OxLDL/ß2GPI/anti­ß2GPI Ab complex induces inflammatory activation via the TLR4/NF­κB pathway in HUVECs.

Patients with antiphospholipid syndrome have been identified to have higher incidence rates of atherosclerosis (AS) due to the elevated levels of anti­ß2­glycoprotein I (ß2GPI) antibody (Ab). Our previous studies revealed that the anti­ß2GPI Ab formed a stable oxidized low­density lipoprotein (oxLDL)/ß2GPI/anti­ß2GPI Ab complex, which accelerated AS development by promoting the accumulation of lipids in macrophages and vascular smooth muscle cell. However, the effects of the complex on endothelial cells, which drive the initiation and development of AS, remain unknown. Thus, the present study aimed to determine the proinflammatory roles of the oxLDL/ß2GPI/anti­ß2GPI Ab complex in human umbilical vein endothelial cells (HUVECs) in an attempt to determine the underlying mechanism. Reverse transcription­quantitative PCR, enzymy­linked immunosorbent assay, western blotting and immunofluorescence staining were performed to detect the expressions of inflammation related factors and adhesion molecules. Monocyte­binding assay was used to investigate the effects of oxLDL/ß2GPI/anti­ß2GPI Ab complex on monocyte adhesion to endothelial cells. The results demonstrated that the oxLDL/ß2GPI/anti­ß2GPI Ab complex upregulated the expression of Toll­like receptor (TLR)4 and the levels of NF­κB phosphorylation in HUVECs, and subsequently enhanced the expression levels of inflammatory cytokines, including TNF­α, IL­1ß and IL­6, as well as those of adhesion molecules, such as intercellular adhesion molecule 1 and vascular adhesion molecule 1. In addition, the complex facilitated the recruitment of monocytes by promoting the secretion of monocyte chemotactic protein 1 in HUVECs. Notably, the described effects of the oxLDL/ß2GPI/anti­ß2GPI Ab complex in HUVECs were abolished by either TLR4 or NF­κB blockade. In conclusion, these findings suggested that the oxLDL/ß2GPI/anti­ß2GPI Ab complex may induce a hyper­inflammatory state in endothelial cells by promoting the secretion of proinflammatory cytokines and monocyte recruitment, which was discovered to be largely dependent on the TLR4/NK­κB signaling pathway.

Activation of plasmacytoid dendritic cells and B cells with two structurally different Toll-like receptor 7 agonists.

Synthetic Toll-like receptor (TLR) 7 agonists have been suggested as immune modulators in a range of conditions. In contrast, self-derived TLR7 activators, such as RNA-containing immune complexes (RNA-IC), can contribute to autoimmune diseases due to endogenous immune activation. The exact difference in immune cell response between synthetic and endogenous TLR7 triggers is only partly known. An understanding of these differences could aid in the development of new therapeutic agents and provide insights into autoimmune disease mechanisms. We therefore compared the stimulatory capacity of two TLR7 agonists, RNA-IC and a synthetic small molecule DSR-6434, on blood leucocytes, plasmacytoid dendritic cells (pDCs) and B cells from healthy individuals. IFN-α, IL-6, IL-8 and TNF levels were measured by immunoassays, and gene expression in pDCs was analysed by an expression array. DSR-6434 triggered 20-fold lower levels of IFN-α by pDCs, but higher production of IL-6, IL-8 and TNF, compared to RNA-IC. Furthermore, IFN-α and TNF production were increased with exogenous IFN-α2b priming, whereas IL-8 synthesis by B cells was reduced for both stimuli. Cocultivation of pDCs and B cells increased the RNA-IC-stimulated IFN-α and TNF levels, while only IL-6 production was enhanced in the DSR-6434-stimulated cocultures. When comparing pDCs stimulated with RNA-IC and DSR-6434, twelve genes were differentially expressed (log2 fold change >2, adjusted P-value <.05). In conclusion, RNA-IC, which mimics an endogenous TLR7 stimulator, and the synthetic TLR7 agonist DSR-6434 trigger distinct inflammatory profiles in immune cells. This demonstrates the importance of using relevant stimuli when targeting the TLR7 pathway for therapeutic purposes.

