Llama-derived Single Variable Domains (Nanobodies) Directed against Chemokine Receptor CXCR7 Reduce Head and Neck Cancer Cell Growth in Vivo

The chemokine receptor CXCR7, belonging to the membrane-bound G protein-coupled receptor superfamily, is expressed in several tumor types. Inhibition of CXCR7 with either small molecules or small interference (si)RNA has shown promising therapeutic benefits in several tumor models. With the increased interest and effectiveness of biologicals inhibiting membrane-bound receptors we made use of the “Nanobody platform” to target CXCR7. Previously we showed that Nanobodies, i.e. immunoglobulin single variable domains derived from naturally occurring heavy chain-only camelids antibodies, represent new biological tools to efficiently tackle difficult drug targets such as G protein-coupled receptors. In this study we developed and characterized highly selective and potent Nanobodies against CXCR7. Interestingly, the CXCR7-targeting Nanobodies displayed antagonistic properties in contrast with previously reported CXCR7-targeting agents. Several high affinity CXCR7-specific Nanobodies potently inhibited CXCL12-induced β-arrestin2 recruitment in vitro. A wide variety of tumor biopsies was profiled, showing for the first time high expression of CXCR7 in head and neck cancer. Using a patient-derived CXCR7-expressing head and neck cancer xenograft model in nude mice, tumor growth was inhibited by CXCR7-targeting Nanobody therapy. Mechanistically, CXCR7-targeting Nanobodies did not inhibit cell cycle progression but instead reduced secretion of the angiogenic chemokine CXCL1 from head and neck cancer cells in vitro, thus acting here as inverse agonists, and subsequent angiogenesis in vivo. Hence, with this novel class of CXCR7 inhibitors, we further substantiate the therapeutic relevance of targeting CXCR7 in head and neck cancer.     

A Combined “Omics” Approach Identifies N-Myc Interactor as a Novel Cytokine-induced Regulator of IRE1α Protein and c-Jun N-terminal Kinase in Pancreatic Beta Cells

Type 1 diabetes is an autoimmune disease with a strong inflammatory component. The cytokines interleukin-1β and interferon-γ contribute to beta cell apoptosis in type 1 diabetes. These cytokines induce endoplasmic reticulum stress and the unfolded protein response (UPR), contributing to the loss of beta cells. IRE1α, one of the UPR mediators, triggers insulin degradation and inflammation in beta cells and is critical for the transition from “physiological” to “pathological” UPR. The mechanisms regulating inositol-requiring protein 1α (IRE1α) activation and its signaling for beta cell “adaptation,” “stress response,” or “apoptosis” remain to be clarified. To address these questions, we combined mammalian protein-protein interaction trap-based IRE1α interactome and functional genomic analysis of human and rodent beta cells exposed to pro-inflammatory cytokines to identify novel cytokine-induced regulators of IRE1α. Based on this approach, we identified N-Myc interactor (NMI) as an IRE1α-interacting/modulator protein in rodent and human pancreatic beta cells. An increased expression of NMI was detected in islets from nonobese diabetic mice with insulitis and in rodent or human beta cells exposed in vitro to the pro-inflammatory cytokines interleukin-1β and interferon-γ. Detailed mechanistic studies demonstrated that NMI negatively modulates IRE1α-dependent activation of JNK and apoptosis in rodent and human pancreatic beta cells. In conclusion, by using a combined omics approach, we identified NMI induction as a novel negative feedback mechanism that decreases IRE1α-dependent activation of JNK and apoptosis in cytokine-exposed beta cells.     

Generation of Human Antigen-Specific Monoclonal IgM Antibodies Using Vaccinated “Human Immune System” Mice

Background

Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the ‘humanization’ of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique.

Methodology/Principal Findings

After transplantation with CD34⁺CD38⁻ human hematopoietic progenitor cells, BALB/c Rag2^−/−IL-2Rγc^−/− mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. “Human Immune System” mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19⁺CD27⁺ B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively.

Conclusion/Significance

This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens.     

