Journal of Immunology Research The latest articles from Hindawi Publishing Corporation © 2016 , Hindawi Publishing Corporation . All rights reserved. Immunogenicity Assessment of Lipegfilgrastim in Patients with Breast Cancer Receiving Chemotherapy Thu, 23 Jun 2016 13:34:59 +0000 Lipegfilgrastim is a long-acting, once-per-cycle, glycopegylated recombinant granulocyte colony-stimulating factor (G-CSF) used to prevent neutropenia in patients receiving myelosuppressive chemotherapy. This integrated analysis examined the immunogenicity of lipegfilgrastim and its potential clinical impact in two double-blind randomized studies (phases II and III) of patients with breast cancer receiving chemotherapy. Serum samples were analyzed using sequential assays for screening, confirmation, antibody titer, and characterization of antidrug antibodies (ADA). Neutropenia-related efficacy measures were reviewed for each ADA-positive patient. Among 255 patients receiving lipegfilgrastim (154 in phase II, 101 in phase III) and 155 patients receiving pegfilgrastim (54 in phase II, 101 in phase III), the incidence of treatment-emergent ADA was low and similar between the lipegfilgrastim (phase II: 1.3%; phase III: 1.0%) and pegfilgrastim (phase II: 1.9%; phase III: 1.0%) arms. None of the treatment-emergent ADA-positive samples exhibited neutralizing activity against lipegfilgrastim, pegfilgrastim, or glycosylated G-CSF in a cell-based neutralizing antibody assay. No changes were observed in neutropenia-related efficacy measures among ADA-positive patients, and no treatment-related hypersensitivity or anaphylaxis occurred. These results indicate that there is no apparent impact of ADA on lipegfilgrastim efficacy and safety. Linglong Zou, Anton Buchner, Martin Roberge, and Patrick M. Liu Copyright © 2016 Linglong Zou et al. All rights reserved. Overcoming the Constraints of Anti-HIV/CD89 Bispecific Antibodies That Limit Viral Inhibition Wed, 22 Jun 2016 14:27:02 +0000 Innovative strategies are necessary to maximize the clinical application of HIV neutralizing antibodies. To this end, bispecific constructs of human antibody F240, reactive with well-conserved gp41 epitope and antibody 14A8, reactive with the IgA receptor (CD89) on effector cells, were constructed. A F240 × 14A8 bispecific single chain variable region (scFv) molecule was constructed by linking two scFvs using a conventional GGGGS linker. Despite immunoreactivity with HIV gp41 and neutrophils, this bispecific scFv failed to inhibit HIV infection. This is in sharp contrast to viral inhibition using a chemical conjugate of the Fab of these two antibodies. Therefore, we constructed two novel Fab-like bispecific antibody molecules centered on fusion of the IgG1 CH1 domain or CH1-hinge domain to the C-terminus of F240scFv and fusion of the kappa chain CL domain to the C-terminus of 14A8scFv. Both Bi-Fab antibodies showed significant ADCVI activity for multiple clade B and clade C isolates by arming the neutrophils to inhibit HIV infection. The approach presented in this study is unique for HIV immunotherapy in that the impetus of neutralization is to arm and mobilize PMN to destroy HIV and HIV infected cells. Xiaocong Yu, Mark Duval, Melissa Gawron, Marshall R. Posner, and Lisa A. Cavacini Copyright © 2016 Xiaocong Yu et al. All rights reserved. Elevated Concentration of Defensins in Hepatitis C Virus-Infected Patients Mon, 20 Jun 2016 12:03:56 +0000 Hepatitis C virus (HCV) is the major etiological agent of human non-A and non-B hepatitis, affecting around 180 million people worldwide. Defensins, small cysteine-rich cationic peptides, are shown to have potent antibacterial, antiviral, and antifungal properties. Defensins can be found in both normal and microbial infected patients, at variable concentrations. Notably, viral infections are often associated with elevated concentrations of defensins. The current study aimed to estimate the concentrations of total, α-, and β-defensins in serum taken from normal and HCV-infected patients. 12 healthy (noninfected) and 34 HCV-infected patients were enrolled. Standardized immunoassay kits were used to obtain serum concentrations of defensins. The obtained results were calibrated against kit standard reagents. Total defensin concentrations in HCV-infected patients were significantly higher (2- to 105-fold) compared to healthy individuals. The concentrations of α-defensins were also significantly elevated in the HCV-infected patients (31–1398 ng/50 μL). However, concentrations of β-defensins ranged from 44.5 ng/50 μL to 1056 ng/50 μL. The results did not reveal differences in serum defensin concentration between male and female HCV-infected patients. A-defensin concentration of ≥250 ng/50 μL was found to contain more β-defensins than total defensins and α-defensins. This study concludes, for the first time, that serum defensin levels are elevated in HCV-infected patients. Ehab H. Mattar, Hussein A. Almehdar, Abdullah A. AlJaddawi, Isam ElDin M. Abu Zeid, and Elrashdy M. Redwan Copyright © 2016 Ehab H. Mattar et al. All rights reserved. Uptake of HLA Alloantigens via CD89 and CD206 Does Not Enhance Antigen Presentation by Indirect Allorecognition Mon, 20 Jun 2016 11:36:13 +0000 In organ transplantation, alloantigens are taken up by antigen presenting cells and presented via the indirect pathway to T-cells which in turn can induce allograft rejection. Monitoring of these T-cells is of major importance; however no reliable assay is available to routinely monitor indirect allorecognition. Recently we showed that HLA monomers can be successfully used to monitor indirect allorecognition. Targeting antigens to endocytic receptors on antigen