Leukocyte Chemotaxis Research Articles

Identification of Important Invasion-Related Genes in Non-functional Pituitary Adenomas.

Non-functioning pituitary adenomas (NFPAs) are locally invasive with high morbidity. The objective of this study was to diagnose important genes and pathways related to the invasiveness of NFPAs and gain more insights into the underlying molecular mechanisms of NFPAs. The gene expression profiles of GSE51618 were downloaded from the Gene Expression Omnibus database with 4 non-invasive NFPA samples, 3 invasive NFPA samples, and 3 normal pituitary gland samples. Differentially expressed genes (DEGs) are screened between invasive NFPA samples and normal pituitary gland samples, followed by pathway and ontology (GO) enrichment analyses. Subsequently, a protein-protein interaction (PPI) network was constructed and analyzed for these DEGs, and module analysis was performed. In addition, a target gene-miRNA network and target gene-TF (transcription factor) network were analyzed for these DEGs. A total of 879 DEGs were obtained. Among them, 439 genes were upregulated and 440 genes were downregulated. Pathway enrichment analysis indicated that the upregulated genes were significantly enriched in cysteine biosynthesis/homocysteine degradation (trans-sulfuration) and PI3K-Akt signaling pathway, while the downregulated genes were mainly associated with docosahexaenoate biosynthesis III (mammals) and chemokine signaling pathway. GO enrichment analysis indicated that the upregulated genes were significantly enriched in animal organ morphogenesis, extracellular matrix, and hormone activity, while the downregulated genes were mainly associated with leukocyte chemotaxis, dendrites, and RAGE receptor binding. Subsequently, ESR1, SOX2, TTN, GFAP, WIF1, TTR, XIST, SPAG5, PPBP, AR, IL1R2, and HIST1H1C were diagnosed as the top hub genes in the upregulated and downregulated PPI networks and modules. In addition, HS3ST1, GPC4, CCND2, and SCD were diagnosed as the top hub genes in the upregulated and downregulated target gene-miRNA networks, while CISH, ISLR, UBE2E3, and CCNG2 were diagnosed as the top hub genes in the upregulated and downregulated target gene-TF networks. The new important DEGs and pathways diagnosed in this study may serve key roles in the invasiveness of NFPAs and indicate more molecular targets for the treatment of NFPAs.

Staphylococcus aureus Secreted Toxins and Extracellular Enzymes.

Staphylococcus aureus is a formidable pathogen capable of causing infections in different sites of the body in a variety of vertebrate animals, including humans and livestock. A major contribution to the success of S. aureus as a pathogen is the plethora of virulence factors that manipulate the host's innate and adaptive immune responses. Many of these immune modulating virulence factors are secreted toxins, cofactors for activating host zymogens, and exoenzymes. Secreted toxins such as pore-forming toxins and superantigens are highly inflammatory and can cause leukocyte cell death by cytolysis and clonal deletion, respectively. Coagulases and staphylokinases are cofactors that hijack the host's coagulation system. Exoenzymes, including nucleases and proteases, cleave and inactivate various immune defense and surveillance molecules, such as complement factors, antimicrobial peptides, and surface receptors that are important for leukocyte chemotaxis. Additionally, some of these secreted toxins and exoenzymes can cause disruption of endothelial and epithelial barriers through cell lysis and cleavage of junction proteins. A unique feature when examining the repertoire of S. aureus secreted virulence factors is the apparent functional redundancy exhibited by the majority of the toxins and exoenzymes. However, closer examination of each virulence factor revealed that each has unique properties that have important functional consequences. This chapter provides a brief overview of our current understanding of the major secreted virulence factors critical for S. aureus pathogenesis.

Taxonomic and Functional Compositions of the Small Intestinal Microbiome in Neonatal Calves Provide a Framework for Understanding Early Life Gut Health.

