Increased drug concentration and repeated eye drop administration as strategies to optimize topical drug delivery: A fluorophotometric study in healthy dogs.

OBJECTIVES: Determine tear film kinetics with different fluorescein concentrations and repeated eye drop administration at various time intervals. ANIMALS STUDIED: Six healthy Beagles. PROCEDURES: Six experiments were conducted on separate days: single eye drop administration (control) or two separate eye drops administered at 30 s, 1, 2, 5, and 10 min intervals. For each experiment, one eye received 0.3% fluorescein solution while the other eye received 1% fluorescein solution, and tear fluid was collected with capillary tubes at 0, 1, 5, 10, 20, 30, 40, 50, 60, 90, 120, and 180 min. Fluorescein concentrations were measured using automated fluorophotometry. RESULTS: Compared with 0.3% solution, eyes receiving 1% fluorescein solution had significantly higher tear film concentrations (p ≤ .046) and the area-under-the-fluorescein-time curve was twofold greater (p = .005). Compared with control: (i) Tear film concentrations were significantly higher for up to 20 min when repeating administration 30 s to 5 min after the first drop (p ≤ .006); (ii) The highest increase in area-under-the-curve was obtained with 2 and 5 min intervals for 0.3% (+109%-130%) and 1% solutions (+153%-157%); (iii) The highest increase in median precorneal retention time (defined as tear film concentration < 5% from baseline values) was obtained with 5 min intervals for 0.3% (55 min vs. 15 min in control) and 2-5 min intervals for 1% solutions (50 min vs. 25 min in control). CONCLUSIONS: Drug delivery to the ocular surface can be enhanced by using more concentrated formulations and/or by repeating eye drop administration 2-5 min after the first dose.

Inhibiting efferocytosis reverses macrophage-mediated immunosuppression in the leukemia microenvironment.

Background: Previous studies show that the spleen and bone marrow can serve as leukemia microenvironments in which macrophages play a significant role in immune evasion and chemoresistance. We hypothesized that the macrophage driven tolerogenic process of efferocytosis is a major contributor to the immunosuppressive leukemia microenvironment and that this was driven by aberrant phosphatidylserine expression from cell turnover and cell membrane dysregulation. Methods: Since MerTK is the prototypic efferocytosis receptor, we assessed whether the MerTK inhibitor MRX2843, which is currently in clinical trials, would reverse immune evasion and enhance immune-mediated clearance of leukemia cells. Results: We found that inhibition of MerTK decreased leukemia-associated macrophage expression of M2 markers PD-L1, PD-L2, Tim-3, CD163 and Arginase-1 compared to vehicle-treated controls. Additionally, MerTK inhibition led to M1 macrophage repolarization including elevated CD86 and HLA-DR expression, and increased production of T cell activating cytokines, including IFN-ß, IL-18, and IL-1ß through activation of NF-κB. Collectively, this macrophage repolarization had downstream effects on T cells within the leukemia microenvironment, including decreased PD-1+Tim-3+ and LAG3+ checkpoint expression, and increased CD69+CD107a+ expression. Discussion: These results demonstrate that MerTK inhibition using MRX2843 altered the leukemia microenvironment from tumor-permissive toward immune responsiveness to leukemia and culminated in improved immune-mediated clearance of AML.

IFITM3 regulates fibrinogen endocytosis and platelet reactivity in nonviral sepsis.

Platelets and megakaryocytes are critical players in immune responses. Recent reports suggest infection and inflammation alter the megakaryocyte and platelet transcriptome to induce altered platelet reactivity. We determined whether nonviral sepsis induces differential platelet gene expression and reactivity. Nonviral sepsis upregulated IFN-induced transmembrane protein 3 (IFITM3), an IFN-responsive gene that restricts viral replication. As IFITM3 has been linked to clathrin-mediated endocytosis, we determined whether IFITM3 promoted endocytosis of α-granule proteins. IFN stimulation enhanced fibrinogen endocytosis in megakaryocytes and platelets from Ifitm+/+ mice, but not Ifitm-/- mice. IFITM3 overexpression or deletion in megakaryocytes demonstrated IFITM3 was necessary and sufficient to regulate fibrinogen endocytosis. Mechanistically, IFITM3 interacted with clathrin and αIIb and altered their plasma membrane localization into lipid rafts. In vivo IFN administration increased fibrinogen endocytosis, platelet reactivity, and thrombosis in an IFITM-dependent manner. In contrast, Ifitm-/- mice were completely rescued from IFN-induced platelet hyperreactivity and thrombosis. During murine sepsis, platelets from Ifitm+/+ mice demonstrated increased fibrinogen content and platelet reactivity, which was dependent on IFN-α and IFITMs. Platelets from patients with nonviral sepsis had increases in platelet IFITM3 expression, fibrinogen content, and hyperreactivity. These data identify IFITM3 as a regulator of platelet endocytosis, hyperreactivity, and thrombosis during inflammatory stress.

