Experimental proliferative vitreoretinopathy in rabbits by delivery of bioactive proteins with gelatin microspheres.

Proliferative vitreoretinopathy (PVR) is a challenging pathological condition, often causing failure of retinal detachment surgery. The purpose of this study was to evaluate the feasibility of a delivery system of bioactive proteins using anionic and cationic gelatin microspheres and to establish a new PVR model in rabbits by intraocular sustained delivery of basic fibroblast growth factor (bFGF) and interferon-beta (IFNß). Anionic and cationic gelatin microspheres were prepared and immersed in bFGF and IFNß solution, respectively, to yield a polyion complex between gelatin matrix and a bioactive protein. The bFGF-impregnated microspheres were injected into the subretinal space in rabbit eyes. At week 2, the IFNß-impregnated microspheres also were injected into the same space. Control eyes received gelatin microspheres without bFGF or IFNß, or both. The eyes then were observed for 8 weeks by ophthalmoscopy, fundus photography, and fluorescein angiography. The eyes also were evaluated histologically. In the group with both bFGF and IFNß, the number of eyes with more severe PVR increased over time. Histologic examination showed retinal folds. In contrast, no proliferative changes were seen in any control groups. Subretinal implantation of bFGF and IFNß-impregnated gelatin microspheres induced reproducible PVR in rabbit eyes. This study guaranteed delivery of bioactive proteins with gelatin microspheres.

Impairment of human neural crest cell migration by prolonged exposure to interferon-beta.

Human cell-based toxicological assays have been used successfully to detect known toxicants, and to distinguish them from negative controls. However, there is at present little experience on how to deal with hits from screens of compounds with yet unknown hazard. As a case study to this issue, we characterized human interferon-beta (IFNß) as potential developmental toxicant affecting neural crest cells (NCC). The protein was identified as a hit during a screen of clinically used drugs in the 'migration inhibition of neural crest' (MINC) assay. Concentration-response studies in the MINC combined with immunocytochemistry and mRNA quantification of cellular markers showed that IFNß inhibited NCC migration at concentrations as low as 20 pM. The effective concentrations found here correspond to levels found in human plasma, and they were neither cytostatic nor cytotoxic nor did they did they affect the differentiation state and overall phenotype of NCC. Data from two other migration assays confirmed that picomolar concentration of IFNß reduced the motility of NCC, while other interferons were less potent. The activation of JAK kinase by IFNß, as suggested by bioinformatics analysis of the transcriptome changes, was confirmed by biochemical methods. The degree and duration of pathway activation correlated with the extent of migration inhibition, and pharmacological block of this signaling pathway before, or up to 6 h after exposure to the cytokine prevented the effects of IFNß on migration. Thus, the reduction of vital functions of human NCC is a hitherto unknown potential hazard of endogenous or pharmacologically applied interferons.

Neuroprotective effect of acute interferon-beta 1B treatment after spinal cord injury.

AIM: The purpose of this trial was to investigate the effect of a well known immunomodulator -interferon beta- on traumatized spinal cord in terms of biochemical and histopathological features. MATERIAL AND METHODS: Twenty-four rats were used in this trial. The rats were divided into 3 groups. In the first group of rats, spinal cord injury was created by the weight drop method and interferon beta was administered. In the second group, physiological saline was administered. Third group was used as control. Rats were sacrificed 24 hours following trauma. Heat shock protein 70 levels were measured in the spinal cord samples and the samples were examined histopathologically. RESULTS: When the rats in the physiological saline and control groups were compared to rats treated with interferon beta 1b, those treated with interferon beta 1b revealed significant increases in the heat shock protein 70 levels in tissues, and histopathological examination revealed decreases in polymorphonuclear leucocyte infiltration, haemorrhage, oedema and necrosis. CONCLUSION: Although, the results of the study indicated that interferon beta might have some healing effects via increasing the cellular heat shock protein 70 on spinal cord injuries, more studies are needed.

Interferon-ß induces a long-lasting antiviral state in human respiratory epithelial cells.