A lack of information on the intestinal microbiome of neonatal calves prevents the use of microbial intervention strategies to improve calf gut health. This study profiled the taxonomic and functional composition of the small intestinal luminal microbiome of neonatal calves using whole-genome sequencing of the metagenome, aiming to understand the dynamics of microbial establishment during early life. Despite highly individualized microbial communities, we identified two distinct taxonomy-based clusters from the collective luminal microbiomes comprising a high level of either Lactobacillus or Bacteroides Among the clustered microbiomes, Lactobacillus-dominant ileal microbiomes had significantly lower abundances of Bacteroides, Prevotella, Roseburia, Ruminococcus, and Veillonella compared to the Bacteroides-dominated ileal microbiomes. In addition, the upregulated ileal genes of the Lactobacillus-dominant calves were related to leukocyte and lymphocyte chemotaxis, the cytokine/chemokine-mediated signaling pathway, and inflammatory responses, while the upregulated ileal genes of the Bacteroides-dominant calves were related to cell adhesion, response to stimulus, cell communication and regulation of mitogen-activated protein kinase cascades. The functional profiles of the luminal microbiomes also revealed two distinct clusters consisting of functions related to either high protein metabolism or sulfur metabolism. A lower abundance of Bifidobacterium and a higher abundance of sulfur-reducing bacteria (SRB) were observed in the sulfur metabolism-dominant cluster (0.2% ± 0.1%) compared to the protein metabolism-dominant cluster (12.6% ± 5.7%), suggesting an antagonistic relationship between SRB and Bifidobacterium, which both compete for cysteine. These distinct taxonomic and functional clusters may provide a framework to further analyze interactions between the intestinal microbiome and the immune function and health of neonatal calves.IMPORTANCE Dietary interventions to manipulate neonatal gut microbiota have been proposed to generate long-term impacts on hosts. Currently, our understanding of the early gut microbiome of neonatal calves is limited to 16S rRNA gene amplicon based microbial profiling, which is a barrier to developing dietary interventions to improve calf gut health. The use of a metagenome sequencing-based approach in the present study revealed high individual animal variation in taxonomic and functional abundance of intestinal microbiome and potential impacts of early microbiome on mucosal immune responses during the preweaning period. During this developmental period, age- and diet-related changes in microbial diversity, richness, density, and the abundance of taxa and functions were observed. A correlation-based approach to further explore the individual animal variation revealed potential enterotypes that can be linked to calf gut health, which may pave the way to developing strategies to manipulate the microbiome and improve calf health.

Hematological and immune changes in Piaractus mesopotamicus in the sepsis induced by Aeromonas hydrophila

The pathogenesis of sepsis involves complex systems and multiple interrelationships between the host and pathogen producing high mortality rates in various animal species. In this study, hematological disturbances, innate immunity and survival during the septic process in Piaractus mesopotamicus inoculated with Aeromonas hydrophila were studied. For this aim, fish blood samples were taken from control and infected groups 1, 3, 6, and 9 h post-inoculation (HPI). Leukogram showed reduction in the number of leukocytes and thrombocytes, followed by cessation of leukocyte chemotaxis 6 HPI and severe morphological changes in leukocytes and erythrocytes. At 3 HPI production of reactive oxygen species increased and at 6 HPI decreased. There was no change in serum lysozyme concentration and lytic activity of the complement system, despite the progressive increase in serum lytic activity and bacterial agglutination. Finally, the changes in clinical signs due to aeromonosis and increasing septicemia resulted in a reduction in survival to 57.14% after 36 HPI. It was possible concluded that these hematological and immune are crucial event in the worsening of sepsis in P. mesopotamicus, and these findings are utility for diagnosing and understanding the pathophysiology sepsis in pacu induced by A. hydrophila.

Vitamin D Binding Protein and Complement Factor C5a Influence Neutrophil Chemotaxis Following HSCT