Assessment of Scavenger Receptor Cysteine-Rich Domain Binding to Bacteria.

The scavenger receptor cysteine-rich SRCR domain is an ancient protein domain found in SR-A and SR-I scavenger receptors, which is characterized by a conserved arrangement of cysteines (Martinez et al., Pharmacol Rev 63(4):967-1000, 2011; Sarrias et al., Crit Rev Immunol 24(1):1-37, 2004; Telfer and Baldwin, Cell Immunol 296(1):76-86, 2015; PrabhuDas et al., J Immunol, 2017. 198(10):3775-3789). SRCR domains are divided into group A and group B SRCR domains by virtue of how many cysteines they contain and the resulting disulfide bonding pattern. Group B SRCR domains, found in WC1, CD163, CD5, CD6, Spα and DMBT1, are approximately 100-110 amino acids long and contain 6-8 cysteines predicted to form 3-4 disulfide bonds. The crystal structure of a CD5 group B SRCR domain predicts a fold of two beta-sheets and an alpha helix (Rodamilans et al., J Biol Chem 282(17):12669-12677, 2007; Wang et al., Mol Immunol 48:801-809, 2011). SRCR domains bind to many different types of chemical compounds found on cells, viruses, and microbes and are usually found in multiples in the extracellular domains of transmembrane proteins or in secreted proteins. Small amino acid differences between these SRCR domains lead to significant differences in binding affinity. In addition, SRCR domain genes contain allelic polymorphisms and can be extensively duplicated. Thus, single and duplicated SRCR domain protein gene loci encode a large tunable binding potential. Binding to pathogen-associated molecular patterns (PAMPs) combined with signaling potential predicts an important role for these molecules in the immune response. WC1 SRCR domains bind to the spirochetes Leptospira and Borrelia (Hsu et al., J Immunol 194(5):2280-2288, 2015). CD6 (Sarrias et al., Proc Natl Acad Sci U S A 104(28):11724-11729, 2007), Spα (Sarrias et al., J Biol Chem 280(42):35391-35398, 2005), CD163A (Fabriek et al., Blood 113(4):887-892, 2009) and DMBT1 (Madsen et al., Eur J Immunol 33(8):2327-2336, 2003) bind to Gram-positive and Gram-negative bacteria; CD5 binds to yeast (Vera et al., Proc Natl Acad Sci U S A 106(5):1506-1511, 2009). Identified ligands include lipoteichoic acid, lipopolysaccharide, poly-phosphorylated, and -sulfated compounds such as dextran sulfate sodium, leucine-rich repeat proteins, and fungal mannose (Sarrias et al., Proc Natl Acad Sci U S A 104(28):11724-11729, 2007; Sarrias et al., J Biol Chem 280(42):35391-35398, 2005; Fabriek et al., Blood 113(4):887-892, 2009; Vera et al., Proc Natl Acad Sci U S A 106(5):1506-1511, 2009; End et al., Eur J Immunol 39(3):833-842, 2009; Loimaranta et al., J Biol Chem 284(28):18614-18623, 2009). A conserved linear binding motif (VEVLXXXXW) in an external loop in the SRCR domain has been identified in CD163A and DMBT1 and can be used as a peptide that aggregates bacteria (Fabriek et al., Blood 113(4):887-892, 2009; Bikker et al., J Biol Chem 279(46):47699-47703, 2004; Leito et al., Biol Chem 389(9):1193-1200, 2008). In contrast, WC1 binding to bacteria is mediated by a noncontinuous motif in the native protein, and mutation of the VEVLXXXXW motif has no effect upon bacterial binding (Hsu et al., J Immunol 194(5):2280-2288, 2015). Thus, bacterial binding studies with WC1 SRCR domains must be done with native, correctly disulfide bonded, protein, ideally posttranslationally modified in mammalian cells.WC1 is found in the genomes of most mammals, reptiles, and birds and is expressed exclusively on γδ T cells in ruminants. The 13 bovine WC1 genes encode up to 11 extracellular SRCR domains, organized in the SRCR domain pattern of a1-[b2-c3-d4-e5-d6]-[b7-c8-d9-e10-d'11], where the alphabet designations indicate homology between genes and across species (Chen et al., BMC Genet 13:86, 2012; Herzig et al., BMC Evol Biol 10:181, 2010; Herzig and Baldwin, BMC Genomics 10:191, 2009). Some of the signaling co-receptor WC1 molecules are required for the γδ T cell response to Leptospira (Wang et al., Mol Immunol 48:801-809, 2011; Rogers et al., J Immunol 174(6):3386-3393, 2005; Wang et al., Eur J Immunol 39(1):254-266, 2009). The WC1 expressed on responsive γδ T cells is correlated with its direct binding to Leptospira via some of its SRCR domains (Hsu et al., J Immunol 194(5):2280-2288, 2015). Because WC1+ γδ T cells share a restriction in their γδ TCRs and WC1 has TCR co-receptor activity, we hypothesize that WC1 co-ligation with the TCR plays the determining role in the activation of WC1+ γδ T cells by pathogens. Classification of the binding of WC1 SRCR domains, their ligands, and their role in the interaction of 𝛾δ T cells with pathogens relevant to the host will allow these cells to be recruited in next-generation vaccines to pathogens that have significant negative economic and health impact.

