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(1→3)-β-D-Glucan (BDG) represents a potent pathogen-associated molecular pattern (PAMP) in triggering the host response to fungal and some bacterial infections. Monocytes play a key role in recognizing BDG and governing the acute host response to infections. However, the mechanisms regulating monocyte's acute response to BDG are poorly understood. We sought to investigate the response of monocytes to BDG at the epigenetic, transcriptomic, and molecular levels. Response of human monocytes to 1, 4, and 24 hours of BDG exposure was investigated using RNA-seq, ATAC-seq, H3K27ac and H3K4me1 ChIP-seq. We show that pathways including glutathione metabolism, pentose phosphate pathway, and citric acid cycle were upregulated at the epigenetic and transcriptomic levels in response to BDG exposure. Strikingly, unlike bacterial lipopolysaccharides, BDG induced intracellular glutathione synthesis. BDG exposure also induced NADP synthesis, increased NADPH/NADP ratio, and increased expression of genes involved in the pentose phosphate pathway in a GSH-dependent manner. By inhibiting GSH synthesis with L-buthionine sulfoximine (BSO) before BDG exposure we show that the GSH pathway promotes cell survival and regulates monocyte's effector functions including NO production, phagocytosis, and cytokine production. In summary, our work demonstrates that BDG induces glutathione synthesis and metabolism in monocytes, which is a major promoter of the acute functional response of monocytes to infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ramendra, Mancini, Ayala, Tung, Isnard, Lin, Routy, Nijnik and Langlais.)

For over 70 years experimental autoimmune encephalomyelitis (EAE) has been induced with myelin autoantigens emulsified in complete Freund's adjuvant (CFA) which has significant side effects such as pain, inflammation, and tissue necrosis at the injection site. β-1,3-d-glucan particles (GPs) are hollow microcapsules prepared from Saccharomyces cerevisiae cell walls that induce potent Th17 cell responses without causing strong injection site tissue reactions. We evaluated the potential of GPs complexed with neuroantigens to induce EAE while avoiding undesirable side effects. GPs loaded with myelin oligodendrocyte glycoprotein 35-55 (MOG 35-55 ) or proteolipid protein 139-151 (PLP 139-151 ) peptides effectively induced EAE in C57BL/6 mice and SJL mice. Disease severity, CNS pathology and immune responses were comparable between GP- and CFA-immunized mice. Importantly, injection with GPs resulted in significantly decreased inflammation compared with CFA. We posit that use of GPs provides an alternative means for inducing EAE that results in comparable disease, but less discomfort to animals., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: During eosinophil differentiation, the granule eosinophil major basic protein 1 (eMBP1) is synthesized as a 32-kDa precursor form, referred to as proMBP1, which is processed into the 14-kDa mature form of eMBP1. The prevalence of these two forms of MBP1 in most pathological conditions remains unknown., Objective: To develop the immunoassays that differentiate mature eMBP1 and proMBP1 and apply them to analyze their levels in biological fluids from patients with eosinophilia and hematologic disorders., Methods: We produced a series of monoclonal antibodies and selected pairs capable of discriminating between the two molecular forms of eMBP1. Radioimmunoassay (RIA) was performed to simultaneously quantitate the levels of mature eMBP1 and proMBP1 in secretions from patients with chronic rhinosinusitis (CRS) and sera from patients with hypereosinophilic syndrome (HES) and other myeloproliferative disorders., Results: The novel immunoassays possessed less than 1% crossreactivity between mature eMBP1 and proMBP1. Mature eMBP1, but not proMBP1, was found in nasal secretions of CRS patients. In contrast, elevated serum levels of mature eMBP1 and proMBP1 were observed in approximately 60% and 90% of HES patients, respectively, with proMBP1 present in greater quantities than mature eMBP1. Patients with several myeloproliferative disorders also showed high serum levels of proMBP1 while mature eMBP1 remained at basal levels., Conclusion: The novel immunoassays successfully differentiated mature eMBP1 and proMBP1 in human biological fluids. Further studies addressing the clinical correlates of these assays will help to develop biomarkers to diagnose and monitor patients with eosinophilia and myeloproliferative disorders., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: The immunoinhibitory receptor Siglec-8 on the surface of human eosinophils and mast cells binds to sialic acid-containing ligands in the local milieu, resulting in eosinophil apoptosis, inhibition of mast cell degranulation, and suppression of inflammation. Siglec-8 ligands were found on postmortem human trachea and bronchi and on upper airways in 2 compartments, cartilage and submucosal glands, but they were surprisingly absent from the epithelium. We hypothesized that Siglec-8 ligands in submucosal glands and ducts are normally transported to the airway mucus layer, which is lost during tissue preparation., Objective: Our aim was to identify the major Siglec-8 sialoglycan ligand on the mucus layer of human airways., Methods: Human upper airway mucus layer proteins were recovered during presurgical nasal lavage of patients at a sinus clinic. Proteins were resolved by gel electrophoresis and blotted, and Siglec-8 ligands detected. Ligands were purified by size exclusion and affinity chromatography, identified by proteomic mass spectrometry, and validated by electrophoretic and histochemical colocalization. The affinity of Siglec-8 binding to purified human airway ligand was determined by inhibition of glycan binding., Results: A Siglec-8-ligand with a molecular weight of approximately 1000 kDa was found in all patient nasal lavage samples. Purification and identification revealed deleted in malignant brain tumors 1 (DMBT1) (also known by the aliases GP340 and SALSA), a large glycoprotein with multiple O-glycosylation repeats. Immunoblotting, immunohistochemistry, and enzyme treatments confirmed that Siglec-8 ligand on the human airway mucus layer is an isoform of DMBT1 carrying O-linked sialylated keratan sulfate chains (DMBT1 S8 ). Quantitative inhibition revealed that DMBT1 S8 has picomolar affinity for Siglec-8., Conclusion: A distinct DMBT1 isoform, DMBT1 S8 , is the major high-avidity ligand for Siglec-8 on human airways., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Legionella pneumophila , an intracellular bacterium, may cause life-threatening pneumonia in immunocompromised individuals. Mononuclear cells and antibodies have been reported to be associated with the host defense response against L. pneumophila. This study is to determine whether Legionella peptidoglycan-associated lipoprotein (PAL)-specific CD8 + T cells are directly associated with protection against L. pneumophila , with a focus on potential epitopes. Synthetic peptides derived from PAL of L. pneumophila were obtained and tested through in vitro and in vivo cytotoxic T lymphocyte (CTL) assays for immunogenicity. PAL DNA vaccines or a peptide epitope with or without CpG-oligodeoxynucleotides (ODN) was evaluated for protection against L. pneumophila infection in animal models. When mice were immunized with DNA vaccines expressing the PAL of L. pneumophila , they were significantly protected against a lethal challenge with L. pneumophila through induction of antigen-specific CD8 + CTLs. Of the 13 PAL peptides tested, PAL 92-100 (EYLKTHPGA) was the most immunogenic and induced the strongest CTL responses. When mice were immunized with the PAL 92-100 peptide plus CpG-ODN, they were protected against the lethal challenge, while control mice died within 3-6 days after the challenge. Consistent with lung tissue histological data, bacterial counts in the lungs of immunized mice were significantly lower than those in control mice. Also, the amino acid sequence of PAL 92-100 peptides is conserved among various Legionella species. To our knowledge, this study is the first to demonstrate that PAL 92-100 -specific CD8 + T cells play a central role in the host defense response against L. pneumophila ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Kim, Sin and Kim.)

