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Objective: Atherogenesis is an inflammatory process accelerated in psoriasis, a chronic inflammatory skin disease associated with premature cardiovascular diseases (CVD). Cholesterol crystal (CC) formation is implicated at onset and progression of atherosclerotic CVD, and we have recently shown CC formation to be upregulated in a murine model of psoriasis-related atherosclerosis. Therefore, we investigated inflammatory CC production in this psoriasis mouse model and also using psoriasis patient derived samples. Methods: K14-Rac1V12 psoriasis mouse aortas were examined for CC formation and structural abnormalities, such as subendothelial spaces, via electron microscopy and histology. Human aortic endothelial cells (HAoEC) were stimulated with sera from healthy and psoriatic volunteers (n=5 each), inflammatory markers TNFα+IFNγ, LDL or combination of TNFα+IFNγ+LDL. The effect on CC formation and lysosomal function was determined by advanced microscopy and flow cytometry. HAoECs were also treated with TNFα+IFNγ+LDL combined with β-cyclodextrin (CD), a stimulant for cholesterol release from cells, and analyzed for CC formation and lysosomal function. Results: Psoriatic mouse aortas showed enlarged subendothelial spaces and 40% increased CC formation under normolipidemic conditions compared to littermate controls. Treatment of HAoECs with psoriasis patient-sera (severe skin disease, low CVD risk score) or TNFα+IFNγ+LDL showed a 2-fold increase in CC formation and 30% increased lysosomal frequency compared to controls. Despite an increase in lysosome frequency, TNFα+IFNγ+LDL treatment decreased overall pH-related lysosomal activity. CD treatment diminished TNFα+IFNγ+LDL-induced CC formation (compared to vehicle), and restored lysosomal function without affecting lysosome frequency. Conclusion: Psoriasis mice displayed accelerated CC formation in the absence of hyperlipidemic conditions suggesting chronic systemic inflammation accelerates CC formation in vivo and in vitro , accompanied by lysosomal dysfunction in ECs. These findings suggest that dyslipidemia and systemic inflammation in psoriasis are important contributors to early atherogenesis and should be treatment targets for CVD risk mitigation.

Visceral leishmaniasis (VL) causes significant mortality and there is no effective vaccine. Previously, we have shown that genetically modified Leishmania donovani parasites, here described as live attenuated parasites, induce a host protective adaptive immune response in various animal models. In this study, we demonstrate an innate immune response upon infection with live attenuated parasites in macrophages from BALB/c mice both in vitro and in vivo. In vitro infection of macrophages with live attenuated parasites (compared to that with wild-type [WT] L. donovani parasites) induced significantly higher production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-12 [IL-12], gamma interferon [IFN-γ], and IL-6), chemokines (monocyte chemoattractant protein 1/CCL-2, macrophage inflammatory protein 1α/CCL-3, and IP-10), reactive oxygen species (ROS), and nitric oxide, while concomitantly reducing anti-inflammatory cytokine IL-10 and arginase-1 activities, suggesting a dominant classically activated/M1 macrophage response. The classically activated response in turn helps in presenting antigen to T cells, as observed with robust CD4 + T cell activation in vitro . Similarly, parasitized splenic macrophages from live attenuated parasite-infected mice also demonstrated induction of an M1 macrophage phenotype, indicated by upregulation of IL-1β, TNF-α, IL-12, and inducible nitric oxide synthase 2 and downregulation of genes associated with the M2 phenotype, i.e., the IL-10, YM1, Arg-1, and MRC-1 genes, compared to WT L. donovani -infected mice. Furthermore, an ex vivo antigen presentation assay showed macrophages from live attenuated parasite-infected mice induced higher IFN-γ and IL-2 but significantly less IL-10 production by ovalbumin-specific CD4 + T cells, resulting in proliferation of Th1 cells. These data suggest that infection with live attenuated parasites promotes a state of classical activation (M1 dominant) in macrophages that leads to the generation of protective Th1 responses in BALB/c mice.

To the Editor: CD146, also called melanoma cell adhesion molecule (MCAM), is a cell surface adhesion molecule on endothelial cells involved in homotypic and heterotypic cell interactions (Bardin et al, 2001). CD146 binding in endothelial cells (ECs) leads to a change in cellular permeability, actin distribution and redistribution of NF-kappa B p50 to the nucleus. CD146 has been shown to be present on 1–3% of circulating peripheral blood T cells in healthy humans (Elshal et al, 2005). CD146+ T cells have an effector memory phenotype, demonstrate up-regulation of a cluster of genes involved with adhesion, migration, homing, and inflammation, and have enhanced binding to endothelial monolayers in vitro (Elshal et al, 2007). These features of the CD146+ T cells in the peripheral circulation have led to speculation that these represent a small pool of cells primed for extravasation and/or homing of activated T cells (Elshal et al, 2007, Guezguez et al, 2007) in response to inflammatory stimuli. Circulating CD146+ T cells are elevated in several inflammatory autoimmune diseases such as sarcoidosis, inflammatory bowel disease, multiple sclerosis, connective tissue disease, and Behcet's disease and produce IL-17 (Dagur et al, 2011, Dagur et al, 2010, LaRochelle et al, 2012). Whether these cells play a role at the site of active inflammation in these diseases remains unknown. Psoriasis, which is associated with increased vascular inflammation (Mehta et al, 2009) and access to both peripheral blood and the disease target tissue (e.g. skin), is ideal to study CD146+ T cell phenotype and function in an inflammatory condition. Here we present findings from a well-characterized patient population with psoriasis using peripheral blood samples and skin biopsies from psoriatic lesions and uninvolved skin. Forty-seven patients with psoriasis and sixty-seven healthy controls were included in this study. Diagnosis of psoriasis was confirmed by a dermatologist and severity was measured by percentage of body surface area (BSA) involved and the validated Psoriasis Area and Severity Index (PASI). Donor demographics and characteristics are presented in Supplemental Table 1. Skin biopsies were isolated from a representative psoriatic target lesion (6 mm) and are identified as lesional psoriatic skin. Non-lesional skin biopsies were obtained from a similar body area at least 10cm away from the nearest psoriasis skin lesion. Frozen sections were obtained from skin lesions for immunofluorescence studies and all patients provided written consent in as part of an IRB-approved study (NCT01778569). Venous blood was collected in sodium heparin vacutainers (Becton Dickinson (BD), San Jose, CA). Cells were stained and flow cytometric analysis was performed as previously described (6). Skin biopsies were digested in Collagenase IV (GIBCO BRL # 17104-019) at 5 mg/ml in RPMI 1640 for 45 min, stained, and then sorted in the same manner as peripheral blood. The following antibodies used for staining were obtained from BD: CD3, CD4, CD8, CD33, CD14, CD19, CD45, CD45-RO, CD146 (Clone P1H12). Anti-IL-17A (clone ebio64DEC17) was purchased from eBiosciences. Immunophenotyping results are expressed as means and standard errors of the mean. RNA was isolated from sorted CD146+ or CD146- T cell subpopulations using RNAquos Micro kits (Ambion) and real time PCR (QRTPCR) was performed using a 7900-sequence detector (PE-Applied Biosystems, Norwalk, CT). Data from a single specimen were considered one experiment (n). A p-value

