were also attracted and the interaction with alternative DC resulted in enhanced secretion of IFNγ and induction of cytotoxic activity. Using leukocytes from cancer patients, it was demonstrated that the monocyte-attracting activity targeted cells with an inflammatory phenotype characterised by high levels of HLA-DR expression.ConclusionsDespite other classes of immune modulatory genes differently expressed in the alternative DC require to be investigated and characterised regarding their functional consequences, the reduced maturation state and chemoattractive properties of the gold standard versus alternative DC clearly promote the necessity to change the clinically used maturation cocktail of DC in order to improve the outcome of patients treated with DC-based vaccines.