IFN-α 2a Research Articles

Successfully Treated Giant Conjunctival Papilloma

An 18-year-old man presented with enlarging masses in his right eye for months. Anterior segment examination revealed multiple pedunculated lesions extending the lower palpebral conjunctiva without corneal involvement (A). The fellow eye was uninvolved. We performed excisional biopsy combined with cryotherapy and injected interferon α-2a (IFN-α 2a) in subconjunctival space during the operation. The histopathological examination revealed papillary, well-differentiated, acanthotic nonkeratinized squamous epithelial cells with fibrovascular cores, which was compatible with conjunctival papilloma (B). After excision (C), we kept topical IFN-α 2a 4 times daily for 2 months. Three years later, no recurrence developed (D). (Magnified version of Figure A-D is available online at www.aaojournal.org).

The potential roles of type I interferon activated neutrophils and neutrophil extracellular traps (NETs) in the pathogenesis of primary Sjögren’s syndrome

ObjectiveNeutrophils and aberrant NETosis have been implicated in the pathogenesis of diverse autoimmune diseases; however, their roles in primary Sjögren’s syndrome (pSS) remain unclear. We aimed to reveal the potential roles of neutrophils and neutrophil extracellular traps (NETs) in pSS.MethodspSS patients were enrolled and NETosis markers were measured in plasma and labial glands using ELISA and immunofluorescence. The gene signatures of neutrophils were assessed by RNA-Seq and RT-PCR. Reactive oxygen species (ROS), mitochondrial ROS (MitoSOX) production, and JC-1 were measured by flow cytometry.ResultsNETosis markers including cell-free DNA (cf-DNA) and myeloperoxidase (MPO) in plasma and labial glands from pSS patients were significantly higher than healthy controls (HCs) and were associated with disease activity. RNA sequencing and RT-qPCR revealed activated type I IFN signaling pathway and higher expression of genes related to type I interferon in pSS neutrophils. Further stimulating with IFN-α 2a in vitro significantly induced ROS production and JC-1 monomer percentage in pSS neutrophils.ConclusionsOur data suggest the involvement of neutrophils and enhanced NETosis in pSS patients. Further mechanism study in vitro revealed that type I IFN activation in pSS neutrophils led to mitochondrial damage and related ROS production which finally result in the generation of NETs.

Influence of gender on cytokine induced depression and treatment: Gender aspects of IFN-α-induced depression

BackgroundCytokine treatment with Interferon-alpha (IFN-α) represents a clinical model of immune associated depression, but it remains unclear if it is of the same entity as major depressive disorder (MDD). The study focuses on possible gender differences in IFN-α induced depression and effects of a pre-emptive antidepressant treatment. MethodsData from 181 patients with chronic hepatitis C infection (cHC) without history of mental illnesses undergoing treatment with IFN-α 2a and ribavirin were re-analyzed for gender effects. Patients with a pre-emptive antidepressant therapy with Escitalopram (n = 90, verum group) to prevent IFN-induced depression were compared to patients who received placebo (n = 91). Depressive symptoms before and during HCV-treatment were assessed using the Montgomery–Asberg Depression Rating Scale (MADRS), Beck's Depression Inventory (BDI) and the Hamilton Anxiety Rating Scale. ResultsWe found significant differences regarding the incidence and severity of depressive symptoms between men and women for patients without antidepressant pre-treatment (placebo group). Significantly more women without pre-emptive antidepressant therapy suffered from clinically relevant depression (MADRS values ≥ 13, p = 0.041) and self-rated depressive symptoms (BDI ≥ 17, p = 0.024). Antidepressant pre-treatment showed comparable effects regarding the reduction of incidence and severity of depression in both women and men. ConclusionsCompared to MDD, IFN-alpha-induced depression in patients with cHC is also characterized by gender differences with an increased risk for women but no gender difference regarding the effects of an antidepressant pre-treatment is found. Our data strengthens the hypothesis that Interferon-induced depression serves as a clinical model for immune related depressive disorders.

Time-Resolved Effect of Interferon-Alpha 2a on Activities of Nuclear Factor Kappa B, Pregnane X Receptor and on Drug Disposition Genes.