Background Vitamin D binding protein (VDBP) is an abundant protein in human serum, related to albumin and alpha-fetoprotein. The VDBP gene is polymorphic, with three major alleles (Gc1F, Gc1S and Gc2) and four frequent genotypes. These genotype differences effect the glycosylation state of the molecule. VDBP has many diverse functions in addition to transporting vitamin D, and only about 4% of VDBP is bound to vitamin D at any one time. Acute inflammation leading to graft-vs-host disease (GVHD) may involve chemotaxis of leukocytes into damaged tissue mediated by chemotactic factors. Complement activation peptide C5a is a potent chemoattractant and together with VDBP has been shown to markedly enhance the motility of leukocytes. In previous work, we have shown that VDBP levels are significantly lower in those with acute GVHD (p=0.026), and 1-year NRM (p=0.03). Here we have reproduced these data analyzing serum from Day 100 post-HSCT with a new 110 patient cohort. Acute GVHD was reduced in those with VDBP levels above the median at Day 100 (0.03 v 0.18, p=0.037). 1-year NRM cumulative incidence (0.02 vs 0.2, p=0.002) was similarly reduced in those with VDBP levels above the median at Day 100, in agreement with our previous findings. In an effort to identify mechanisms whereby VDBP improves survival, we hypothesized that higher levels of VDBP will have a beneficial effect on neutrophil chemotaxis following HSCT. Previous VDBP animal model studies show chemotaxis of neutrophils to lung injuries can exacerbate pulmonary inflammation, but too little chemotaxis and the animals can succumb to infection. Here we observe a similar paradoxical effect with VDBP and chemotaxis. Methods VDBP was measured in serum at Day 100 in 110 transplant recipients using a polyclonal VDBP ELISA. Genotypes were determined by Taqman allele discrimination. Neutrophils from whole blood were negatively enriched from a normal adult donor and incubated in 10% patient serum in HBSS for 1 hr. For modified Transwell assay, put 4 × 105 cells in a 3-um-pore, 12-mm-diameter Transwell and place on top of 20 nM C5a in HBSS in a fibrinogen coated 12-well plate. Allow neutrophils to migrate for 2 hrs then count cells by hemocytometer. Results VDBP levels above the median at Day 100 were associated with significantly lower cumulative incidence of both NRM and GVHD 1-year post transplant (Fig 1 & 2). High levels of VDBP in patients with GcS1/GcS1 genotype decelerate chemotaxis towards a C5a bait. However, VDBP of a similar level but different genotype gives contrasting results; high VDBP levels with a GcS1/Gc2 genotype increase chemotaxis (Fig 3). Conclusions Our data demonstrate that VDBP-containing serum from children post-BMT modifies chemotaxis, potentially in a genotype dependent manner. We are extending our studies to test additional samples and genotypes. Moreover, we are exploring the effect of VDBP genotype on other transplant outcomes.

Circulating levels of chemokines in patients with psoriasis vulgaris and their association with disease severity: A case-control study from North India.

Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation and incomplete differentiation of epidermis, and accumulation of neutrophils and proinflammatory T cells in epidermis and dermis. Chemokines are believed to be the main players mediating the chemotaxis of leucocytes to the lesional site. Previous studies have established the role of various chemokine ligands and receptors at the lesional site in psoriasis. In this study, we have compared the serum levels of various chemokines, namely, inducible protein-10 (IP-10) (CXCL10), MCP-1 (CCL-2), monokine induced by gamma interferon (MIG) (CXCL-9), RANTES (CCL5), interleukin (IL)-8, and eotaxin in patients with chronic plaque psoriasis with that of healthy controls. We also studied whether the chemokine levels varied within different patient groups based on various clinical and demographic parameters, and if any of these chemokines correlated with disease activity. We studied 40 patients with chronic plaque psoriasis from a single center. Their clinical and demographic details were recorded in predesigned prforma. Patients with unstable forms of psoriasis like guttate, erythrodermic, or pustular psoriasis were excluded. The serum chemokine levels were measured by flow cytometry-based bead array set system. The serum levels of the patients were compared with that of 25 healthy controls. A subgroup analysis was also done to study the correlation of chemokine levels with age, sex, duration, and severity of disease. We observed a significant decrease in serum level of all these chemokines in patients, when compared with that of healthy controls. We also found that MIG levels showed a positive correlation with disease severity based on Psoriasis Area and Severity Index. The major limitation of the study is lack of data on the lesional chemokine levels compared to serum chemokines. The inflammatory process in psoriasis is orchestrated through chemokines. MIG is a potential serum biomarker for assessing disease severity.

Oral Doxycycline as an alternative initial treatment for desquamative gingivitis

Oral Doxycycline as an alternative initial treatment for desquamative gingivitis

Transcriptomic analysis revealing hepcidin expression in Oryzias melastigma regulated through the JAK-STAT signaling pathway upon exposure to BaP