Involvement of condensin in cellular senescence through gene regulation and compartmental reorganization.

Senescence is induced by various stimuli such as oncogene expression and telomere shortening, referred to as oncogene-induced senescence (OIS) and replicative senescence (RS), respectively, and accompanied by global transcriptional alterations and 3D genome reorganization. Here, we demonstrate that the human condensin II complex participates in senescence via gene regulation and reorganization of euchromatic A and heterochromatic B compartments. Both OIS and RS are accompanied by A-to-B and B-to-A compartmental transitions, the latter of which occur more frequently and are undergone by 14% (430 Mb) of the human genome. Mechanistically, condensin is enriched in A compartments and implicated in B-to-A transitions. The full activation of senescence genes (SASP genes and p53 targets) requires condensin; its depletion impairs senescence markers. This study describes that condensin reinforces euchromatic A compartments and promotes B-to-A transitions, both of which are coupled to optimal expression of senescence genes, thereby allowing condensin to contribute to senescent processes.

The role of human serum albumin and neurotoxin associated proteins in the formulation of BoNT/A products.

Botulinum neurotoxin (BoNT) is synthesized as a progenitor toxin complex (PTC) by Clostridium botulinum. This PTC comprises, in addition to the neurotoxin itself, neurotoxin associated proteins (NAPs) which are composed of three hemagglutinins and one non-toxic, non-hemagglutinin protein. After oral ingestion, these NAPs protect the neurotoxin from the low pH and proteases in the gastrointestinal tract and play a role in the entry via the intestinal barrier. Two of the three therapeutically used botulinum neurotoxin serotype A (BoNT/A) products (onabotulinumtoxinA and abobotulinumtoxinA) contain different amounts of NAPs, while incobotulinumtoxinA, lacks these proteins. In addition, human serum albumin (HSA) that is supposed to stabilize BoNT/A is added at different concentrations. Up to now, the function of the NAPs and HSA after parenteral therapeutic application is not completely understood. To investigate the influence of NAPs and HSA on potency of BoNT/A, we used the ex vivo mouse phrenic nerve hemidiaphragm assay. Increasing doses of HSA resulted dose-dependently in a more pronounced effect of BoNT/A. Though, a plateau was reached with concentrations of 0.8 mg/ml HSA and higher, the accessory addition of NAPs in a relevant amount (4 ng/ml) did not further enhance the effect of BoNT/A. In conclusion, in our ex vivo assay an adequate concentration of HSA prevented BoNT/A from loss of effect and supplementary NAPs did not alter this effect. A confirmation of these data in an in vivo assay is still lacking. However, it might be supposed that even in clinically applied BoNT/A products an increase of HSA accompanied by the avoidance of NAPs could potentially reduce the injected dose and, thus, the risk of unwanted side effects, the treatment costs as well as the risk of a secondary therapy failure due to BoNT/A neutralizing antibodies.

Assessment of bioactivities of the human milk lactoferrin-osteopontin complex in vitro.

Lactoferrin (LF) and osteopontin (OPN) are both multi-functional whey proteins present at high levels in human milk. These two proteins have a high affinity for each other due to their opposite charges; LF is a basic glycoprotein while OPN is an acidic phosphorylated glycoprotein. LF and OPN were identified to bind to each other over a decade ago, but potential functions of their complex remain unexplored. In this work, we investigated the characteristics of the LF-OPN complex with a focus on its bioactivities. Our results reveal a stronger stability of the LF-OPN complex towards in vitro digestion and more effective binding and uptake by human intestinal cells (HIEC) than LF or OPN alone show. Moreover, the LF-OPN complex promotes proliferation and differentiation of intestinal cells significantly more than the individual proteins do and shows an effect on anti-bacterial function and immune-stimulatory activities intermediate between those of LF and OPN. Thus, by forming a complex in human milk, LF and OPN may protect each other against proteolysis and enhance their individual bioactivities.