presenting cells may further enhance the presentation of antigens via HLA class II and improve the efficiency of this assay. In the current study we explored targeting of HLA monomers to either CD89 expressing monocytes or mannose receptor expressing dendritic cells. Monomer-antibody complexes were generated using biotin-labeled monomers and avidin labeling of the antibodies. We demonstrate that targeting the complexes to these receptors resulted in a dose-dependent HLA class II mediated presentation to a T-cell clone. The immune-complexes were efficiently taken up and presented to T-cells. However, the level of T-cell reactivity was similar to that when only exogenous antigen was added. We conclude that HLA-A2 monomers targeted for presentation through CD89 on monocytes or mannose receptor on dendritic cells lead to proper antigen presentation but do not enhance indirect allorecognition via HLA-DR. Eytan Breman, Jurjen M. Ruben, Kees L. Franken, Mirjam H. M. Heemskerk, Dave L. Roelen, Frans H. Claas, and Cees van Kooten Copyright © 2016 Eytan Breman et al. All rights reserved. Aptamers: A Feasible Technology in Cancer Immunotherapy Mon, 20 Jun 2016 10:55:18 +0000 Aptamers are single-chained RNA or DNA oligonucleotides (ODNs) with three-dimensional folding structures which allow them to bind to their targets with high specificity. Aptamers normally show affinities comparable to or higher than that of antibodies. They are chemically synthesized and therefore less expensive to manufacture and produce. A variety of aptamers described to date have been shown to be reliable in modulating immune responses against cancer by either blocking or activating immune receptors. Some of them have been conjugated to other molecules to target the immune system and reduce off-target side effects. Despite the success of first-line treatments against cancer, the elevated number of relapsing cases and the tremendous side effects shown by the commonly used agents hinder conventional treatments against cancer. The advantages provided by aptamers could enhance the therapeutic index of a given strategy and therefore enhance the antitumor effect. Here we recapitulate the provided benefits of aptamers with immunomodulatory activity described to date in cancer therapy and the benefits that aptamer-based immunotherapy could provide either alone or combined with first-line treatments in cancer therapy. M. M. Soldevilla, H. Villanueva, and F. Pastor Copyright © 2016 M. M. Soldevilla et al. All rights reserved. A Modified NK Cell Degranulation Assay Applicable for Routine Evaluation of NK Cell Function Mon, 20 Jun 2016 10:54:18 +0000 Natural killer (NK) cells play important role in innate immunity against tumors and viral infections. Studies show that lysosome-associated membrane protein-1 (LAMP-1, CD107a) is a marker for degranulation of NK and cytotoxic T cells and its expression is a sensitive marker for the cytotoxic activity determination. The conventional methods of determination of CD107a on NK cells involve use of peripheral blood mononuclear cells (PBMC) or pure NK cells and K562 cells as stimulants. Thus, it requires large volume of blood sample which is usually difficult to obtain in pediatric patients and patients with cytopenia and also requires specialized laboratory for maintaining cell line. We have designed a flow cytometric assay to determine CD107a on NK cells using whole blood, eliminating the need for isolation of PBMC or isolate NK cells. This assay uses phorbol-12-myristate-13-acetate (PMA) and calcium ionophore (Ca2+-ionophore) instead of K562 cells for stimulation and thus does not require specialized cell culture laboratory. CD107a expression on NK cells using modified NK cell degranulation assay compared to the conventional assay was significantly elevated (). It was also validated by testing patients diagnosed with familial hemophagocytic lymphohistiocytosis (FHL) with defect in exocytosis. This assay is rapid, cost effective, and reproducible and requires significantly less volume of blood which is important for clinical evaluation of NK cells. Snehal Shabrish, Maya Gupta, and Manisha Madkaikar Copyright © 2016 Snehal Shabrish et al. All rights reserved. Immune Regulation and Antitumor Effect of TIM-1 Mon, 20 Jun 2016 10:42:55 +0000 T cells play an important role in antitumor immunity, and the T cell immunoglobulin domain and the mucin domain protein-1 (TIM-1) on its surface, as a costimulatory molecule, has a strong regulatory effect on T cells. TIM-1 can regulate and enhance type 1 immune response of tumor association. Therefore, TIM-1 costimulatory pathways may be a promising therapeutic target in future tumor immunotherapy. This review describes the immune regulation and antitumor effect of TIM-1. Peng Du, Ruihua Xiong, Xiaodong Li, and Jingting Jiang Copyright © 2016 Peng Du et al. All rights reserved. The Mannose Receptor Is Involved in the Phagocytosis of Mycobacteria-Induced Apoptotic Cells Mon, 20 Jun 2016 07:28:30 +0000 Upon Mycobacterium tuberculosis infection, macrophages may undergo apoptosis, which has been considered an innate immune response. The pathways underlying the removal of dead cells in homeostatic apoptosis have been extensively studied, but little is known regarding how cells that undergo apoptotic death during mycobacterial infection are removed. This study shows that macrophages induced to undergo apoptosis with mycobacteria cell wall proteins are engulfed by J-774A.1 monocytic cells through the mannose receptor. This demonstration was achieved through assays in which phagocytosis was inhibited with a blocking anti-mannose receptor antibody and with mannose receptor competitor