Leptin Receptor Blockade Attenuates Hypertension, but Does Not Affect Ventilatory Response to Hypoxia in a Model of Polygenic Obesity

BackgroundObesity can cause hypertension and exacerbates sleep-disordered breathing (SDB). Leptin is an adipocyte-produced hormone, which increases metabolic rate, suppresses appetite, modulates control of breathing, and increases blood pressure. Obese individuals with high circulating levels of leptin are resistant to metabolic and respiratory effects of leptin, but they appear to be sensitive to hypertensive effects of this hormone. Obesity-induced hypertension has been associated with hyperleptinemia. New Zealand obese (NZO) mice, a model of polygenic obesity, have high levels of circulating leptin and hypertension, and are prone to develop SDB, similarly to human obesity. We hypothesize that systemic leptin receptor blocker Allo-aca will treat hypertension in NZO mice without any effect on body weight, food intake, or breathing.MethodsMale NZO mice, 12–13 weeks of age, were treated with Allo-aca (n = 6) or a control peptide Gly11 (n = 12) for 8 consecutive days. Doses of 0.2 mg/kg were administered subcutaneously 2×/day, at 10 AM and 6 PM. Blood pressure was measured by telemetry for 48 h before and during peptide infusion. Ventilation was assessed by whole-body barometric plethysmography, control of breathing was examined by assessing the hypoxic ventilatory response (HVR), and polysomnography was performed during light-phase at baseline and during treatment. Heart rate variability analyses were performed to estimate the cardiac autonomic balance.ResultsSystemic leptin receptor blockade with Allo-aca did not affect body weight, body temperature, and food intake in NZO mice. Plasma levels of leptin did not change after the treatment with either Allo-aca or the control peptide Gy11. NZO mice were hypertensive at baseline and leptin receptor blocker Allo-aca significantly reduced the mean arterial pressure from 134.9 ± 3.1 to 124.9 ± 5.7 mmHg during the light phase (P < 0.05), whereas the control peptide had no effect. Leptin receptor blockade did not change the heart rate or cardiac autonomic balance. Allo-aca did not affect minute ventilation under normoxic or hypoxic conditions and HVR. Ventilation, apnea index, and oxygen desaturation during NREM and REM sleep did not change with leptin receptor blockade.ConclusionSystemic leptin receptor blockade attenuates hypertension in NZO mice, but does not exacerbate obesity and SDB. Thus, leptin receptor blockade represents a potential pharmacotherapy for obesity-associated hypertension.

POS0395 ANTI-ACETYLATED PROTEIN ANTIBODIES IN RHEUMATOID ARTHRITIS (RA): CLUES FOR THE STARTING POINT OF AUTOANTIBODY RESPONSES IN RA

Background:Rheumatoid arthritis (RA) is characterized by autoantibodies such as rheumatoid factor (RF) and anti-modified protein autoantibodies (AMPAs) like anti-citrullinated protein antibodies (ACPA) and anti-carbamylated protein antibodies (anti-CarP). Recently, another AMPA: anti-acetylated protein antibodies (AAPA) have been found in RA patients [1]. The prevalence of AAPA antibodies and their isotypes have yet to be determined. Since isotype profiles reflect the breadth of an immune response, the prevalence of AAPA isotypes in arthritis patients with and without RA can help to understand the relevance of this autoantibody response in RA.Objectives:To describe the prevalence of AAPA isotypes in arthritis patients with and without RA.Methods:In 650 RA patients fulfilling the 1987 RA criteria and 555 non-RA arthritis patients from the Leiden Early Arthritis Cohort, baseline serum samples were screened by ELISA for IgG, IgM and IgA to an acetylated- and control peptide that was based upon the CCP-2 backbone. The cutoff for positivity was based on 80 controls (mean + 2SD). A sample was considered positive if it was above the cutoff and was 0.1 optical density higher on the acetylated peptide than on the control peptide.Results:AAPA IgG was found in 36% of RA patients versus 6.7% of non-RA arthritis patients (figure 1a). Within RA patients, AAPA IgG antibodies were mostly present in the ACPA-(CCP-2) positive group (64% in ACPA-positive, compared to 5% in ACPA-negative). Levels of AAPA IgG and IgA were higher in RA patients than in healthy controls and non-RA arthritis patients (figure 1b), however, surprisingly, no difference in levels was found for IgM.When isotype profiles in AAPA- positive arthritis patients were compared, patients with RA were more often positive for two or more isotypes then patients without RA, and thus displayed considerably more overlap in AAPA isotypes compared to non-RA patients (table 1). Intriguingly, IgM AAPA was the most prevalent isotype in non-RA patients, versus IgG in RA patients.Table 1.Anti-acetylated protein antibody (AAPA) isotype overlap in AAPA positive patients.AAPA isotypeRA patients (=310) n (%)Non-RA arthritis patients (n=106) n (%)IgG+IgM-IgA-115 (37.1)28 (5.1)IgG-IgM+IgA-52 (16.8)48 (8.7)IgG-IgM-IgA+14 (4.5)13 (2.3)IgG+IgM+IgA-24 (7.7)3 (0.5)IgG+IgM-IgA+37 (11.9)4 (0.7)IgG-IgM+IgA+9 (2.9)8 (1.4)IgG+IgM+IgA+59 (19.0)2 (0.4)AAPA: anti-acetylated protein antibodies, RA: rheumatoid arthritisConclusion:AAPA are detected in one third of RA patients, and mainly in the ACPA-positive subgroup. The predominance of IgM AAPA in non-RA arthritis patients and healthy controls suggests that healthy persons can develop AAPA IgM without the development of RA. These results also suggest that in healthy individuals, AAPA responses can occur, but do not mature past the IgM-stage, while in RA patients, the AAPA-response does mature and might form a “starting point” for development of other AMPA leading to the concurrent present of several AMPA in disease.References:[1]Juarez, M., et al., Identification of novel antiacetylated vimentin antibodies in patients with early inflammatory arthritis. Ann Rheum Dis, 2016. 75(6): p. 1099-107.Disclosure of Interests:None declared

