Induction of CD73 prevents death after emergency open aortic surgery for a ruptured abdominal aortic aneurysm: a randomized, double-blind, placebo-controlled study.

Mortality remains high after emergency open surgery for a ruptured abdominal aortic aneurysm (RAAA). The aim of the present study was to assess, if intravenous (IV) Interferon (IFN) beta-1a improve survival after surgery by up-regulating Cluster of differentiation (CD73). This is a multi-center phase II double-blind, 2:1 randomized, parallel group comparison of the efficacy and safety of IV IFN beta-1a vs. placebo for the prevention of death after open surgery for an infra-renal RAAA. All study patients presented a confirmed infra-renal RAAA, survived the primary emergency surgery and were treated with IFN beta-1a (10 µg) or matching placebo for 6 days after surgery. Major exclusion criteria included fatal hemorrhagic shock, chronic renal replacement therapy, diagnosed liver cirrhosis, severe congestive heart failure, advanced malignant disease, primary attempt of endovascular aortic repair (EVAR), and per-operative suprarenal clamping over 30 min. Main outcome measure was all-cause mortality at day 30 (D30) from initial emergency aortic reconstruction. The study was pre-maturely stopped due to a reported drug-drug interaction and was left under-powered. Out of 40 randomized patients 38 were included in the outcome analyses (27 IFN beta-1a and 11 placebo). There was no statistically significant difference between treatment groups at baseline except more open-abdomen and intestinal ischemia was present in the IFN beta-1a arm. D30 all-cause mortality was 22.2% (6/27) in the IFN beta-1a arm and 18.2% (2/11) in the placebo arm (OR 1.30; 95% CI 0.21-8.19). The most common adverse event relating to the IFN beta-1a was pyrexia (20.7% in the IFN beta-1a arm vs. 9.1% in the placebo arm). Patients with high level of serum CD73 associated with survival (P = 0.001) whereas the use of glucocorticoids and the presence of IFN beta-1a neutralizing antibodies associated with a poor CD73 response and survival. The initial aim of the trial, if postoperative INF beta-1a treatment results on better RAAA survival, could not be demonstrated. Nonetheless the anticipated target mechanism up-regulation of CD73 was associated with 100% survival. According to present results the INF beta-1a induced up-regulation of serum CD73 was blocked with both use of glucocorticoids and serum IFN beta-1a neutralizing antibodies. The study was pre-maturely stopped due to interim analysis after a study concerning the use if IV IFN beta-1a in ARDS suggested that the concomitant use of glucocorticoids and IFN beta-1a block the CD73 induction. Trial registration: ClinicalTrials.gov NCT03119701. Registered 19/04/2017 (retrospectively registered).

Effects of human recombinant type I IFNs (IFN-α2b and IFN-ß1a) on growth and migration of primary endometrial stromal cells from women with deeply infiltrating endometriosis: A preliminary study.

OBJECTIVE(S): Endometriosis is a major cause of infertility and disability for women, caused by the presence of inflammatory endometrial implants in extrauterine locations. Among the constituents involved in the immune response during the development of endometriosis, several chemokines, including interferons (IFNs) may have a role in the pathogenesis of this disease. The aim of this preliminary study was to investigate the anti-proliferative and anti-migratory activities of type I IFNs (IFN-α2b and IFN-ß1a) in primary endometrial stromal cells (ESCs) isolated from women with deeply infiltrating endometriosis (DIE). STUDY DESIGN: The study subjects included 7 women ranged in the age from 27 to 37 years with diagnosis of DIE (Stage III and IV). Collected primary ESC monolayers, isolated from endometriotic nodules, were incubated with various concentrations (from 1 to 1000 IU/ml) of IFN-α2b or IFN-ß1a. RESULT(S): IFN-ß1a had a significantly higher activity in hampering the proliferation of cells compared to IFN-α2b. This effect could be related to the induction of apoptosis and cell cycle arrest in S phase, observed in ESCs during incubation with IFN-ß1a. Moreover, IFN-ß1a was more potent than IFN-α2b in inhibiting migration and EGF-induced ERK activity of primary ESCs. CONCLUSION(S): The inhibitory in vitro effect on ESC proliferation and migration of IFN-ß1a was much more potent than IFN-α2b. These preliminary data offer the rationale for future preclinical and clinical trials using IFN-ß1a as a new tool for the therapy and tertiary prevention in patients with DIE.

The cross-reactivity of binding antibodies with different interferon beta formulations used as disease-modifying drugs in multiple sclerosis patients.

Interferon beta (IFNb) preparations are commonly used as first-line therapy in relapsing-remitting multiple sclerosis (RRMS). They are, however, characterized by limited efficacy, partly due to the formation of anti-IFNb antibodies in patients.In this pilot study, we assessed with the ELISA method the presence of the binding antibodies (BAbs) against interferon beta after 2 years of therapy with subcutaneous interferon beta 1a (Rebif) in 49 RRMS patients. Antibody levels were established again within 1 year after treatment withdrawal. We used 3 interferons that are commercially available for MS therapy, namely Avonex (Biogen Idec Limited), Rebif (Merck Serono), and Betaferon (Bayer Pharma AG), as antigens.BAbs reacting with Rebif were found in 24.4% to 55% of patients, depending on the units of their expression. The levels of anti-Rebif antibodies remained high in 8 patients and in 4 patients they dropped significantly. Strong correlations were obtained in all assays (anti-Rebif-anti-Avonex, anti-Rebif-anti-Betaferon, and anti-Betaferon-anti-Avonex) and the existence of cross-reactivity in the formation of antibodies against all the tested formulations of interferon beta was confirmed. The levels of BAbs remain significant in the clinical context, and their assessment is the first choice screening; however, methods of BAbs evaluation can be crucial for further decisions. More studies are needed to confirm our results; specifically it would be of interest to evaluate methods of neutralizing antibodies identification, as we only assessed the binding antibodies. Nevertheless, our results support the concept that in interferon nonresponders, that are positive for binding antibodies, switching the therapy to alternative disease-modifying agent (for example glatiramer acetate, fingolimod, or natalizumab) is justified, whereas the switch to another interferon formulation will probably be of no benefit.