Targeting Insulin-Like Growth Factor-I and Extracellular Matrix Interactions in Melanoma Progression.

Insulin-like growth factor (IGF)-I binds to the ECM protein vitronectin (VN) through IGF binding proteins (IGFBPs) to enhance proliferation and migration of skin keratinocytes and fibroblasts. Although evidence exists for the role of individual components of the complex (IGF-I, IGFBP-3 and VN), the cellular functions stimulated by these proteins together as a complex remains un-investigated in melanoma cells. We report here that the IGF-I:IGFBP-3:VN trimeric complex stimulates a dose-dependent increase in the proliferation and migration of WM35 and Sk-MEL28 melanoma cells. In 3D Matrigel™ and hydrogel cultures, both cell lines formed primary tumor-like spheroids, which increased in size in a dose-dependent manner in response to the trimeric complex. Furthermore, we reveal IGFBP-3:VN protein complexes in malignant melanoma and squamous cell carcinoma patient tissues, where the IGFBP-3:VN complex was seen to be predominantly tumor cell-associated. Peptide antagonists designed to target the binding of IGF-I:IGFBP-3 to VN were demonstrated to inhibit IGF-I:IGFBP-3:VN-stimulated cell migration, invasion and 3D tumor cell growth of melanoma cells. Overall, this study provides new data on IGF:ECM interactions in skin malignancies and demonstrates the potential usefulness of a growth factor:ECM-disrupting strategy for abrogating tumor progression.

Inhibition of plasmin generation in plasma by heparin, low molecular weight heparin, and a covalent antithrombin-heparin complex.

: The clinical limitations of unfractionated heparin (UFH) and low molecular weight heparin (LMWH) led to the development of an antithrombin-heparin covalent complex (ATH), which displays superior anticoagulant abilities compared with UFH. A recent study investigating its interaction with fibrinolysis showed that ATH inhibited free and fibrin bound plasmin and decreased plasmin generation on fibrin clots. These studies were conducted using purified components and did not elucidate the interaction of ATH with plasmin in the presence of its natural inhibitors α2-antiplasmin (α2-AP) and α2-macroglobulin (α2-M). The aim of this study was to determine the effects of ATH, UFH, and LMWH on plasmin generation in plasma, under more physiological conditions. Plasmin generation in plasma in the absence and presence of anticoagulants was initiated by tissue plasminogen activator and soluble fibrin fragments, and plasmin and plasmin-α2-M complexes generated over time were quantified chromogenically. Generation of plasmin-α2-AP complexes and consumption of plasminogen were quantified by ELISA. Plasmin generation was decreased in the presence of UFH and ATH, whereas LMWH had no effect. Neither plasminogen consumption nor generation of plasmin-α2-AP complexes were affected by UFH or ATH. However, plasmin-α2-M complexes were slightly reduced by ATH suggesting that ATH may be able to compete with α2-M for plasmin. Plasmin generation may be mildly inhibited by heparin-based anticoagulants; however, heparin-catalyzed antithrombin activity is not a major inhibitor of plasmin, as compared to its natural inhibitors α2-AP and α2-M. This adds to our understanding of ATH mechanisms of action and aids in its development for clinical use.