Interferon-alpha (IFN-α) is suggested to cause pharmacokinetic drug interactions by lowering expression of drug disposition genes through affecting the activities of nuclear factor kappa B (NF-ĸB) and pregnane X receptor (PXR). The time-resolved impact of IFN-α 2a (1000 U/mL; 5000 U/mL; 2 h to 30 h) on the activities of NF-ĸB and PXR and mRNA expression (5000 U/mL; 24 h, 48 h) of selected drug disposition genes and on cytochrome P450 (CYP3A4) activity in LS180 cells (5000 U/mL; 24 h, 48 h) was evaluated using luciferase-based reporter gene assays, reverse transcription polymerase chain reaction, and luminescence-based CYP3A4 activity assays. The cross-talk between NF-ĸB activation and PXR suppression was evaluated by NF-ĸB blockage (10 µM parthenolide). IFN-α 2a initially (2 h, 6 h) enhanced NF-ĸB activity 2-fold and suppressed PXR activity by 30%. mRNA of CYP3A4 was halved, whereas UGT1A1 was increased (1.35-fold) after 24 h. After 48 h, ABCB1 expression was increased (1.76-fold). CYP3A4 activity remained unchanged after 24 h, but was enhanced after 48 h (1.35-fold). IFN-α 2a demonstrated short-term suppressive effects on PXR activity and CYP3A4 mRNA expression, likely mediated by activated NF-ĸB. Longer exposure enhanced CYP3A4 activity. Clinical trials should evaluate the relevance by investigating the temporal effects of IFN-α on CYP3A4 using a sensitive marker substrate.

Site-specific monoPEGylated interferon alpha2a mediated by microbial transglutaminase

PEGylation is considered one of the most successful techniques to improve the characteristics of protein drugs including to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. One known PEG modification method is to attach PEG to the free amino group, typically at lysine residues or at the N-terminal amino acid with no selectivity, resulting in a heterogeneous product mixture. This lack of selectivity can present problems when a therapeutic PEGylated protein is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic PEGylation of proteins is one route to overcome this limitation. Transglutaminases (TGase) are enzyme candidates for site-specific PEGylation. We use human interferon alpha 2a (IFN α2a) as a test case, and predict that the potential modification residues are Gln101 by computational approach as it contains 12 potential PEGylation sites. IFN α2a was PEGylated by Y shaped PEG40k-NH2 mediated by microbial transglutaminase. Our results show that the microbial transglutaminase mediated PEGylation of IFN α2a was site-specific only at the site of Gln101 in IFN α2a, yielding the single mono-conjugate PEG-Gln101-IFN α2a with a mass of 59 374.66 Da. Circular dichroism studies showed that PEG-Gln101-IFN α2a preserved the same secondary structures as native IFN α2a. As expected, the bioactivity and pharmacokinetic profile in rats of PEG-Gln101-IFN α2a revealed a significant improvement to unmodified IFN α2a, and better than PEGASYS.

Delivery of TLR7 agonist to monocytes and dendritic cells by DCIR targeted liposomes induces robust production of anti-cancer cytokines.

Cancer immunotherapy is a powerful new tool in the oncologist's therapeutic arsenal, with our increased knowledge of anti-tumor immunity providing many new targets for intervention. Monocytes and dendritic cells (DCs) are attractive targets for enhancing the anti-tumor immune response, but systemic delivery of immunomodulators has proven to be associated with a high risk of fatal adverse events due to the systemic activation of the immune system. We address this important obstacle by targeting the delivery of an immunomodulator, a Toll-like receptor agonist, to DCs and monocytes in the bloodstream. We thus focus the activation, potentially avoiding the above-mentioned adverse effects, and demonstrate greatly increased ability of the agonist to induce secretion of anti-cancer cytokines.

Combination Treatment with RG7112 and Pegylated Interferon α 2a Specifically Targets JAK2V617F Positive Progenitor Cells From Patients with Myeloproliferative Neoplasms.