Inflammation is critical to atherogenesis. Psoriasis is a chronic inflammatory skin disease that accelerates atherosclerosis in humans and provides a compelling model to understand potential pathways linking these diseases. A murine model capturing the vascular and metabolic diseases in psoriasis would accelerate our understanding and provide a platform to test emerging therapies. We aimed to characterize a new murine model of skin inflammation (Rac1V12) from a cardiovascular standpoint to identify novel atherosclerotic signaling pathways modulated in chronic skin inflammation. The RacV12 psoriasis mouse resembled the human disease state, including presence of systemic inflammation, dyslipidemia, and cardiometabolic dysfunction. Psoriasis macrophages had a proatherosclerotic phenotype with increased lipid uptake and foam cell formation, and also showed a 6-fold increase in cholesterol crystal formation. We generated a triple-genetic K14-RacV12-/+/Srb1-/-/ApoER61H/H mouse and confirmed psoriasis accelerates atherogenesis (~7-fold increase). Finally, we noted a 60% reduction in superoxide dismutase 2 (SOD2) expression in human psoriasis macrophages. When SOD2 activity was restored in macrophages, their proatherogenic phenotype reversed. We demonstrate that the K14-RacV12 murine model captures the cardiometabolic dysfunction and accelerates vascular disease observed in chronic inflammation and that skin inflammation induces a proatherosclerotic macrophage phenotype with impaired SOD2 function, which associated with accelerated atherogenesis.

Purpose Autoimmune retinopathy (AIR) is a retinopathy associated with unexplained vision loss presumably linked to circulating antiretinal antibodies; currently, however, there are no standardized criteria regarding the diagnosis, treatment strategy, or pathogenesis of this disease. The importance of B-lymphocyte immunophenotyping in the classification of AIR is unknown. Methods We utilized 15-color multiparametric flow cytometry to identify aberrations in B cell subsets that may contribute to the pathophysiology of AIR. Luminex cytokine analysis was also performed on plasma samples from AIR patients. Results Significant differences in AIR patients compared to individuals with other inflammatory conditions or healthy donors were found in the B cell memory compartment, including an increase in naive B cells and a decrease in switched and unswitched memory B cells, which correlated with alterations in immunoglobulin secretion. Conclusions These findings suggest that the maturation process of B cells may be impaired and that B cell immunophenotyping may help in understanding disease process in AIR.

Background Low-density lipoprotein receptor–related protein 1 (LRP-1) is a scavenger receptor that regulates adaptive immunity and inflammation. LRP-1 is not known to modulate the pathogenesis of allergic asthma. Objective We sought to assess whether LRP-1 expression by dendritic cells (DCs) modulates adaptive immune responses in patients with house dust mite (HDM)–induced airways disease. Methods LRP-1 expression on peripheral blood DCs was quantified by using flow cytometry. The role of LRP-1 in modulating HDM-induced airways disease was assessed in mice with deletion of LRP-1 in CD11c + cells ( Lrp1 fl/fl ; CD11c -Cre) and by adoptive transfer of HDM-pulsed CD11b + DCs from Lrp1 fl/fl ; CD11c -Cre mice to wild-type (WT) mice. Results Human peripheral blood myeloid DC subsets from patients with eosinophilic asthma have lower LRP-1 expression than cells from healthy nonasthmatic subjects. Similarly, LRP-1 expression by CD11b + lung DCs was significantly reduced in HDM-challenged WT mice. HDM-challenged Lrp1 fl/fl ; CD11c-Cre mice have a phenotype of increased eosinophilic airway inflammation, allergic sensitization, T H 2 cytokine production, and mucous cell metaplasia. The adoptive transfer of HDM-pulsed LRP-1–deficient CD11b + DCs into WT mice generated a similar phenotype of enhanced eosinophilic inflammation and allergic sensitization. Furthermore, CD11b + DCs in the lungs of Lrp1 fl/fl ; CD11c -Cre mice have an increased ability to take up HDM antigen, whereas bone marrow–derived DCs display enhanced antigen presentation capabilities. Conclusion This identifies a novel role for LRP-1 as a negative regulator of DC-mediated adaptive immune responses in the setting of HDM-induced eosinophilic airway inflammation. Furthermore, the reduced LRP-1 expression by circulating myeloid DCs in patients with eosinophilic asthma suggests a possible role for LRP-1 in modulating type 2–high asthma.

Objective: Fluid-phase pinocytosis is a receptor-independent mechanism of endocytosis that occurs in all mammalian cells and may be a mechanism for the uptake of LDL by macrophages. As there are currently no methods for the measurement of fluid-phase pinocytosis by individual aortic cells in vivo, we sought to identify a suitable method. Methods: ApoE-/- mice were retro-orbitally injected with AngioSPARK fluorescent nanoparticles specifically designed to not interact with cells. After 24 h, mice were sacrificed, and the aortas were isolated and then digested to analyze aortic cell uptake of AngioSPARK by flow cytometry. Results: CD11b-expressing aortic macrophages from mice injected with AngioSPARK showed high levels of fluid-phase pinocytosis compared to aortic cells not expressing CD11b (4,393.7 vs. 408.3 mean fluorescence intensity [MFI], respectively). Conclusion: This new technique allows for the measurement of fluid-phase pinocytosis by aortic cells in vivo, making it possible to examine the cell-signaling molecules and drugs that affect this process. Published by S. Karger AG, Basel