La proteína tuberina: diana terapéutica para activar autofagia y mitofagia evitando la progresión a la diabetes tipo 2 / Tuberin protein: therapeutic target to activate autophagy and mitophagy avoiding the progression to type 2 diabetes

El control de calidad citoplasmático es esencial en el mantenimiento de la viabilidad celular, y particularmente, de las células β pancreáticas, debido a su gran capacidad de síntesis proteica. En este contexto, juega un papel esencial la activación de un proceso fisiológico denominado autofagia, conduciendo a la eliminación de agregados proteicos y/o orgánulos, induciendo la atenuación del estrés de retículo celular. El complejo formado por las proteínas hamartina y tuberina (TSC1-TSC2) ha emergido como un núcleo de integración de la señalización de factores de crecimiento y del estado energético celular. Este complejo funciona como un inhibidor de la actividad de la vía del complejo mecanicístico diana de la rapamicina (mTORC1) y un activador de la autofagia. En este proyecto queremos profundizar en el estudio de nuevos mecanismos moleculares de regulación de TSC2, así como sobre dichos mecanismos de control de calidad citoplasmático, autofagia y mitofagia. Proponemos que el estado de acetilación en lisinas de TSC2, mediado por la actividad desacetilasa de la sirtuina1 (SIRT1), modula la estabilidad y actividad de la misma afectando a la homeostasis celular (AU)

Cytoplasmic quality control is essential in maintaining cell viability, and articularly, β pancreatic cells, due to its huge protein synthesis capacity. In this context, it is important the activation of a physiological process called autophagy, in order to eliminate protein aggregates and damaged organelles, resulting in a reduction in the reticulum cell stress. The complex formed by hamartin and tuberin (TSC1-TSC2) has emerged as a central signal, energy status and nutrient-integrating node within the cell. This complex negatively regulates the mechanistic target of rapamycin complex 1 (mTORC1), and activates autophagy. In this proyect we aimed to further investigate new molecular mechanisms of TSC2 regulation, aswell as cytoplasmic quality control, autophagy and mitophagy ones. We propose that the TSC2 acetylation status, mediated by the deacetylation activity of sirtuin1 (SIRT1), modulates its stability and protein activity, affecting cell homeostasis

Alpha-synuclein activates BV2 microglia dependent on its aggregation state.

Synucleinopathies such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are defined by the presence of intracellular alpha-synuclein aggregates in neurons and/or oligodendrocytes. In addition, post mortem tissue analysis revealed profound changes in microglial morphology, indicating microglial activation and neuroinflammation. Thus, alpha-synuclein may directly activate microglia, leading to increased production of key pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1ß), which in turn modulates the disease progression. The distinct alpha-synuclein species, which mediates the activation of microglia, is not well defined. We hypothesized that microglial activation depends on a specific aggregation state of alpha-synuclein. Here, we show that primarily human fibrillar alpha-synuclein increased the production and secretion of pro-inflammatory cytokines by microglial BV2 cells compared to monomeric and oligomeric alpha-synuclein. BV2 cells also preferentially phagocytosed fibrillar alpha-synuclein compared to alpha-synuclein monomers and oligomers. Microglial uptake of alpha-synuclein fibrils and the consequent activation were time- and concentration-dependent. Moreover, the degree of fibrillization determined the efficiency of microglial internalization. Taken together, our study highlights the specific crosstalk of distinct alpha-synuclein species with microglial cells.

Therapeutic Targeting of the IL-6 Trans-Signaling/Mechanistic Target of Rapamycin Complex 1 Axis in Pulmonary Emphysema.