Abstract 2865▪▪This icon denotes a clinically relevant abstractWe have recently reported that MDM2 levels are increased in PV CD34+ cells as a consequence of JAK2617F, while the p53 levels are reduced in CD34+ cells from both patients with PV and PMF. These observations led us to explore therapeutic approaches by which to up-regulate p53 as a strategy to treat MPN patients. We previously reported that combination treatment with low doses of Peg IFNα 2a and Nutlin-3, an antagonist of MDM2, induced PV CD34+ apoptosis and inhibited PV colony formation significantly. The combination of these agents also decreased the proportion of JAK2V617F-positive HPCs. We demonstrated that these two drugs affect p53 through different pathways with Peg IFNα 2a activating p38 MAP kinase and STAT1 leading to increased p53 transcription while nutlin-3 prevents the degradation of p53 (Lu et al, Blood, 2012; In Press).RG7112 is an orally bioactivity small molecule inhibitor of p53-MDM2 binding which activates the p53 pathway. RG7112 is currently being evaluated in several phase 1 cancer trials. Based on our previous data, we hypothesized that RG7112 would be an effective drug to combine with low doses of IFN alpha to treat MPN patients. In order to evaluate the therapeutic effects of RG7112 alone or in combination with Peg IFNα 2a on hematopoietic progenitor cells from MPN patients, CD34+ cells were isolated from patients with PV and PMF patients and were treated with varying of doses of RG7112 alone or/and in combination with a low dose of Peg IFNα 2a and its effects on PV CD34+ cell proliferation, apoptosis and colony formation was evaluated. The effects of this drug on MPN CD34+ cells was compared to its effects on cord blood CD34+ cells After treatment with RG7112 at doses ranging from 100 nM to 5000 nM for 4 days, the numbers of PV CD34+ cells were reduced to a far greater degree (about 60%) than that observed with cord blood CD34+ cells (90% and 80%) at the doses between 200 and 500 nM indicating that low doses of RG7112 selectively affects MPN HPCs. 200nM of RG7112 and 200 U/ml of Peg-IFNα 2a alone induced cell cycle arrest of PV CD34+ cells, and combination treatment significantly promoted apoptosis of MPN CD34+ cells (2.5 folds). This observation leads us to examine the possibility that low doses of RG7112 might be used in combination with Peg-IFNα 2a to treat MPN patients. Treatment with 200 nM of RG7112 alone decreased PV CFU-GM and BFU-E-derived colony formation by 20% while combination treatment with 200 U/ml of Peg-IFNα 2a decreased PV CFU-GM and BFU-E-derived colony formation by 60% and 70%, respectively. Similarly, treatment with 200 nM of RG7112 alone decreased PMF CFU-GM and BFU-E-derived colony formation by 20% and 40%, respectively, while combination treatment with 200 U/ml of Peg-IFNα 2a decreased PMF CFU-GM and BFU-E-derived colony formation by 60% and 80%, respectively. We also found that treatment with both agents in combination also decreased the size of the hematopoietic colonies. By contrast, treatment with the same low doses of RG7112 and Peg-IFNα 2a alone or in combination did not significantly affect the ability of normal CD34+ cells to generate CFU-GM and BFU-E-derived colonies. Combined treatment with low doses of RG7112 and Peg-IFNα 2a decreased the total number of JAKV617F-positive PV HPC by 9% - 25% (p=0.014) leading to an increased proportion of colonies with wild type JAK2. In conclusion, these data demonstrate that IFNα plus RG7112 specifically target malignant MPN HPC by activating p53 in vitro, which we propose can be exploited therapeutically to eliminate JAK2V617F positive HPC in both PV and PMF patients. These results strongly suggest that combinations of low doses of IFNα and RG7112 might serve as a novel therapeutic strategy for the long term treatment of MPN patients. Disclosures:No relevant conflicts of interest to declare.

P53 as a target in myeloproliferative neoplasms

The Philadelphia chromosome negative myeloproliferative neoplasms (MPN) originate at the level of the pluripotent hematopoietic stem cell (HSC). JAK2V617F is present in virtually all patients with polycythemia vera (PV) as well as 50-60% of patients with primary myelofibrosis (PMF) and essential thrombocythemia (ET) [1]. Recently, Nakatake and coworkers demonstrated that JAK2V617F alters p53 responses to DNA damage by upregulating La antigen that increases MDM2 protein translation [2]. p53 plays a pivotal role in the maintenance of cell integrity in response to a variety of stresses by controlling target genes that regulate cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. Based on these roles, p53 has been proposed as a promising molecular target for the treatment of a variety of cancers. Although about 50 % of cancers contain mutated forms of p53 which lead to loss of function, wild type p53 is universally present in PV, by contrast, p53 mutations have been identified exclusively in patients undergoing transformation to acute leukemia. The cellular levels of p53 are controlled by the rate at which it is degraded. MDM2 is the master regulator of p53, it regulates p53 levels through a negative feedback loop. MDM2 not only facilitates p53 degradation, but also binds p53 and inhibits its transcription. We have recently shown that MDM2 levels are increased in PV CD34+ cells while p53 mRNA levels are lower [3]. These observations lead to the exploration of therapeutic strategies to up-regulate p53 for the treatment of PV patients. Nutlins are small-molecule antagonists of MDM2, which specifically bind to MDM2, blocking MDM2-p53 interactions, resulting in p53 stabilization, accumulation and activation. This approach has been shown to inhibit tumor growth in a non-genotoxic manner in xenograft murine tumor models [4, 5]. MDM2 antagonists have the potential to be potent weapons to treat cancers containing wild type p53. Recently, small-molecule inhibitors of JAK2 inhibitors have been shown to be effective in treating patients with advanced forms of myelofibrosis resulting in a reduction in the degree of splenomegaly and improvement in systemic symptoms but unfortunately the progeny of the malignant clone has not been documented to be substantially affected [6]. By contrast, interferon (IFNα) has been reported to reverse morphological marrow abnormalities, eliminate cytogenetic abnormalities, reduce or eliminate cells with JAK2V617F and result in the re-establishment of polyclonal hematopoiesis in selected patients with PV, essential thrombocythemia (ET) and early forms of primary myelofibrosis (PMF) [7]. We previously determined that IFNα specific targets PV JAK2V617F positive hematopoietic progenitor cells (HPC). IFNα activates a p38 mitogen-activated protein kinase (MAP kinase) resulting in apoptosis of PV HPC [8]. IFNα binds to the type I IFN receptor, and activates the JAK/TYK/STAT pathway, leading to multiple downstream events. Both the STAT1 and p38 MAPK pathways activate p53 [9]. Frequently protracted therapy of PV patients with IFNα is not possible due to a variety of adverse events necessitating its discontinuation. Since many of these adverse events are dose dependent, the identification of drugs which could be combined together with low doses of IFN would potentially provide a means of treating greater numbers of PV patients for longer periods of time. We recently reported that combination treatment with sub-therapeutic doses of Peg IFNα 2a and Nutlin-3 significantly inhibited the proliferation and induced apoptosis in PV CD34+ cells as compared to each agent alone [3]. We also found that the combination of these agents at low doses decreased the proportion of JAK2V617F-positive HPCs. Both of these drugs affect p53 through two distinct pathways with Peg IFNα 2a activating p38 MAP kinase and STAT1 leading to increased p53 transcription and nutlin-3 prevents the degradation of p53 [3, 8]. These results strongly suggest that combinations of low doses of IFNα and nutlin-3 might serve as a novel therapeutic strategy for the long term treatment of PV patients. RG7112 is a novel drug which acts as a selective inhibitor of p53-MDM2 binding and frees p53 from negative control, activating the p53 pathway in cancer cells. RG7112 is currently being evaluated in several clinical trials [10]. We predict that combination treatment with low doses of RG7112 or other second generation MDM2 antagonists will provide a promising strategy to treat a variety of blood cancers including PV.