Apolipoprotein A-I (apoA-I) is an important component of high-density lipoprotein particles that mediates reverse cholesterol transport out of cells by interacting with the ATP-binding cassette transporter 1 (ABCA1). apoA-I has also been shown to attenuate neutrophilic airway inflammation in experimental ovalbumin (OVA)-induced asthma by reducing the expression of granulocyte colony-stimulating factor (G-CSF). Here, we hypothesized that overexpression of the ABCA1 transporter might similarly attenuate OVA-induced neutrophilic airway inflammation. Tie2-human ABCA1 (hABCA1) mice expressing human ABCA1 under the control of the Tie2 promoter, which is primarily expressed by vascular endothelial cells, but can also be expressed by macrophages, received daily intranasal OVA challenges, 5 d/wk for 5 weeks. OVA-challenged Tie2-hABCA1 mice had significant reductions in total bronchoalveolar lavage fluid (BALF) cells that reflected a decrease in neutrophils, as well as reductions in peribronchial inflammation, OVA-specific IgE levels, and airway epithelial thickness. The reduced airway neutrophilia in OVA-challenged Tie2-hABCA1 mice was associated with significant decreases in G-CSF protein levels in pulmonary vascular endothelial cells, alveolar macrophages, and BALF. Intranasal administration of recombinant murine G-CSF to OVA-challenged Tie2-hABCA1 mice for 5 days increased BALF neutrophils to a level comparable to that of OVA-challenged wild-type mice. We conclude that ABCA1 suppresses OVA-induced airway neutrophilia by reducing G-CSF production by vascular endothelial cells and alveolar macrophages. These findings suggest that ABCA1 expressed by vascular endothelial cells and alveolar macrophages may play important roles in attenuating the severity of neutrophilic airway inflammation in asthma.

Peptidoglycan recognition protein (Pglyrp) 1 is a pattern-recognition protein that mediates antibacterial host defense. Because we had previously shown that Pglyrp1 expression is increased in the lungs of house dust mite (HDM)-challenged mice, we hypothesized that it might modulate the pathogenesis of asthma. Wild-type and Pglyrp1(-/-) mice on a BALB/c background received intranasal HDM or saline, 5 days/week for 3 weeks. HDM-challenged Pglyrp1(-/-) mice showed decreases in bronchoalveolar lavage fluid eosinophils and lymphocytes, serum IgE, and mucous cell metaplasia, whereas airway hyperresponsiveness was not changed when compared with wild-type mice. T helper type 2 (Th2) cytokines were reduced in the lungs of HDM-challenged Pglyrp1(-/-) mice, which reflected a decreased number of CD4(+) Th2 cells. There was also a reduction in C-C chemokines in bronchoalveolar lavage fluid and lung homogenates from HDM-challenged Pglyrp1(-/-) mice. Furthermore, secretion of CCL17, CCL22, and CCL24 by alveolar macrophages from HDM-challenged Pglyrp1(-/-) mice was markedly reduced. As both inflammatory cells and airway epithelial cells express Pglyrp1, bone marrow transplantation was performed to generate chimeric mice and assess which cell type promotes HDM-induced airway inflammation. Chimeric mice lacking Pglyrp1 on hematopoietic cells, not structural cells, showed a reduction in HDM-induced eosinophilic and lymphocytic airway inflammation. We conclude that Pglyrp1 expressed by hematopoietic cells, such as alveolar macrophages, mediates HDM-induced airway inflammation by up-regulating the production of C-C chemokines that recruit eosinophils and Th2 cells to the lung. This identifies a new family of innate immune response proteins that promotes HDM-induced airway inflammation in asthma.

Summary 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.

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.

The hyperparathyroidism–jaw tumor syndrome (HPT–JT) is a familial cancer syndrome that can result from germline inactivation of HRPT2/CDC73, a putative tumor suppressor gene that encodes parafibromin, a component of the transcriptional regulatory PAF1 complex with homology to the yeast protein Cdc73p. The vast majority of HRPT2/CDC73 germline mutations identified have been truncation or frameshift mutations, and loss of function due to missense mutation is rare. We report here a kindred with HPT–JT due to a germline L95P missense mutation in parafibromin. The mutant parafibromin was studied in vitro to understand the basis of its presumed loss-of-function. When transfected in cultured cells, the L95P mutant was expressed to a lower level than wild-type (wt) parafibromin, a difference that was not overcome by inhibition of the proteasomal degradation pathway. The L95P mutant parafibromin retained the ability to assemble with endogenous PAF1 complex components as evidenced by co-immunoprecipitation. Analysis of subcellular localization showed that the L95P mutant was markedly deficient in nucleolar localization compared to the wt, an impairment likely resulting from disruption of a putative nucleolar localization signal immediately upstream of the L95P mutation. Transfection of the L95P parafibromin mutant, but not the wt, enhanced cell cycle progression and increased cell survival in NIH-3T3 and HEK 293 cells, resulting apparently from dominant interference with endogenous parafibromin action. The simultaneous loss of nucleolar localization and acquisition of a growth stimulatory phenotype with the L95P mutation raise the possibility that parafibromin must interact with targets in the nucleolus to fully execute its tumor suppressor functions.

Human peripheral blood is often studied by flow cytometry in both the research and clinical laboratories. The methods for collection, storage, and preparation of peripheral blood will vary depending on the cell lineage to be examined as well as the type of assay to be performed. This unit presents protocols for collection of blood, separation of leukocytes from whole blood by lysis of erythrocytes, isolating mononuclear cells by density gradient separation, and assorted non-flow sorting methods, such as magnetic bead separations, for enriching specific cell populations, including monocytes, T lymphocytes, B lymphocytes, neutrophils,, , and platelets prior to flow cytometric analysis. A protocol is also offered for cryopreservation of cells since clinical research often involves retrospective flow cytometric analysis of samples stored over a period of months or years.

DNA-dependent protein kinase (DNA-PK) mediates double stranded DNA break repair, V(D)J recombination, and immunoglobulin class switch recombination, as well as innate immune and pro-inflammatory responses. However, there is limited information regarding the role of DNA-PK in adaptive immunity mediated by dendritic cells (DCs), which are the primary antigen-presenting cells in allergic asthma. Here we show that house dust mite induces DNA-PK phosphorylation, which is a marker of DNA-PK activation, in DCs via the generation of intracellular reactive oxygen species. We also demonstrate that pharmacological inhibition of DNA-PK, as well as the specific deletion of DNA-PK in DCs, attenuates the induction of allergic sensitization and Th2 immunity via a mechanism that involves the impaired presentation of mite antigens. Furthermore, pharmacological inhibition of DNA-PK following antigen priming similarly reduces the manifestations of mite-induced airway disease. Collectively, these findings suggest that DNA-PK may be a potential target for treatment of allergic asthma.

