Antioxidant and anti-inflammatory protective effects of yellowtail (Seriola quinqueradiata) milt hydrolysates on human intestinal epithelial cells in vitro and dextran sodium sulphate-induced mouse colitis in vivo.

Milt is an underutilized fish processing by-product containing valuable nutrients for human health. Here, a gastrointestinal hydrolysate of degreased yellowtail (Seriola quinqueradiata) milt contained 70.6% arginine-rich protein, 20% nucleic acids, 7.1% minerals and 2.3% carbohydrates. Yellowtail milt hydrolysates (YMH) effectively attenuated the H2O2-induced burst of intracellular reactive oxygen species, plasma membrane impairment, loss of cell viability, interleukin 8 production and the expression of claudin-4 and occludin in Caco-2 cells with its protein fraction playing a greater antioxidant role than its nucleic acid fraction. YMH also significantly counteracted the tumor necrosis factor α- and interleukin 1ß-stimulated interleukin 8 production and cyclooxygenase-2 and inducible nitric oxide synthase expression in Caco-2 cells and inhibited the production of nitric oxide and proinflammatory mediators in lipopolysaccharide-stimulated RAW 264.7 cells depending on its protein fraction, rather than its nucleic acid fraction. YMH and a positive drug 5-aminosalicylic acid were intragastrically administered to C57BL/6 mice daily for 7 days during and after 4-day dextran sodium sulphate exposure. Based on clinical signs, colon histopathology and biochemical analysis of colonic tight junction proteins, mucus compositions and goblet cells, YMH ameliorated mouse colitis symptoms and intestinal epithelial barrier dysfunction more effectively than 5-aminosalicylic acid. According to myeloperoxidase activity, proinflammatory cytokines and NF-κB, YMH and 5-aminosalicylic acid exerted equivalent inhibitory effects on colonic and systemic inflammation. Overall, YMH have considerable antioxidant and anti-inflammatory efficacies to maintain gut health.

Research Progress on Exosomes in Osteonecrosis of the Femoral Head.

Osteonecrosis of the femoral head (ONFH) is a progressive disease that often necessitates hip replacement if hip preservation therapy fails. ONFH places a heavy economic burden and severe psychological pressure on patients. At present, ONFH is treated by either surgical or non-surgical methods. In clinical practice, stem cells combined with surgery has achieved some positive results, but many problems remain to be resolved. Exosomes are small vesicles of 30-150 nm, which are rich in various nucleic acids, proteins, and small molecules depending on the cells from which they are derived. A growing number of studies have found that exosomes play an important role in tissue damage repair. In comparison with stem cells, exosomes have lower immunogenicity. Also, exosomes can promote cell proliferation and inhibit tumor growth. In addition, exosomes can also be used as natural carriers of drugs. Many studies have shown that exosomes have therapeutic effects in hormone-induced ONFH. Exosomes have the effect of promoting vascular regeneration and show good application prospects in ONFH. Here, we present a review of studies on the application of exosomes in ONFH to provide a reference for future research.

SIDT1 plays a key role in type I IFN responses to nucleic acids in plasmacytoid dendritic cells and mediates the pathogenesis of an imiquimod-induced psoriasis model.

BACKGROUND: Type I IFN (IFN-I) is a family of cytokines involved in the pathogenesis of autoimmune and autoinflammatory diseases such as psoriasis. SIDT1 is an ER-resident protein expressed in the lymphoid lineage, and involved in anti-viral IFN-I responses in vivo, through an unclear mechanism. Herein we have dissected the role of SIDT1 in the natural IFN-producing cells, the plasmacytoid dendritic cells (pDC). METHODS: The function of SIDT1 in pDC was determined by silencing its expression in human primary pDC and GEN2.2 cell line. SIDT1 role in vivo was assessed using the imiquimod-induced psoriasis model in the SIDT1-deficient mice (sidt1-/-). FINDINGS: Silencing of SIDT1 in GEN2.2 led to a blockade of the IFN-I response after stimulation of TLR7 and TLR9, without affecting the pro-inflammatory responses or upregulation of maturation markers. We found that SIDT1 migrates from the ER to the endosomal and lysosomal compartments together with TLR9 after CpG stimulation, participating in the access of the TLR9-CpG complex to lysosome-related vesicles, and therefore mediating the activation of TBK1 and the nuclear migration of IRF7, but not of NF-κB. sidt1-/- mice showed a significant decrease in severity parameters of the imiquimod-induced acute psoriasis-like model, associated with a decrease in the production of IFN-I and IFN-dependent chemokines. INTERPRETATION: Our findings indicate that SIDT1 is at the cross-road between the IFN-I and the proinflammatory pathways and constitutes a promising drug target for psoriasis and other diseases mediated by IFN-I responses. FUNDING: This work was supported by the Consejería de Salud y Familias de la Junta de Andalucía (PIER_S1149 and C2_S0050) and Instituto de Salud Carlos III (PI18/00082 and PI21/01151), partly supported by European FEDER funds, and prior funding to MEAR from the Alliance for Lupus Research and the Swedish Research Council.