Combination treatment in vitro with Nutlin, a small-molecule antagonist of MDM2, and pegylated interferon-α 2a specifically targets JAK2V617F-positive polycythemia vera cells

AbstractInterferon (IFN-α) is effective therapy for polycythemia vera (PV) patients, but it is frequently interrupted because of adverse events. To permit the long-term use of IFN, we propose combining low doses of IFN with Nutlin-3, an antagonist of MDM2, which is also capable of promoting PV CD34+ cell apoptosis. Combination treatment with subtherapeutic doses of Peg IFN-α 2a and Nutlin-3 inhibited PV CD34+ cell proliferation by 50% while inhibiting normal CD34+ cells by 30%. Combination treatment with Nutlin-3 and Peg IFN-α 2a inhibited PV colony formation by 55%-90% while inhibiting normal colony formation by 22%-30%. The combination of these agents also decreased the proportion of JAK2V617F-positive hematopoietic progenitor cells in 6 PV patients studied. Treatment with low doses of Peg IFN-α 2a combined with Nutlin-3 increased phospho-p53 and p21 protein levels in PV CD34+ cells and increased the degree of apoptosis. These 2 reagents affect the tumor suppressor p53 through different pathways with Peg IFN-α 2a activating p38 MAP kinase and STAT1, leading to increased p53 transcription, whereas Nutlin-3 prevents the degradation of p53. These data suggest that treatment with low doses of both Nutlin-3 combined with Peg IFN-α 2a can target PV hematopoietic progenitor cells, eliminating the numbers of malignant hematopoietic progenitor cells.

Release pathways of interferon α2a molecules from lipid twin screw extrudates revealed by single molecule fluorescence microscopy

The pathways of interferon α2a release from a triglyceride based implant system were studied by single molecule fluorescence microscopy. The protein was labeled with a stable fluorescent dye ATTO647N, freeze-dried and embedded into the lipid matrix via twin-screw extrusion. The implant system consisted of a pore-forming agent (water soluble PEG 6000) and two types of triglycerides with different melting ranges which allowed the production of the implants at moderate temperatures and without the use of organic solvents. Single molecule microscopy and single particle tracking of labeled proteins contained in these implants revealed that two populations of diffusing proteins were present. Moreover, proteins were not only released via water-filled pores (created by dissolution of the pore-former), but surprisingly also through diffusion in a phase of molten lipid. Diffusion coefficients of IFNα 2a derived by tracking of individual protein molecules within the implant system were similar to diffusion coefficients obtained from control measurements in pure molten lipid and highly concentrated solutions of PEG 6000. In conclusion, tracking of individual protein molecules was successfully used to elucidate the release pathways of proteins from a relevant lipid based implant system.

PMO-173 District general hospital networks can provide safe and effective hepatitis C treatment: results of a 4-year audit

IntroductionChronic Hepatitis C (CHC) treatment is well described in the context of Randomised Controlled Trials (RCTs).1 Whether these findings can be extrapolated to treatment programmes delivered by nurse specialists working...

