Pradeep K. Dagur

Hypomethylation of the IL17RC promoter associates with age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population worldwide. Although recent studies have demonstrated strong genetic associations between AMD and SNPs in a number of genes, other modes of regulation are also likely to play a role in the etiology of this disease. We identified a significantly decreased level of methylation on the IL17RC promoter in AMD patients. Furthermore, we showed that hypomethylation of the IL17RC promoter in AMD patients led to an elevated expression of its protein and messenger RNA in peripheral blood as well as in the affected retina and choroid, suggesting that the DNA methylation pattern and expression of IL17RC may potentially serve as a biomarker for the diagnosis of AMD and likely plays a role in disease pathogenesis.

Baseline Levels and Temporal Stability of 27 Multiplexed Serum Cytokine Concentrations in Healthy Subjects

Background Cytokines are humoral molecules that elicit regulatory function in immunologic pathways. The level and type of cytokine production has become critical in distinguishing physiologic from pathologic immune conditions. Cytokine profiling has become an important biomarker discovery tool in monitoring of the immune system. However, the variations in cytokine levels in individual subjects over time in healthy individuals have not been extensively studied. In this study, we use multiplex bead arrays to evaluate 27 analytes in paired serum samples taken seven days apart from 144 healthy individuals in order to assess variations over a short time period. Methods Fluorescent bead-based immunoassay (Luminex) was used to measure 27 analytes in serum samples. Measurements were performed on matched samples from 144 healthy donors. To assess inter-plate variability, one arbitrarily selected serum sample was analyzed on each of the first ten plates as bridge sample. Results Using the bridge sample, we showed minimal inter-plate variations in the measurement of most analytes. In measurement of cytokines from the 144 patients at two time points, we found that three cytokines (IL-2, IL-15 and GM-CSF) were undetectable and five analytes (RANTES, MCP-1, VEGF, MIP-1β and PDGF-BB) showed significant difference in concentrations at Day 0 compared to Day 7. Conclusions The current study demonstrated higher variations in cytokine levels among individuals than were observed for samples obtained one week apart from identical donors. These data suggest that a serum sample from each subject for use as a baseline measurement is a better control for clinical trials rather than sera from a paired cohort.

5A, an Apolipoprotein A-I Mimetic Peptide, Attenuates the Induction of House Dust Mite-Induced Asthma

New treatment approaches are needed for patients with asthma. Apolipoprotein A-I (apoA-I), the major structural protein of high-density lipoproteins, mediates reverse cholesterol transport and has atheroprotective and anti-inflammatory effects. In this study, we hypothesized that an apoA-I mimetic peptide might be effective at inhibiting asthmatic airway inflammation. A 5A peptide, which is a synthetic, bihelical apoA-I mimetic, was administered to wild-type A/J mice via osmotic mini-pump prior to the induction of house dust mite (HDM)-induced asthma. HDM-challenged mice that received the 5A apoA-I mimetic peptide had significant reductions in the number of bronchoalveolar lavage fluid eosinophils, lymphocytes, and neutrophils, as well as in histopathological evidence of airway inflammation. The reduction in airway inflammation was mediated by a reduction in the expression of Th2- and Th17-type cytokines, as well as in chemokines that promote T cell and eosinophil chemotaxis, including CCL7, CCL17, CCL11, and CCL24. Furthermore, the 5A apoA-I mimetic peptide inhibited the alternative activation of pulmonary macrophages in the lungs of HDM-challenged mice. It also abrogated the development of airway hyperresponsiveness and reduced several key features of airway remodeling, including goblet cell hyperplasia and the expression of collagen genes (Col1a1 and Col3a1). Our results demonstrate that the 5A apoA-I mimetic peptide attenuates the development of airway inflammation and airway hyperresponsiveness in an experimental murine model of HDM-induced asthma. These data support the conclusion that strategies using apoA-I mimetic peptides, such as 5A, might be developed further as a possible new treatment approach for asthma.

Live Attenuated Leishmania donovani p27 Gene Knockout Parasites Are Nonpathogenic and Elicit Long-Term Protective Immunity in BALB/c Mice

Leishmaniasis causes significant morbidity and mortality worldwide, and no vaccines against this disease are available. Previously, we had shown that the amastigote-specific protein p27 (Ldp27) is a component of an active cytochrome c oxidase complex in Leishmania donovani and that upon deletion of its gene the parasite had reduced virulence in vivo. In this study, we have shown that Ldp27−/− parasites do not survive beyond 20 wk in BALB/c mice and hence are safe as an immunogen. Upon virulent challenge, mice 12 wk postimmunization showed significantly lower parasite burden in the liver and spleen. When mice were challenged 20 wk postimmunization, a significant reduction in parasite burden was still noted, suggesting long-term protection by Ldp27−/− immunization. Immunization with Ldp27−/− induced both pro- and anti-inflammatory cytokine responses and activated splenocytes for enhanced leishmanicidal activity in association with NO production. Protection in both short- and long-term immunized mice after challenge with the wild-type parasite correlated with the stimulation of multifunctional Th1-type CD4 and CD8 T cells. Adoptive transfer of T cells from long-term immunized mice conferred protection against virulent challenge in naive recipient mice, suggesting involvement of memory T cell response in protection against Leishmania infection. Immunization of mice with Ldp27−/−also demonstrated cross-protection against Leishmania major and Leishmania braziliensis infection. Our data show that genetically modified live attenuated Ldp27−/− parasites are safe, induce protective immunity even in the absence of parasites, and can provide protection against homologous and heterologous Leishmania species.