sugars. Moreover, elimination of the mannose receptor by a specific siRNA significantly diminished the expression of the mannose receptor and the phagocytosis of apoptotic cells. As shown by immunofluorescence, engulfed apoptotic bodies are initially located in Rab5-positive phagosomes, which mature to express the phagolysosome marker LAMP1. The phagocytosis of dead cells triggered an anti-inflammatory response with the production of TGF-β and IL-10 but not of the proinflammatory cytokines IL-12 and TNF-α. This study documents the previously unreported participation of the mannose receptor in the removal of apoptotic cells in the setting of tuberculosis (TB) infection. The results challenge the idea that apoptotic cell phagocytosis in TB has an immunogenic effect. Teresa Garcia-Aguilar, Patricia Espinosa-Cueto, Alejandro Magallanes-Puebla, and Raúl Mancilla Copyright © 2016 Teresa Garcia-Aguilar et al. All rights reserved. Immunoregulatory Role of HLA-G in Allergic Diseases Mon, 20 Jun 2016 06:30:49 +0000 Allergic diseases are sustained by a T-helper 2 polarization leading to interleukin-4 secretion, IgE-dependent inflammation, and mast cell and eosinophil activation. HLA-G molecules, both in membrane-bound and in soluble forms, play a central role in modulation of immune responses. Elevated levels of soluble HLA-G (sHLA-G) molecules are detected in serum of patients with allergic rhinitis to seasonal and perennial allergens and correlate with allergen-specific IgE levels, clinical severity, drug consumption, and response to allergen-specific immunotherapy. sHLA-G molecules are also found in airway epithelium of patients with allergic asthma and high levels of sHLA-G molecules are detectable in plasma and bronchoalveolar lavage of asthmatic patients correlating with allergen-specific IgE levels. Finally, HLA-G molecules are expressed by T cells, monocytes-macrophages, and Langerhans cells infiltrating the dermis of atopic dermatitis patients. Collectively, although at present it is difficult to completely define the role of HLA-G molecules in allergic diseases, it may be suggested that they are expressed and secreted by immune cells during the allergic reaction in an attempt to suppress allergic inflammation. Giuseppe Murdaca, Paola Contini, Simone Negrini, Giorgio Ciprandi, and Francesco Puppo Copyright © 2016 Giuseppe Murdaca et al. All rights reserved. Preexisting Antibodies to an F(ab′)2 Antibody Therapeutic and Novel Method for Immunogenicity Assessment Thu, 16 Jun 2016 12:12:51 +0000 Anti-therapeutic antibodies (ATAs) may impact drug exposure and activity and induce immune complex mediated toxicity; therefore the accurate measurement of ATA is important for the analysis of drug safety and efficacy. Preexisting ATAs to the hinge region of anti-Delta like ligand 4 (anti-DLL4) F(ab′)2, a potential antitumor therapeutic, were detected in cynomolgus monkey serum, which presented a challenge in developing assays for detecting treatment induced ATA. A total ATA assay was developed using a bridging ELISA that detected both anti-CDR and anti-framework ATA including anti-hinge reactivity. A competition assay that could detect 500 ng/mL of anti-CDR ATA in the presence of preexisting ATA was also developed to determine ATA specific to the anti-DLL4 F(ab′)2 CDR using anti-DLL4 F(ab′)2 and a control F(ab′)2. We used these assay methods in a cynomolgus monkey in vivo study to successfully evaluate total and anti-CDR ATA. The preexisting anti-hinge reactivity was also observed to a lesser extent in human serum, and a similar approach could be applied for specific immunogenicity assessment in clinical trials. Jane Ruppel, Ann Brady, Rebecca Elliott, Cecilia Leddy, Marco Palencia, Daniel Coleman, Jessica A. Couch, and Eric Wakshull Copyright © 2016 Jane Ruppel et al. All rights reserved. Identification of Novel Vaccine Candidates against Campylobacter through Reverse Vaccinology Thu, 16 Jun 2016 09:23:25 +0000 Campylobacteriosis is the most prevalent bacterial foodborne gastroenteritis affecting humans in the European Union. Human cases are mainly due to Campylobacter jejuni or Campylobacter coli, and contamination is associated with the handling and/or consumption of poultry meat. In fact, poultry constitutes the bacteria’s main reservoir. A promising way of decreasing the incidence of campylobacteriosis in humans would be to decrease avian colonization. Poultry vaccination is of potential for this purpose. However, despite many studies, there is currently no vaccine available on the market to reduce the intestinal Campylobacter load in chickens. It is essential to identify and characterize new vaccine antigens. This study applied the reverse vaccinology approach to detect new vaccine candidates. The main criteria used to select immune proteins were localization, antigenicity, and number of B-epitopes. Fourteen proteins were identified as potential vaccine antigens. In vitro and in vivo experiments now need to be performed to validate the immune and protective power of these newly identified antigens. Marine Meunier, Muriel Guyard-Nicodème, Edouard Hirchaud, Alberto Parra, Marianne Chemaly, and Daniel Dory Copyright © 2016 Marine Meunier et al. All rights reserved. Expression of Pentraxin 3 and Thrombospondin 1 in Gingival Crevicular Fluid during Wound Healing after Gingivectomy in Postorthodontic Patients Tue, 14 Jun 2016 12:19:35 +0000 Background. Wound healing is a tissue repair process after an injury, and two of its main components are inflammation and angiogenesis, in which course a cascade of mediators is involved. The aim of this research was to evaluate the involvement of Pentraxin 3 and Thrombospondin 1 in