Our previous study revealed that an antimicrobial peptide hepcidin, can be significantly up-regulated either with LPS challenge or upon exposure to Benzo[a]pyrene (BaP) in red sea bream, but the molecular mechanism involved in whether the transcriptional expression of hepcidin induced by LPS or BaP is regulated through a similar signaling pathway is not yet known. To elucidate the underlying molecular mechanism, the marine model fish Oryzias melastigma was exposed to 1 μg/L BaP as well as challenged with 5 μg of LPS per fish. Samples at 3 h post-LPS challenge, and 2 d and 3 d post-BaP exposure were separately collected for transcriptome analysis. General analysis of the predicted immune-associated unigenes based on the transcriptomic data showed that the percentages of modulated immune-associated genes were 7% with LPS challenge, and 3% and 7% with BaP exposure at 2 and 3 days, respectively. Genes involved in functions like antimicrobial activity, neutrophil activation, and leukocyte chemotaxis were up-regulated with LPS challenge, whereas more than half of the immune associated genes including the KLF family were down-regulated upon BaP exposure, indicating a difference in the modulated immune genes between LPS challenge and BaP exposure. Specific comparative analyses of the immune-associated signal pathways NOD, TOLL, NF-κB and JAK-STAT with LPS challenge or upon exposure to BaP, indicated that most of the modulated genes in association with the NOD, TOLL and NF-κB pathways were induced with LPS challenge but only a few after exposure to BaP, suggesting that BaP exposure was generally not associated with any of the three signal pathways. Interestingly, further transcriptomic analysis revealed that 5 of the 8 modulated genes associated with the JAK-STAT pathway were down-regulated, while 2 inhibiting genes were up-regulated after BaP exposure for 2 days whereas LPS challenge resulted in only less than half modulated, suggesting the possibility of down-regulation caused by BaP exposure through JAK-STAT pathway. Further testing using an EPC cell culture demonstrated that expression of the hepcidin1 gene was less involved in the known signal pathways, such as c/EBP, BMP, and NF-κB, but instead mostly in association with the JAK-STAT pathway upon BaP exposure.

Endoplasmic reticulum stress mediates inflammatory response triggered by ultra-small superparamagnetic iron oxide nanoparticles in hepatocytes

Ultra-small superparamagnetic iron oxide nanoparticles (USPIO-NPs) are widely used as clinical magnetic resonance imaging contrast agents for hepatic diseases diagnosis. USPIO-NPs often damage the hepatocytes and affect the function of liver but its mechanism of action remains unclear. In the present study, USPIO-NPs caused higher cytotoxicity and lactate dehydrogenase (LDH) leakage in hepatic L02 cells than SPIO-NPs. Subsequently, USPIO-NPs affected more genes’ expression than SPIO-NPs analyzed through microarray and bioinformatics analysis. The affected genes were involved in several biological processes, including calcium ion homeostasis, inflammatory response-related leukocyte chemotaxis, and migration. In addition, the level of endoplasmic reticulum (ER) calcium ion was increased by USPIO-NPs. USPIO-NPs also upregulated the genes related to acute-phase inflammation, including IL1B, IL6, IL18, TNFSF12, TNFRSF12, SAA1, SAA2, JAK1, STAT5B, and CXCL14. Furthermore, interleukin-6 (IL-6) secretion was elevated by USPIO-NPs as detected using ELISA. On the other hand, USPIO-NPs changed the morphology of ER and triggered the ER stress and unfolded protein response PERK/ATF4 pathway. Furthermore, blocking ER stress with inhibitor or ATF4 small interfering RNA counteracted IL-6-related acute-phase inflammation and cytotoxicity caused by USPIO-NPs. Taken together, we found that the USPIO-NPs could trigger stronger IL-6-related acute-phase inflammation than SPIO-NPs in hepatocytes. We demonstrated, for the first time, that IL-6-related acute-phase inflammation caused by NPs was regulated by PERK/ATF4 signaling. The PERK/ATF4 pathway explored in this study could be a candidate for diagnostic and therapeutic target against NPs-induced liver injury and cytotoxicity, which would be helpful for USPIO-NPs medical application.

Modeling the complete chemokine-receptor interaction.

Chemokines are soluble, secreted proteins that induce chemotaxis of leukocytes and other cells. Migratory cells can sense the chemokine concentration gradient following chemokine binding and activation of chemokine receptors, a subset of the G protein-coupled receptor (GPCR) superfamily. Chemokine receptor signaling plays a central role in cell migration during inflammatory responses as well as in cancer and other diseases. Given their important role in mediating essential pathologic and physiologic processes, chemokines and their receptors are attractive targets for therapeutic development. A better understanding of the molecular basis of chemokine-GPCR interactions will aid in the understanding of the mechanistic basis for chemokine function in disease-related processes, as well as aid in the design of new therapeutics. High resolution protein structures are critical for determining these mechanisms and investigating the interactions between approximately 50 chemokines and 20 chemokine receptors. Currently, three unique structures of chemokine-GPCR complexes have been determined and have greatly broadened our knowledge of this large protein-protein interaction. While these structures represent only a small fraction of clinically relevant chemokines and receptors, they can be exploited as scaffolds for homology modeling to understand the chemokine-GPCR interactions. This chapter presents a specialized methodology to construct and validate models of chemokine-GPCR complexes using the Rosetta software suite.