Absence of Pannexin 1 Stabilizes Hippocampal Excitability After Intracerebral Treatment With Aβ (1-42) and Prevents LTP Deficits in Middle-Aged Mice

Beta-amyloid protein [Aβ(1-42)] plays an important role in the disease progress and pathophysiology of Alzheimer's disease (AD). Membrane properties and neuronal excitability are altered in the hippocampus of transgenic AD mouse models that overexpress amyloid precursor protein. Although gap junction hemichannels have been implicated in the early pathogenesis of AD, to what extent Pannexin channels contribute to Aβ(1-42)-mediated brain changes is not yet known. In this study we, therefore, investigated the involvement of Pannexin1 (Panx1) channels in Aβ-mediated changes of neuronal membrane properties and long-term potentiation (LTP) in an animal model of AD. We conducted whole-cell patch-clamp recordings in CA1 pyramidal neurons 1 week after intracerebroventricular treatments of adult wildtype (wt) and Panx1 knockout (Panx1-ko) mice with either oligomeric Aβ(1-42), or control peptide. Panx1-ko hippocampi treated with control peptide exhibited increased neuronal excitability compared to wt. In addition, action potential (AP) firing frequency was higher in control Panx1-ko slices compared to wt. Aβ-treatment reduced AP firing frequency in both cohorts. But in Aβ-treated wt mice, spike frequency adaptation was significantly enhanced, when compared to control wt and to Aβ-treated Panx1-ko mice. Assessment of hippocampal LTP revealed deficits in Aβ-treated wt compared to control wt. By contrast, Panx1-ko exhibited LTP that was equivalent to LTP in control ko hippocampi. Taken together, our data show that in the absence of Pannexin1, hippocampi are more resistant to the debilitating effects of oligomeric Aβ. Both Aβ-mediated impairments in spike frequency adaptation and in LTP that occur in wt animals, are ameliorated in Panx1-ko mice. These results suggest that Panx1 contributes to early changes in hippocampal neuronal and synaptic function that are triggered by oligomeric Aβ.

Identification and Characterization of CMV-Specific, HLA-C*07:02 Restricted Antibodies

In patients with insufficient T-cell responses to the human cytomegalovirus (HCMV), reactivation can occur which significantly contributes to morbidity and mortality after allogeneic stem cell transplantation (HSCT). Despite the event of new therapeutics like letermovir, HCMV infection remains a major complication after stem cell and solid organ transplantation. HCMV infected cells process HCMV proteins intracellularly and present HCMV-peptides on the cell surface in the context of HLA class I complexes. Antibodies naturally do not bind to peptides that are presented in HLA complexes and immunity against HCMV is mostly dependent on T-cells recognizing HLA I/HCMV-peptide complexes via their T-cell receptor. Antibodies that are generated to bind to peptides that are presented in HLA complexes are termed T-cell receptor like (TCR-like) antibodies. It has recently been shown that the T-cell response against an HLA-C*07:02 presented HCMV-peptide (CRVLCCYVL) derived from the IE-1 antigen is particularly strong. Also, HLA-C*07:02 is less susceptible to viral immune evasion than HLA-B and HLA-A molecules making it an attractive target for TCR-like antibodies. Here, we set out to obtain and characterize HLA-C*07:02 restricted, HCMV-specific antibodies. Antibody Fab fragments (Fabs) specific for the HLA-C*07:02 restricted HCMV-peptide CRVLCCYVL were identified using phage display technology. For selection, phages were incubated for 1 hour with biotinylated HLA-C*07:02/CRVLCCYVL complexes at decreasing concentrations (15, 5 and 1 µg/ml). Before each round a pre-absorption using HLA-C*07:02 complexes folded with control-peptide was performed. Selected phages were eluted and used to infect E. coli bacteria of the strain TG1 to produce soluble Fabs, which were then tested for binding to HCMV-peptide (CRVLCCYVL) and control-peptide pulsed lymphoblastoid cell lines (LCLs) and lymphocytes expressing HLA-C*07:02. As control, selected Fabs were tested for binding to HCMV-peptide (CRVLCCYVL) and control-peptide loaded LCLs and lymphocytes expressing different HLA-C alleles. By this, we were able to identify 3 different Fabs (4/4, 7/3 and 13/2) that bound to HLA-C*07:02 expressing LCLs and blood lymphocytes loaded with the HCMV-peptide CRVLCCYVL. All Fabs proofed to be highly specific since they showed no binding properties towards HLA-C*07:02 positive LCLs and lymphocytes that were loaded with no peptide or a control peptide. Furthermore, only LCLs and lymphocytes expressing the HLA-C*07:02 allele could be stained by our Fabs after HCMV-peptide loading. LCLs and lymphocytes of different HLA-C alleles that were pulsed with the HCMV-peptide CRVLCCYVL or a control peptide were not bound by the selected Fabs. Using phage display technology, we identified 3 TCR-like antibody fragments that target specifically HLA-C*07:02 positive LCLs and lymphocytes when loaded with the IE-1 derived HCMV-peptide CRVLCCYVL. The Fabs 4/4, 7/3 and 13/2 seem to be highly specific and to our knowledge the described Fabs are the first TCR-like antibodies that target an HLA-C presented peptide. Further studies are underway to determine binding properties to HCMV infected fibroblasts and affinity of the identified HLA-C*07:02 restricted and HCMV-specific Fabs and to explore their therapeutic potential. Disclosures Held: Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Roche: Consultancy, Other: Travel support, Research Funding; Amgen: Research Funding; Acrotech: Research Funding; MSD: Consultancy. Stilgenbauer:AbbVie, AstraZeneca, Celgene, Gilead Sciences, Inc., GSK, Hoffmann La-Roche, Janssen, Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Preclinical verification of the efficacy by targeting peptide-linked liposomal nanoparticles for hepatocellular carcinoma therapy