wound healing after periodontal surgery (gingivectomy) for gingival overgrowth during orthodontic treatment with or without magnification devices, by assessing their levels in GCF. Methods. From 19 patients with gingival overgrowth as a result of fixed orthodontic treatment, the overgrown gingiva was removed by gingivectomy, from one half of the mandibular arch without magnification and from the other under magnification. Pentraxin 3 and Thrombospondin 1 were determined from gingival crevicular fluid by ELISA tests. Results. Statistically significant differences () and correlations between levels of the two biomarkers were analyzed. Statistically significant differences were established between levels of the two biomarkers at different time points, with significant positive correlation at the point of 24 hours. Conclusions. Within the limitations of this study, the results seem to sustain the involvement of Pentraxin 3 and Thrombospondin 1 in the processes of inflammation and angiogenesis in wound healing of patients with postorthodontic gingivectomy. The dynamics of Pentraxin 3 and Thrombospondin 1 levels could suggest a reduced inflammation and a faster angiogenesis using microsurgery. Anne Marie Rauten, Isabela Silosi, Stefan Ioan Stratul, Liliana Foia, Adrian Camen, Vasilica Toma, Daniel Cioloca, Valeriu Surlin, Petra Surlin, and Maria Bogdan Copyright © 2016 Anne Marie Rauten et al. All rights reserved. Low-Dose Oxygen Enhances Macrophage-Derived Bacterial Clearance following Cigarette Smoke Exposure Tue, 14 Jun 2016 08:50:33 +0000 Background. Chronic obstructive pulmonary disease (COPD) is a common, smoking-related lung disease. Patients with COPD frequently suffer disease exacerbations induced by bacterial respiratory infections, suggestive of impaired innate immunity. Low-dose oxygen is a mainstay of therapy during COPD exacerbations; yet we understand little about whether oxygen can modulate the effects of cigarette smoke on lung immunity. Methods. Wild-type mice were exposed to cigarette smoke for 5 weeks, followed by intratracheal instillation of Pseudomonas aeruginosa (PAO1) and 21% or 35–40% oxygen. After two days, lungs were harvested for PAO1 CFUs, and bronchoalveolar fluid was sampled for inflammatory markers. In culture, macrophages were exposed to cigarette smoke and oxygen (40%) for 24 hours and then incubated with PAO1, followed by quantification of bacterial phagocytosis and inflammatory markers. Results. Mice exposed to 35–40% oxygen after cigarette smoke and PAO1 had improved survival and reduced lung CFUs and inflammation. Macrophages from these mice expressed less TNF- and more scavenger receptors. In culture, macrophages exposed to cigarette smoke and oxygen also demonstrated decreased TNF- secretion and enhanced phagocytosis of PAO1 bacteria. Conclusions. Our findings demonstrate a novel, protective role for low-dose oxygen following cigarette smoke and bacteria exposure that may be mediated by enhanced macrophage phagocytosis. William G. Bain, Ashutosh Tripathi, Pooja Mandke, Jonathan H. Gans, Franco R. D’Alessio, Venkataramana K. Sidhaye, and Neil R. Aggarwal Copyright © 2016 William G. Bain et al. All rights reserved. New Alternatives for Autoimmune Disease Treatments: Physicochemical and Clinical Comparability of Biosimilar Etanercept Sun, 12 Jun 2016 09:29:50 +0000 Etanercept is a recombinant fusion protein approved for the treatment of TNF-α mediated diseases such as rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, and ankylosing spondylitis. Herein, we present an evaluation of the physicochemical and biological properties of a biosimilar etanercept and its reference product followed by a clinical study in patients diagnosed with RA intended to demonstrate comparability of their immunomodulatory activity. Identity analyses showed a total correspondence of the primary and higher-order structure between the two products. In regard to intrinsic heterogeneity, both products showed to be highly heterogenous; however the biosimilar etanercept exhibited similar charge and glycan heterogeneity intervals compared to the reference product. Apoptosis inhibition assay also showed that, despite the high degree of heterogeneity exhibited by both products, no significant differences exist in their in vitro activity. Finally, the clinical assessment conducted in RA-diagnosed patients did not show significant differences in the evaluated pharmacodynamic markers of both products. Collectively, the results from the comparability exercise provide convincing evidence that the evaluated biosimilar etanercept can be considered an effective alternative for the treatment of RA. Mariana P. Miranda-Hernández, Carlos A. López-Morales, Francisco C. Perdomo-Abúndez, Rodolfo D. Salazar-Flores, Nancy D. Ramírez-Ibanez, Nestor O. Pérez, Aarón Molina-Pérez, Jorge Revilla-Beltri, Luis F. Flores-Ortiz, and Emilio Medina-Rivero Copyright © 2016 Mariana P. Miranda-Hernández et al. All rights reserved. Monocyte Differentiation towards Protumor Activity Does Not Correlate with M1 or M2 Phenotypes Wed, 08 Jun 2016 06:36:56 +0000 Macrophages facilitate breast cancer progression. Macrophages were initially classified as M1 or M2 based on their distinct metabolic programs and then expanded to include antitumoral (M1) and protumoral (M2) activities. However, it is still uncertain what markers define the pro- and antitumoral phenotypes and what conditions lead to their formation. In this study, monocytic cell lines and primary monocytes were subjected to commonly reported protocols of M1/M2 polarization and conditions known to engage monocytes into protumoral functions. The results showed that only IDO enzyme