A subset of T cells defined by the cell surface expression of MCAM (CD146) has been identified in the peripheral circulation of healthy individuals. These cells comprise approximately 3% of the pool of circulating T cells, have an effector memory phenotype, and are capable of producing several cytokines. Notably, the MCAM positive cells are enhanced for IL-17 production compared to MCAM negative effector memory T cells. These cells are committed to IL-17 production and do not require in vitro polarization with exogenous cytokines. MCAM positive T cells also demonstrate an increased ability to bind to endothelial monolayers. In numerous autoimmune diseases these cells are found at increased proportions in the peripheral circulation, and at the sites of active inflammation in patients with autoimmune disease, these cells appear in large numbers and are major contributors to IL-17 production. Studies to date have been performed with human subjects and it is uncertain if appropriate mouse models exist for this cell type. These cells could represent early components of the adaptive immune response and serve as targets of therapy in these diseases, although much work remains to be performed in order to discern the exact nature and function of these cells.

Genomic engineering by the guide RNA (gRNA)-directed CRISPR/CAS9 is rapidly becoming a method of choice for various biological systems. However, pressing concerns remain regarding its off-target activities and wide variations in efficacies. While next generation sequencing (NGS) has been primarily used to evaluate the efficacies and off-target activities of gRNAs, it only detects the imperfectly repaired double strand DNA breaks (DSB) by the error-prone non-homologous end joining (NHEJ) mechanism and may not faithfully represent the DSB activities because the efficiency of NHEJ-mediated repair varies depending on the local chromatin environment. Here we describe a reporter system for unbiased detection and comparison of DSB activities that promises to improve the chance of success in genomic engineering and to facilitate large-scale screening of CAS9 activities and gRNA libraries. Additionally, we demonstrated that the tolerances to mismatches between a gRNA and the corresponding target DNA can occur at any position of the gRNA, and depend on both specific gRNA sequences and CAS9 constructs used.

Previously, we showed that genetically modified live-attenuated Leishmania donovani parasite cell lines (LdCen−/− and Ldp27−/−) induce a strong cellular immunity and provide protection against visceral leishmaniasis in mice. In this study, we explored the mechanism of cross-protection against cutaneous lesion-causing Leishmania mexicana. Upon challenge with wild-type L. mexicana, mice immunized either for short or long periods showed significant protection. Immunohistochemical analysis of ears from immunized/challenged mice exhibited significant influx of macrophages, as well as cells expressing MHC class II and inducible NO synthase, suggesting an induction of potent host-protective proinflammatory responses. In contrast, substantial inhibition of IL-10, IL-4, and IL-13 expression and the absence of degranulated mast cells and less influx of eosinophils within the ears of immunized/challenged mice suggested a controlled anti-inflammatory response. L. mexicana Ag–stimulated lymph node cell culture from the immunized/challenged mice revealed induction of IFN-γ secretion by the CD4 and CD8 T cells compared with non-immunized/challenged mice. We also observed suppression of Th2 cytokines in the culture supernatants of immunized/challenged lymph nodes compared with non-immunized/challenged mice. Adoptively transferred total T cells from immunized mice conferred strong protection in recipient mice against L. mexicana infection, suggesting that attenuated L. donovani can provide protection against heterologous L. mexicana parasites by induction of a strong T cell response. Furthermore, bone marrow–derived dendritic cells infected with LdCen−/− and Ldp27−/− parasites were capable of inducing a strong proinflammatory response leading to the proliferation of Th1 cells. These studies demonstrate the potential of live-attenuated L. donovani parasites as pan–Leishmania species vaccines.

The very low density lipoprotein receptor (VLDLR) is a member of the low-density lipoprotein receptor family that binds multiple ligands and plays a key role in brain development. Although the VLDLR mediates pleiotropic biological processes, only a limited amount of information is available regarding its role in adaptive immunity. In this study, we identify an important role for the VLDLR in attenuating house dust mite (HDM)-induced airway inflammation in experimental murine asthma. We show that HDM-challenged Vldlr−/− mice have augmented eosinophilic and lymphocytic airway inflammation with increases in Th2 cytokines, C-C chemokines, IgE production, and mucous cell metaplasia. A genome-wide analysis of the lung transcriptome identified that mRNA levels of CD209e (DC-SIGNR4), a murine homolog of DC-SIGN, were increased in the lungs of HDM-challenged Vldlr−/− mice, which suggested that the VLDLR might modify dendritic cell (DC) function. Consistent with this, VLDLR expression by human monocyte-derived DCs was increased by HDM stimulation. In addition, 55% of peripheral blood CD11c+ DCs from individuals with allergy expressed VLDLR under basal conditions. Lastly, the adoptive transfer of HDM-pulsed, CD11c+ bone marrow–derived DCs (BMDCs) from Vldlr−/− mice to the airways of wild type recipient mice induced augmented eosinophilic and lymphocytic airway inflammation upon HDM challenge with increases in Th2 cytokines, C-C chemokines, IgE production, and mucous cell metaplasia, as compared with the adoptive transfer of HDM-pulsed, CD11c+ BMDCs from wild type mice. Collectively, these results identify a novel role for the VLDLR as a negative regulator of DC-mediated adaptive immune responses in HDM-induced allergic airway inflammation.

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 Behcet's 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.

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.