PAR2 Pepducin-Based Suppression of Inflammation and Itch in Atopic Dermatitis Models

PAR2 has been proposed to contribute to lesion formation and intense itch in atopic dermatitis. Here, we tested the ability of a cell-penetrating pepducin, PZ-235, to mitigate the potentially deleterious effects of PAR2 in models of atopic dermatitis. PZ-235 significantly inhibited PAR2-mediated expression of inflammatory factors NF-κB, TSLP, TNF-α, and differentiation marker K10 by 94%-98% (P < 0.001) in human keratinocytes and suppressed IL-4 and IL-13 by 68%-83% (P < 0.05) in mast cells. In delayed pepducin treatment models of oxazolone- and DNFB-induced dermatitis, PZ-235 significantly attenuated skin thickening by 43%-100% (P < 0.01) and leukocyte crusting by 57% (P< 0.05), and it inhibited exvivo chemotaxis of leukocytes toward PAR2 agonists. Daily PZ-235 treatment of filaggrin-deficient mice exposed to dust mite allergens for 8 weeks significantly suppressed total leukocyte and T-cell infiltration by 50%-68%; epidermal thickness by 60%-77%; and skin thickening, scaling, excoriation, and total lesion severity score by 46%-56%. PZ-235 significantly reduced itching caused by wasp venom peptide degranulation of mast cells in mice by 51% (P < 0.05), which was comparable to the protective effects conferred by PAR2 deficiency. Taken together, these results suggest that a PAR2 pepducin may confer broad therapeutic benefits as a disease-modifying treatment for atopic dermatitis and itch.

CNS-border associated macrophages respond to acute ischemic stroke attracting granulocytes and promoting vascular leakage

The central nervous system (CNS) contains several types of immune cells located in specific anatomic compartments. Macrophages reside at the CNS borders surrounding the brain vessels, in leptomeningeal spaces and the choroid plexus, where they interact with the vasculature and play immunological surveillance and scavenging functions. We investigated the phenotypic changes and role of these macrophages in response to acute ischemic stroke. Given that CD163 expression is a hallmark of perivascular and meningeal macrophages in the rat and human brain, we isolated CD163+ brain macrophages by fluorescence activated cell sorting. We obtained CD163+ cells from control rats and 16 h following transient middle cerebral artery occlusion, after verifying that infiltration of CD163+ peripheral myeloid cells is negligible at this acute time point. Transcriptome analysis of the sorted CD163+ cells identified ischemia-induced upregulation of the hypoxia inducible factor-1 pathway and induction of genes encoding for extracellular matrix components and leukocyte chemoattractants, amongst others. Using a cell depletion strategy, we found that CNS border-associated macrophages participate in granulocyte recruitment, promote the expression of vascular endothelial growth factor (VEGF), increase the permeability of pial and cortical blood vessels, and contribute to neurological dysfunction in the acute phase of ischemia/reperfusion. We detected VEGF expression surrounding blood vessels and in some CD163+ perivascular macrophages in the brain tissue of ischemic stroke patients deceased one day after stroke onset. These findings show ischemia-induced reprogramming of the gene expression profile of CD163+ macrophages that has a rapid impact on leukocyte chemotaxis and blood-brain barrier integrity, and promotes neurological impairment in the acute phase of stroke.

Comparative proteomics of respiratory exosomes in cystic fibrosis, primary ciliary dyskinesia and asthma

Cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) are pulmonary genetic disorders associated with inflammation and heterogeneous progression of the lung disease. We hypothesized that respiratory exosomes, nanovesicles circulating in the respiratory tract, may be involved in the progression of inflammation-related lung damage. We compared proteomic content of respiratory exosomes isolated from bronchoalveolar lavage fluid in CF and PCD to asthma (A), a condition also associated with inflammation but with less severe lung damage.BALF were obtained from 3 CF, 3 PCD and 6 A patients. Exosomes were isolated from BALF by ultracentrifugations and characterized using immunoelectron microscopy and western-blot. Exosomal protein analysis was performed by high-resolution mass spectrometry using label-free quantification.Exosome enrichment was validated by electron microscopy and immunodetection of CD9, CD63 and ALIX. Mass spectrometry analysis allowed the quantification of 665 proteins, of which 14 were statistically differential according to the disease. PCD and CF exosomes contained higher levels of antioxidant proteins (Superoxide-dismutase, Glutathione peroxidase-3, Peroxiredoxin-5) and proteins involved in leukocyte chemotaxis. All these proteins are known activators of the NF-KappaB pathway.Our results suggest that respiratory exosomes are involved in the pro-inflammatory propagation during the extension of CF or PCD lung diseases. SignificanceThe mechanism of local propagation of lung disease in cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) is not clearly understood. Differential Proteomic profiles of exosomes isolated from BAL from CF, PCD and asthmatic patients suggest that they carry pro-inflammatory proteins that may be involved in the progression of lung damage.