XoxF-Type Methanol Dehydrogenase from the Anaerobic Methanotroph “Candidatus Methylomirabilis oxyfera”

“Candidatus Methylomirabilis oxyfera” is a newly discovered anaerobic methanotroph that, surprisingly, oxidizes methane through an aerobic methane oxidation pathway. The second step in this aerobic pathway is the oxidation of methanol. In Gram-negative bacteria, the reaction is catalyzed by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH). The genome of “Ca. Methylomirabilis oxyfera” putatively encodes three different MDHs that are localized in one large gene cluster: one so-called MxaFI-type MDH and two XoxF-type MDHs (XoxF1 and XoxF2). MxaFI MDHs represent the canonical enzymes, which are composed of two PQQ-containing large (α) subunits (MxaF) and two small (β) subunits (MxaI). XoxF MDHs are novel, ecologically widespread, but poorly investigated types of MDHs that can be phylogenetically divided into at least five different clades. The XoxF MDHs described thus far are homodimeric proteins containing a large subunit only. Here, we purified a heterotetrameric MDH from “Ca. Methylomirabilis oxyfera” that consisted of two XoxF and two MxaI subunits. The enzyme was localized in the periplasm of “Ca. Methylomirabilis oxyfera” cells and catalyzed methanol oxidation with appreciable specific activity and affinity (V_max of 10 μmol min⁻¹ mg⁻¹ protein, K_m of 17 μM). PQQ was present as the prosthetic group, which has to be taken up from the environment since the known gene inventory required for the synthesis of this cofactor is lacking. The MDH from “Ca. Methylomirabilis oxyfera” is the first representative of type 1 XoxF proteins to be described.     

α-Actinin-4-Mediated FSGS: An Inherited Kidney Disease Caused by an Aggregated and Rapidly Degraded Cytoskeletal Protein

Focal segmental glomerulosclerosis (FSGS) is a common pattern of renal injury, seen as both a primary disorder and as a consequence of underlying insults such as diabetes, HIV infection, and hypertension. Point mutations in theα-actinin-4 gene ACTN4 cause an autosomal dominant form of human FSGS. We characterized the biological effect of these mutations by biochemical assays, cell-based studies, and the development of a new mouse model. We found that a fraction of the mutant protein forms large aggregates with a high sedimentation coefficient. Localization of mutant α-actinin-4 in transfected and injected cells, as well as in situ glomeruli, showed aggregates of the mutant protein. Video microscopy showed the mutant α-actinin-4 to be markedly less dynamic than the wild-type protein. We developed a “knockin” mouse model by replacing Actn4 with a copy of the gene bearing an FSGS-associated point mutation. We used cells from these mice to show increased degradation of mutant α-actinin-4, mediated, at least in part, by the ubiquitin–proteasome pathway. We correlate these findings with studies of α-actinin-4 expression in human samples. “Knockin” mice with a disease-associated Actn4 mutation develop a phenotype similar to that observed in humans. Comparison of the phenotype in wild-type, heterozygous, and homozygous Actn4 “knockin” and “knockout” mice, together with our in vitro data, suggests that the phenotypes in mice and humans involve both gain-of-function and loss-of-function mechanisms.     

Diversity of “Candidatus Liberibacter asiaticus,” Based on the omp Gene Sequence

Huanglongbing (yellow dragon disease) is a destructive disease of citrus. The etiological agent is a noncultured, phloem-restricted alpha-proteobacterium, “Candidatus Liberibacter africanus” in Africa and “Candidatus Liberibacter asiaticus” in Asia. In this study, we used an omp-based PCR-restriction fragment length polymorphism (RFLP) approach to analyze the genetic variability of “Ca. Liberibacter asiaticus” isolates. By using five different enzymes, each the 10 isolates tested could be associated with a specific combination of restriction profiles. The results indicate that the species “Ca. Liberibacter asiaticus,” even within a given region, may comprise several different variants. Thus, omp-based PCR-RFLP analysis is a simple method for detecting and differentiating “Ca. Liberibacter asiaticus” isolates.     

β1-Integrin Cytoplasmic Subdomains Involved in Dominant Negative Function

The β1-integrin cytoplasmic domain consists of a membrane proximal subdomain common to the four known isoforms (“common” region) and a distal subdomain specific for each isoform (“variable” region). To investigate in detail the role of these subdomains in integrin-dependent cellular functions, we used β1A and β1B isoforms as well as four mutants lacking the entire cytoplasmic domain (β1TR), the variable region (β1COM), or the common region (β1ΔCOM-B and β1ΔCOM-A). By expressing these constructs in Chinese hamster ovary and β1 integrin-deficient GD25 cells (Wennerberg et al., J Cell Biol 132, 227–238, 1996), we show that β1B, β1COM, β1ΔCOM-B, and β1ΔCOM-A molecules are unable to support efficient cell adhesion to matrix proteins. On exposure to Mn⁺⁺ ions, however, β1B, but none of the mutants, can mediate cell adhesion, indicating specific functional properties of this isoform. Analysis of adhesive functions of transfected cells shows that β1B interferes in a dominant negative manner with β1A and β3/β5 integrins in cell spreading, focal adhesion formation, focal adhesion kinase tyrosine phosphorylation, and fibronectin matrix assembly. None of the β1 mutants tested shows this property, indicating that the dominant negative effect depends on the specific combination of common and B subdomains, rather than from the absence of the A subdomain in the β1B isoform.     