Development and characterization of 5-FU bearing ferritin appended solid lipid nanoparticles for tumour targeting

Ferritin coupled solid lipid nanoparticles were investigated for tumour targeting. Solid lipid nanoparticles were prepared using HSPC, cholesterol, DSPE and triolien. The SLNs without ferritin which has similar lipid composition were used for comparison. SLNs preparations were characterized for shape, size and percentage entrapment. The average size of SLNs was found to be in the range 110–152 nm and maximum drug entrapment was found to be 34.6–39.1%. In vitro drug release from the formulations is obeying fickian release kinetics. Cellular uptake and IC50 values of the formulation were determined in vitro in MDA-MB-468 breast cancer cells. In vitro cell binding of Fr-SLN exhibits 7.7-folds higher binding to MDA-MB-468 breast cancer cells in comparison to plain SLNs. Ex-vivo cytotoxicity assay on targeted nanoparticles gave IC50 of 1.28 µM and non-targeted nanoparticles gave IC50 of 3.56 µM. In therapeutic experiments, 5-FU, SLNs and Fr-SLNs were administered at the dose of 10 mg 5-FU/kg body weight to MDA-MB-468 tumour bearing Balb/c mice. Administration of Fr-SLNs formulation results in effective reduction in tumour growth as compared with free 5-FU and plain SLNs. The result demonstrates that this delivery system possessed an enhanced anti-tumour activity. The results warrant further evaluation of this delivery system.

High Dimensional Flow Cytometry for Comprehensive Leukocyte Immunophenotyping (CLIP) in Translational Research

New paradigms in translational research are focused on deep understanding of all aspects of the human immune system in response to diseases or perturbations such as vaccination or therapy. To obtain this knowledge, coordinated, comprehensive assessments by genomics, proteomics, and cytomics are necessary. One component of this assessment is comprehensive leukocyte immunophenotyping (CLIP) that not only provides a deep and broad description of the entire immune system at any given moment, but also encompasses all leukocyte lineages, including activation states, functional markers, and signaling molecules. As envisioned, a CLIP panel could study nearly 400 antigens utilizing 17-parameter flow cytometry. The CLIP panel is structured in a manner that tubes are grouped by lineage and, within lineage each of the tubes, while having some redundant markers, characterize distinct populations. To date, a preliminary 10 tube CLIP panel has been developed with the following 17 parameter tubes: T(reg), T(h₁₇), T(h₁/₂), B(general), B(naive/memory), B(intracellular), NK₁, NK₂, myeloid/monocyte, and dendritic cells (DC). Together these tubes have the potential to identify over 28,000 subsets of leukocytes. The feasibility of developing these tubes has been demonstrated, as well as their utility in describing complex alterations of the immune system in the context of disease and vaccination. The plethora of data accrued in the preliminary CLIP panel highlights the need for novel data analysis and reduction strategies, while at the same time illustrates the power of CLIP.

Apolipoprotein A-I Attenuates Ovalbumin-Induced Neutrophilic Airway Inflammation via a Granulocyte Colony–Stimulating Factor–Dependent Mechanism

Apolipoprotein A-I (apoA-I) is a key component of high-density lipoproteins that mediates reverse cholesterol transport from cells and reduces vascular inflammation. We investigated whether endogenous apoA-I modulates ovalbumin (OVA)-induced airway inflammation in mice. We found that apoA-I expression was significantly reduced in the lungs of OVA-challenged, compared with saline-challenged, wild-type (WT) mice. Next, to investigate the role of endogenous apoA-I in the pathogenesis of OVA-induced airway inflammation, WT and apoA-I(-/-) mice were sensitized by intraperitoneal injections of OVA and aluminum hydroxide, followed by multiple nasal OVA challenges for 4 weeks. OVA-challenged apoA-I(-/-) mice exhibited a phenotype of increased airway neutrophils compared with WT mice, which could be rescued by an administration of a 5A apoA-I mimetic peptide. Multiple pathways promoted neutrophilic inflammation in OVA-challenged apoA-I(-/-) mice, including the up-regulated expression of (1) proinflammatory cytokines (IL-17A and TNF-α), (2) CXC chemokines (CXCL5), (3) vascular adhesion molecules (i.e., vascular cell adhesion molecule-1), and (4) granulocyte colony-stimulating factors (G-CSF). Because concentrations of G-CSF in bronchoalveolar lavage fluid (BALF) were markedly increased in OVA-challenged apoA-I(-/-) mice, we hypothesized that enhanced G-CSF expression may represent the predominant pathway mediating increased neutrophilic inflammation. This was confirmed by the intranasal administration of a neutralizing anti-G-CSF antibody, which significantly reduced BALF neutrophilia by 72% in OVA-challenged apoA-I(-/-) mice, compared with mice that received a control antibody. We conclude that endogenous apoA-I negatively regulates OVA-induced neutrophilic airway inflammation, primarily via a G-CSF-dependent mechanism. Furthermore, these findings suggest that apoA-I may play an important role in modulating the severity of neutrophilic airway inflammation in asthma.