The purpose of this study was to investigate the efficacy of targeting peptides chemotherapy to overcome adverse event in the conventional chemotherapy for human hepatocellular carcinoma. Previously we reported several cancer-targeting peptides that bind specifically to cancer cells and their vascular endothelia: L-peptide (anti-cancer cell membrane), RLLDTNRPLLPY; SP-94-peptide (anti-hepatoma cell membrane), SFSHHTPILP; PC5-52-peptide (anti-tumor endothelia), SVSVGMKPSPRP; and control peptide, RLLDTNRGGGGG. In this study, these peptides were linked to liposomal iron oxide nanoparticles to localize the targeted tumor cells and endothelia, and to dextran-coated liposomal doxorubicin (L-D) to treat nonobese diabetic severe combined immunodeficient mice bearing hepatoma xenografts. Our results showed that L-peptide-linked liposomal doxorubicin could inhibit tumor growth with very mild adverse events. Use of the control peptide led to a decrease in the xenograft size but also led to marked apoptotic change in the visceral organ. In conclusion, L-peptide-linked liposomal doxorubicin, SP-94-peptide, and PC5-52-peptide can be used for the treatment of hepatoma xenografts in nonobese diabetic severe combined immunodeficient mice with minimal adverse events.

Abstract 580: Acidic Domain of ApoB-100 and Other Short Negatively Charged Peptides Anchored to Lipoproteins Cause Hypertriglyceridemia in Mice

Human Apolipoprotein B-100 (ApoB-100) is the main protein of VLDL and LDL particles in plasma. It is a large 4536 amino acid protein with various structural domains, such as the LDL-receptor binding motif and multiple proteoglycan binding sites, all of which have a high positive charge. A search of the primary amino acid sequence of ApoB-100 revealed the presence of the following acidic domain: DMDEDDD (AA 3985-3991). We hypothesized that this motif could affect the interaction of ApoB-containing lipoproteins with liver and or possibly GPIHBP1, which also contains an acidic domain that interacts with the positive charged motifs on VLDL. We synthesized a fluorescent tagged DMDEDDD peptide and conjugated it at the N-terminus with two molecules of alpha-tocopherol to serve as an anchor to lipoprotein particles. After adding the peptide to human plasma, it was found to readily bind to all lipoproteins, as determined by FPLC analysis. VLDL incubated with the peptide showed an increased negative charge and lost its ability to bind to heparin as determined by chromatography on a HiTrap TM heparin column. The peptide injected in LDLr-KO mice (30 mg/kg) caused a rapid 3-fold increase in plasma triglycerides within 1 h and the triglyceride elevation persisted for at least 24 h. A similarly designed negative charged peptide attached to tocopherol (EEEEEEEE) also raised triglycerides in mice but a neutral charged control peptide (KEKEKEKE) did not. To understand the mechanism, we tested the above peptides for their ability to inhibit LPL, using an in vitro assay with intralipid as a substrate and found that negative charged peptides inhibited lipolysis (IC 50= 50 uM), but the neutral control peptide did not. We are now investigating other potential mechanisms for how acidic peptides may cause hypertriglyceridemia. In summary, a peptide based on an acidic domain on ApoB-100 appears to cause hypertriglyceridemia in mice by inhibition of LPL and possibly by interfering with proteoglycan binding of lipoproteins. These results may be relevant in understanding VLDL metabolism and for the design of therapeutic apolipoprotein mimetic peptides.