Safety and Efficacy of 48 Weeks REP 2139 or REP 2165, Tenofovir Disoproxil, and Pegylated Interferon Alfa-2a in Patients With Chronic HBV Infection Naïve to Nucleos(t)ide Therapy.

BACKGROUND & AIMS: Nucleic acid polymers (NAPs) inhibit assembly and secretion of hepatitis B virus (HBV) subviral particles. We performed an open-label, phase 2 study of the safety and efficacy of the NAPs REP 2139 or REP 2165 combined with tenofovir disoproxil fumarate (TDF) and pegylated interferon alfa-2a (pegIFN) in patients with chronic HBV infection who were negative for hepatitis B e antigen. METHODS: Following 24 weeks TDF therapy, 40 patients were randomly assigned to groups that received 48 weeks of experimental therapy (TDF + pegIFN + REP 2139-Mg or REP 2165-Mg) or 24 weeks of control therapy (TDF + pegIFN) followed by 48 weeks of experimental therapy. Patients were then followed for a treatment-free period of 48 weeks. Primary outcomes were the safety and tolerability of REP 2139-Mg or REP 2165-Mg in combination with TDF + pegIFN compared with TDF + pegIFN alone through the first 48 weeks of therapy and subsequently throughout 48 weeks of NAP-based combination therapy (treatment weeks 24-72 in the experimental group and weeks 48-96 in the control group). Secondary outcomes were reductions in hepatitis B surface antigen (HBsAg) in control and experimental groups over the first 48 weeks of the study and throughout 48 weeks of combination therapy and virologic control (HBsAg positive, HBV DNA below 2000 IU/mL, normal level of alanine aminotransferase) or functional cure (HBsAg below 0.05 IU/mL, HBV DNA target not detected, normal level of alanine aminotransferase) after removal of all therapy. RESULTS: Levels of HBsAg, anti-HBs, and HBV DNA did not differ significantly between the groups given REP 2139 vs REP 2165. PegIFN-induced thrombocytopenia (P = .299 vs controls) and neutropenia (P = .112 vs controls) were unaffected by NAPs (REP 2139 vs REP 2165). Increases in levels of transaminases were significantly more frequent (P < .001 vs controls) and greater (P = .002 vs controls) in the NAP groups (but did not produce symptoms), correlated with initial decrease in HBsAg, and normalized during therapy and follow-up. During the first 24 weeks of TDF and pegIFN administration, significantly higher proportions of patients in NAP groups had decreases in HBsAg to below 1 IU/mL (P < .001 vs control) and HBsAg seroconversion (P = .046 vs control). At the time patients completed the TDF + pegIFN + NAP regimen, HBsAg levels were 0.05 IU/mL or lower in 24/40 participants (all with seroconversion up to 233,055 mIU/mL). During 48 weeks of treatment-free follow-up, virologic control persisted in 13 of 40 participants (2 lost to follow-up after 24 weeks), whereas functional cure persisted in 14 of 40 participants (all completing 48 weeks of follow-up) with persistent HBsAg seroconversion. One participant had a viral rebound during follow-up with hepatic decompensation and was placed on TDF therapy. CONCLUSIONS: In a phase 2 randomized trial, we found that addition of NAPs to TDF + pegIFN did not alter tolerability and significantly increased rates of HBsAg loss and HBsAg seroconversion during therapy and functional cure after therapy. Clinicaltrials.gov no: NCT02565719.

Beyond Pegylated Interferon-Alpha: New Treatments for Hepatitis Delta.