Atopic cataracts develop under the ages of 40 years, after which visual acuity rapidly declines. However, the mechanism underlying the development of atopic cataracts is not yet clear. We focused on the eosinophil granule major basic protein (MBP), which was detected in the aqueous humor of atopic cataracts previously, and which was cytotoxic. Specifically, we investigated its origin in this fluid and its effects on lens epithelial cells (LECs). MBP immunostaining was positive in atopic cataract-derived LECs, but negative in age-related cataract-derived LECs. MBP mRNA was not detected in either type of cataract, but protein was detected in the aqueous humor. Furthermore, the flare values associated with atopic cataracts were higher than those with age-related cataracts. When MBP was purified from eosinophils or recombinant MBP was added to LEC culture medium, cell viability decreased in a concentration-dependent manner, but an MBP antibody neutralized the cytotoxic effect of this protein towards these cells. These results were consistent with the flow of MBP into the aqueous humor from the blood due to a compromised blood-aqueous barrier. Thus, MBP could further penetrate the lens capsule and adhere to LECs, resulting in decreased cell viability and the development of atopic cataracts.

Proteoglycans are a specific subset of glycoproteins found at the cell surface and in the extracellular matrix, where they interact with a plethora of proteins involved in metabolic homeostasis and meta-inflammation. Over the last decade, new insights have emerged on the mechanism and biological significance of these interactions in the context of diet-induced disorders such as obesity and type-2 diabetes. Complications of energy metabolism drive most diet-induced metabolic disorders, which results in low-grade chronic inflammation, thereby affecting proper function of many vital organs involved in energy homeostasis, such as the brain, liver, kidney, heart and adipose tissue. Here, we discuss how heparan, chondroitin and keratan sulfate proteoglycans modulate obesity-induced metabolic dysfunction and low-grade inflammation that impact the initiation and progression of obesity-associated morbidities., (Copyright © 2020 Pessentheiner, Ducasa and Gordts.)

Over the past few decades, understanding how tumor cells evade the immune system and their communication with their tumor microenvironment, has been the subject of intense investigation, with the aim of developing new cancer immunotherapies. The current therapies against cancer such as monoclonal antibodies against checkpoint inhibitors, adoptive T-cell transfer, cytokines, vaccines, and oncolytic viruses have managed to improve the clinical outcome of the patients. However, in some tumor entities, the response is limited and could benefit from the identification of novel therapeutic targets. It is known that tumor-extracellular matrix interplay and matrix remodeling are necessary for anti-tumor and pro-tumoral immune responses. Proteoglycans are dominant components of the extracellular matrix and are a highly heterogeneous group of proteins characterized by the covalent attachment of a specific linear carbohydrate chain of the glycosaminoglycan type. At cell surfaces, these molecules modulate the expression and activity of cytokines, chemokines, growth factors, adhesion molecules, and function as signaling co-receptors. By these mechanisms, proteoglycans influence the behavior of cancer cells and their microenvironment during the progression of solid tumors and hematopoietic malignancies. In this review, we discuss why cell surface proteoglycans are attractive pharmacological targets in cancer, and we present current and recent developments in cancer immunology and immunotherapy utilizing proteoglycan-targeted strategies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