Endothelial cells in the peripheral circulation are rare events that require technically rigorous approaches for detection by flow cytometry. Visualization of these cells has been even more demanding, as this has historically required extensive enrichment and processing prior to attempting imaging. As a result, few, if any, examples exist on images of peripheral blood circulating endothelial cells (CECs) that include verification of the cell lineage both phenotypically and genomically. In this study, we have devised a method whereby CECs can be directly visualized after lysis of red blood cells and staining, without pre-enrichment or additional processing. Peripheral blood is stained with CD45, CD146, CD3, Hoechst, and DAPI to permit identification of CD146 positive, nonleukocyte, nucleated, and live cells that fit the description of CECs. These cells are imaged using the Amnis ImageStream(X), an imaging flow cytometer. Genomic verification of the endothelial nature of these cells is accomplished by using an aliquot of the same stained samples for sorting CECs using similar gating strategies. This proof of principle of direct imaging of CECs by imaging flow cytometry will permit studies to be conducted heretofore not possible, as the ImageStream(X) has the capability of detecting additional fluorochromes other than those used to identify the CECs. Such potential investigations include antigen colocalization or capping, autophagy and apoptosis, morphologic changes in response to therapy, and others. Thus, this method will enable a broad range of novel studies to be conducted using CECs as surrogates of the endothelium.

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.

Cell heterogeneity and the inherent complexity due to the interplay of multiple molecular processes within the cell pose difficult challenges for current single-cell biology. We introduce an approach that identifies a disease phenotype from multiparameter single-cell measurements, which is based on the concept of "supercell statistics", a single-cell-based averaging procedure followed by a machine learning classification scheme. We are able to assess the optimal tradeoff between the number of single cells averaged and the number of measurements needed to capture phenotypic differences between healthy and diseased patients, as well as between different diseases that are difficult to diagnose otherwise. We apply our approach to two kinds of single-cell datasets, addressing the diagnosis of a premature aging disorder using images of cell nuclei, as well as the phenotypes of two non-infectious uveitides (the ocular manifestations of Behçet's disease and sarcoidosis) based on multicolor flow cytometry. In the former case, one nuclear shape measurement taken over a group of 30 cells is sufficient to classify samples as healthy or diseased, in agreement with usual laboratory practice. In the latter, our method is able to identify a minimal set of 5 markers that accurately predict Behçet's disease and sarcoidosis. This is the first time that a quantitative phenotypic distinction between these two diseases has been achieved. To obtain this clear phenotypic signature, about one hundred CD8+ T cells need to be measured. Although the molecular markers identified have been reported to be important players in autoimmune disorders, this is the first report pointing out that CD8+ T cells can be used to distinguish two systemic inflammatory diseases. Beyond these specific cases, the approach proposed here is applicable to datasets generated by other kinds of state-of-the-art and forthcoming single-cell technologies, such as multidimensional mass cytometry, single-cell gene expression, and single-cell full genome sequencing techniques., Instituto de Física de Líquidos y Sistemas Biológicos

Background Visceral leishmaniasis (VL) caused by the protozoan parasite Leishmania donovani causes severe disease. Age appears to be critical in determining the clinical outcome of VL and at present there is no effective vaccine available against VL for any age group. Previously, we showed that genetically modified live attenuated L. donovani parasites (LdCen-/-) induced a strong protective innate and adaptive immune response in young mice. In this study we analyzed LdCen-/- parasite mediated modulation of innate and adaptive immune response in aged mice (18 months) and compared to young (2 months) mice. Methodology Analysis of innate immune response in bone marrow derived dendritic cells (BMDCs) from both young and aged mice upon infection with LdCen-/- parasites, showed significant enhancement of innate effector responses, which consequently augmented CD4+ Th1 cell effector function compared to LdWT infected BMDCs in vitro. Similarly, parasitized splenic dendritic cells from LdCen-/- infected young and aged mice also revealed induction of proinflammatory cytokines (IL-12, IL-6, IFN-γ and TNF) and subsequent down regulation of anti-inflammatory cytokine (IL-10) genes compared to LdWT infected mice. We also evaluated in vivo protection of the LdCen-/- immunized young and aged mice against virulent L. donovani challenge. Immunization with LdCen-/- induced higher IgG2a antibodies, lymphoproliferative response, pro- and anti-inflammatory cytokine responses and stimulated splenocytes for heightened leishmanicidal activity associated with nitric oxide production in young and aged mice. Furthermore, upon virulent L. donovani challenge, LdCen-/- immunized mice from both age groups displayed multifunctional Th1-type CD4 and cytotoxic CD8 T cells correlating to a significantly reduced parasite burden in the spleen and liver compared to naïve mice. It is interesting to note that even though there was no difference in the LdCen-/- induced innate response in dendritic cells between aged and young mice; the adaptive response specifically in terms of T cell and B cell activation in aged animals was reduced compared to young mice which correlated with less protection in old mice compared to young mice. Conclusions Taken together, LdCen-/- immunization induced a significant but diminished host protective response in aged mice after challenge with virulent L. donovani parasites compared to young mice., Author Summary Visceral leishmaniasis (VL) is caused by the protozoan parasite Leishmania donovani. There is no effective vaccine available against VL for any age group and importantly, there are no previous studies regarding immune responses against experimental Leishmania vaccines tested in aged animals. We have reported earlier that immunization with a live attenuated L. donovani parasites (LdCen-/-) induced protective immune response in young animals viz, mice, hamsters and dogs. In this study we analyzed LdCen-/- mediated modulation of innate and adaptive responses in aged mice and compared to young mice. We observed that LdCen-/- infected dendritic cells from young and aged mice resulted in enhanced innate effector functions compared to LdWT parasites both in vitro and in vivo. Further, upon virulent challenge, LdCen-/- immunized young and aged mice displayed protective Th1 immune response which correlated with a significantly reduced parasite burden in the visceral organs compared with naïve challenged mice. Although there was no difference in the LdCen-/- induced dendritic cell response between aged and young mice; adaptive response in aged was reduced, compared to young which correlated with less protection in aged compared to young mice. This study supports the potential use of LdCen-/- as vaccine candidate across all age groups against VL.

Increased numbers of T regulatory (T(reg)) cells are found in B-chronic lymphocytic leukemia, but the nature and function of these T(regs) remains unclear. Detailed characterization of the T(regs) in chronic lymphocytic leukemia has not been performed and the degree of heterogeneity of among these cells has not been studied to date. Using 15-color flow cytometry we show that T(reg) cells, defined using CD4, CD25, and forkhead box P3 (FOXP3), can be divided into multiple complex subsets based on markers used for naive, memory, and effector delineation as well as markers of T(reg) activation. Furthermore FOXP3(+) cells can be identified among CD4(+)CD25(-) as well as CD8(+)CD4(-) populations in increased proportions in patients with chronic lymphocytic leukemia compared with healthy donors. Significantly different frequencies of naive and effector T(regs) populations are found in healthy donor controls compared with donors with chronic lymphocytic leukemia. A population of CCR7(+)CD39(+) T(regs) was significantly associated with chronic lymphocytic leukemia. This population demonstrated slightly reduced suppressive activity compared with total T(regs) or T(regs) of healthy donors. These data suggest that FOXP3-expressing cells, particularly in patients with chronic lymphocytic leukemia are much more complex for T(reg) sub-populations and transitions than previously reported. These findings demonstrate the complexity of regulation of T-cell responses in chronic lymphocytic leukemia and illustrate the use of high-dimensional analysis of cellular phenotypes in facilitating understanding of the intricacies of cellular immune responses and their dysregulation in cancer.