Persistent coinfection with the hepatitis B/D viruses (HDV) represents the most severe form of viral hepatitis. Hepatitis D often leads to liver cirrhosis, hepatic decompensation, and hepatocellular carcinoma. The current treatment options are limited as only pegylated interferon-alpha (PEG-IFNa) has efficacy against HDV. However, treatment response is still unsatisfactory with 25-40% HDV RNA suppression after 1-2 years. In addition, late HDV RNA relapses have been described during long-term follow-up. Fortunately, new treatment options for patients with chronic hepatitis delta are now on the horizon. The hepatocyte entry inhibitor bulevirtide (formerly myrcludex B) and the farnesyl transferase inhibitor lonafarnib are currently explored in patients with chronic hepatitis delta in Phase 3 clinical studies. The nucleic acid inhibitor REP-2139-Ca and PEG-IFN-lambda are studied in Phase 2 trials. We here summarize data on the efficacy of these new antiviral drugs and the existing safety data on the treatment of HDV infection.

Subpollens delivery of Platanus acerifolia pollen allergen Pla a3 and nucleic acid into lungs and cells.

Pollen allergy is a very serious seasonal respiratory disease. However, there has been a lack of understanding how pollen allergens enter the body and act on cells. This study focused on the release, transport and characteristic of Pla a3 allergen of the Platanus acerifolia pollen. Pla a3 protein was purified by prokaryotic expression system for preparation of polyclonal antibody. The distribution and release of Pla a3 protein in pollen were observed by immunohistochemistry. Mice were immunized with purified Pla a3 protein and SPPs, respectively. The pathological examination of mouse lung tissue proved that SPPs, as a fine particle in the range of 0.1-1µm, can enter the deep part of the lung directly through the respiratory tract and led to inflammation. Furthermore, DAPI staining confirmed a certain amount of nucleic acids in SPPs. After incubation with SPPs for 6 h, the Pla a3 mRNA could be detected in A549 cells by PCR. This suggests that nucleic acid wrapped in SPPs could be delivered into A549 cells. These results could provide a new clue and experimental data accumulation for further study on the mechanism of pollen sensitization.

Structure and Composition Define Immunorecognition of Nucleic Acid Nanoparticles.

Nucleic acid nanoparticles (NANPs) have evolved as a new class of therapeutics with the potential to detect and treat diseases. Despite tremendous advancements in NANP development, their immunotoxicity, one of the major impediments in clinical translation of traditional therapeutic nucleic acids (TNAs), has never been fully characterized. Here, we describe the first systematically studied immunological recognition of 25 representative RNA and DNA NANPs selected to have different design principles and physicochemical properties. We discover that, unlike traditional TNAs, NANPs used without a delivery carrier are immunoquiescent. We show that interferons (IFNs) are the key cytokines triggered by NANPs after their internalization by phagocytic cells, which agrees with predictions based on the experiences with TNAs. However, in addition to type I IFNs, type III IFNs also serve as reliable biomarkers of NANPs, which is usually not characteristic of TNAs. We show that overall immunostimulation relies on NANP shapes, connectivities, and compositions. We demonstrate that, like with traditional TNAs, plasmacytoid dendritic cells serve as the primary interferon producers among all peripheral blood mononuclear cells treated with NANPs, and scavenger receptor-mediated uptake and endosomal Toll-like receptor signaling are essential for NANP immunorecognition. The TLR involvement, however, is different from that expected for traditional TNA recognition. Based on these results, we suggest that NANP technology may serve as a prototype of auxiliary molecular language for communication with the immune system and the modulation of immune responses.

Bacillus Calmette-Guerin, polysaccharide nucleic acid in the treatment of cutaneous and oral lichen planus.

The aim of this study was to evaluate the safety and efficacy of Bacillus Calmette-Guerin, polysaccharide nucleic acid (BCG-PSN) therapy in the treatment of oral and cutaneous LP. Twenty-four LP patients were included in this study and classified randomly into; Oral LP group (OLP), 11 patients and Cutaneous LP group (CLP), 13 patients. All patients received intradermal injections of BCG-PSN, twice weekly for three weeks. Patients with complete response were followed up for 3 months. The assessment in OLP was based on the reduction in the treated area, (Reticulation/Erythema/Ulceration) REU scoring system and numerical rating scale (NRS). CLP evaluated by the response to treatment as (complete, partial and no response) and visual analogue scale (VAS). There were highly significant differences in the diminution of lesion areas (p < .006), NRS scores (p < .001), REU score (p < .011), and VAS (p < .001) after treatment. The majority of patients achieved complete response after 3-week management. The BCG-PNS is safe and effective in the treatment of oral and cutaneous LP.

Safety and efficacy of REP 2139 and pegylated interferon alfa-2a for treatment-naive patients with chronic hepatitis B virus and hepatitis D virus co-infection (REP 301 and REP 301-LTF): a non-randomised, open-label, phase 2 trial.