While recent research points to the importance of glycans in cancer immunity, knowledge on functional mechanisms is lacking. In lung carcinoma among other tumors, anti-tumor immunity is suppressed; and while some recent therapies boost T-cell mediated immunity by targeting immune-checkpoint pathways, robust responses are uncommon. Augmenting tumor antigen-specific immune responses by endogenous dendritic cells (DCs) is appealing from a specificity standpoint, but challenging. Here, we show that restricting a heparan sulfate (HS) loss-of-function mutation in the HS sulfating enzyme Ndst1 to predominantly conventional DCs (Ndst1f/f CD11cCre+ mutation) results in marked inhibition of Lewis lung carcinoma growth along with increased tumor-associated CD8+ T cells. In mice deficient in a major DC HS proteoglycan (syndecan-4), splenic CD8+ T cells showed increased anti-tumor cytotoxic responses relative to controls. Studies examining Ndst1f/f CD11cCre + mutants revealed that mutation was associated with an increase in anti-tumor cytolysis using either splenic CD8+ T cells or tumor-infiltrating (TIL) CD8+ T cells purified ex-vivo, and tested in pooled effector-to-target cytolytic assays against tumor cells from respective animals. On glycan compositional analysis, HS purified from Ndst1f/f CD11cCre + mutant DCs had reduced overall sulfation, including reduced sulfation of a tri-sulfated disaccharide species that was intriguingly abundant on wildtype DC HS. Interestingly, antigen presentation in the context of major histocompatibility complex class-I (MHC-I) was enhanced in mutant DCs, with more striking effects in the setting of HS under-sulfation, pointing to a likely regulatory role by sulfated glycans at the antigen/MHC-I - T-cell interface; and possibly future opportunities to improve antigen-specific T cell responses by immunologic targeting of HS proteoglycans in cancer., (Published by Elsevier Inc.)

Reactivation of functionally-impaired anticancer T cells by programmed cell death protein 1 (PD-1) and programmed cell death receptor ligand-1 (PD-L1)-blocking antibodies shows prominent therapeutic benefit in advanced melanoma and patients with non-small cell lung cancer. However, current PD-L1-blocking antibodies lack intrinsic tumor selectivity. Therefore, efficacy may be reduced resulting from on-target and off-tumor binding to PD-L1-expressing normal cells. This may lead to indiscriminate activation of antigen-experienced T cells, including those implicated in autoimmune-related adverse events. To direct PD-L1 blockade to chondroitin sulfate proteoglycan 4 (CSPG4)-expressing cancers and to reactivate anticancer T cells more selectively, we constructed bispecific antibody PD-L1xCSPG4. CSPG4 is an established target antigen that is selectively overexpressed on malignant melanoma and various other difficult-to-treat cancers. PD-L1xCSPG4 showed enhanced capacity for CSPG4-directed blockade of PD-L1 on cancer cells. Importantly, treatment of mixed cultures containing primary patient-derived CSPG4-expressing melanoma cells and autologous tumor-infiltrating lymphocytes with PD-L1xCSPG4 significantly enhanced activation status, IFN-γ production, and cytolytic activity of anticancer T cells. In conclusion, tumor-directed blockade of PD-L1 by PD-L1xCSPG4 may improve efficacy and safety of PD-1/PD-L1 checkpoint blockade for treatment of melanoma and other CSPG4-overexpressing malignancies., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Canine distemper virus (CDV) causes a fatal demyelinating leukoencephalitis in young dogs resembling human multiple sclerosis. Astrocytes are the main cellular target of CDV and undergo reactive changes already in pre-demyelinating brain lesions. Based on their broad range of beneficial and detrimental effects in the injured brain reactive astrogliosis is in need of intensive investigation. The aim of the study was to characterize astrocyte plasticity during the course of CDV-induced demyelinating leukoencephalitis by the aid of immunohistochemistry, immunofluorescence and gene expression analysis. Immunohistochemistry revealed the presence of reactive glial fibrillary acidic protein (GFAP) + astrocytes with increased survivin and reduced aquaporin 4, and glutamine synthetase protein levels, indicating disturbed blood brain barrier function, glutamate homeostasis and astrocyte maladaptation, respectively. Gene expression analysis revealed 81 differentially expressed astrocyte-related genes with a dominance of genes associated with neurotoxic A1-polarized astrocytes. Accordingly, acyl-coA synthetase long-chain family member 5 + /GFAP + , and serglycin + /GFAP + cells, characteristic of A1-astrocytes, were found in demyelinating lesions by immunofluorescence. In addition, gene expression revealed a dysregulation of astrocytic function including disturbed glutamate homeostasis and altered immune function. Observed findings indicate an astrocyte polarization towards a neurotoxic phenotype likely contributing to lesion initiation and progression in canine distemper leukoencephalitis.

Proteoglycan 4 (PRG4), first identified in synovial fluid, is an extracellular matrix structural protein in the joint implicated in reducing shear at the cartilage surface as well as controlling adhesion-dependent synovial growth and regulating bulk protein deposition onto the cartilage. However, recent evidence suggests that it can bind to and effect downstream signaling of a number of cell surface receptors implicated in regulating the inflammatory response. Therefore, we pose the hypothesis: Does PRG4 regulate the inflammatory response and maintain tissue homeostasis? Based on these novel findings implicating PRG4 as an inflammatory signaling molecule, we will present and discuss several hypotheses regarding potential mechanisms by which PRG4 may be able to regulate inflammation. If future studies can demonstrate that PRG4 is a potent inflammatory mediator, this will change current paradigms in the musculoskeletal and ophthalmological fields regarding the how the inflammatory microenvironment is regulated in these tissues and potentially others., (© 2018 WILEY Periodicals, Inc.)

It has emerged that neutrophils can play important roles in the host response following infection with helminth parasites. Mice infected with the tapeworm, Hymenolepis diminuta, are protected from some inflammatory conditions, accompanied by reduced neutrophil tissue infiltration. Thus, the ability of a phosphate-buffered saline-soluble extract of the worm (H. diminuta extract [HdE]) was tested for (1) its ability to activate murine neutrophils (Ca2+ mobilization, reactive oxygen species (ROS) and cytokine production); and (2) affect neutrophil chemotaxis in vitro to the penta-peptide, WKYMVm, the chemokine, KC, and leukotriene B4. HdE was not cytotoxic to neutrophils, elicited a Ca2+ response and ROS, but not, cytokine (KC, interleukin-10, tumour necrosis factor-α) generation. HdE is not a chemotactic stimulus for murine neutrophils. However, a heat- and trypsin-sensitive, acid-insensitive proteoglycan (sensitive to sodium metaperiodate) in the HdE significantly reduced neutrophil chemotaxis towards WKYMVm or KC, but not LTB4. The latter suggested that the HdE interfered with p38 mitogen-activated protein kinase signalling, which is important in WKYMVm chemotaxis. Corroborating this, immunoblotting revealed reduced phosphorylated p38, and the downstream signal heat-shock protein-27, in protein extracts from HdE + WkYMVm treated cells compared to those exposed to the penta-peptide only. We speculate that HdE can be used to modify the outcome of neutrophilic disease and that purification of the bioactive proteoglycan(s) from the extract could be used as a template to design immunomodulatory drugs targeting neutrophils., (© 2018 The Author(s) Published by S. Karger AG, Basel.)