Treatment with Nutlin-3, a Small-Molecule Antagonist of MDM2, in Combination with Low Doses of Pegylated Interferon Alpha 2a Targets Hematopoietic Progenitor Cells From Polycythemia Vera (PV) Patients

Abstract 796▪FN2▪This icon denotes a clinically relevant abstractThe chronic myeloproliferative neoplasms (MPN) originate at the level of the hematopoietic stem cell (HSC). The prolonged and frequently unpredictable natural history of the MPNs has limited the use of many therapeutic approaches some of which are associated with unacceptable toxicities. IFNα therapy reverses morphological marrow abnormalities, eliminates cytogenetic abnormalities, reduces the JAK2V617F allele burden and re-establishes polyclonal hematopoiesis in patients with PV. Prolonged IFNα therapy is, however also frequently difficult due to drug related toxicity. We hypothesized that the addition of a non-genotoxic therapeutic agent to lower doses of IFNα might be better tolerated and be effective in eliminating the PV clone. The tumor suppressor, p53 gene plays an important role in the control of DNA repair, cell cycling, and apoptosis and is frequently inactivated in many cancers. The cellular level of p53 is controlled by a master regulator, MDM2. Nutlin-3, a small-molecule antagonist of MDM2 which activates the p53 pathway in cancer cells with wild-type p53 and is currently being evaluated in clinical trials. In MPN, p53 mutations and heterozygous deletions have been identified in a limited number of patients undergoing transformation to acute leukemia. We have previously reported that IFNα acts through p38 MAP kinase (Lu M, et al. Exp Hematol. 2010;38:472-80) while others have shown that activating this pathway leads to increased p53 (Sancéau J, Oncogene. 2000;19:3372-83). We hypothesized that targeting alternative pathways which can up-regulate p53 might serve as a novel therapeutic strategy for treating MPN patients. In this study, the therapeutic effects of nutlin-3 combined with Pegylated interferon alpha 2a on PV and normal CD34+ cells were investigated. FACS and western blot analysis demonstrated that the p53 protein levels in PV CD34+ cells were lower than normal CD34+ cells. CD34+ cells isolated from 8 PV patients and 7 normal donors were exposed to low doses of Peg IFNa 2a (200 U/ml) and/or nutlin-3 (200 nM) for 4 days and the number of hematopoietic progenitor cells (HPC) assayed. A dose dependent anti-proliferative effect of nutlin-3 on PV CD34+ cells was documented. PV CD34+ cells treated with nutlin-3 at doses ranging from 100 nM to 1000 nM, led to a progressively greater inhibition of colony formation from 10% to 80%. Treatment of normal CD34+ cells with 200 nM of nutlin-3 did not affect BFU-E or CFU-GM numbers, but inhibited PV CFU-GM by 20% and BFU-E by 50%. Simultaneous treatment with low doses of both nutlin-3 and Peg IFNa 2a inhibited PV CFU-GM by 55% and BFU-E by 90%, while normal cord blood CFU-GM and BFU-E were inhibited by 18% and 22%, respectively. This inhibition of PV HPC was far greater than that observed following the treatment with either agent alone. The combination of drugs also further decreased the proportion of JAK2V617F positive HPCs, as compared to CD34+ cells treated with Peg IFNa 2a alone (between 12.5% and 30% greater reduction). In addition, in a case of JAK2V617F negative myelofibrosis with a deletion of the long arm of chromosome 20 del(20) (q11–q12), Peg IFNα 2a and Nutlin-3 treatment of CD34+ cells cultured in the presence of SCF, TPO, IL-3 and FLT3 ligand reduced the number of cytogenetically abnormal total cells to 1% as compared to 15% observed following Peg IFNa 2a treatment alone. These data suggest that Peg IFNα 2a combined with Nutlin-3 treatment can substantially reduce the number of malignant MPN HPCs. The mechanism by which nutlin-3 and Peg IFNa 2a act on PV HPC was explored by FACS and Western Blot analysis using a JAK2V617F positive erythroleukemia cell line (HEL cells) and K562 cells. Peg IFNa 2a and nutlin-3 treatment each individually increased the p53 protein level and nuclear translocation in HEL but not in K562 cells. A further increase in p53 was observed when HEL cells were exposed to the combination of Peg IFNa 2a and nutlin-3. These data suggest that Peg IFNa 2a leads to increased p53 production while nutlin-3 results in reduced p53 degradation thereby providing a means of up regulating wild type p53 in PV cells. It is anticipated that the completion of these studies will provide a rationale for a phase I clinical trial evaluating the effects of nutlin3 alone or in combination with low doses of IFNα for the treatment of MPN patients. Disclosures:No relevant conflicts of interest to declare.