RATIONALE: The potent immunomodulatory cytokine IL-6 is consistently up-regulated in human lungs with emphysema and in mouse emphysema models; however, the mechanisms by which IL-6 promotes emphysema remain obscure. IL-6 signals using two distinct modes: classical signaling via its membrane-bound IL-6 receptor (IL-6R), and trans-signaling via a naturally occurring soluble IL-6R. OBJECTIVES: To identify whether IL-6 trans-signaling and/or classical signaling contribute to the pathogenesis of emphysema. METHODS: We used the gp130F/F genetic mouse model for spontaneous emphysema and cigarette smoke-induced emphysema models. Emphysema in mice was quantified by various methods including in vivo lung function and stereology, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to assess alveolar cell apoptosis. In mouse and human lung tissues, the expression level and location of IL-6 signaling-related genes and proteins were measured, and the levels of IL-6 and related proteins in sera from emphysematous mice and patients were also assessed. MEASUREMENTS AND MAIN RESULTS: Lung tissues from patients with emphysema, and from spontaneous and cigarette smoke-induced emphysema mouse models, were characterized by excessive production of soluble IL-6R. Genetic blockade of IL-6 trans-signaling in emphysema mouse models and therapy with the IL-6 trans-signaling antagonist sgp130Fc ameliorated emphysema by suppressing augmented alveolar type II cell apoptosis. Furthermore, IL-6 trans-signaling-driven emphysematous changes in the lung correlated with mechanistic target of rapamycin complex 1 hyperactivation, and treatment of emphysema mouse models with the mechanistic target of rapamycin complex 1 inhibitor rapamycin attenuated emphysematous changes. CONCLUSIONS: Collectively, our data reveal that specific targeting of IL-6 trans-signaling may represent a novel treatment strategy for emphysema.

Association of MTOR Mutations With Developmental Brain Disorders, Including Megalencephaly, Focal Cortical Dysplasia, and Pigmentary Mosaicism.

IMPORTANCE: Focal cortical dysplasia (FCD), hemimegalencephaly, and megalencephaly constitute a spectrum of malformations of cortical development with shared neuropathologic features. These disorders are associated with significant childhood morbidity and mortality. OBJECTIVE: To identify the underlying molecular cause of FCD, hemimegalencephaly, and diffuse megalencephaly. DESIGN, SETTING, AND PARTICIPANTS: Patients with FCD, hemimegalencephaly, or megalencephaly (mean age, 11.7 years; range, 2-32 years) were recruited from Pediatric Hospital A. Meyer, the University of Hong Kong, and Seattle Children's Research Institute from June 2012 to June 2014. Whole-exome sequencing (WES) was performed on 8 children with FCD or hemimegalencephaly using standard-depth (50-60X) sequencing in peripheral samples (blood, saliva, or skin) from the affected child and their parents and deep (150-180X) sequencing in affected brain tissue. Targeted sequencing and WES were used to screen 93 children with molecularly unexplained diffuse or focal brain overgrowth. Histopathologic and functional assays of phosphatidylinositol 3-kinase-AKT (serine/threonine kinase)-mammalian target of rapamycin (mTOR) pathway activity in resected brain tissue and cultured neurons were performed to validate mutations. MAIN OUTCOMES AND MEASURES: Whole-exome sequencing and targeted sequencing identified variants associated with this spectrum of developmental brain disorders. RESULTS: Low-level mosaic mutations of MTOR were identified in brain tissue in 4 children with FCD type 2a with alternative allele fractions ranging from 0.012 to 0.086. Intermediate-level mosaic mutation of MTOR (p.Thr1977Ile) was also identified in 3 unrelated children with diffuse megalencephaly and pigmentary mosaicism in skin. Finally, a constitutional de novo mutation of MTOR (p.Glu1799Lys) was identified in 3 unrelated children with diffuse megalencephaly and intellectual disability. Molecular and functional analysis in 2 children with FCD2a from whom multiple affected brain tissue samples were available revealed a mutation gradient with an epicenter in the most epileptogenic area. When expressed in cultured neurons, all MTOR mutations identified here drive constitutive activation of mTOR complex 1 and enlarged neuronal size. CONCLUSIONS AND RELEVANCE: In this study, mutations of MTOR were associated with a spectrum of brain overgrowth phenotypes extending from FCD type 2a to diffuse megalencephaly, distinguished by different mutations and levels of mosaicism. These mutations may be sufficient to cause cellular hypertrophy in cultured neurons and may provide a demonstration of the pattern of mosaicism in brain and substantiate the link between mosaic mutations of MTOR and pigmentary mosaicism in skin.

Host cell mTORC1 is required for HCV RNA replication.