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.

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 , NK 1 , NK 2 , 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.

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 (30)DETD(33) 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.

Present study investigates the role of Mycobacterium leprae (M. leprae) antigens on TCR- and TCR/CD28-induced signalling leading to T-cell activation and further correlates these early biochemical events with T-cell anergy, as prevailed in advanced stages of leprosy. We observed that both whole cell lystae (WCL) and soluble fraction of M. leprae sonicate (MLSA) not only inhibited TCR, thapsigargin and ionomycin induced calcium fluxes by diminishing the opening of calcium channels, but also TCR- or TCR/CD28-induced proximal signalling events like phosphorylation of Zap-70 and protein kinase-C (PKC) activity. Study of TCR- and TCR/CD28-induced downstream signals revealed that M. leprae antigens curtail phosphorylation of both Erk1/2 and p38MAPK, consequently altering terminal signalling events like reduced binding of NFAT on IL-2 promoter and transcription of IL-2 gene, diminished expression of activation markers (CD25 and CD69). Furthermore, M. leprae fractions significantly inhibited IL-2 secretion and T-cell blastogenesis in healthy individuals. Altogether, results suggest that M. leprae interferes with TCR/CD28-induced upstream as well as downstream signalling events resulting in reduced IL-2 production and thus inhibition in T-cell proliferation, which might be responsible for T-cell unresponsiveness leading to stage of immunosuppression and consequently, for the progression of disease.

Twenty-eight synovial effusions (SE) were obtained from 24 patients, paired samples of peripheral blood (PB) from 10 of these patients, and PB from 36 healthy individuals for analysis of CD146 on T-lymphocytes by flow cytometry. CD146+ or CD146- T-lymphocytes were sorted from three SE to study gene expression profiles and selected genes revalidated using QPCR assays. We found more CD3+CD146+ and CD4+CD146+ T-lymphocytes in PB from patients compared with PB of healthy individuals (4.71% +/- 2.48% vs. 2.53% +/- 1.08%, P = 0.028) and (6.29% +/- 2.74% vs. 2.41% +/- 0.96%, P = 0.0017), respectively, whereas CD8+CD146+ T-lymphocytes were not significantly different (2.55% +/- 1.65% vs. 3.18% +/- 2.59%, P = 0.5008). SE displayed CD146 staining on 16.32% +/- 6.06% of CD3+ cells. This expression was skewed toward CD4+ T-lymphocytes, with CD146 present on 24.06% +/- 8.20% of the CD4+ T-lymphocytes compared with 6.19% +/- 5.22% of the CD8+ T-lymphocytes. CD146 on CD3+, CD4+ and CD8+ T-lymphocytes in SE was significantly higher compared with PB in patients (P < 0.0001, P < 0.0001 and P = 0.0036, respectively). Gene expression profiles of sorted CD146+CD4+CD3+ vs. CD146-CD4+CD3+ T-lymphocytes (n = 2) and CD2+CD146+ vs. CD2+CD 146- (n = 1) from SE, displayed increased CD146, LAIR2, CXCL13, CD109, IL6ST, IL6R, TNFRsf18, and TNFRsf4 genes, whereas decreased CCR7, CCL5, and cytotoxicity-associated genes including granzymes b, h, and k, perforin were found with the CD146- T-lymphocytes. By QPCR higher mRNA expression of CXCL13, CD146 and CD109 was also noted in the CD146+ subset, compared with the CD146- subset, in PB of healthy individuals and in PB and SE from patients. Our study establishes increased CD146+ T-lymphocytes in diseases with joint effusions, and demonstrates pro-inflammatory gene profiles in these cells.

Childhood tuberculosis is difficult to diagnose. A rapid, simple and relatively inexpensive diagnostic test will be crucial to future control efforts. Therefore, efficacy and diagnostic potential of different secretory antigens of Mycobacterium tuberculosis (CFP-10, Ag85complex, Ag85 A, B, C) and their combinations along with ESAT-6 in the detection of antibody profiles of childhood tuberculosis cases were evaluated using ELISA technique and reactivity was compared with the gold standards (smear, culture and IS6110 targeted PCR). In the present study, 88 fresh, untreated childhood tuberculosis (TB) cases, 17 children undergoing anti-tubercular therapy, 17 children having disease other than TB and 25 healthy children were included. ROC curve analysis was used to calculate the sensitivity and specificity of each antigen for antibody detection. Ag85C was found to be showing highest sensitivity of 89.77% and specificity of 92% among all the antigens used (P < 0.0001). Positivity with antigen was 95% in smear and culture negative patients. Antibody reactivity was noted in 92.62% of patients who were positive for IS6110 by PCR. Cocktail of all the antigens showed 67.1% sensitivity and 80% of specificity. Sensitivity of 29.55%, 57.95%, 64.77% and specificity of 80%, 72%, 64% was observed using CFP-10, Ag85complex and Ag85B. Low reactivity of 31.82% in patients and least specificity of 24% was noted with Ag85A. Our finding demonstrates the potential of Ag85C in the detection of antibody in childhood TB cases and this antigen showed good concordance with PCR positivity.