Ultra-Rapid Virological Response, Young Age, Low γ-GT/ALT-Ratio, and Absence of Steatosis Identify a Subgroup of HCV Genotype 3 Patients Who Achieve SVR with IFN-α2a Monotherapy

Background and AimsThe standard treatment regimen for chronic HCV genotype 3 (HCV-G3) hepatitis consists of PEGylated interferon-α (IFN-α) and ribavirin at varying doses ranging from 400 to 1,200 mg and results in response rates of 80%. However, this therapy has substantial side-effects including anemia, is teratogenic, and costly. To reduce the side-effects of therapy, the role of monotherapy consisting of only IFN-α was investigated.MethodsA retrospective analysis of individual therapy courses of HCV-G3-infected patients who were treated with IFN-α2a monotherapy or a combination therapy with attention to the treatment outcome and the presence of IL28B rs12979860 and IL28B rs8099917 single-nucleotide polymorphism genotypes was performed. Conventional prognostic features in each case were assessed as well.ResultsIn the study, 15/30 (50%) of patients treated with IFN-α2a monotherapy and 32/36 (89%) treated with combination therapy achieved a sustained virological response (SVR). In addition, 7/11 (64%) of those treated initially with monotherapy and subsequently with combination therapy achieved an SVR. An “ultra-rapid” virological response occurring within 2 weeks of initiation of therapy (p = 0.005), young age (<40; p < 0.001) and low initial γ-GT/ALT-ratio (p = 0.03) were associated with a SVR to IFN-α2a monotherapy. An SVR in those treated with combination therapy was found to be associated with a rapid virological response (RVR) (p = 0.03). The absence of histologic steatosis was associated with SVR in all patient groups (p = 0.01). Therapy duration (24 vs. 48 weeks) did not affect the SVR in either group. As expected, combination therapy resulted in more hematological side-effects than did monotherapy.ConclusionsAn “ultra-rapid” virological response, young age, low initial γ-GT/ALT-ratio and absence of steatosis were each associated with an SVR in those receiving IFN-α2a monotherapy. Therefore, monotherapy in these patients should still be discussed independently of the existence of the IL28B polymorphisms.

Does the antiviral therapy of patients with chronic hepatitis exert nephrotoxic effects?

Introduction: HBV and HCV chronic hepatitis can be accompanied by secondary renal disease. In addition, these patients receive antiviral drugs with potential nephrotoxicity. It is known that interferon (IFN) therapy in HCV-infected kidney transplant recipients is followed by rejection of the transplant in 50% of the cases. Ribavirin is contraindicated in hemodialyzed patients and in patients with a GFR <50 ml/min/1.73 m2. IFN therapy requires dosage reduction and close monitoring in patients with a GFR <50 ml/min/1.73 m2 and in patients with end stage renal disease. The aim of our study was to assess the nephrotoxicity of antiviral drugs in patients with chronic hepatitis by measuring three renal biomarkers: urinary albumin, N-acetyl-β-d-glucosaminidase (NAG) and α 1-microglobulin, as well as glomerular filtration rate (GFR-MDRD4) before and at 6 months of therapy.Methods: Fifty-five patients (28 male and 27 female, with a mean age of 47.85 ± 12.03 years) with chronic hepatitis (40 patients with HCV, 13 patients with HBV, 1 patient with HBV+HCV, and 1 patient with HBV+HDV) were enrolled into the study. Different antiviral drug associations were used on a case-by-case basis. The 40 patients with HCV chronic hepatitis received either Peg-IFN-α 2a+Ribavirin (37 patients) or Peg-IFN-α 2b+Ribavirin (3 patients). The 13 patients with HBV chronic hepatitis received Peg-IFN-α 2a (9 patients), Lamivudine (2 patients), Entecavir (1 patient), or Adefovir (1 patient). The patient with HBV+HCV chronic hepatitis received Peg-IFN-α 2a+Ribavirin. The patient with HBV+HDV chronic hepatitis received IFN-α 2a. Urinary albumin (ELISA), NAG (colorimetrical method), α 1-microglobulin (ELISA), and serum creatinine were measured before and at 6 months of antiviral therapy. Urinary markers were expressed as either mg/gCr (for albumin and α 1-microglobulin) or U/gCr (for NAG). Statistical analysis (Pearson’s correlation coefficient, paired t-test and χ2-test) was performed.Results: At 6 months of therapy urinary albumin/gCr did not increase significantly: 16.58 ± 23.39 vs. 15.85 ± 24.96 mg/gCr before therapy, p = 0.87. Urinary NAG/gCr did not increase significantly: 4.21 ± 3.37 vs. 3.83 ± 3.2 U/gCr before therapy, p = 0.53. Urinary α 1-microglobulin/gCr was almost unchanged: 4.38 ± 4.47 vs. 4.38 ± 3.57 mg/gCr before therapy, p = 0.99. The GFR did not decline significantly: 92.41 ± 22.21 vs. 94.59 ± 36.1 ml/min/1.73 m2 before therapy, p = 0.7. Ten patients (18.18%) were albuminuric before therapy, and 14 patients (25.45%) were albuminuric at 6 months of therapy, a non-significant increase (p = 0.35). We found a correlation between urinary albumin/gCr and NAG/gCr and between urinary albumin/gCr and α 1-microglobulin/gCr both at baseline and at 6 months of therapy: r = 0.54, p = 0.0005; r = 0.29, p = 0.03; r = 0.51, p = 0.0005; and r = 0.4, p = 0.002, respectively. In the patient receiving Adefovir, a known nephrotoxic drug, two of the three biomarkers (urinary albumin/gCr and NAG/gCr) increased, most notably NAG/gCr. Both HCV and HBV chronic hepatitis therapy were associated with non-significant changes in renal biomarker excretion and GFR.Conclusions: With the exception of Adefovir, all of the drug associations used in this study were safe.