OBJECTIVE: Chronically HCV-infected orthotopic liver transplantation (OLT) recipients appear to have improved outcomes when their immunosuppressive regimen includes a mammalian target of rapamycin (mTOR) inhibitor. The mechanism underlying this observation is unknown. DESIGN: We used virological assays to investigate mTOR signalling on the HCV replication cycle. Furthermore, we analysed HCV RNA levels of 42 HCV-positive transplanted patients treated with an mTOR inhibitor as part of their immunosuppressive regimen. RESULTS: The mTOR inhibitor rapamycin was found to be a potent inhibitor for HCV RNA replication in Huh-7.5 cells as well as primary human hepatocytes. Half-maximal inhibition was observed at 0.01 µg/mL, a concentration that is in the range of serum levels seen in transplant recipients and does not affect cell proliferation. Early replication cycle steps such as cell entry and RNA translation were not affected. Knockdown of raptor, an essential component of mTORC1, but not rictor, an essential component of mTORC2, inhibited viral RNA replication. In addition, overexpression of raptor led to higher viral RNA replication, demonstrating that mTORC1, but not mTORC2, is required for HCV RNA replication. In 42 HCV-infected liver-transplanted or kidney-transplanted patients who were switched to an mTOR inhibitor, we could verify that mTOR inhibition decreased HCV RNA levels in vivo. CONCLUSIONS: Our data identify mTORC1 as a novel HCV replication factor. These findings suggest an underlying mechanism for the observed benefits of mTOR inhibition in HCV-positive OLT recipients and potentiate further investigation of mTOR-containing regimens in HCV-positive recipients of solid organ transplants.

Effects of heterodimeric bone morphogenetic protein-2/7 on osteogenesis of human adipose-derived stem cells.

OBJECTIVE: Roles of bone morphogenetic proteins (BMPs) on osteogenesis of human adipose-derived stem cells (hASCs) remain ambiguous. In this study, we evaluated in vitro and in vivo functional characteristics of BMPs of different dimerization types, with the aim of determining osteoinductive efficiency of heterodimeric BMP-2/7 on osteogenesis of hASCs. MATERIALS AND METHODS: We explored osteoinductive effects of three different BMPs by using cell DNA assay, alkaline phosphatase (ALP) activity assay, alizarin red staining and mineralization assay, and real-time PCR, in vitro. Also, we examined ectopic bone formation in nude mice by using soft X-ray, histomorphometric and immunohistochemical analyses in vivo. RESULTS: In our dose-response study, we showed that BMPs with both dimerization types did not induce in vitro osteogenesis of hASCs without osteogenic medium (OM). In the presence of OM, BMPs significantly enhanced hASC osteogenesis in a dose-dependent manner. In in vivo experiments, our analyses indicated that BMPs promoted osteogenesis of hASCs without in vitro osteogenic induction. However, both in vitro and in vivo, there were no significant differences in hASC osteogenic induction between heterodimeric and homodimeric BMPs. CONCLUSIONS: Heterodimeric BMP-2/7 significantly promoted osteogenesis of hASCs in vitro and in vivo. However, BMP-2/7 was not found to be a stronger inducer of osteogenesis compared to homodimeric either BMP-2 or BMP-7.

IL-15 Superagonist-Mediated Immunotoxicity: Role of NK Cells and IFN-γ.