The respiratory pathogen Moraxella catarrhalis is a human-specific commensal that frequently causes acute otitis media in children and stimulates acute exacerbations in chronic obstructive pulmonary disease patients. The exact molecular mechanisms defining host-pathogen interactions promoting pathogenesis are not clearly understood. Limited knowledge hampers vaccine and immunotherapeutic development required to treat this emerging pathogen. In this study, we reveal in detail a novel antibacterial role displayed by short leucine-rich proteoglycans (SLRPs) in concert with complement. We show that fibromodulin (FMOD), osteoadherin (OSAD), and biglycan (BGN) but not decorin (DCN) enhance serum killing of M. catarrhalis. Our results suggest that M. catarrhalis binding to SLRPs is a conserved feature, as the overwhelming majority of clinical and laboratory strains bound all four SLRPs. Furthermore, we resolve the binding mechanism responsible for this interaction and highlight the role of the ubiquitous surface protein (Usp) A2/A2H in mediating binding to host SLRPs. A conserved immune evasive strategy used by M. catarrhalis and other pathogens is the surface acquisition of host complement inhibitors such as C4b-binding protein (C4BP). We observed that FMOD, OSAD, and BGN competitively inhibit binding of C4BP to the surface of M. catarrhalis , resulting in increased C3b/iC3b deposition, membrane attack complex (MAC) formation, and subsequently decreased bacterial survival. Furthermore, both OSAD and BGN promote enhanced neutrophil killing in vitro, both in a complement-dependent and independent fashion. In summary, our results illustrate that SLRPs, FMOD, OSAD, and BGN portray complement-modulating activity enhancing M. catarrhalis killing, defining a new antibacterial role supplied by SLRPs., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Background: Endothelial cell specific molecule-1, also called as endocan, is a dermatan sulfate proteoglycan, which is expressed by endothelial cells in alveolar walls of the lung and kidney. High endocan levels are found associated with endothelial dysfunction and inflammation. We hypothesize that endocan level is also high in COPD due to systemic inflammation and endothelial dysfunction. We aimed to investigate the expression of endocan in patients with stable COPD., Material and Methods: The study included patients with COPD and control subjects. COPD patients were classified according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2017 criteria. Demographics, body mass index, smoking history, and comorbidities were recorded. Endocan levels of COPD patients and controls were compared., Results: Totally, 88 subjects (47 stable COPD patients, 41 controls) were evaluated. Endocan levels were significantly higher in COPD patients than control group (860.1±259.8 vs 647.3±316.9 pg/mL, P =0.001). There was no relationship between GOLD COPD categories and endocan levels. Also endocan levels were similar between COPD patients with or without hypoxemia., Conclusion: Serum endocan level was significantly higher in patients with stable COPD. Further studies should be performed to better understand the relationship between endocan and COPD., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Proteoglycans are glycosylated proteins which have covalently attached highly anionic glycosaminoglycans. They can be located on the extracellular matrix, cell membrane or intracellular granules. To date, few studies have reported the presence of proteoglycans in human dental pulp., Objective: The aim of this study was, therefore, to analyze the expression of lumican, versican and glypican proteoglycans in deciduous and permanent human dental pulp by real-time polymerase chain reaction (q-PCR) and immunofluorescence., Design: Healthy human dental pulps were used: 13 from permanent teeth (group 1) and eight from deciduous teeth (group 2). Versican, lumican and glypican (glypican-1 to 6) gene expressions were quantitatively evaluated by real-time PCR technique, using the expression of the endogenous gene GAPDH as control. Pulp sections were submitted to immunostaining procedure with fluorescence labelling, the tissues being fixed and incubated with well-characterized monoclonal and polyclonal antibodies against proteoglycan epitopes, including anti-versican and anti-lumican. Comparisons among the groups of the quantitative scores for each proteoglycan were analyzed using the t-test and ANOVA (P < 0.05)., Results: The real-time PCR analysis showed expression of versican and lumican proteoglycans in the two groups, with significant predominance of lumican gene (P = 0.03). Considering the glypican genes, glypican-3 was the proteoglycan most significantly expressed in permanent pulps (P < 0.001), while glypican-2 was not expressed in this tissue. The immunofluorescence quantification exhibited no significant differences between lumican and versican among the pulps and groups., Conclusions: The lumican gene was more expressed than versican and glypican-3 was the isoform more expressed in permanent pulp compared to deciduous., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Proteoglycans (PGs) are complex, multifaceted molecules that participate in diverse interactions vital for physiological and pathological processes. As structural components, they provide a scaffold for cells and structural organization that helps define tissue architecture. Through interactions with water, PGs enable molecular and cellular movement through tissues. Through selective ionic interactions with growth factors, chemokines, cytokines, and proteases, PGs facilitate the ability of these soluble ligands to regulate intracellular signaling events and to influence the inflammatory response. In addition, recent findings now demonstrate that PGs can activate danger-associated molecular patterns (DAMPs) and other signaling pathways to influence production of many of these soluble ligands, indicating a more direct role for PGs in influencing the immune response and tissue inflammation. This review will focus on PGs that are selectively expressed during lung inflammation and will examine the novel emerging concept of PGs as immunomodulatory regulators of the innate immune responses in lungs.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a poor prognosis. There is a clinical need for effective, targeted therapy strategies that destroy both differentiated TNBC cells and TNBC cancer initiating cells (CICs), as the latter are implicated in the metastasis and recurrence of TNBC. Chondroitin sulfate proteoglycan 4 (CSPG4) is overexpressed on differentiated tumor cells and CICs obtained from TNBC patient specimens, suggesting that CSPG4 may be a clinically relevant target for the imaging and therapy of TNBC. The purpose of this study was to determine whether α-particle radioimmunotherapy (RIT) targeting TNBC cells using the CSPG4-specific monoclonal antibody (mAb) 225.28 as a carrier was effective at eliminating TNBC tumors in preclinical models. To this end, mAb 225.28 labeled with 212 Pb ( 212 Pb-225.28) as a source of α-particles for RIT was used for in vitro Scatchard assays and clonogenic survival assays with human TNBC cells (SUM159 and 2LMP) grown as adherent cells or non-adherent CIC-enriched mammospheres. Immune-deficient mice bearing orthotopic SUM159 or 2LMP xenografts were injected i.v. with the targeted (225.28) or irrelevant isotype-matched control (F3-C25) mAbs, labeled with 99m Tc, 125 I, or 212 Pb for in vivo imaging, biodistribution, or tumor growth inhibition studies. 212 Pb-225.28 bound to adherent SUM159 and 2LMP cells and to CICs from SUM159 and 2LMP mammospheres with a mean affinity of 0.5 nM. Nearly ten times more binding sites per cell were present on SUM159 cells and CICs compared with 2LMP cells. 212 Pb-225.28 was six to seven times more effective than 212 Pb-F3-C25 at inhibiting SUM159 cell and CIC clonogenic survival ( p < 0.05). Radiolabeled mAb 225.28 showed significantly higher uptake than radiolabeled mAb F3-C25 in SUM159 and 2LMP xenografts ( p < 0.05), and the uptake of 212 Pb-225.28 in TNBC xenografts was correlated with target epitope expression. 212 Pb-225.28 caused dose-dependent growth inhibition of SUM159 xenografts; 0.30 MBq 212 Pb-225.28 was significantly more effective than 0.33 MBq 212 Pb-F3-C25 at inhibiting tumor growth ( p < 0.01). These results suggest that CSPG4-specific 212 Pb-225.28 is a useful reagent for RIT of CSPG4-expressing tumors, including metastatic TNBC., Competing Interests: The authors declare no conflict of interest.

Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology. A characteristic feature of RA is the presence of anti-citrullinated protein antibodies (ACPA). Since ACPAs are highly specific for RA and are often present before the onset of RA symptoms, they have become valuable diagnostic and prognostic. As a result, several assays for detection of ACPAs exist, which vary in sensitivity and specificity. In this study, we analyzed the reactivity of RA sera to selected peptides by solid-phase immunoassays in order to develop an ACPA assay with improved sensitivity and specificity. ACPA levels were determined with respect to sensitivity and specificity in 332 serum samples using the newly developed peptide panel, which was compared to the commercial assays CCPlus (Eurodiagnostica) and CCP3.1 (Inova Diagnostics). A primary panel (peptides 814, 33062 and 33156) was identified, which obtained a sensitivity of 71%, while the complete peptide panel reacted with 79% of RA sera screened. Total specificities of 89% and 80% were obtained for the primary peptide panel and the complete peptide panel. Sensitivities for the commercial assays ranged between 71% and 76% and specificities between 88% and 90%. These findings indicate that the generated peptide panel is optimal for ACPA detection and able to compete with commercial available assays. Collectively, this study may contribute to characterize autoimmunity towards citrullinated proteins and to the development of new and improved diagnostic assays for detection of ACPA and determination of RA., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Chondroitin sulfate proteoglycans (CSPGs) are the main active component of perineuronal nets (PNNs). Digestion of the glycosaminoglycan chains of CSPGs with chondroitinase ABC or transgenic attenuation of PNNs leads to prolongation of object recognition memory and activation of various forms of plasticity in the adult central nervous system. The inhibitory properties of the CSPGs depend on the pattern of sulfation of their glycosaminoglycans, with chondroitin 4-sulfate (C4S) being the most inhibitory form. In this study, we tested a number of candidates for functional blocking of C4S, leading to selection of an antibody, Cat316, which specifically recognizes C4S and blocks its inhibitory effects on axon growth. It also partly blocks binding of semaphorin 3A to PNNs and attenuates PNN formation. We asked whether injection of Cat316 into the perirhinal cortex would have the same effects on memory as chondroitinase ABC treatment. We found that masking C4S with the Cat316 antibody extended long-term object recognition memory in normal wild-type mice to 24 hours, similarly to chondroitinase or transgenic PNN attenuation. We then tested Cat316 for restoration of memory in a neurodegeneration model. Mice expressing tau with the P301S mutation showed profound loss of object recognition memory at 4 months of age. Injection of Cat316 into the perirhinal cortex normalized object recognition at 3 hours in P301S mice. These data indicate that Cat316 binding to C4S in the extracellular matrix can restore plasticity and memory in the same way as chondroitinase ABC digestion. Our results suggest that antibodies to C4S could be a useful therapeutic to restore memory function in neurodegenerative disorders., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: The dog represents an excellent large animal model for translational cell-based studies. Importantly, the properties of canine multipotent stromal cells (cMSCs) and the ideal tissue source for specific translational studies have yet to be established. The aim of this study was to characterize cMSCs derived from synovium, bone marrow, and adipose tissue using a donor-matched study design and a comprehensive series of in-vitro characterization, differentiation, and immunomodulation assays., Methods: Canine MSCs were isolated from five dogs with cranial cruciate ligament rupture. All 15 cMSC preparations were evaluated using colony forming unit (CFU) assays, flow cytometry analysis, RT-PCR for pluripotency-associated genes, proliferation assays, trilineage differentiation assays, and immunomodulation assays. Data were reported as mean ± standard deviation and compared using repeated-measures analysis of variance and Tukey post-hoc test. Significance was established at p < 0.05., Results: All tissue samples produced plastic adherent, spindle-shaped preparations of cMSCs. Cells were negative for CD34, CD45, and STRO-1 and positive for CD9, CD44, and CD90, whereas the degree to which cells were positive for CD105 was variable depending on tissue of origin. Cells were positive for the pluripotency-associated genes NANOG, OCT4, and SOX2. Accounting for donor and tissue sources, there were significant differences in CFU potential, rate of proliferation, trilineage differentiation, and immunomodulatory response. Synovium and marrow cMSCs exhibited superior early osteogenic activity, but when assessing late-stage osteogenesis no significant differences were detected. Interestingly, bone morphogenic protein-2 (BMP-2) supplementation was necessary for early-stage and late-stage osteogenic differentiation, a finding consistent with other canine studies. Additionally, synovium and adipose cMSCs proliferated more rapidly, displayed higher CFU potential, and formed larger aggregates in chondrogenic assays, although proteoglycan and collagen type II staining were subjectively decreased in adipose pellets as compared to synovial and marrow pellets. Lastly, cMSCs derived from all three tissue sources modulated murine macrophage TNF-α and IL-6 levels in a lipopolysaccharide-stimulated coculture assay., Conclusions: While cMSCs from synovium, marrow, and adipose tissue share a number of similarities, important differences in proliferation and trilineage differentiation exist and should be considered when selecting cMSCs for translational studies. These results and associated methods will prove useful for future translational studies involving the canine model.