and CD86 M1 markers were upregulated correlating with M1 polarization. TNF-α, CCR7, IL-10, arginase I, CD36, and CD163 were expressed indistinguishably from M1 or M2 polarization. Similarly, protumoral engaging resulted in upregulation of both M1 and M2 markers, with conditioned media from the most aggressive breast cancer cell line promoting the greatest changes. In spite of the mixed phenotype, M1-polarized macrophages exhibited the highest expression/secretion of inflammatory mediators, many of which have previously been associated with breast cancer aggressiveness. These data argue that although the existence of protumoral macrophages is unquestionable, their associated phenotypes and the precise conditions driving their formation are still unclear, and those conditions may need both M1 and M2 stimuli. G. Karina Chimal-Ramírez, Nancy Adriana Espinoza-Sánchez, Luis Chávez-Sánchez, Lourdes Arriaga-Pizano, and Ezequiel M. Fuentes-Pananá Copyright © 2016 G. Karina Chimal-Ramírez et al. All rights reserved. Genetic Variation in the IL-6 and HLA-DQB1 Genes Is Associated with Spontaneous Clearance of Hepatitis C Virus Infection Thu, 02 Jun 2016 11:12:15 +0000 Background. Millions of people are infected with hepatitis C virus (HCV) worldwide and 30% spontaneously clear the infection. Reasons for HCV clearance without antiviral treatment are not well understood. Methods. Blood was collected for DNA analysis from patients with chronic HCV infection or evidence of spontaneous clearance. To overcome anticipated limitations of small sample size, primary analyses consisted of a candidate gene analysis of 12 preselected genes based on known association with host immunologic response to HCV infection. To further reduce the impact of multiple testing on power, a single likelihood ratio test was conducted for each gene using all associated SNPs assayed on the Illumina Quad 610/660W chip. Step-down permutation methods were used to adjust for multiple testing in all analyses. Results. Ninety-five and 62 patients with HCV chronic infection or spontaneous clearance, respectively, were included for analysis. HLA-DQB1 () and IL-6 () genes were significantly associated with spontaneous HCV clearance. IL-28B was not significantly associated with spontaneous clearance (). Conclusion. Our whole-gene analytic strategy identified a previously unreported association of IL-6 with spontaneous clearance of HCV infection. We also confirmed the finding that HLA-DQB1 is associated with spontaneous resolution of HCV infection. Paul Ravi Waldron, Ilana Belitskaya-Lévy, Aarthi Chary, Johann Won, Mark Winters, Alexander Monto, James Ryan, Laura C. Lazzeroni, and Mark Holodniy Copyright © 2016 Paul Ravi Waldron et al. All rights reserved. Immunotherapy in the Treatment of Human Solid Tumors: Basic and Translational Aspects Wed, 01 Jun 2016 09:01:18 +0000 D. Olive, B. Savoldo, F. Pastorino, and R. Castriconi Copyright © 2016 D. Olive et al. All rights reserved. A Fusion Protein Consisting of the Vaccine Adjuvant Monophosphoryl Lipid A and the Allergen Ovalbumin Boosts Allergen-Specific Th1, Th2, and Th17 Responses In Vitro Wed, 01 Jun 2016 07:04:12 +0000 Background. The detoxified TLR4-ligand Monophosphoryl Lipid A (MPLA) is the first approved TLR-agonist used as adjuvant in licensed vaccines but has not yet been explored as part of conjugated vaccines. Objective. To investigate the immune-modulating properties of a fusion protein consisting of MPLA and Ovalbumin (MPLA : Ova). Results. MPLA and Ova were chemically coupled by stable carbamate linkage. MPLA : Ova was highly pure without detectable product-related impurities by either noncoupled MPLA or Ova. Light scattering analysis revealed MPLA : Ova to be aggregated. Stimulation of mDC and mDC : DO11.10 CD4+ TC cocultures showed a stronger activation of both mDC and Ova-specific DO11.10 CD4+ TC by MPLA : Ova compared to the mixture of both components. MPLA : Ova induced both strong proinflammatory (IL-1β, IL-6, and TNF-α) and anti-inflammatory (IL-10) cytokine responses from mDCs while also boosting allergen-specific Th1, Th2, and Th17 cytokine secretion. Conclusion. Conjugation of MPLA and antigen enhanced the immune response compared to the mixture of both components. Due to the nonbiased boost of Ova-specific Th2 and Th17 responses while also inducing Th1 responses, this fusion protein may not be a suitable vaccine candidate for allergy treatment but may hold potential for the treatment of other diseases that require a strong stimulation of the host’s immune system (e.g., cancer). Stefan Schülke, Lothar Vogel, Ann-Christine Junker, Kay-Martin Hanschmann, Adam Flaczyk, Stefan Vieths, and Stephan Scheurer Copyright © 2016 Stefan Schülke et al. All rights reserved. Understanding the Supersensitive Anti-Drug Antibody Assay: Unexpected High Anti-Drug Antibody Incidence and Its Clinical Relevance Tue, 31 May 2016 11:32:57 +0000 Numbers of biotherapeutic products in development have increased over past decade. Despite providing significant benefits to patients with unmet needs, almost all protein-based biotherapeutics could induce unwanted immunogenicity, which result in a loss of efficacy and/or increase the risk of adverse reactions, such as infusion reactions, anaphylaxis, and even life-threatening response to endogenous proteins. Recognizing these possibilities, regulatory agencies request that immunogenicity be assessed as part of the approval process for biotherapeutics. Great efforts have been made to reduce drug immunogenicity through protein engineering. Accordingly the immunogenicity incidence has been reduced from around 80% in murine derived products to 0–10% in fully human