γδ T cells in artiodactyls: Focus on swine.

Vaccination is the most effective medical strategy for disease prevention but there is a need to improve livestock vaccine efficacy. Understanding the structure of the immune system of swine, which are considered a γδ T cell "high" species, and thus, particularly how to engage their γδ T cells for immune responses, may allow for development of vaccine optimization strategies. The propensity of γδ T cells to home to specific tissues, secrete pro-inflammatory and regulatory cytokines, exhibit memory or recall responses and even function as antigen-presenting cells for αß T cells supports the concept that they have enormous potential for priming by next generation vaccine constructs to contribute to protective immunity. γδ T cells exhibit several innate-like antigen recognition properties including the ability to recognize antigen in the absence of presentation via major histocompatibility complex (MHC) molecules enabling γδ T cells to recognize an array of peptides but also non-peptide antigens in a T cell receptor-dependent manner. γδ T cell subpopulations in ruminants and swine can be distinguished based on differential expression of the hybrid co-receptor and pattern recognition receptors (PRR) known as workshop cluster 1 (WC1). Expression of various PRR and other innate-like immune receptors diversifies the antigen recognition potential of γδ T cells. Finally, γδ T cells in livestock are potent producers of critical master regulator cytokines such as interferon (IFN)-γ and interleukin (IL)-17, whose production orchestrates downstream cytokine and chemokine production by other cells, thereby shaping the immune response as a whole. Our knowledge of the biology, receptor expression and response to infectious diseases by swine γδ T cells is reviewed here.

Subpopulations of swine γδ T cells defined by TCRγ and WC1 gene expression.

γδ T cells constitute a major portion of lymphocytes in the blood of both ruminants and swine. Subpopulations of swine γδ T cells have been distinguished by CD2 and CD8α expression. However, it was not clear if they have distinct expression profiles of their T-cell receptor (TCR) or WC1 genes. Identifying receptor expression will contribute to understanding the functional differences between these subpopulations and their contributions to immune protection. Here, we annotated three genomic assemblies of the swine TCRγ gene locus finding four gene cassettes containing C, J and V genes, although some haplotypes carried a null TRGC gene (TRGC4). Genes in the TRGC1 cassette were homologs of bovine TRGC5 cassette while the others were not homologous to bovine genes. Here we evaluated three principal populations of γδ T cells (CD2+/SWC5-, CD2-/SWC5+, and CD2-/SWC5-). Both CD2- subpopulations transcribed WC1 co-receptor genes, albeit with different patterns of gene expression but CD2+ cells did not. All subpopulations transcribed TCR genes from all four cassettes, although there were differences in expression levels. Finally, the CD2+ and CD2- γδ T-cell populations differed in their representation in various organs and tissues, presumably at least partially reflective of different ligand specificities for their receptors.