Introduction Inflammation is increased and related to early mortality in patients with sickle cell disease.(1) This inflammation is associated with upregulation of Toll-like receptor 4 and iron regulated genes in human sickle cell peripheral blood mononuclear cells. In sickle cell mice, heme released during intravascular hemolysis augments pro-inflammatory Toll-like receptor 4 signalling and this results in subsequent organ damage and death. In this study we evaluated whether heme in human sickle cell monocytes is associated with increased Toll-like receptor 4 mediated pro-inflammatory cytokine production. Methods Fresh whole blood from patients (n=10) and controls (n=10) was used for a calcein assay to measure intracellular iron or was incubated with combinations of vehicle, a Toll-like receptor 4 ligand (lipopolysaccharide, 100 ng/ml) and/or an iron chelator (0.1 mM deferasirox (Exjade)). After three hours, the percentage of monocytes with detectable levels of intracellular interleukin-6 and tumor necrosis factor-alpha was quantified by flow cytometry. In an additional experiment fresh whole blood of patients (n=8) was incubated with combinations of vehicle, a Toll-like receptor 4 ligand (lipopolysaccharide, 1 ng/ml) and/or 20uM heme. Results Intracellular monocyte iron was correlated (R-spearman and P-value) positively with plasma levels of C-reactive protein in patients and controls (R=0.454, P=0.044), confirming that high intracellular iron in monocytes is associated with a pro-inflammatory state in vivo. Compared to incubation with lipopolysaccharide alone, co-incubation of fresh human sickle cell blood with lipopolysaccharide and heme increased the absolute percentage of monocytes producing interleukin-6 with a median 8.5% (interquartile range -5.6-22.1, p=0.17) and tumor necrosis factor-alpha with 15.2% (2.0-21.2, p=0.025). Incubation of fresh sickle cell monocytes with heme alone did not increase interleukin-6 and tumor necrosis factor-alpha production significantly (respectively 0.1% and 0.0%). Compared to incubation with lipopolysaccharide alone, co-incubation of lipopolysaccharide with the iron chelator deferasirox significantly decreased the absolute percentage of interleukin-6 producing monocytes with 20.4% (15.2-26.3) (P=0.004), further supporting the involvement of intracellular monocyte iron in Toll-like receptor 4 response. Conclusion We show that levels of intracellular monocyte iron correlate to markers of inflammation in human sickle cell patients. In an additional ex vivo experiment we show that the same monocytes have an increased Toll-like receptor 4 mediated inflammatory response when exposed to heme and a decreased inflammatory response when treated with an iron chelator. We suggest that heme bound iron which is released during intravascular hemolysis and scavenged by monocytes, is a cause of monocyte activation and pro-inflammatory state in sickle cell disease, by augmenting Toll-like receptor 4 signaling. Iron chelation might be an interesting therapeutic option to decrease this pro-inflammatory effect of heme. Figure Monocyte Toll-like receptor 4 dependent pro-inflammatory cytokine production is augmented by heme and inhibited by iron chelation. (A) Compared to incubation of fresh human sickle cell blood with the Toll-like receptor 4 ligand lipopolysaccharide alone, co-incubation of lipopolysaccharide together with the iron chelator deferasirox significantly decreased the absolute percentage of interleukin-6 producing monocytes with 20.4% (15.2-26.3) (P=0.004) (B) In contrast, compared to incubation with lipopolysaccharide alone, co-incubation lipopolysaccharide together with heme increased the absolute percentage of monocytes producing interleukin-6 with a median 8.5% (interquartile range -5.6-22.1, p=0.17) and tumor necrosis factor-alpha with 15.2% (2.0-21.2, p=0.025). *** p 1. van Beers EJ, Yang Y, Raghavachari N, Tian X, Allen DT, Nichols JS, e.a. Iron, inflammation, and early death in adults with sickle cell disease. Circ Res. 16 januari 2015;116(2):298-306. Figure 1. Figure 1. Disclosures van Beers: Novartis: Research Funding.

T regulatory cells (Tregs) are a subset of CD4 T cells, which are capable of suppressing immune responses of other T cells. In the peripheral blood of healthy individuals, these constitute approximately 3–5% of CD4 T cells. Tregs have been identified by a number of differing approaches, involving some, or all, of the following marker expressions: CD25high, Forkhead box protein 3 (FoxP3), and CD127low (1–3). In the current panel, all of these markers were used, as well as additional markers such as CD39 that have been reported to be associated with high suppressive activity among Tregs (4). Antibodies to identify naive and memory T cells, including CD45RA, CD27, and CD197 (CCR7) have been added to enable classification of Tregs in this manner (5–7). Finally, additional markers for gating (live/dead, CD45, CD3), and for cell activation (CD38, HLA-DR, CD103) were added to this panel (Supporting Information Table 2).

Background Advanced stages of leprosy show T cell unresponsiveness and lipids of mycobacterial origin are speculated to modulate immune responses in these patients. Present study elucidates the role of phenolicglycolipid (PGL-1) and Mannose-capped lipoarabinomannan (Man-LAM) on TCR- and TCR/CD28- mediated signalling. Results We observed that lipid antigens significantly inhibit proximal early signalling events like Zap-70 phosphorylation and calcium mobilization. Interestingly, these antigens preferentially curtailed TCR-triggered early downstream signalling events like p38 phosphorylation whereas potentiated that of Erk1/2. Further, at later stages inhibition of NFAT binding, IL-2 message, CD25 expression and T-cell blastogenesis by PGL-1 and Man-LAM was noted. Conclusion Altogether, we report that Man-LAM and PGL-1 preferentially interfere with TCR/CD28-triggered upstream cell signalling events, leading to reduced IL-2 secretion and T-cell blastogenesis which potentially could lead to immunosupression and thus, disease exacerbation, as noted in disease spectrum.

Germline mutation of the tumor suppressor gene CDC73 confers susceptibility to the hyperparathyroidism-jaw tumor syndrome associated with a high risk of parathyroid malignancy. Inactivating CDC73 mutations have also been implicated in sporadic parathyroid cancer, but are rare in sporadic benign parathyroid tumors. The molecular pathways that distinguish malignant from benign parathyroid transformation remain elusive. We previously showed that a hypomorphic allele of hyrax (hyx), the Drosophila homolog of CDC73, rescues the loss-of-ventral-eye phenotype of lobe, encoding the fly homolog of Akt1s1/ PRAS40. We report now an interaction between hyx and Tor, a central regulator of cell growth and autophagy, and show that eukaryotic translation initiation factor 4E-binding protein (EIF4EBP), a translational repressor and effector of mammalian target of rapamycin (mTOR), is a conserved target of hyx/CDC73. Flies heterozygous for Tor and hyx, but not Mnn1, the homolog of the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor associated with benign parathyroid tumors, are starvation resistant with reduced basal levels of Thor/4E-BP. Human peripheral blood cell levels of EIF4EBP3 were reduced in patients with CDC73, but not MEN1, heterozygosity. Chromatin immunoprecipitation demonstrated occupancy of EIF4EBP3 by endogenous parafibromin. These results show that EIF4EBP3 is a peripheral marker of CDC73 function distinct from MEN1-regulated pathways, and suggest a model whereby starvation resistance and/or translational de-repression contributes to parathyroid malignant transformation.