products. However, recent improvements in immunogenicity assays have led to unexpectedly high immunogenicity rates, even in fully human products, leading to new challenges in assessing immunogenicity and its clinical relevance. These new immunogenicity assays are becoming supersensitive and able to detect more of anti-drug antibodies (ADA) than with earlier assays. This paper intends to review and discuss our understanding of the supersensitive ADA assay and the unexpected high ADA incidence and its potential clinical relevance. Sam Song, Lili Yang, William L. Trepicchio, and Timothy Wyant Copyright © 2016 Sam Song et al. All rights reserved. NKG2C+CD57+ Natural Killer Cell Expansion Parallels Cytomegalovirus-Specific CD8+ T Cell Evolution towards Senescence Sun, 29 May 2016 09:31:20 +0000 Objective. Measuring NKG2C+CD57+ natural killer (NK) cell expansion to investigate NK responses against human cytomegalovirus (HCMV) and assessing relationships with adaptive immunity against HCMV. Methods. Expansion of NKG2C+CD57+ NK was measured in peripheral blood mononuclear cells (PBMC) from groups distinguished by HCMV and human immunodeficiency virus (HIV) infection status. Anti-HCMV antibody levels against HCMV-infected MRC-5 cell lysate were assessed by ELISA and HCMV-specific CD8+ T cell responses characterized by intracellular flow cytometry following PBMC stimulation with immunodominant HCMV peptides. Results. Median NK, antibody, and CD8+ T cell responses against HCMV were significantly greater in the HCMV/HIV coinfected group than the group infected with CMV alone. The fraction of CMV-specific CD8+ T cells expressing CD28 correlated inversely with NKG2C+CD57+ NK expansion in HIV infection. Conclusion. Our data reveal no significant direct relationships between NK and adaptive immunity against HCMV. However, stronger NK and adaptive immune responses against HCMV and an inverse correlation between NKG2C+CD57+ NK expansion and proliferative reserve of HCMV-specific CD8+ T cells, as signified by CD28 expression, indicate parallel evolution of NK and T cell responses against HCMV in HIV infection. Similar aspects of chronic HCMV infection may drive both NK and CD8+ T cell memory inflation. John Heath, Nicholas Newhook, Emilie Comeau, Maureen Gallant, Neva Fudge, and Michael Grant Copyright © 2016 John Heath et al. All rights reserved. Function of G-Protein-Coupled Estrogen Receptor-1 in Reproductive System Tumors Sun, 29 May 2016 09:29:39 +0000 The G-protein-coupled estrogen receptor-1 (GPER-1), also known as GPR30, is a novel estrogen receptor mediating estrogen receptor signaling in multiple cell types. The progress of estrogen-related cancer is promoted by GPER-1 activation through mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinase (PI3K), and phospholipase C (PLC) signaling pathways. However, this promoting effect of GPER-1 is nonclassic estrogen receptor (ER) dependent manner. In addition, clinical evidences revealed that GPER-1 is associated with estrogen resistance in estrogen-related cancer patients. These give a hint that GPER-1 may be a novel therapeutic target for the estrogen-related cancers. However, preclinical studies also found that GPER-1 activation of its special agonist G-1 inhibits cancer cell proliferation. This review aims to summarize the characteristics and complex functions of GPER-1 in cancers. Hongyan Qian, Jingxiu Xuan, Yuan Liu, and Guixiu Shi Copyright © 2016 Hongyan Qian et al. All rights reserved. Diagnostic Utility of Total IgE in Foods, Inhalant, and Multiple Allergies in Saudi Arabia Wed, 25 May 2016 11:18:12 +0000 Objective. To assess the diagnostic significance of total IgE in foods, inhalant, and multiple allergies. Methods. Retrospective review of the laboratory records of patients who presented with clinical suspicion of food or inhalant allergy between January 2013 and December 2014. Total IgE level was defined as positive for a value >195 kU/L; and diagnosis was confirmed by the detection of specific IgE (golden standard) for at least one food or inhalant allergen and at least two allergens in multiple allergies. Results. A total of 1893 (male ratio = 0.68, mean age = 39.0 ± 19.2 years) patients were included. Total IgE had comparable sensitivity (55.8% versus 59.6%) and specificity (83.9% versus 84.4%) in food versus inhalant allergy, respectively, but a superior PPV in inhalant allergy (79.1% versus 54.4%). ROC curve analysis showed a better diagnostic value in inhalant allergies (AUC = 0.817 (95% CI = 0.796–0.837) versus 0.770 (95% CI = 0.707–0.833)). In multiple allergies, total IgE had a relatively good sensitivity (78.6%), while negative IgE testing (<195 kU/L) predicted the absence of multiple allergies with 91.5% certitude. Conclusion. Total IgE assay is not efficient as a diagnostic test for foods, inhalant, or multiple allergies. The best strategy should refer to specific IgE testing guided by a comprehensive atopic history. Jamil A. Al-Mughales Copyright © 2016 Jamil A. Al-Mughales. All rights reserved. Prolactin Rescues Immature B-Cells from Apoptosis Induced by B-Cell Receptor Cross-Linking Tue, 24 May 2016 09:02:20 +0000 Prolactin has an immunomodulatory effect and has been associated with B-cell-triggered autoimmune diseases, such as systemic lupus erythematosus (SLE). In mice that develop SLE, the PRL receptor is expressed in early bone marrow B-cells, and increased levels of PRL hasten disease manifestations, which are correlated with a reduction in the absolute number of immature B-cells. The aim of this work was to determine the effect of PRL in an in vitro system of B-cell tolerance using WEHI-231 cells and immature B-cells from lupus prone MRL/lpr