B Cell Receptor Signaling-Based Index as a Biomarker for the Loss of Peripheral Immune Tolerance in Autoreactive B Cells in Rheumatoid Arthritis

This study examines the loss of peripherally induced B cell immune tolerance in Rheumatoid arthritis (RA) and establishes a novel signaling-based measure of activation in a subset of autoreactive B cells - the Induced tolerance status index (ITSI). Naturally occurring naïve autoreactive B cells can escape the “classical” tolerogenic mechanisms of clonal deletion and receptor editing, but remain peripherally tolerized through B cell receptor (BCR) signaling inhibition (postdevelopmental “receptor tuning” or anergy). ITSI is a statistical index that numerically determines the level of homology between activation patterns of BCR signaling intermediaries in B cells that are either tolerized or activated by auto antigen exposure, and thus quantifies the level of peripheral immune tolerance. The index is based on the logistic regression analysis of phosphorylation levels in a panel of BCR signaling proteins. Our results demonstrate a new approach to identifying autoreactive B cells based on their BCR signaling features.     

Superparamagnetic Iron Oxide Nanoparticles Labeling of Bone Marrow Stromal (Mesenchymal) Cells Does Not Affect Their “Stemness”

Superparamagnetic iron oxide nanoparticles (SPION) are increasingly used to label human bone marrow stromal cells (BMSCs, also called “mesenchymal stem cells”) to monitor their fate by in vivo MRI, and by histology after Prussian blue (PB) staining. SPION-labeling appears to be safe as assessed by in vitro differentiation of BMSCs, however, we chose to resolve the question of the effect of labeling on maintaining the “stemness” of cells within the BMSC population in vivo. Assays performed include colony forming efficiency, CD146 expression, gene expression profiling, and the “gold standard” of evaluating bone and myelosupportive stroma formation in vivo in immuncompromised recipients. SPION-labeling did not alter these assays. Comparable abundant bone with adjoining host hematopoietic cells were seen in cohorts of mice that were implanted with SPION-labeled or unlabeled BMSCs. PB+ adipocytes were noted, demonstrating their donor origin, as well as PB+ pericytes, indicative of self-renewal of the stem cell in the BMSC population. This study confirms that SPION labeling does not alter the differentiation potential of the subset of stem cells within BMSCs.     

Induction of Humoral and Cell-Mediated Immune Responses by Hepatitis B Virus Epitope Displayed on the Virus-Like Particles of Prawn Nodavirus

Hepatitis B virus (HBV) is a deadly pathogen that has killed countless people worldwide. Saccharomyces cerevisiae-derived HBV vaccines based upon hepatitis B surface antigen (HBsAg) is highly effective. However, the emergence of vaccine escape mutants due to mutations on the HBsAg and polymerase genes has produced a continuous need for the development of new HBV vaccines. In this study, the “a” determinant within HBsAg was displayed on the recombinant capsid protein of Macrobrachium rosenbergii nodavirus (MrNV), which can be purified easily in a single step through immobilized metal affinity chromatography (IMAC). The purified protein self-assembled into virus-like particles (VLPs) when observed under a transmission electron microscope (TEM). Immunization of BALB/c mice with this chimeric protein induced specific antibodies against the “a” determinant. In addition, it induced significantly more natural killer and cytotoxic T cells, as well as an increase in interferon gamma (IFN-γ) secretion, which are vital for virus clearance. Collectively, these findings demonstrated that the MrNV capsid protein is a potential carrier for the HBV “a” determinant, which can be further extended to display other foreign epitopes. This paper is the first to report the application of MrNV VLPs as a novel platform to display foreign epitopes.     