The hyperparathyroidism-jaw tumor syndrome (HPT-JT) is a familial cancer syndrome that results from germline loss-of-function mutation of HRPT2/CDC73, a putative tumor suppressor gene that encodes parafibromin, a component of the transcriptional regulatory PAF1 complex with homology to the yeast protein Cdc73p. The vast majority of HRPT2/CDC73 germline mutations identified have been truncation or frameshift mutations, and loss-of-function due to missense mutation is rare. We report here a kindred with HPT-JT due to a germline L95P missense mutation in parafibromin. The mutant parafibromin was studied in vitro to understand the basis of its presumed loss-of-function. When transfected in cultured cells the L95P mutant was expressed to a lower level than wild-type parafibromin, a difference that was not overcome by inhibition of the proteasome degradation pathway. The L95P mutant parafibromin retained the ability to assemble with endogenous PAF1 complex components as evidenced by co-immunoprecipitation. Analysis of subcellular localization showed that the L95P mutant was markedly deficient in nucleolar localization compared to the wild-type, an impairment likely resulting from disruption of a nucleolar localization signal immediately upstream of the L95P mutation. Transfection of the L95P parafibromin mutant, but not the wild type, increased HEK 293 cell survival and stimulated cell-cycle progression in NIH-3T3 cells, properties that appear to result from dominant interference with endogenous parafibromin signaling pathways. The simultaneous loss of nucleolar localization and acquisition of a growth stimulatory phenotype with the L95P mutation raise the possibility that parafibromin must interact with targets in the nucleolus to fully execute its tumor suppressor functions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-10.

Elimination of tuberculosis (TB) largely depends upon definitive rapid diagnosis and treatment. Widely used diagnostic tests do not qualify for use in a developing country due to lack of either desired accuracy or their cost. In the present study an enzyme-linked immunosorbent assay was used to evaluate the diagnostic potential of an immuno-dominant 30/32-kDa mycolyl transferase complex (Ag85 complex) and Mycobacterium tuberculosis-specific proteins (ESAT-6 and CFP-10) of the RD1 region. Higher sensitivity (84.1%) with Ag85 complex was observed compared with ESAT-6 (64.9%) and CFP-10 (66%), with almost similar specificity (Ag85: 85.2%, ESAT-6: 88.9%, CFP-10: 85.2%), whereas the individual components of Ag85 complex, i.e. Ag85A, Ag85B, and Ag85C, showed sensitivities of 44.6, 34, and 80.9% and specificities of 55.6, 74.1, and 40.7% respectively. A cocktail of Ag85 complex, ESAT-6, CFP-10, Ag85A, Ag85B, and Ag85C antigens also could not help in increasing either sensitivity (51.1%) or specificity (85.2%). Furthermore, immunoblot analysis using clinical isolates as well as a standard strain (H37Rv) of M. tuberculosis also showed strong reactivity of sera from TB patients to Ag85 complex and, to a lesser extent, also to ESAT-6. To conclude, use of Ag85 complex along with ESAT-6 and CFP-10 seems to be promising in minimizing the heterogeneous sero-responses of adult TB cases.

Background Adipose tissue normally contains immune cells that regulate adipocyte function and contribute to metabolic disorders including obesity and diabetes mellitus. Psoriasis is associated with increased risk for metabolic disease, which may in part be due to adipose dysfunction, which has not been investigated in psoriasis. There is currently no standardized method for immunophenotyping human adipose tissue. In prior studies, characteristic phenotypic markers of immune cell populations identified in animal models or in other human tissues have been applied in a similar manner to human adipose tissue. Rarely have these populations been verified with confirmatory methodologies or functional studies. Thus, we performed a comprehensive phenotypic and functional analysis of immune cell populations in psoriatic adipose tissue. Methods Conventional and imaging flow cytometry were used to define immune cell populations in biopsy specimens of psoriatic adipose tissue (n = 30) including T cells, B cells, NK cells, NKT cells, neutrophils, and macrophages. Relationships between adipose immune cell types and body mass index were determined using Spearman regression analysis, and multivariate linear regression analysis was performed to adjust for cardiometabolic disease risk factors. Results These analyses revealed a wide range of cell surface receptors on adipose tissue macrophages, which may serve a dual purpose in immunity and metabolism. Further, both CD16+CD56Lo and CD16-CD56Hi NK cells were found to correlate inversely with body mass index. The relationship between the predominant CD16+CD56Lo NK cell population and body mass index persisted after adjusting for age, sex, diabetes, and tobacco use. Conclusions Together, these studies enhance our understanding of adipose immune cell phenotype and function, and demonstrate that examination of adipose tissue may provide greater insight into cardiometabolic pathophysiology in psoriasis. Electronic supplementary material The online version of this article (doi:10.1186/s12967-014-0258-2) contains supplementary material, which is available to authorized users.

Abstract 1644 Poster Board I-670 CD4+ regulatory T cells (Treg) act to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens. Tregs have been shown to be increased in the peripheral circulation of patients with various types of cancer including CLL and have been postulated to play a role in inhibition of anti-tumor immune responses. 15 color staining was performed on mononuclear cells isolated from peripheral blood from 33 healthy controls and 12 patients with chronic lymphoid leukemia (CLL). Treg cells were defined as viable CD45+ CD3+ CD8- CD4+ CD25+ Forkhead box P3+ (FoxP3)+ mononuclear cells. Among the Treg population we defined 4 sub-populations; Naïve Treg (CD45-RA+ CD27+ CD197+), Effector Treg (CD45-RA- CD27- CD197-), Central memory Treg (CD45-RA- CD27+ CD197+) and Effector memory Treg (CD45-RA- CD27+ CD197-). We report level of expression of activation markers CD39, HLA-DR and CD38 and IL-7 receptor CD127 on Treg subsets as defined above. The percentage of Treg was increased significantly in CLL patients compared with healthy controls 12.8±1.3% vs 5.7±0.3% (p Disclosures No relevant conflicts of interest to declare.