mice. WEHI-231 cells express the long isoform of the PRL receptor, and PRL rescued the cells from cell death by decreasing the apoptosis induced by the cross-linking of the B-cell antigen receptor (BCR) as measured by Annexin V and active caspase-3. This decrease in apoptosis may have been due to the PRL and receptor interaction, which increased the relative expression of antiapoptotic Bcl-xL and decreased the relative expression of proapoptotic Bad. In immature B-cells from MRL/lpr mice, PRL increased the viability and decreased the apoptosis induced by the cross-linking of BCR, which may favor the maturation of self-reactive B-cells and contribute to the onset of disease. Rocio Flores-Fernández, Francisco Blanco-Favela, Ezequiel M. Fuentes-Pananá, Luis Chávez-Sánchez, Patricia Gorocica-Rosete, Alberto Pizaña-Venegas, and Adriana Karina Chávez-Rueda Copyright © 2016 Rocio Flores-Fernández et al. All rights reserved. Effector, Memory, and Dysfunctional CD8+ T Cell Fates in the Antitumor Immune Response Sun, 22 May 2016 09:26:18 +0000 The adaptive immune system plays a pivotal role in the host’s ability to mount an effective, antigen-specific immune response against tumors. CD8+ tumor-infiltrating lymphocytes (TILs) mediate tumor rejection through recognition of tumor antigens and direct killing of transformed cells. In growing tumors, TILs are often functionally impaired as a result of interaction with, or signals from, transformed cells and the tumor microenvironment. These interactions and signals can lead to transcriptional, functional, and phenotypic changes in TILs that diminish the host’s ability to eradicate the tumor. In addition to effector and memory CD8+ T cells, populations described as exhausted, anergic, senescent, and regulatory CD8+ T cells have been observed in clinical and basic studies of antitumor immune responses. In the context of antitumor immunity, these CD8+ T cell subsets remain poorly characterized in terms of fate-specific biomarkers and transcription factor profiles. Here we discuss the current characterization of CD8+ T cell fates in antitumor immune responses and discuss recent insights into how signals in the tumor microenvironment influence TIL transcriptional networks to promote CD8+ T cell dysfunction. John Reiser and Arnob Banerjee Copyright © 2016 John Reiser and Arnob Banerjee. All rights reserved. Regulatory T Cells: Molecular Actions on Effector Cells in Immune Regulation Thu, 19 May 2016 14:08:52 +0000 T regulatory cells play a key role in the control of the immune response, both in health and during illness. While the mechanisms through which T regulatory cells exert their function have been extensively described, their molecular effects on effector cells have received little attention. Thus, this revision is aimed at summarizing our current knowledge on those regulation mechanisms on the target cells from a molecular perspective. Asiel Arce-Sillas, Diana Denisse Álvarez-Luquín, Beatriz Tamaya-Domínguez, Sandra Gomez-Fuentes, Abel Trejo-García, Marlene Melo-Salas, Graciela Cárdenas, Juan Rodríguez-Ramírez, and Laura Adalid-Peralta Copyright © 2016 Asiel Arce-Sillas et al. All rights reserved. CAR T Cell Therapy: A Game Changer in Cancer Treatment Thu, 19 May 2016 09:12:37 +0000 The development of novel targeted therapies with acceptable safety profiles is critical to successful cancer outcomes with better survival rates. Immunotherapy offers promising opportunities with the potential to induce sustained remissions in patients with refractory disease. Recent dramatic clinical responses in trials with gene modified T cells expressing chimeric antigen receptors (CARs) in B-cell malignancies have generated great enthusiasm. This therapy might pave the way for a potential paradigm shift in the way we treat refractory or relapsed cancers. CARs are genetically engineered receptors that combine the specific binding domains from a tumor targeting antibody with T cell signaling domains to allow specifically targeted antibody redirected T cell activation. Despite current successes in hematological cancers, we are only in the beginning of exploring the powerful potential of CAR redirected T cells in the control and elimination of resistant, metastatic, or recurrent nonhematological cancers. This review discusses the application of the CAR T cell therapy, its challenges, and strategies for successful clinical and commercial translation. Hilde Almåsbak, Tanja Aarvak, and Mohan C. Vemuri Copyright © 2016 Hilde Almåsbak et al. All rights reserved. Adjuvant Autologous Melanoma Vaccine for Macroscopic Stage III Disease: Survival, Biomarkers, and Improved Response to CTLA-4 Blockade Wed, 18 May 2016 14:30:26 +0000 Background. There is not yet an agreed adjuvant treatment for melanoma patients with American Joint Committee on Cancer stages III B and C. We report administration of an autologous melanoma vaccine to prevent disease recurrence. Patients and Methods. 