Herpesvirus-Associated Lymphadenitis Distorts Fibroblastic Reticular Cell Microarchitecture and Attenuates CD8 T Cell Responses to Neurotropic Infection in Mice Lacking the STING-IFNα/ß Defense Pathways.

Type I IFN (IFN-α/ß)-driven immune responses to acute viral infection are critical to counter replication and prevent dissemination. However, the mechanisms underlying host resistance to HSV type 1 (HSV-1) are incompletely understood. In this study, we show that mice with deficiencies in IFN-α/ß signaling or stimulator of IFN genes (STING) exhibit exacerbated neurovirulence and atypical lymphotropic dissemination of HSV-1 following ocular infection. Synergy between IFN-α/ß signaling and efficacy of early adaptive immune responses to HSV-1 were dissected using bone marrow chimeras and adoptive cell transfer approaches to profile clonal expansion, effector function, and recruitment of HSV-specific CD8(+) T cells. Lymphotropic viral dissemination was commensurate with abrogated CD8(+) T cell responses and pathological alterations of fibroblastic reticular cell networks in the draining lymph nodes. Our results show that resistance to HSV-1 in the trigeminal ganglia during acute infection is conferred in part by STING and IFN-α/ß signaling in both bone marrow-derived and -resident cells, which coalesce to support a robust HSV-1-specific CD8(+) T cell response.

Immunogenicity of Recombinant Human Interferon Beta-1b in Immune-Tolerant Transgenic Mice Corresponds with the Biophysical Characteristics of Aggregates.

Determining to what extent biophysical characteristics of aggregates affect immunogenicity of therapeutic interferon beta-1b. Three recombinant human interferon beta-1b (rhIFNß-1b) samples with different levels of aggregates generated by copper oxidation, thermal stress, or left untreated, as well as Avonex(®) drug substance and Betaferon(®) drug product, were injected intraperitoneally in nontransgenic and interferon beta transgenic FVB/N mice 5 times per week for 3 weeks. Antibodies against interferon beta were measured using enzyme-linked immunosorbent assay. UV and fluorescence spectroscopy, dynamic light scattering, size exclusion chromatography, reversed-phase high-performance liquid chromatography (RP-HPLC), fluid imaging microscopy, and resonant mass measurement, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, were used to characterize and quantitate aggregates in the 3 rhIFNß preparations, to correlate biophysical characteristics with immunogenicity. In immune-tolerant interferon beta transgenic FVB/N mice, Betaferon drug product showed the highest immunogenicity, while Avonex drug substance showed the lowest level of immunogenicity. Of the 3 forms of rhIFNß-1b, copper-oxidized rhIFNß-1b showed lower immunogenicity than thermally stressed rhIFNß-1b, despite containing larger aggregates. Both copper-oxidized rhIFNß-1b and thermally stressed rhIFNß-1b exhibited changes in protein structure as shown using fluorescence spectroscopy and RP-HPLC. Nontransgenic, nonimmune-tolerant FVB/N mice generated high antibody titers against all interferon beta samples tested. The level of immunogenicity and the breaking of tolerance in FVB/N transgenic mice are not only related to the level of aggregation but also depend on the size and structure of the aggregates.

[Preparation of monoclonal antibody against recombinant human interferon-ß1a].

OBJECTIVE: To prepare the monoclonal antibody (mAb) against recombinant human interferon-ß1a (rhIFN-ß1a). METHODS: BALB/c mice were immunized with purified rhIFN-ß1a. The spleen lymphocyte cells from the one whose blood specific antibody titer exceeded 1:10 000 were fused with Sp2/0 cells. The fused cells went through HAT selection, serial dilution, and positive selection until stable hybridoma cell lines were obtained. The equilibrium dissociation constant (K(D)) of antibodies was calculated based on ELISA result. The cell line with the best KD value was cultured in a stirred-tank bioreactor with a fed-batch strategy to produce secreted anti-rhIFN-ß1a monoclonal antibody. The mAb released into the cell culture supernatant were purified by ultrafiltration and protein-G affinity chromatography. RESULTS: Thirteen hybridoma cell lines which stably produced anti-rhIFN-ß1a antibody were retrieved from standard hybridoma fusion and selection procedures. One of the 13 cell lines, 1E8 with the KD value of 4.6 × 10⁻9 mol/L was cultured and antibody titer in the cell culture supernatant reached 1:5000. The mAb with 90% purity was recovered from the two-steps purification. CONCLUSION: The anti-rhIFN-ß1a mAb with high purity has been successfully obtained.