BACKGROUND: REP 2139 clears circulating hepatitis B virus (HBV) surface antigen (HBsAg), enhancing the restoration of functional control of HBV infection by immunotherapy. We assessed the safety and efficacy of REP 2139 and pegylated interferon alfa-2a in patients with chronic HBV and hepatitis D virus (HDV) co-infection. METHODS: In this open-label, non-randomised, phase 2 trial, patients aged 18-55 years, who were treatment naive, hepatitis B e antigen [HBeAg] negative, anti-hepatitis D antigen [HDAg] positive, and HDV RNA positive, with serum HBsAg concentrations of more than 1000 IU/mL, and a history of HDV infection for 6 months or more before treatment, were recruited at Toma Ciorba Hospital of Infectious Diseases in Chișinau, Moldova. Patients were excluded if they had HDV superinfection, liver infections other than HBV and HDV, or liver cirrhosis. Patients received 500 mg intravenous REP 2139 once per week for 15 weeks, followed by combined therapy with 250 mg intravenous REP 2139 and 180 µg subcutaneous pegylated interferon alfa-2a once per week for 15 weeks, then monotherapy with 180 µg pegylated interferon alfa-2a once per week for 33 weeks. The primary endpoints assessed at the end of treatment were the safety and tolerability of the treatment regimen, analysed in the intention-to-treat population. Secondary outcomes included the proportion of patients with serum HBsAg less than 50 IU/mL, the proportion of patients with suppressed HBV DNA, and the proportion of patients who maintained these responses through follow-up. The REP 301 trial is registered with ClinicalTrials.gov, number NCT02233075. We also did an additional follow-up at 1 year after the end of treatment, as an interim analysis of the REP 301-LTF trial (planned duration 3 years), registered with ClinicalTrials.gov, number NCT02876419, which is ongoing but not recruiting patients. FINDINGS: Between Sept 8, 2014, and Jan 27, 2015, we enrolled 12 patients into the REP 301 study. All 12 patients experienced at least one adverse event during treatment: two (17%) patients experienced anaemia, eight (67%) neutropenia, and ten (83%) thrombocytopenia. Five (42%) patients had raised alanine aminotransferase levels, four (33%) had raised aspartate aminotransferase levels, and two (17%) had increased bilirubin concentrations. Four (33%) patients had a serious adverse event, and 12 (100%) patients had treatment-emergent lab abnormalities. Six patients had HBsAg levels less than 50 IU/mL by the end of treatment (all <0·05 IU/mL); five maintained this level of suppression at the end of 1 year follow-up. Six patients had hepatitis B surface antibody (anti-HBs) titres above 10 mIU/mL at the end of treatment (five had maximum anti-HBs concentrations of 7681-86 532 mIU/mL during treatment), which were maintained at the end of 1 year follow-up in these five patients. Elevated alanine and aspartate aminotransferase concentrations and profound elevations of anti-HBs titres were restricted to patients who had HBsAg levels of less than <1 IU/mL before the introduction of pegylated interferon alfa-2a. Nine patients had suppressed HBV DNA (<10 IU/mL]) at the end of treatment, which was maintained by seven patients and newly established in an eighth patient at the end of 1 year follow-up. 11 patients became HDV RNA negative during treatment, with nine remaining HDV RNA negative at the end of treatment; seven of these patients remained HDV RNA negative by the end of 1 year follow-up. By the end of 1 year follow-up, normalisation of serum aminotransferases occurred in nine of 12 patients. INTERPRETATION: Combined REP 2139 and pegylated interferon alfa-2a therapy is safe, well tolerated, and establishes functional control of HBV and HDV co-infection and normalisation of serum aminotransferases in a high proportion of patients 1 year after therapy. This combination therapy approach might provide a new treatment option for patients with HBV and HDV co-infection. FUNDING: Replicor.

Therapeutic Effects of Ribunucleinate (Ribonucleotides) in Immuno-Inflammatory and Arthritic Diseases.

Ribonucleic acids from different organs and from yeast have been used for the treatment of chronic and degenerative diseases in the context of naturopathic medicine in the last 60 years. This chapter provides general information about ribonucleinates as therapeutic agents. Past and present pharmacological and clinical investigations are discussed in the field of the central nervous system, sensory organs, cancer and degenerative diseases of joints and vertebra.

Strategy for selecting nanotechnology carriers to overcome immunological and hematological toxicities challenging clinical translation of nucleic acid-based therapeutics.