Recombinant human antibodies to IFN-α from the immune repertoires of MG/thymoma donors (83.13)

Abstract A serum titer of autoantibodies to IFN-α has been reported in several autoimmune conditions and in many cases has been shown to neutralize IFN-α. The IFN-α antibody repertoire from 10 patients with autoimmune myasthenia gravis and associated thymoma (MG/T) was characterized. Surface IgG positive memory B-cells from peripheral blood mononuclear cells (PBMC) were cultured in 384-well plates to produce assayable quantities of antibody. Sensitive antibody microarray and AlphaScreen™ binding assays were used to identify antibodies reactive with IFN-α(2a). A range of IFN-α subtype specificity profiles was observed in competition assays for binding to the 12 subtypes of IFN-α. A subset of these antibodies was expressed recombinantly after recovery of heavy and light chain variable regions by RT-PCR and these recombinant antibodies recapitulated the activities demonstrated in the B-cell culture supernatants. These antibodies fell into 2 groups, blocking binding of IFN-α(2a) to either the IFNAR2 or IFNAR1 subunit of the Type 1 interferon receptor. The ability of these antibodies to neutralize the activity of IFN-α(2a) in a Daudi cell proliferation assay was confirmed. The antibody repertoires of MG/T patients include high affinity antibodies with both broad and narrow specificities for the different subtypes of IFN-α. These function-blocking human antibodies may have therapeutic utility in IFN-α dependent diseases such as SLE.

Interferon-α targets JAK2V617F-positive hematopoietic progenitor cells and acts through the p38 MAPK pathway

Interferon-alpha (IFNalpha) therapy leads to hematological remissions and a reduction of the JAK2V617F allele burden in patients with polycythemia vera (PV). In this study, the cellular target by which IFNalpha affects hematopoiesis in PV patients was evaluated. CD34(+) cells were isolated from normal bone marrow and the peripheral blood of patients with PV and were treated in vitro with each of the three commercially available forms of IFNalpha: IFNalpha 2b, pegylated IFNalpha 2a (Peg-IFNalpha 2a), and pegylated IFNalpha 2b (Peg-IFNalpha 2b). Each form of IFNalpha was equally potent in suppressing hematopoietic colony formation by normal CD34(+) cells, but Peg-IFNalpha 2a and IFNalpha 2b were more effective than Peg-IFNalpha 2b in inhibiting burst-forming unit erythroid-derived colony formation by PV CD34(+) cells. In addition, exposure of PV CD34(+) cells to equal doses of Peg-IFNalpha 2a and IFNalpha 2b resulted in a 38% to 40% reduction in the proportion of JAK2V617F-positive hematopoietic progenitor cells (HPC), while equivalent doses of Peg-IFNalpha 2b did not reduce the number of malignant HPC. Further studies explored the mechanism by which IFNalpha induced PV HPC growth inhibition. Treatment of Peg-IFNalpha 2a increased the rate of apoptosis of PV CD34(+) cells and the phosphorylation/activation of p38 mitogen-activated protein kinase in PV CD34(+) cells, while the p38-specific inhibitor SB203580 reversed the growth inhibition and apoptosis induced by Peg-IFNalpha 2a. These data suggest that low doses of IFNalpha selectively and directly suppress PV JAK2V617F HPC and that these agents act through the p38 mitogen-activated protein kinase pathway.

Chronic hepatitis C treatment in näive patients

Hepatitis C (HCV) is a major public health problem worldwide and it is considered that there are about 180 millions of infected people. The natural history of hepatitis C shows that, of those individuals affected by a primo-infection, 55 to 95% evolve into chronicity. The objective of treatment for chronic hepatitis C is to prevent in the long term the complications and death that this disease may cause. In a short term the most important aim is the sustained virological response (SVR), considered a virological response, normalization of the serum ALT level, histological improvement, improvement in patients’ quality of life and the risk of transmission reduction.The association Peginterferon alpha - Ribavirin (PEG IFNα-RBV), at the moment, is the standard of care of patients with chronic hepatitis C and compensated cirrhosis. Two PEG IFNα are licensed, PEG IFNα 2a and PEG IFNα 2b. Pegylation is a procedure that allows the union of polyethylene glycol moieties (PEG) to pharmacologic active proteins; in this case, IFNα. Pegylation of the IFNα 2a and 2b provoke important modifications in these proteins: slower absorption, different distribution, slower elimination, and longer half life with major exposure to the drug and lesser antigenicity. The two pegylated interferons available are dissimilar between them. The SVR in chronic hepatitis C patients who were treated with PEG IFNα-RBV in registration trials was 54 to 61%. Patients with genotypes 1 and 4 must be treated 48 weeks and those with genotypes 2 and 3, 24 weeks. In some situations patients could be treated lesser or longer time. Results obtained from the association of PEG IFNα - RBV - Amantadine in chronic hepatitis C patients are controversial.Meta-analysis comparing both PEG IFNs alpha shows a better SVR with PEG IFNα 2a. Therapies in patients with mild chronic hepatitis C have a similar SVR that those with more advanced liver disease and could be treated in this phase of the disease.