Influence of Schirmer strip wetness on volume absorbed, volume recovered, and total protein content in canine tears.

OBJECTIVE: Determine the protein content and volume of tears sampled by Schirmer strips wetness ranging from 20 to 35 mm. ANIMALS STUDIED: Ten healthy beagle dogs. PROCEDURES: Each dog underwent 20 tear collections per day (10 sessions in each eye, spaced by ≥1 h) for 4 separate days, providing 200 tear samples for each length of wetness evaluated: 20, 25, 30, and 35 mm. A Schirmer strip was placed in each eye until the selected mm-mark was reached, calculating the volume absorbed (VA) as the difference between the post- and pre-collection weight (assuming 1 mg~1 µL for tear fluid), and the volume recovered (VR) as the amount pipetted from the tube following centrifugation. Total protein content (TPC) was measured with infrared spectroscopy. Outcome measures were compared with the Kruskal-Wallis test. RESULTS: Median values for VA (µL), VR (µL) and TPC (mg/mL) were as follows: 20 mm (18, 10, 5.94), 25 mm (22, 12.5, 5.97), 30 mm (25.5, 16, 5.89), and 35 mm (31, 22.5, 7.13). Both VA and VR were significantly greater (p < .001) for Schirmer strips wetness of 35¼30¼25¼20 mm. TPC was significantly greater (p < .001) for 35 > 20-30 mm, but not among other groups (p = 1.000). CONCLUSIONS: The study established normative data to consider when canine studies use Schirmer strips to collect tears for bioanalytical purposes (eg, proteomics, pharmacokinetics). Although 35 mm yielded higher VA and VR, the higher TPC could be explained by greater disruption of ocular surface homeostasis. Absorption to 20-30 mm is the suggested length of strip wetness for bioanalytical tear collection in dogs.

Platelet electrical resistance for measuring platelet activation and adhesion in human health and disease.

BACKGROUND: The ability to measure changes in platelet reactivity is important to identify novel aspects of platelet biology and develop targeted therapeutics to prevent bleeding or thrombosis. Current platelet function testing allows for single agonist analysis at a time. The ability to phenotype platelets in a single assay with multiple agonists and adhesion substrates could yield more insights into altered pathways than are feasible with current approaches. We hypothesized platelet electrical resistance (PER) could be used for more comprehensive phenotyping of platelets. METHODS: Platelets were isolated from male and female healthy donors (age 39.6 ± 6.9) and septic patients (age 44.0 ± 13.5). PER 96-well plates were coated with various substrates, including fibrinogen and collagen. Platelets were added to the coated plates in the presence or absence of thrombin or convulxin. Platelet activation and spreading was monitored by measuring changes in electrical impedance. RESULTS: Platelets adhesion to fibrinogen and collagen increased impedance. In addition, impedance increased in response to thrombin or convulxin. No changes in impedance were observed in the absence of platelets or when wells were uncoated, indicating changes in impedance were directly due to platelet adhesion and activation. Inhibiting integrin αIIbß3 decreased impedance when fibrinogen was used as a substrate, consistent with platelet-dependent effects. Platelets from septic patients caused increased impedance compared to healthy donors, demonstrating this assay can be used to assess platelet hyperreactivity. CONCLUSION: PER can be applied as a high throughput tool to measure platelet reactivity in health and disease, where platelet activation is increased.

Impact of diurnal variation, sex, tear collection method, and disease state on tear protein levels in dogs.