IL-15 is currently undergoing clinical trials to assess its efficacy for treatment of advanced cancers. The combination of IL-15 with soluble IL-15Rα generates a complex termed IL-15 superagonist (IL-15 SA) that possesses greater biological activity than IL-15 alone. IL-15 SA is considered an attractive antitumor and antiviral agent because of its ability to selectively expand NK and memory CD8(+) T (mCD8(+) T) lymphocytes. However, the adverse consequences of IL-15 SA treatment have not been defined. In this study, the effect of IL-15 SA on physiologic and immunologic functions of mice was evaluated. IL-15 SA caused dose- and time-dependent hypothermia, weight loss, liver injury, and mortality. NK (especially the proinflammatory NK subset), NKT, and mCD8(+) T cells were preferentially expanded in spleen and liver upon IL-15 SA treatment. IL-15 SA caused NK cell activation as indicated by increased CD69 expression and IFN-γ, perforin, and granzyme B production, whereas NKT and mCD8(+) T cells showed minimal, if any, activation. Cell depletion and adoptive transfer studies showed that the systemic toxicity of IL-15 SA was mediated by hyperproliferation of activated NK cells. Production of the proinflammatory cytokine IFN-γ, but not TNF-α or perforin, was essential to IL-15 SA-induced immunotoxicity. The toxicity and immunological alterations shown in this study are comparable to those reported in recent clinical trials of IL-15 in patients with refractory cancers and advance current knowledge by providing mechanistic insights into IL-15 SA-mediated immunotoxicity.

Looking into the "Garden of the Hesperides": new drugs for hepatocellular carcinoma.

Advanced hepatocellular carcinoma remains an entity that is hard to approach therapeutically and has shown disappointing results in terms of survival. For many years, the only accepted option for this setting was the use of a multikinase inhibitor, sorafenib. Nevertheless, through the years, a deeper knowledge has arisen about how pathogenic pathways correlate with hepatocellular carcinoma. In this review, we provide an update of the most recent data regarding new agents under investigation and new possible targets for future treatments.

Alum and squalene-oil-in-water emulsion enhance the titer and avidity of anti-Aß antibodies induced by multimeric protein antigen (1-11)E2, preserving the Igg1-skewed isotype distribution.

The development of active immunotherapy for Alzheimer's disease (AD) requires the identification of immunogens that can ensure a high titer antibody response toward Aß, while minimizing the risks of adverse reactions. Multimeric protein (1-11)E2 induces a robust and persistent antibody response to Aß in mice, when formulated in Freund's adjuvant. The goal of this translational study was to evaluate the immunogenicity of (1-11)E2 formulated in alum (Alhydrogel 2%), or in a squalene oil-in-water emulsion (AddaVax), or without adjuvant. A IgG1-skewed isotype distribution was observed for the anti-Aß antibodies generated in mice immunized with either the non-adjuvanted or the adjuvanted vaccine, indicating that (1-11)E2 induces a Th2-like response in all tested conditions. Both Alhydrogel 2% and AddaVax enhanced the titer and avidity of the anti-Aß response elicited by (1-11)E2. We conclude that (1-11)E2 is a promising candidate for anti-Aß immunization protocols that include alum or squalene-oil-in-water emulsion, or no adjuvant.

Immunization with a functional protein complex required for erythrocyte invasion protects against lethal malaria.

An essential step in the invasion of red blood cells (RBCs) by Plasmodium falciparum (Pf) merozoites is the binding of rhoptry neck protein 2 (RON2) to the hydrophobic groove of apical membrane antigen 1 (AMA1), triggering junction formation between the apical end of the merozoite and the RBC surface to initiate invasion. Vaccination with AMA1 provided protection against homologous parasites in one of two phase 2 clinical trials; however, despite its ability to induce high-titer invasion-blocking antibodies in a controlled human challenge trial, the vaccine conferred little protection even against the homologous parasite. Here we provide evidence that immunization with an AMA1-RON2 peptide complex, but not with AMA1 alone, provided complete protection against a lethal Plasmodium yoelii challenge in mice. Significantly, IgG from mice immunized with the complex transferred protection. Furthermore, IgG from PfAMA1-RON2-immunized animals showed enhanced invasion inhibition compared with IgG elicited by AMA1 alone. Interestingly, this qualitative increase in inhibitory activity appears to be related, at least in part, to a switch in the proportion of IgG specific for certain loop regions in AMA1 surrounding the binding site of RON2. Antibodies induced by the complex were not sufficient to block the FVO strain heterologous parasite, however, reinforcing the need to include multiallele AMA1 to cover polymorphisms. Our results suggest that AMA1 subunit vaccines may be highly effective when presented to the immune system as an invasion complex with RON2.