Autoimmune and other chronic inflammatory diseases (AID) are prevalent diseases which can severely impact the quality of life of those that suffer from the disease. In most cases, the etiology of these conditions have remained unclear. Immune responses that take place e.g. during natural infection or after vaccination are often linked with the development or exacerbation of AID. It is highly debated if vaccines induce or aggravate AID and in particular adjuvants are mentioned as potential cause. Since vaccines are given on a large scale to healthy individuals but also to elderly and immunocompromised individuals, more research is warranted. Non-specific induction of naïve or memory autoreactive T cells via bystander activation is one of the proposed mechanisms of how vaccination might be involved in AID. During bystander activation, T cells unrelated to the antigen presented can be activated without (strong) T cell receptor (TCR) ligation, but via signals derived from the ongoing response directed against the vaccine-antigen or adjuvant at hand. In this study we have set up a TCR transgenic T cell transfer mouse model by which we were able to measure local bystander activation of transferred and labeled CD4+ T cells. Intramuscular injection with the highly immunogenic Complete Freund's Adjuvant (CFA) led to local in vivo proliferation and activation of intravenously transferred CD4+ T cells in the iliac lymph node. This local bystander activation was also observed after CFA prime and Incomplete Freund's Adjuvant (IFA) boost injection. Furthermore, we showed that an antigen specific response is sufficient for the induction of a bystander activation response and the general, immune stimulating effect of CFA or IFA does not appear to increase this effect. In other words, no evidence was obtained that adjuvation of antigen specific responses is essential for bystander activation.

Background: Determining the expression levels of neuroglial antigen 2 (NG2) in glioma cell lines and to evaluate the potential contribution of NG2 to cilengitide response were aimed., Materials and Methods: Endogenous expression level of NG2 was determined using quantitative reverse transcription polymerase chain reaction and immunoblotting. Cilengitide responses of the cells were monitored to determine half maximal inhibitory concentration values. Whether the suppression of NG2 expression alters the response of A172 cells to cilengitide was examined., Results: The effect of cilengitide on inducing apoptosis of the cells was determined by TUNEL staining. High mRNA and protein expression of NG2 was detected in A172 and U-87MG cells, while T98G, M059K and M059J cells demonstrated low levels of NG2. A172, U-87MG and positive control MG-63 were relatively sensitive to cilengitide compared to T98G, M059K and M059J. MG-63, A172 and U-87MG were unexpectedly found to be more susceptible to cilengitide. In addition, NG2 knock-down showed no significant difference in cell death between small interfering RNA (siRNA)-transfected and cilengitide-treated groups. The results showed that cilengitide caused detachment and subsequently initiated apoptosis. Glioma cell lines express variable levels of NG2 and differ in their responses to cilengitide. Although increased numbers of apoptotic cells were found in untransfected cells compared to siRNA-transfected cells upon exposed to cilengitide, the difference was not documented to be significant between two groups., Conclusion: It may be proposed that the combination therapy of NG2 suppression and cilengitide treatment showed no considerable effect on glioblastoma compared to cilengitide therapy alone. Response to therapy may be further improved by targeting other factors act in concert in this signaling pathway.