Trace Amine-Associated Receptors as Novel Therapeutic Targets for Immunomodulatory Disorders.

Trace amines and their receptors (trace amine-associated receptors; TAARs) are an emerging pharmacological target for the treatment of human disorders. While most studies have focused on their therapeutic potential for neurologic and psychiatric disorders, TAARs are also expressed throughout the periphery, including prominent expression in human leukocytes. Furthermore, recent independent, unbiased metabolomic studies have consistently identified one or more TAAR ligands as potential etiologic factors in inflammatory bowel disease (IBD). The putative role of TAARs in diseases such as IBD that are associated with hyperactive immune responses has not, however, previously been systematically addressed. Here, we review the current state of the knowledge of the effects of TAARs on leukocyte function, in particular in the context of mucosal epithelial cells that interface with the environment; developing a model whereby TAARs may be considered as a novel therapeutic target for disorders associated with dysregulated immune responses to environmental factors. In this model, we hypothesize that altered trace amine homeostasis results in hyperactivity of the immune system. Such loss of homeostasis can occur through many different mechanisms including TAAR polymorphisms and altered trace amine load due to changes in host synthesis and/or degradative enzymes, diet, or microbial dysbiosis. The resulting alterations in TAAR functioning can then lead to a loss of homeostasis of leukocyte chemotaxis, differentiation, and activation, as well as an altered ability of members of the microbiota to adhere to and penetrate the epithelial cell layers. Such changes would generate a pro-inflammatory state at mucosal epithelial barrier layers that can manifest as clinical symptomatology such as that seen in IBD. These alterations may also have the potential to induce systemic effects, which could possibly contribute to immunomodulatory disorders in other systems, including neurological diseases.

Abstract LB-345: Unique molecular identifiers of indolent versus aggressive phenotyping in non-Hodgkin lymphoma: Spotlight on SMZL

Abstract Splenic marginal zone lymphoma (SMZL) is an indolent, non-Hodgkin's lymphoma that lacks clear genotypic classification. Approximately 30% of all SMZL cases will become aggressive or undergo transformation to a more lethal lymphoma. Hence, the main purpose of this study was to broadly characterize the molecular profiles of SMZL cases, compared with healthy B-cell controls and other B-cell lymphomas (BCLs), in an effort to delineate the variable malignant presentations. We aimed to identify a unique indolent and aggressive gene expression signature among SMZL cases in order to potentially provide prognostic value. We conducted a meta-analysis of previously published gene expression data to identify genes most dysregulated in SMZL. We aimed to delineate the “indolent phenotype” and the “aggressive phenotype” by comparing SMZL to more aggressive BCLs as well as healthy B-cell controls, in order to extract a prognostic gene expression profile specific to SMZL. Markers potentially demonstrating an “aggressive” signature were those significantly up or down regulated in SMZL samples when compared to BCLs and healthy controls. The most upregulated genes in SMZL included PPBP, CXCL6, HBB, KAZN, NTN4, SLC40A1, MAFB, ATP1B1, and C1QB, while the most downregulated genes were RASSF6, RGS13, ELL3, LHFPL2, and SYB. Pathways impacted include immune defense (inclusive of leukocyte chemotaxis), motility (inclusive of lamellipodium assembly), cell proliferation, and many other mechanisms. Contrarily, 19 key markers of potential oncogenic indolency (i.e. down regulated compared with other, aggressive B-cell lymphomas, but upregulated compared with healthy B-cells) were also identified: ABI3BP, GPNMB, LUM, CCL19, ADAMDEC1, GJA1, MGP, SERPINF1, COL6A3, C3, RARRES1, FN1, CCL18, PRRX1, RGS5, CHI3L1, COL5A2, LOXL1, and ANKRD29. Additionally, a transcription factor, ZC3H7B, known to play a role in BCL6 repression as a result of a fusion with BCOR, is uniquely down regulated in SMZL compared with normal B controls while being up regulated when compared to similar BCLs. This exclusive expression of ZC3H7B represents a unique marker for SMZL identification compared to other BCLs. The aforementioned gene expression profile provides SMZL identification potential, and more importantly, these unique gene signatures potentially identify markers of indolent progression due to their differential expression from more aggressive B-cell lymphoma counterparts. Further investigations into the translatability of these gene expressions and their resultant impacted pathways will be evaluated for diagnostic and prognostic significance using immunohistochemistry, and hypothesized targets will be evaluated in vitro. Ultimately, we expect that the unique gene expression profile identified in this study will provide translatable results to assist with patient staging in SMZL. Citation Format: Jacob E. Robinson, Christine E. Cutucache. Unique molecular identifiers of indolent versus aggressive phenotyping in non-Hodgkin lymphoma: Spotlight on SMZL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-345.