Effects of High Dose Ribavirin, Alinia® (Nitazoxanide) and Pegylated Interferon alfa-2a in Attaining Sustained Viral Response in Treatment of Chronic Hepatitis C (ERAIS-C trial) - Interim Results in Naïve Genotype 1 Patients

Purpose: Pegylated interferon alfa (PEG IFN-a) and Ribavirin(RBV) are the backbone of therapy of Chronic Hepatitis C with Sustained Virological Response (SVR) ˜50%, globally in all genotypes. A recent study reveals correlation of rapid viral response(RVR) to SVR. Alinia® (nitazoxanide) is an interferon (IFN) inducer that may increase SVR when used in combination with IFN and RBV. More rapid response or Very Rapid Viral Response (VRVR) defined as undetectable viral load at 14 days after initiation of therapy may predict even higher SVR rates. We assessed Nitazoxanide (NTZ) in combination with high dose RBV and PEG IFN-α 2a in achieving VRVR, RVR and Early Viral Response (EVR) in naive Chronic HCV genotype 1 (GT1) patients. Methods: Prospective, open-label pilot trial of naïve hepatitis-C GT1 patients. Exclusions: Uncontrolled diabetes mellitus (Alc>7.5), decompensated cirrhosis, active use of illicit drugs, excessive alcohol intake (>40 g/day), poorly motivated patients and co-morbid conditions (i.e. severe depression, anemia, thrombocytopenia, HIV). Patients received a lead in dose of NTZ 500 mg bid for first 2 weeks; RBV 800 mg in AM with 600 mg at night added to NTZ for next 2 weeks; subcutaneous PEG IFN-a2a 180 mcg weekly added to RBV and NTZ at the same dose (triple regimen) for next 12 weeks. Patients who achieved EVR continued PEG IFN-α2a and RBV only for the next 12 weeks at the same dose.VRVR, RVR, EVR and viral load at the end of 24 weeks were assessed (COBAS TaqMan HCV Test v2.0, Roche Diagnostics). Hematologic growth factors were used as needed. Results: Total patients: N=23: Caucasians (n=9) 39%, Hispanic (n=4) 17%, blacks (n=5) 21% and Asians (n=5) 21%. Study patients had mean BMI of 27.9% and a mean viral load <600,000 IU/ml. Most had fibrotic score-F2 (57%). Epoetin (52%), Elthrombopag (8.6%) and filgrastim (30%) were used to minimize dose reductions. See table. 11/23 (47.8%)- neutropenia requiring Filgrastim 12/23 (52%)—anemia requiring epoetin 9/23 (39%)—got both Filgrastim and Epoetin.Table: Table. Virologic response (interim results)Conclusion: This regimen of NTZ, high-dose RBV and PEG IFN-a2a enhances RVR over historical controls and offers promise for an accelerated SVR with this interim analysis. Precise efficacy of this regimen could be elucidated with SVR analysis.

Viral Evasion of the Interferon System

Viral Evasion of the Interferon System

A case of polymyositis in a chronic hepatitis C patient treated with peg-interferon-alpha 2b and ribavirin therapy.

Hepatitis C virus (HCV) infection may result in progression to chronic hepatitis, cirrhosis and hepatocellular carcinoma. Interferon-based treatment in patients with chronic hepatitis C may achieve viral clearance, and as a consequence improve liver histology and prevent progression to hepatocellular carcinoma. At present, the recommended therapy for chronic hepatitis C is peg-interferon-alpha (PEG-IFN-α) in conjunction with the oral nucleoside analog ribavirin. In the current study, we report a case of polymyositis associated with chronic hepatitis C following PEG-IFN-α and ribavirin therapy. The patient, a 64-year-old female who was treated with combination therapy, demonstrated elevated serum CPK, AST, ALT and LDH levels at 28weeks after treatment onset. As there was an elevation of the serum HCV-RNA levels, combination treatment was ceased at 24weeks. The patient had received IFN therapy twice previously (IFN-α 2a and IFN-α 2b with ribavirin therapy); however, no adverse side effects were observed. Further laboratory examination, muscle biopsy and imaging data suggested polymyositis, possibly triggered by the PEG-IFN-α treatment. The patient was subsequently administered prednisolone and the dose tapered over 7months. As a result the polymyositis has remained in remission. Although many autoimmune diseases have been associated with IFN therapy, the development of polymyositis is extremely rare.