Polysaccharopeptide (PSP), from Coriolus versicolor, has been used widely as an adjuvant to chemotherapy with demonstrated anti-tumor and broad immunomodulating effects. While PSP's mechanism of action still remains unknown, its enhanced immunomodulatory potential with acacia gum is of great interest. Acacia gum, which also contains polysaccharides and glycoproteins, has been demonstrated to be immunopotentiating. To elucidate whether PSP directly activates T-cell-dependent B-cell responses in vivo, we used a well-established hapten carrier system (Nitrophenyl-chicken gamma globulin (NP-CGG)). 6-week C57BL/6 male mice were immunised with 50 μg of NP25-CGG alum precipitate intraperitoneally. Mice were gavaged daily with 50 mg/kg PSP in a vehicle containing acacia gum and sacrificed at days 0, 4, 7, 10, 14 and 21. ELISA was used to measure the total and relative hapten-specific anti-NP IgA, IgM and IgG titre levels compared to the controls. It was found that PSP, combined with acacia gum, significantly increased total IgG titre levels at day 4 (P< 0.05), decreased IgM titre levels at days 4 and 21 (P< 0.05) with no alterations observed in the IgA or IgE titre levels at any of the time points measured. Our results suggest that while PSP combined with acacia gum appears to exert weak immunological effects through specific T-cell dependent B-cell responses, they are likely to be broad and non-specific which supports the current literature on PSP. We report for the first time the application of a well-established hapten-carrier system that can be used to characterise and delineate specific T-cell dependent B-cell responses of potential immunomodulatory glycoprotein-based herbal medicines combinations in vivo.

Background: Lubricin/proteoglycan-4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes. PRG4 has a homeostatic multifaceted role in the joint. PRG4 intra-articular treatment retards progression of cartilage degeneration in pre-clinical posttraumatic osteoarthritis models. The objective of this study is to evaluate the binding of recombinant human PRG4 (rhPRG4) and native human PRG4 (nhPRG4) to toll-like receptors 2 and 4 (TLR2 and TLR4) and whether this interaction underpins a PRG4 anti-inflammatory role in synovial fluid (SF) from patients with osteoarthritis (OA) and rheumatoid arthritis (RA)., Methods: rhPRG4 and nhPRG4 binding to TLR2 and TLR4 was evaluated using a direct enzyme linked immunosorbent assay (ELISA). Association of rhPRG4 with TLR2 and TLR4 overexpressing human embryonic kidney (HEK) cells was studied by flow cytometry. Activation of TLR2 and TLR4 on HEK cells by agonists Pam3CSK4 and lipopolysaccharide (LPS) was studied in the absence or presence of nhPRG4 at 50, 100 and 150 μg/ml. Activation of TLR2 and TLR4 by OA SF and RA SF and the effect of nhPRG4 SF treatment on receptor activation was assessed. PRG4 was immunoprecipitated from pooled OA and RA SF. TLR2 and TLR4 activation by pooled OA and RA SF with or without PRG4 immunoprecipitation was compared., Results: rhPRG4 and nhPRG4 exhibited concentration-dependent binding to TLR2 and TLR4. rhPRG4 associated with TLR2- and TLR4-HEK cells in a time-dependent manner. Co-incubation of nhPRG4 (50, 100 and 150 μg/ml) and Pam3CSK4 or LPS reduced TLR2 or TLR4 activation compared to Pam3CSK4 or LPS alone (p <0.05). OA SF and RA SF activated TLR2 and TLR4 and nhPRG4 treatment reduced SF-induced receptor activation (p <0.001). PRG4 depletion by immunoprecipitation significantly increased TLR2 activation by OA SF and RA SF (p <0.001)., Conclusion: PRG4 binds to TLR2 and TLR4 and this binding mediates a novel anti-inflammatory role for PRG4.

Copper (Cu) is essential for multiple cellular functions. Cellular uptake of Cu(+) is carried out by the Ctr1 high-affinity Cu transporter. The mobilization of endosomal Cu pools is regulated by a protein structurally similar to Ctr1, called Ctr2. It was recently shown that ablation of Ctr2 caused an increase in the concentration of Cu localized to endolysosomes. However, the biological significance of excess endolysosomal Cu accumulation has not been assessed. In this study, we addressed this issue by investigating the impact of Ctr2 deficiency on mast cells, a cell type unusually rich in endolysosomal organelles (secretory granules). We show that Ctr2(-/-) mast cells have increased intracellular Cu concentrations and that the absence of Ctr2 results in increased metachromatic staining, the latter indicating an impact of Ctr2 on the storage of proteoglycans in the secretory granules. In agreement with this, the absence of Ctr2 caused a skewed ratio between proteoglycans of heparin and chondroitin sulfate type, with increased amounts of heparin accompanied by a reduction of chondroitin sulfate. Moreover, transmission electron microscopy analysis revealed a higher number of electron-dense granules in Ctr2(-/-) mast cells than in wild-type cells. The increase in granular staining and heparin content is compatible with an impact of Ctr2 on mast cell maturation and, in support of this, the absence of Ctr2 resulted in markedly increased mRNA expression, storage, and enzymatic activity of tryptase. Taken together, the present study introduces Ctr2 and Cu as novel actors in the regulation of mast cell maturation and granule homeostasis., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The extracellular matrix is a dynamic repository harboring instructive cues that embody substantial regulatory dominance over many evolutionarily conserved intracellular activities, including proliferation, apoptosis, migration, motility, and autophagy. The matrix also coordinates and parses hierarchical information, such as angiogenesis, tumorigenesis, and immunological responses, typically providing the critical determinants driving each outcome. We provide the first comprehensive review focused on proteoglycan receptors, that is, signaling transmembrane proteins that use secreted proteoglycans as ligands, in addition to their natural ligands. The majority of these receptors belong to an exclusive subset of receptor tyrosine kinases and assorted cell surface receptors that specifically bind, transduce, and modulate fundamental cellular processes following interactions with proteoglycans. The class of small leucine-rich proteoglycans is the most studied so far and constitutes the best understood example of proteoglycan-receptor interactions. Decorin and biglycan evoke autophagy and immunological responses that deter, suppress, or exacerbate pathological conditions such as tumorigenesis, angiogenesis, and chronic inflammatory disease. Basement membrane-associated heparan sulfate proteoglycans (perlecan, agrin, and collagen XVIII) represent a unique cohort and provide proteolytically cleaved bioactive fragments for modulating cellular behavior. The receptors that bind the genuinely multifactorial and multivalent proteoglycans represent a nexus in understanding basic biological pathways and open new avenues for therapeutic and pharmacological intervention.