INTRODUCTION: Clinical translation of nucleic acid-based therapeutics (NATs) is hampered by assorted challenges in immunotoxicity, hematotoxicity, pharmacokinetics, toxicology and formulation. Nanotechnology-based platforms are being considered to help address some of these challenges due to the nanoparticles' ability to change drug biodistribution, stability, circulation half-life, route of administration and dosage. Addressing toxicology and pharmacology concerns by various means including NATs reformulation using nanotechnology-based carriers has been reviewed before. However, little attention was given to the immunological and hematological issues associated with nanotechnology reformulation. AREAS COVERED: This review focuses on application of nanotechnology carriers for delivery of various types of NATs, and how reformulation using nanoparticles affects immunological and hematological toxicities of this promising class of therapeutic agents. EXPERT OPINION: NATs share several immunological and hematological toxicities with common nanotechnology carriers. In order to avoid synergy or exaggeration of undesirable immunological and hematological effects of NATs by a nanocarrier, it is critical to consider the immunological compatibility of the nanotechnology platform and its components. Since receptors sensing nucleic acids are located essentially in all cellular compartments, a strategy for developing a nanoformulation with reduced immunotoxicity should first focus on precise delivery to the target site/cells and then on optimizing intracellular distribution.

Efficacy and safety of MCNA in patients with nonmuscle invasive bladder cancer at high risk for recurrence and progression after failed treatment with bacillus Calmette-Guérin.

PURPOSE: Patients with high risk recurrences after bacillus Calmette-Guérin failure have limited options. We performed an open label study to evaluate the efficacy and safety of intravesical MCNA in this setting. MATERIALS AND METHODS: Patients were treated intravesically with 8 mg MCNA weekly for 6 weeks followed by 3 weekly instillations at months 3, 6, 12, 18 and 24. Cystoscopy and cytology were performed every 3 months for 2 years with mandatory biopsy at 6 months and as clinically indicated thereafter. The primary efficacy end point was the disease-free survival rate at 1 year. RESULTS: A total of 129 patients were enrolled in study, including 91 with carcinoma in situ with or without papillary disease and 38 with papillary only tumors. Most patients had high risk disease. A total of 107 cases were bacillus Calmette-Guérin refractory and 2 or more prior bacillus Calmette-Guérin induction courses had been given in 68. Median followup in all patients was 34.7 months. The overall disease-free survival rate was 25.0% at 1 year and 19.0% at 2 years. In patients with papillary only tumors the disease-free survival rate was 35.1% and 32.2% at 1 and 2 years, respectively. The median disease-free duration in the 30 responders was 32.7 months. The progression-free survival rate was 87.3%, 79.8% and 77.7% at 1, 2 and 3 years, respectively, with a progression event in 28 patients. MCNA was well tolerated and few adverse events led to treatment discontinuation. CONCLUSIONS: Intravesical MCNA achieved significant activity in patients at high risk with nonmuscle invasive bladder cancer in whom bacillus Calmette-Guérin treatment failed, especially those with papillary only tumors and those with bacillus Calmette-Guérin relapse. A durable response was seen, particularly in patients with a response at 1 year. MCNA offers an option for patients who are not candidates for or who refuse cystectomy.

Nucleic acids-based therapeutics in the battle against pathogenic viruses.

For almost three decades, researchers have studied the possibility to use nucleic acids as antiviral therapeutics. In theory, compounds such as antisense oligonucleotides, ribozymes, DNAzymes, and aptamers can be designed to trigger the sequence-specific inhibition of particular mRNA transcripts, including viral genomes. However, difficulties with their efficiency, off-target effects, toxicity, delivery, and stability halted the development of nucleic acid-based therapeutics that can be used in the clinic. So far, only a single antisense drug, Vitravene for the treatment of CMV-induced retinitis in AIDS patients, has made it to the clinic. Since the discovery of RNA interference (RNAi), there is a renewed interest in the development of nucleic acid-based therapeutics. Antiviral RNAi approaches are highly effective in vitro and in animal models and are currently being tested in clinical trials. Here we give an overview of antiviral nucleic acid-based therapeutics. We focus on antisense and RNAi-based compounds that have been shown to be effective in animal model systems.

Safety considerations for nucleic acid vaccines.

Whilst it is premature to formulate guidelines for the manufacturing requirements of a nucleic acid vaccine, it can never be too early to discuss and debate the issues surrounding the use of a novel biotherapeutic. The major safety issues posed by nucleic acid vaccination include the possibility of transformation (or tumorigenic) events in recipients of a DNA vaccine, the potential formation of anti-DNA antibodies, and unexpected and untoward effects of long-term expression of a foreign antigen. This paper examines the extent to which these points impinge on the safety of DNA vaccines.