Robo1 and vimentin regulate radiation-induced motility of human glioblastoma cells.

Glioblastoma is a primary brain tumor with a poor prognosis despite of many treatment regimens. Radiotherapy significantly prolongs patient survival and remains the most common treatment. Slit2 and Robo1 are evolutionarily conserved proteins involved in axon guidance, migration, and branching of neuronal cells. New studies have shown that Slit2 and Robo1 could play important roles in leukocyte chemotaxis and glioblastoma cell migration. Therefore, we investigated whether the Slit2/Robo1 complex has an impact on the motility of glioblastoma cells and whether irradiation with therapeutic doses modulates this effect. Our results indicate that photon irradiation increases the migration of glioblastoma cells in vitro. qPCR and immunoblotting experiments in two different glioblastoma cell lines (U-373 MG and U-87 MG) with different malignancy revealed that both Slit2 and Robo1 are significantly lower expressed in the cell populations with the highest motility and that the expression was reduced after irradiation. Overexpression of Robo1 significantly decreased the motility of glioblastoma cells and inhibited the accelerated migration of wild-type cells after irradiation. Immunoblotting analysis of migration-associated proteins (fascin and focal adhesion kinase) and of the epithelial-mesenchymal-transition-related protein vimentin showed that irradiation affected the migration of glioblastoma cells by increasing vimentin expression, which can be reversed by the overexpression of Slit2 and Robo1. Our findings suggest that Robo1 expression might counteract migration and also radiation-induced migration of glioblastoma cells, a process that might be connected to mesenchymal-epithelial transition.

Myeloid Kdm6b deficiency results in advanced atherosclerosis

Background and aimsAtherosclerosis is a lipid-driven chronic inflammatory disorder of the arteries, and monocytes and macrophages play a central role in this process. Within the atherosclerotic lesion, macrophages can scavenge modified lipids and become the so-called foam cells. We previously reported that the epigenetic enzyme Kdm6b (also known as Jmjd3) controls the pro-fibrotic transcriptional profile of peritoneal foam cells. Given the importance of these cells in atherosclerosis, we now studied the effect of myeloid Kdm6b on disease progression. MethodsBone marrow of myeloid Kdm6b deficient (Kdm6bdel) mice or wild type littermates (Kdm6bwt) was transplanted to lethally irradiated Ldlr−/− mice fed a high fat diet for 9 weeks to induce atherosclerosis. ResultsLesion size was similar in Kdm6bwt and Kdm6bdel transplanted mice. However, lesions of Kdm6bdel mice contained more collagen and were more necrotic. Pathway analysis on peritoneal foam cells showed that the pathway involved in leukocyte chemotaxis was most significantly upregulated. Although macrophage and neutrophil content was similar after 9 weeks of high fat diet feeding, the relative increase in collagen content and necrosis revealed that atherosclerotic lesions in Kdm6bdel mice progress faster. ConclusionMyeloid Kdm6b deficiency results in more advanced atherosclerosis.