Interleukin-15 Dendritic Cells Harness NK Cell Cytotoxic Effector Function in a Contact- and IL-15-Dependent Manner

The contribution of natural killer (NK) cells to the treatment efficacy of dendritic cell (DC)-based cancer vaccines is being increasingly recognized. Much current efforts to optimize this form of immunotherapy are therefore geared towards harnessing the NK cell-stimulatory ability of DCs. In this study, we investigated whether generation of human monocyte-derived DCs with interleukin (IL)-15 followed by activation with a Toll-like receptor stimulus endows these DCs, commonly referred to as “IL-15 DCs”, with the capacity to stimulate NK cells. In a head-to-head comparison with “IL-4 DCs” used routinely for clinical studies, IL-15 DCs were found to induce a more activated, cytotoxic effector phenotype in NK cells, in particular in the CD56^bright NK cell subset. With the exception of GM-CSF, no significant enhancement of cytokine/chemokine secretion was observed following co-culture of NK cells with IL-15 DCs. IL-15 DCs, but not IL-4 DCs, promoted NK cell tumoricidal activity towards both NK-sensitive and NK-resistant targets. This effect was found to require cell-to-cell contact and to be mediated by DC surface-bound IL-15. This study shows that DCs can express a membrane-bound form of IL-15 through which they enhance NK cell cytotoxic function. The observed lack of membrane-bound IL-15 on “gold-standard” IL-4 DCs and their consequent inability to effectively promote NK cell cytotoxicity may have important implications for the future design of DC-based cancer vaccine studies.     

Diverse Phage-Encoded Toxins in a Protective Insect Endosymbiont

The lysogenic bacteriophage APSE infects “Candidatus Hamiltonella defensa,” a facultative endosymbiont of aphids and other sap-feeding insects. This endosymbiont has established a beneficial association with aphids, increasing survivorship following attack by parasitoid wasps. Although APSE and “Ca. Hamiltonella defensa” are effectively maternally transmitted between aphid generations, they can also be horizontally transferred among insect hosts, which results in genetically distinct “Ca. Hamiltonella defensa” strains infecting the same aphid species and sporadic distributions of both APSE and “Ca. Hamiltonella defensa” among hosts. Aphids infected only with “Ca. Hamiltonella defensa” have significantly less protection than those infected with both “Ca. Hamiltonella defensa” and APSE. This protection has been proposed to be connected to eukaryote-targeted toxins previously discovered in the genomes of two characterized APSE strains. In this study, we have sequenced partial genomes from seven additional APSE strains to address the evolution and extent of toxin variation in this phage. The APSE lysis region has been a hot spot for nonhomologous recombination of novel virulence cassettes. We identified four new toxins from three protein families, Shiga-like toxin, cytolethal distending toxin, and YD-repeat toxins. These recombination events have also resulted in reassortment of the downstream lysozyme and holin genes. Analysis of the conserved APSE genes flanking the variable toxin cassettes reveals a close phylogenetic association with phage sequences from two other facultative endosymbionts of insects. Thus, phage may act as a conduit for ongoing gene exchange among heritable endosymbionts.     

Distinct Licensing of IL-18 and IL-1β Secretion in Response to NLRP3 Inflammasome Activation

Inflammasome activation permits processing of interleukins (IL)-1β and 18 and elicits cell death (pyroptosis). Whether these responses are independently licensed or are “hard-wired” consequences of caspase-1 (casp1) activity has not been clear. Here, we show that that each of these responses is independently regulated following activation of NLRP3 inflammasomes by a “non-canonical” stimulus, the secreted Listeria monocytogenes (Lm) p60 protein. Primed murine dendritic cells (DCs) responded to p60 stimulation with reactive oxygen species (ROS) production and secretion of IL-1β and IL-18 but not pyroptosis. Inhibitors of ROS production inhibited secretion of IL-1β, but did not impair IL-18 secretion. Furthermore, DCs from caspase-11 (casp11)-deficient 129S6 mice failed to secrete IL-1β in response to p60 but were fully responsive for IL-18 secretion. These findings reveal that there are distinct licensing requirements for processing of IL-18 versus IL-1β by NLRP3 inflammasomes.     

Ecdysteroids: A novel class of anabolic agents?