OBJECTIVE: To investigate the effects of various biological factors on total protein concentration (TPC) and serum albumin levels in canine tears. ANIMALS STUDIED: 10 healthy beagles (5 female, 5 male) were used. PROCEDURES: Experiments were conducted on separate days, collecting tears with either capillary tubes or Schirmer strips, as follows: (i) Tear collection at 3 hours intervals (from 6 am to 12 am); and (ii) Tear collection before and 20 minutes following topical histamine application (1, 10, 375 mg/mL) to induce mild, moderate, and severe conjunctivitis, respectively. TPC and serum albumin were measured with infrared spectroscopy and ELISA, respectively. RESULTS: Tear film TPC and serum albumin ranged from 9.7-26.1 mg/mL and 6.4-1662.6 µg/mL, respectively. Protein levels did not differ significantly among time points (P ≥ .080). Median coefficient of variation (CV%) was lower with Schirmer strips compared to capillary tubes for both TPC (12% vs 15%, P = .020) and serum albumin (57% vs 78%, P = .232). TPC (P < .001), but not serum albumin was greater in male vs. female dogs. Serum albumin, but not TPC (P ≥ .099), increased significantly with each grade of conjunctivitis severity (P < .001), with no differences between collection devices (P ≥ .322); median increase was 106%, 1389%, and 2871% in eyes with mild, moderate, and severe conjunctivitis, respectively. CONCLUSIONS: There is no apparent diurnal variation in canine tear protein levels. Blood-tear barrier breakdown with conjunctivitis allows serum albumin to leak into the tear film at high concentrations. Schirmer strips compare well with capillary tubes for bioanalytical purposes in healthy and diseased eyes, and this collection method may offer improved reproducibility for protein quantification.

γδ T cells in livestock: Responses to pathogens and vaccine potential.

The immediate objective of our research is to understand the molecular mechanisms underlying activation and potentiation of the protective functional response of WC1+ γδ T cells to pathogens afflicting livestock species. The long-term goal is to incorporate stimulation of these cells into the next generation of vaccine constructs. γδ T cells have roles in the immune response to many infectious diseases including viral, bacterial, protozoan and worm infections, and their functional responses overlap with those of canonical αß T cells, for example they produce cytokines including interferon-γ and IL-17. Stimulation of non-conventional lymphocytes including γδ T cells and αß natural killer T (NKT) cells has been shown to contribute to protective immunity in mammals, bridging the gap between the innate and adaptive immune responses. Because of their innate-like early response, understanding how to engage γδ T-cell responses has the potential to optimize strategies of those that aim to induce pro-inflammatory responses as discussed here.

MERTK inhibition alters the PD-1 axis and promotes anti-leukemia immunity.

MERTK is ectopically expressed and promotes survival in acute lymphoblastic leukemia (ALL) cells and is thus a potential therapeutic target. Here we demonstrate both direct therapeutic effects of MERTK inhibition on leukemia cells and induction of anti-leukemia immunity via suppression of the coinhibitory PD-1 axis. A MERTK-selective tyrosine kinase inhibitor, MRX-2843, mediated therapeutic anti-leukemia effects in immunocompromised mice bearing a MERTK-expressing human leukemia xenograft. In addition, inhibition of host MERTK by genetic deletion (Mertk-/- mice) or treatment with MRX-2843 significantly decreased tumor burden and prolonged survival in immune-competent mice inoculated with a MERTK-negative ALL, suggesting immune-mediated therapeutic activity. In this context, MERTK inhibition led to significant decreases in expression of the coinhibitory ligands PD-L1 and PD-L2 on CD11b+ monocytes/macrophages in the leukemia microenvironment. Furthermore, although T cells do not express MERTK, inhibition of MERTK indirectly decreased PD-1 expression on CD4+ and CD8+ T cells and decreased the incidence of splenic FOXP3+ Tregs at sites of leukemic infiltration, leading to increased T cell activation. These data demonstrate direct and immune-mediated therapeutic activities in response to MERTK inhibition in ALL models and provide validation of a translational agent targeting MERTK for modulation of tumor immunity.

Dental anxiety in patients attending a student dental clinic.