Characterization of the rat cerebrospinal fluid proteome following acute cerebral ischemia using an aptamer-based proteomic technology

The limited accessibility to the brain has turned the cerebrospinal fluid (CSF) into a valuable source that may contribute to the complete understanding of the stroke pathophysiology. Here we have described the CSF proteome in the hyper-acute phase of cerebral ischemia by performing an aptamer-based proteomic assay (SOMAscan) in CSF samples collected before and 30 min after male Wistar rats had undergone a 90 min Middle Cerebral Artery Occlusion (MCAO) or sham-surgery. Proteomic results indicated that cerebral ischemia acutely increased the CSF levels of 716 proteins, mostly overrepresented in leukocyte chemotaxis and neuronal death processes. Seven promising candidates were further evaluated in rat plasma and brain (CKB, CaMK2A, CaMK2B, CaMK2D, PDXP, AREG, CMPK). The 3 CaMK2 family-members and CMPK early decreased in the infarcted brain area and, together with AREG, co-localized with neurons. Conversely, CKB levels remained consistent after the insult and specifically matched with astrocytes. Further exploration of these candidates in human plasma revealed the potential of CKB and CMPK to diagnose stroke, while CaMK2B and CMPK resulted feasible biomarkers of functional stroke outcome. Our findings provided insights into the CSF proteome following cerebral ischemia and identified new outstanding proteins that might be further considered as potential biomarkers of stroke.

A PKC-MARCKS-PI3K regulatory module links Ca2+ and PIP3 signals at the leading edge of polarized macrophages

The leukocyte chemosensory pathway detects attractant gradients and directs cell migration to sites of inflammation, infection, tissue damage, and carcinogenesis. Previous studies have revealed that local Ca2+ and PIP3 signals at the leading edge of polarized leukocytes play central roles in positive feedback loop essential to cell polarization and chemotaxis. These prior studies showed that stimulation of the leading edge Ca2+ signal can strongly activate PI3K, thereby triggering a larger PIP3 signal, but did not elucidate the mechanistic link between Ca2+ and PIP3 signaling. A hypothesis explaining this link emerged, postulating that Ca2+-activated PKC displaces the MARCKS protein from plasma membrane PIP2, thereby releasing sequestered PIP2 to serve as the target and substrate lipid of PI3K in PIP3 production. In vitro single molecule studies of the reconstituted pathway on lipid bilayers demonstrated the feasibility of this PKC-MARCKS-PI3K regulatory module linking Ca2+ and PIP3 signals in the reconstituted system. The present study tests the model predictions in live macrophages by quantifying the effects of: (a) two pathway activators—PDGF and ATP that stimulate chemoreceptors and Ca2+ influx, respectively; and (b) three pathway inhibitors—wortmannin, EGTA, and Go6976 that inhibit PI3K, Ca2+ influx, and PKC, respectively; on (c) four leading edge activity sensors—AKT-PH-mRFP, CKAR, MARCKSp-mRFP, and leading edge area that report on PIP3 density, PKC activity, MARCKS membrane binding, and leading edge expansion/contraction, respectively. The results provide additional evidence that PKC and PI3K are both essential elements of the leading edge positive feedback loop, and strongly support the existence of a PKC-MARCKS-PI3K regulatory module linking the leading edge Ca2+ and PIP3 signals. As predicted, activators stimulate leading edge PKC activity, displacement of MARCKS from the leading edge membrane and increased leading edge PIP3 levels, while inhibitors trigger the opposite effects. Comparison of the findings for the ameboid chemotaxis of leukocytes with recently published findings for the mesenchymal chemotaxis of fibroblasts suggests that some features of the emerging leukocyte leading edge core pathway (PLC-DAG-Ca2+-PKC-MARCKS-PIP2-PI3K-PIP3) may well be shared by all chemotaxing eukaryotic cells, while other elements of the leukocyte pathway may be specialized features of these highly optimized, professional gradient-seeking cells. More broadly, the findings suggest a molecular mechanism for the strong links between phospho-MARCKS and many human cancers.

Tissue Damage Signaling Is a Prerequisite for Protective Neutrophil Recruitment to Microbial Infection in Zebrafish

Tissue damage and infection are deemed likewise triggers of innate immune responses. But whereas neutrophil responses to microbes are generally protective, neutrophil recruitment into damaged tissues without infection is deleterious. Why neutrophils respond to tissue damage and not just to microbes is unknown. Is it a flaw of the innate immune system that persists because evolution did not select against it, or does it provide a selective advantage? Here we dissect the contribution of tissue damage signaling to antimicrobial immune responses in a live vertebrate. By intravital imaging of zebrafish larvae, a powerful model for innate immunity, we show that prevention of tissue damage signaling upon microbial ear infection abrogates leukocyte chemotaxis andreduces animal survival, at least in part, through suppression of cytosolic phospholipase A2 (cPla2), which integrates tissue damage- and microbe-derived cues. Thus, microbial cues are insufficient, and damage signaling is essential for antimicrobial neutrophil responses in zebrafish.