Increasing numbers of dietary supplements with ecdysteroids are marketed as “natural anabolic agents”. Results of recent studies suggested that their anabolic effect is mediated by estrogen receptor (ER) binding. Within this study the anabolic potency of ecdysterone was compared to well characterized anabolic substances. Effects on the fiber sizes of the soleus muscle in rats as well the diameter of C2C12 derived myotubes were used as biological readouts. Ecdysterone exhibited a strong hypertrophic effect on the fiber size of rat soleus muscle that was found even stronger compared to the test compounds metandienone (dianabol), estradienedione (trenbolox), and SARM S 1, all administered in the same dose (5 mg/kg body weight, for 21 days). In C2C12 myotubes ecdysterone (1 µM) induced a significant increase of the diameter comparable to dihydrotestosterone (1 µM) and IGF 1 (1.3 nM). Molecular docking experiments supported the ERβ mediated action of ecdysterone. To clarify its status in sports, ecdysterone should be considered to be included in the class “S1.2 Other Anabolic Agents” of the list of prohibited substances of the World Anti-Doping Agency.     

Modulation of Recombinant Antigenic Constructs Containing Multi-Epitopes towards Effective Reduction of Atherosclerotic Lesion in B6;129S-Ldlrtm1HerApobtm2Sgy/J Mice

Atherosclerosis is increasingly recognized as a complex chronic inflammatory disease. Many more studies have extended vaccination against atherosclerosis by using epitopes from self-antigens or beyond and demonstrated that vaccination with antigens or derivatives could reduce the extent of the lesions in atherosclerosis-prone mice. Our previous study has demonstrated that construct AHHC [ApoB100^688-707 + hHSP60^303-312 + hHSP60^153-163 + Cpn derived peptide (C)] significantly reduced atherosclerotic lesion. The aim of this study was to investigate whether AHHC can be modulated towards increased lesion reduction in mice by creating two other derivatives with a sequential epitope-substitution named RHHC in which A was replaced by an “R” (C5aR^1-31) and RPHC with a further “H” (hHSP60^303-312) conversion into “P” (protease-activated receptor-1^42-55) in mice. Antigenic epitopes were incorporated into a dendroaspin scaffold. Immunization of B6;129S-Ldlr^tm1HerApob^tm2Sgy/J mice with three constructs elicited production of high levels of antibodies against each epitope (apart from hHSP60^153-163 and P which induced a low antibody response). Histological analyses demonstrated that the mice immunized with either RPHC or RHHC showed significant reductions in the size of atherosclerostic lesions compared to those with AHHC (69.5±1.1% versus 55.7±3.4%, P<0.01 or 65.6±1.3% versus 55.7±3.4%, P<0.01). Reduction of plaque size in the aortic sinus and descending aorta correlated with alterations in cellular immune responses when compared with controls. We conclude that a recombinant construct RPHC may provide new antigenic and structural features which are favorable for significant reduction in atherosclerotic lesion formation. This approach offers a novel strategy for developing anti-atherosclerotic agents.     

Thermoanaerobacter thermohydrosulfuricus WC1 Shows Protein Complement Stability during Fermentation of Key Lignocellulose-Derived Substrates

Thermoanaerobacter spp. have long been considered suitable Clostridium thermocellum coculture partners for improving lignocellulosic biofuel production through consolidated bioprocessing. However, studies using “omic”-based profiling to better understand carbon utilization and biofuel producing pathways have been limited to only a few strains thus far. To better characterize carbon and electron flux pathways in the recently isolated, xylanolytic strain, Thermoanaerobacter thermohydrosulfuricus WC1, label-free quantitative proteomic analyses were combined with metabolic profiling. SWATH-MS proteomic analysis quantified 832 proteins in each of six proteomes isolated from mid-exponential-phase cells grown on xylose, cellobiose, or a mixture of both. Despite encoding genes consistent with a carbon catabolite repression network observed in other Gram-positive organisms, simultaneous consumption of both substrates was observed. Lactate was the major end product of fermentation under all conditions despite the high expression of gene products involved with ethanol and/or acetate synthesis, suggesting that carbon flux in this strain may be controlled via metabolite-based (allosteric) regulation or is constrained by metabolic bottlenecks. Cross-species “omic” comparative analyses confirmed similar expression patterns for end-product-forming gene products across diverse Thermoanaerobacter spp. It also identified differences in cofactor metabolism, which potentially contribute to differences in end-product distribution patterns between the strains analyzed. The analyses presented here improve our understanding of T. thermohydrosulfuricus WC1 metabolism and identify important physiological limitations to be addressed in its development as a biotechnologically relevant strain in ethanologenic designer cocultures through consolidated bioprocessing.