BACKGROUND: This study investigated the expectations and experiences of a sample of new patients visiting an Australian regional university Student Dental Clinic with regard to anxiety provoking and alleviating stimuli in the clinical environment. Differences in anxiety levels were examined by age, gender and the type of procedure undergone. METHODS: The number of dental patients who participated in the study was 102 (56 males, 43 females). The study used a pre-treatment/post-treatment design to assess the effect of the dental procedure on anxiety levels of patients. The Modified Dental Anxiety Scale (MDAS) was used to measure anxiety levels in patients at pre-treatment. Questions were also asked about factors which may increase (length of the appointment, invasiveness of procedure) or decrease (perceived student interpersonal skills and clinical ability) dental fear. RESULTS: Females reported higher total MDAS scores (M = 11.93) compared to males (M = 9.94). Younger patients (M = 12.15) had higher dental anxiety than older patients (M = 9.34). There was a reduction in dental anxiety from pre-treatment (M = 1.92) to post-treatment (M = 1.23) on the single item anxiety measure though most of the treatment being undergone by patients was for less complex procedures. CONCLUSIONS: Patients' anticipatory experience of anxiety was higher than the anxiety experience after having undergone treatment at the student dental clinic. Student interpersonal skills and clinical ability as perceived by the patient can lessen dental anxiety in patients. Clinical Supervisor-student ratios need to be more equivalent in order to reduce the time length of appointments which currently are associated with increased patient anxiety levels in student dental clinics.

UNC2025, a MERTK Small-Molecule Inhibitor, Is Therapeutically Effective Alone and in Combination with Methotrexate in Leukemia Models.

Purpose: MERTK tyrosine kinase is ectopically expressed in 30% to 50% of acute lymphoblastic leukemias (ALL) and more than 80% of acute myeloid leukemias (AML) and is a potential therapeutic target. Here, we evaluated the utility of UNC2025, a MERTK tyrosine kinase inhibitor, for treatment of acute leukemia.Experimental Design: Preclinical in vitro and in vivo assays using cell lines and primary leukemia patient samples were used to evaluate antileukemic effects of UNC2025.Results: UNC2025 potently inhibited prosurvival signaling, induced apoptosis, and reduced proliferation and colony formation in MERTK-expressing ALL and AML cell lines and patient samples. Approximately 30% of primary leukemia patient samples (78 of 261 total) were sensitive to UNC2025. Sensitive samples were most prevalent in the AML, T-ALL, and minimally differentiated (M0) AML subsets. UNC2025 inhibited MERTK in bone marrow leukemia cells and had significant therapeutic effects in xenograft models, with dose-dependent decreases in tumor burden and consistent two-fold increases in median survival, irrespective of starting disease burden. In a patient-derived AML xenograft model, treatment with UNC2025 induced disease regression. In addition, UNC2025 increased sensitivity to methotrexate in vivo, suggesting that addition of MERTK-targeted therapy to current cytotoxic regimens may be particularly effective and/or allow for chemotherapy dose reduction.Conclusions: The broad-spectrum activity mediated by UNC2025 in leukemia patient samples and xenograft models, alone or in combination with cytotoxic chemotherapy, supports continued development of MERTK inhibitors for treatment of leukemia. Clin Cancer Res; 23(6); 1481-92. ©2016 AACR.

Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia.

Mer and Flt3 receptor tyrosine kinases have been implicated as therapeutic targets in acute myeloid leukemia (AML). In this manuscript we describe UNC1666, a novel ATP-competitive small molecule tyrosine kinase inhibitor, which potently diminishes Mer and Flt3 phosphorylation in AML. Treatment with UNC1666 mediated biochemical and functional effects in AML cell lines expressing Mer or Flt3 internal tandem duplication (ITD), including decreased phosphorylation of Mer, Flt3 and downstream effectors Stat, Akt and Erk, induction of apoptosis in up to 98% of cells, and reduction of colony formation by greater than 90%, compared to treatment with vehicle. These effects were dose-dependent, with inhibition of downstream signaling and functional effects correlating with the degree of Mer or Flt3 kinase inhibition. Treatment of primary AML patient samples expressing Mer and/or Flt3-ITD with UNC1666 also inhibited Mer and Flt3 intracellular signaling, induced apoptosis, and inhibited colony formation. In summary, UNC1666 is a novel potent small molecule tyrosine kinase inhibitor that decreases oncogenic signaling and myeloblast survival, thereby validating dual Mer/Flt3 inhibition as an attractive treatment strategy for AML.