Circulating complexes of the vitamin D binding protein with G-actin induce lung inflammation by targeting endothelial cells.

This study investigated the actin scavenger function of the vitamin D binding protein (DBP) in vivo using DBP null (-/-) mice. Intravenous injection of G-actin into wild-type (DBP+/+) and DBP-/- mice showed that contrary to expectations, DBP+/+ mice developed more severe acute lung inflammation. Inflammation was restricted to the lung and pathological changes were clearly evident at 1.5 and 4h post-injection but were largely resolved by 24h. Histology of DBP+/+ lungs revealed noticeably more vascular leakage, hemorrhage and thickening of the alveolar wall. Flow cytometry analysis of whole lung homogenates showed significantly increased neutrophil infiltration into DBP+/+ mouse lungs at 1.5 and 4h. Increased amounts of protein and leukocytes were also noted in bronchoalveolar lavage fluid from DBP+/+ mice 4h after actin injection. In vitro, purified DBP-actin complexes did not activate complement or neutrophils but induced injury and death of cultured human lung microvascular endothelial cells (HLMVEC) and human umbilical vein endothelial cells (HUVEC). Cells treated with DBP-actin showed a significant reduction in viability at 4h, this effect was reversible if cells were cultured in fresh media for another 24h. However, a 24-h treatment with DBP-actin complexes showed a significant increase in cell death (95% for HLMVEC, 45% for HUVEC). The mechanism of endothelial cell death was via both caspase-3 dependent (HUVEC) and independent (HLMVEC) pathways. These results demonstrate that elevated levels and/or prolonged exposure to DBP-actin complexes may induce endothelial cell injury and death, particularly in the lung microvasculature.

Strand breakage of a (6-4) photoproduct-containing DNA at neutral pH and its repair by the ERCC1-XPF protein complex.

The (6-4) photoproduct is one of the major UV-induced lesions in DNA. We previously showed that hydrolytic ring opening of the 5' base and subsequent hydrolysis of the glycosidic bond of the 3' component occurred when this photoproduct was treated with aqueous NaOH. In this study, we found that another product was obtained when the (6-4) photoproduct was heated at 90 °C for 6 h, in a 0.1 M solution of N,N'-dimethyl-1,2-ethanediamine adjusted to pH 7.4 with acetic acid. An analysis of the chemical structure of this product revealed that the 5' base was intact, whereas the glycosidic bond at the 3' component was hydrolyzed in the same manner. The strand break was detected for a 30-mer oligonucleotide containing the (6-4) photoproduct upon treatment with the above solution or other pH 7.4 solutions containing biogenic amines, such as spermidine and spermine. In the case of spermidine, the rate constant was calculated to be 1.4 × 10(-8) s(-1) at 37 °C. The strand break occurred even when the oligonucleotide was heated at 90 °C in 0.1 M sodium phosphate (pH 7.0), although this treatment produced several types of 5' fragments. The Dewar valence isomer was inert to this reaction. The product obtained from the (6-4) photoproduct-containing 30-mer was used to investigate the enzymatic processing of the 3' end bearing the damaged base and a phosphate. The ERCC1-XPF complex removed several nucleotides containing the damaged base, in the presence of replication protein A.

Free and protein-bound angiotensin II(1-7)in the regulation of drinking behavior and hemodynamics in rats.

We compared physiological activity of synthetic analogues of endogenous protein-peptide compounds, complexes of angiotensin II(1-7)with functionally different proteins (transport protein, serum albumin; and neurospecific Ca(2+)-binding protein, S100b). Physiological activity of angiotensin II(1-7)was shown to depend on the type of a carrier protein. Our results suggest that complexes of angiotensins with BSA and S100b are strong factors for the integration of central and peripheral functions at the homeostatic and behavioral level.