Neutrophil-specific granule deficiency (SGD) is a rare autosomal recessive primary immunodeficiency characterized by neutrophil dysfunction, bilobed neutrophil nuclei and lack of neutrophil-specific granules. Defects in a myeloid-specific transcription factor, CCAAT/enhancer binding protein-ε (C/EBPε), have been identified in two cases in which homozygous frameshift mutations led to loss of the leucine zipper domain. In this study, we report a 55-y-old woman affected with SGD caused by a novel homozygous 2-aa deletion (ΔRS) in the leucine zipper domain of the C/EBPε gene. The patient showed characteristic neutrophil abnormalities and recurrent skin infections; however, there was no history of deep organ infections. Biochemical analysis revealed that, in contrast to the two frameshift mutations, the ΔRS mutant maintained normal cellular localization, DNA-binding activity, and dimerization, and all three mutants exhibited marked reduction in transcriptional activity. The ΔRS mutant was defective in its association with Gata1 and PU.1, as well as aberrant cooperative transcriptional activation of eosinophil major basic protein. Thus, the ΔRS likely impairs protein-protein interaction with other transcription factors, resulting in a loss of transcriptional activation. These results further support the importance of the leucine zipper domain of C/EBPε for its essential function, and indicate that multiple molecular mechanisms lead to SGD., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Objectives: To explore the immunosuppressive effect and mechanism of action of intraperitoneal (ip) and intra-articular (ia) mesenchymal stem cell (MSC) injection in proteoglycan induced arthritis (PGIA)., Methods: MSC were administered ip or ia after establishment of arthritis. We used serial bioluminescence imaging (BLI) to trace luciferase-transfected MSC. Mice were sacrificed at different time points to examine immunomodulatory changes in blood and secondary lymphoid organs., Results: Both ip and local ia MSC injection resulted in a beneficial clinical and histological effect on established PGIA. BLI showed that MSC ip and ia in arthritic mice are largely retained for several weeks in the peritoneal cavity or injected joint respectively, without signs of migration. Following MSC treatment pathogenic PG-specific IgG2a antibodies in serum decreased. The Th2 cytokine IL-4 was only upregulated in PG-stimulated lymphocytes from spleens in ip treated mice and in lymphocytes from draining lymph nodes in ia treated mice. An increase in production of IL-10 was seen with equal distribution. Although IFN-γ was also elevated, the IFN-γ/IL-4 ratio in MSC treated mice was opposite to the ratio in (untreated) active PGIA., Conclusions: MSC treatment, both ip and ia, suppresses PGIA, a non-collagen induced arthritis model. MSC are largely retained for weeks in the injection region. MSC treatment induced at the region of injection a deviation of PG-specific immune responses, suggesting a more regulatory phenotype with production of IL-4 and IL-10, but also of IFN-γ, and a systemic decrease of pathogenic PG-specific IgG2a antibodies. These findings underpin the potential of MSC treatment in resistant arthritis., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: Effective treatments for glioblastoma multiforme (GBM) are lacking due, in part, to cellular heterogeneity. Consequently, single-target therapeutic strategies are unlikely to succeed. Simultaneous targeting of different neoplastic cell populations within the same tumour may, therefore, prove of value. Neuron-glia 2 (NG2), a transmembrane chondroitin sulphate proteoglycan, present on developing glial cells, and GD3(A), a ganglioside expressed on developing migratory glia, are re-expressed in GBM., Materials and Methods: The aims of this study were to conduct 'proof of concept' experiments in human GBM cell lines to show that proliferative high NG2-expressing cells and high GD3(A) -expressing migratory cells could be effectively ablated using a Mab-Zap saporin immunotoxin system., Results: The combinatorial ablation of both NG2 and GD3(A)-expressing cells resulted in significant reduction in GBM cell viability compared to single epitope targeting and controls (p<0.0001); non-neoplastic astrocytes were not affected., Conclusion: Multiple targeting of GBM sub-populations may, therefore, help inform novel therapeutic approaches., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Introduction: Proteoglycans are critical molecules involved in multiple physiological cell functions, but also key players in cancer development and progression. In particular, chondroitin sulfate proteoglycan 4 (CSPG4) is recognized as an attractive target for antibody-based approaches because of its high expression on cancer cells in several types of human malignancies and its restricted distribution in normal tissues., Areas Covered: Adoptive transfer of genetically modified T cells is emerging as a powerful therapeutic approach in cancer patients. In this regard, the selection of the appropriate antigen to be targeted in solid tumors becomes a critical aspect in promoting potent antitumor effects while preventing toxicities. This review summarizes the authors' current knowledge on the expression and function of CSPG4 in normal tissues and malignant tumors, with a particular focus on the potential use of CSPG4 as a target for antigen-specificity redirected T cells., Expert Opinion: T cells expressing a CSPG4-specific chimeric antigen receptor (CAR) offer the possibility to target a broad spectrum of solid tumors for which no curative treatment is currently available. In addition, since CSPG4 is also selectively up-regulated on tumor-associated pericytes, targeting this antigen may also contribute to tumor regression via inhibition of neoangiogenesis. Preclinical experiments to date justify the clinical translation of CSPG4-specific CAR-T cells.

In reply to internal or external danger stimuli, the body orchestrates an inflammatory response. The endogenous triggers of this process are the damage-associated molecular patterns (DAMPs). DAMPs represent a heterogeneous group of molecules that draw their origin either from inside the various compartments of the cell or from the extracellular space. Following interaction with pattern recognition receptors in cross-talk with various non-immune receptors, DAMPs determine the downstream signaling outcome of septic and aseptic inflammatory responses. In this review, the diverse nature, structural characteristics, and signaling pathways elicited by DAMPs will be critically evaluated., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)