126 patients received eight doses of irradiated autologous melanoma cells conjugated to dinitrophenyl and mixed with BCG. Delayed type hypersensitivity (DTH) response to unmodified melanoma cells was determined on the vaccine days 5 and 8. Gene expression analysis was performed on 35 tumors from patients with good or poor survival. Results. Median overall survival was 88 months with a 5-year survival of 54%. Patients attaining a strong DTH response had a significantly better () 5-year overall survival of 75% compared with 44% in patients without a strong response. Gene expression array linked a 50-gene signature to prognosis, including a cluster of four cancer testis antigens: CTAG2 (NY-ESO-2), MAGEA1, SSX1, and SSX4. Thirty-five patients, who received an autologous vaccine, followed by ipilimumab for progressive disease, had a significantly improved 3-year survival of 46% compared with 19% in nonvaccinated patients treated with ipilimumab alone (). Conclusion. Improved survival in patients attaining a strong DTH and increased response rate with subsequent ipilimumab suggests that the autologous vaccine confers protective immunity. Michal Lotem, Sharon Merims, Stephen Frank, Tamar Hamburger, Aviram Nissan, Luna Kadouri, Jonathan Cohen, Ravid Straussman, Galit Eisenberg, Shoshana Frankenburg, Einat Carmon, Bilal Alaiyan, Shlomo Shneibaum, Zeynep Ozge Ayyildiz, Murat Isbilen, Kerem Mert Senses, Ilan Ron, Hanna Steinberg, Yoav Smith, Eitan Shiloni, Ali Osmay Gure, and Tamar Peretz Copyright © 2016 Michal Lotem et al. All rights reserved. The Emerging Role of HMGB1 in Neuropathic Pain: A Potential Therapeutic Target for Neuroinflammation Wed, 18 May 2016 11:52:20 +0000 Neuropathic pain (NPP) is intolerable, persistent, and specific type of long-term pain. It is considered to be a direct consequence of pathological changes affecting the somatosensory system and can be debilitating for affected patients. Despite recent progress and growing interest in understanding the pathogenesis of the disease, NPP still presents a major diagnostic and therapeutic challenge. High mobility group box 1 (HMGB1) mediates inflammatory and immune reactions in nervous system and emerging evidence reveals that HMGB1 plays an essential role in neuroinflammation through receptors such as Toll-like receptors (TLR), receptor for advanced glycation end products (RAGE), C-X-X motif chemokines receptor 4 (CXCR4), and N-methyl-D-aspartate (NMDA) receptor. In this review, we present evidence from studies that address the role of HMGB1 in NPP. First, we review studies aimed at determining the role of HMGB1 in NPP and discuss the possible mechanisms underlying HMGB1-mediated NPP progression where receptors for HMGB1 are involved. Then we review studies that address HMGB1 as a potential therapeutic target for NPP. Wenbin Wan, Lan Cao, Ramin Khanabdali, Bill Kalionis, Xiantao Tai, and Shijin Xia Copyright © 2016 Wenbin Wan et al. All rights reserved. Systemic Administration of Proteoglycan Protects BALB/c Retired Breeder Mice from Experimental Arthritis Wed, 18 May 2016 10:52:56 +0000 This study was undertaken to evaluate the prophylactic potential of proteoglycan (PG) administration in experimental arthritis. Female BALB/c retired breeder mice received two (2xPG50 and 2xPG100 groups) or three (3xPG50 group) intraperitoneal doses of bovine PG (50 μg or 100 μg) every three days. A week later the animals were submitted to arthritis induction by immunization with three i.p. doses of bovine PG associated with dimethyldioctadecylammonium bromide adjuvant at intervals of 21 days. Disease severity was daily assessed after the third dose by score evaluation. The 3xPG50 group showed significant reduction in prevalence and clinical scores. This protective effect was associated with lower production of IFN-γ and IL-17 and increased production of IL-5 and IL-10 by spleen cells restimulated in vitro with PG. Even though previous PG administration restrained dendritic cells maturation this procedure did not alter the frequency of regulatory Foxp3+ T cells. Lower TNF-α and IL-6 levels and higher expression of ROR-γ and GATA-3 were detected in the paws of protected animals. A delayed-type hypersensitivity reaction confirmed specific tolerance induction. Taken together, these results indicate that previous PG inoculation determines a specific tolerogenic effect that is able to decrease severity of subsequently induced arthritis. Larissa Lumi Watanabe Ishikawa, Priscila Maria Colavite, Thais Fernanda de Campos Fraga-Silva, Luiza Ayumi Nishiyama Mimura, Thais Graziela Donegá França, Sofia Fernanda Gonçalves Zorzella-Pezavento, Fernanda Chiuso-Minicucci, Larissa Doddi Marcolino, Camila Marques, Maura Rosane Valerio Ikoma, and Alexandrina Sartori Copyright © 2016 Larissa Lumi Watanabe Ishikawa et al. All rights reserved. The Microbiota Determines Susceptibility to Experimental Autoimmune Uveoretinitis Tue, 17 May 2016 11:23:22 +0000 The microbiota is a crucial modulator of the immune system. Here, we evaluated how its absence or reduction modifies the inflammatory response in the murine model of experimental autoimmune uveoretinitis (EAU). We induced EAU in germ-free (GF) or conventionally housed (CV) mice and in CV mice treated with a combination of broad-spectrum antibiotics either from the day of EAU induction or from one week prior to induction of disease. The severity of the inflammation was assessed by fundus biomicroscopy or by histology, including immunohistology. The immunophenotyping of T cells in local and distant lymph nodes was performed by flow cytometry. We found that GF mice and mice where the microbiota was reduced one week before EAU induction were protected from severe autoimmune inflammation. GF mice had lower numbers of infiltrating macrophages and significantly less T cell infiltration in the retina than CV mice with EAU. GF mice also had reduced numbers of IFN-γ and IL-17-producing T cells and increased numbers of regulatory T cells in the eye-draining lymph nodes. These data suggest that the presence of microbiota during autoantigen recognition regulates the inflammatory response by influencing the adaptive immune response. Jarmila Heissigerova, Petra Seidler Stangova, Aneta Klimova, Petra Svozilkova, Tomas Hrncir, Renata Stepankova, Miloslav Kverka, Helena Tlaskalova-Hogenova, and John V. Forrester Copyright © 2016 Jarmila Heissigerova et al. All rights reserved.