Drug‐induced allergic hepatitis develops in mice when myeloid‐derived suppressor cells are depleted prior to halothane treatment

Clinical evidence suggests that many cases of serious idiosyncratic drug‐induced liver injury are mediated by the adaptive immune system in response to hepatic drug‐protein adducts, also referred to as “drug‐induced allergic hepatitis”; but detailed mechanistic proof has remained elusive due to the lack of animal models. We have hypothesized that drug‐induced allergic hepatitis is as rare in animals as it is in humans due at least in part to the tolerogenic nature of the liver. We provide evidence that immune tolerance can be overcome in a murine model of halothane‐induced liver injury initiated by trifluoroacetylated protein adducts of halothane formed in the liver. Twenty‐four hours after female Balb/cJ mice were initially treated with halothane, perivenous necrosis and an infiltration of CD11b+Gr‐1high cells were observed in the liver. Further study revealed a subpopulation of myeloid‐derived suppressor cells within the CD11b+Gr‐1high cell fraction that inhibited the proliferation of both CD4+ and CD8+ T cells. When CD11b+Gr‐1high cells were depleted from the liver with Gr‐1 antibody treatment, enhanced liver injury was observed at 9 days after halothane rechallenge. Toxicity was associated with increased serum levels of interleukin‐4 and immunoglobulins G1 and E directed against hepatic trifluoroacetylated protein adducts, as well as increased hepatic infiltration of eosinophils and CD4+ T cells, all features of an allergic reaction. When hepatic CD4+ T cells were depleted 5 days after halothane rechallenge, trifluoroacetylated protein adduct–specific serum immunoglobulin and hepatotoxicity were reduced. Conclusion: Our data provide a rational approach for developing animal models of drug‐induced allergic hepatitis mediated by the adaptive immune system and suggest that impaired liver tolerance may predispose patients to this disease. (Hepatology 2015;62:546–557

Caspase-7 Cleavage of Kaposi Sarcoma-associated Herpesvirus ORF57 Confers a Cellular Function against Viral Lytic Gene Expression

Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 is a viral early protein essential for KSHV multiplication. We found that B cells derived from cavity-based B cell lymphoma with lytic KSHV infection display activation of caspase-8 and cleavage of ORF57 in the cytoplasm by caspase-7 at the aspartate residue at position 33 from the N terminus. Caspase-7 cleavage of ORF57 is prevented by pan-caspase inhibitor z-VAD, caspase-3 and caspase-7 inhibitor z-DEVD, and caspase-7 small interfering RNAs. The caspase-7 cleavage site 30DETD33 in ORF57 is not cleavable by caspase-3, although both enzymes use DEXD as a common cleavage site. B cells with lytic KSHV infection and caspase-7 activation exhibited a greatly reduced level of ORF57. A majority of the cells expressing active caspase-7 appeared to have no detectable ORF57 and vice versa. Upon cleavage with caspase-7, ORF57 was deficient in promoting the expression of viral lytic genes. Inhibiting caspase-7 cleavage of ORF57 in KSHV+ BCBL-1 cells by z-VAD, z-DEVD, or caspase-7 small interfering RNA led to increased expression of viral lytic genes and production of cell-free virus particles. Collectively, our data provide the first compelling evidence that caspase cleavage of ORF57 may represent a cellular function against lytic KSHV infection.

Secretion of interleukin-17 by CD8+ T cells expressing CD146 (MCAM).

Interleukin-17 (IL-17) has been associated with the pathogenesis of numerous autoimmune diseases. CD4+ T cells secreting IL-17 are termed Th17 cells. CD8+ T cells, designated Tc17 cells, are also capable of secreting IL-17. Here we describe a population of Tc17 cells characterized by the expression of surface CD146, an endothelial adhesion molecule. These cells display signatures of a human Tc17 genotype and phenotype. Circulating CD8+CD146+ T cells are present in low levels in healthy adults. Elevations in CD8+CD146+ T cells are found in Behcets disease and birdshot retinochoroidopathy, which have been reported to have HLA class I associations. Sarcoidosis does not have a class I association and displays an increase in CD4+ CD146+ T cells but not in CD8+CD146+ T cells. CD146 on these cells may facilitate their ability to bind to, and migrate through, endothelium, as has been reported for CD4+CD146+ T cells.