OBJECTIVES: Interferon-ß (IFNß) induces strong antiviral effects and is therefore an attractive agent to prevent or reduce the incidence of virus-mediated exacerbations in asthmatic or chronic obstructive pulmonary disease (COPD) patients. We therefore investigated the effects of prophylactic IFNß on respiratory epithelial cells infected with rhinovirus (RV). METHODS: A549 cells and primary bronchial epithelial cells (PBECs) were exposed for 18 h to IFNß. Then, IFNß was either removed or maintained in the supernatant for the rest of the experiment and cells were infected with RV-1B at t = 0 or 72 h after the initial exposure to IFNß. RESULTS: Viral RNA levels were decreased in both cell types. Furthermore, both viral RNA and infectious virus levels in the supernatant of infected A549 cells were still significantly reduced at 72 h after removal of IFNß. This pronounced antiviral pre-treatment effect was associated with increased expression of the antiviral genes IFN-stimulated protein of MR15000 (ISG15) and Myxovirus resistance 1 (Mx1) and the effect was maintained even when IFNß levels in the supernatant of A549 cells were undetectable. CONCLUSIONS: These data show that IFNß has not only a strong, but also a long-lasting protective effect against RV infection of respiratory epithelium.

[Quality of life measurement: a suitable method for benefit-risk assessment?]. / Messung der Lebensqualität als Methode der Nutzen-Schaden-Abwägung?

Measurement of health-related quality of life can play a major role in benefit-risk assessment. Preference-based methods for measuring quality of life where data are aggregated into a single quality of life index are particularly well suited for use in quantitative analysis. Using two examples from research on cancer and multiple sclerosis, this paper compares approaches in which quality of life data from genuine studies or from external sources is used. It will be demonstrated that quality of life data can provide relevant information for quantitative benefit-risk assessment in applied research. In addition, quality of life data may be included in more complex approaches of multi-criteria decision analysis.

Cooperative contributions of interferon regulatory factor 1 (IRF1) and IRF8 to interferon-γ-mediated cytotoxic effects on oligodendroglial progenitor cells.

BACKGROUND: Administration of exogenous interferon-γ (IFNγ) aggravates the symptoms of multiple sclerosis (MS), whereas interferon-ß (IFNß) is used for treatment of MS patients. We previously demonstrated that IFNγ induces apoptosis of oligodendroglial progenitor cells (OPCs), suggesting that IFNγ is more toxic to OPCs than IFNß. Thus we hypothesized that a difference in expression profiles between IFNγ-inducible and IFNß-inducible genes in OPCs would predict the genes responsible for IFNγ-mediated cytotoxic effects on OPCs. We have tested this hypothesis particularly focusing on the interferon regulatory factors (IRFs) well-known transcription factors up-regulated by IFNs. METHODS: Highly pure primary rat OPC cultures were treated with IFNγ and IFNß. Cell death and proliferation were assessed by MTT reduction, caspase-3-like proteinase activity, Annexin-V binding, mitochondrial membrane potential, and BrdU-incorporation. Induction of all nine IRFs was comprehensively compared by quantitative PCR between IFNγ-treated and IFNß-treated OPCs. IRFs more strongly induced by IFNγ than by IFNß were selected, and tested for their ability to induce OPC apoptosis by overexpression and by inhibition by dominant-negative proteins or small interference RNA either in the presence or absence of IFNγ. RESULTS: Unlike IFNγ, IFNß did not induce apoptosis of OPCs. Among nine IRFs, IRF1 and IRF8 were preferentially up-regulated by IFNγ. In contrast, IRF7 was more robustly induced by IFNß than by IFNγ. Overexpressed IRF1 elicited apoptosis of OPCs, and a dominant negative IRF1 protein partially protected OPCs from IFNγ-induced apoptosis, indicating a substantial contribution of IRF1 to IFNγ-induced OPC apoptosis. On the other hand, overexpression of IRF8 itself had only marginal proapoptotic effects. However, overexpressed IRF8 enhanced the IFNγ-induced cytotoxicity and the proapoptotic effect of overexpressed IRF1, and down-regulation of IRF8 by siRNA partially but significantly reduced preapoptotic cells after treatment with IFNγ, suggesting that IRF8 cooperatively enhances IFNγ-induced OPC apoptosis. CONCLUSIONS: This study has identified that IRF1 and IRF8 mediate IFNγ-signaling leading to OPC apoptosis. Therapies targeting at these transcription factors and their target genes could reduce IFNγ-induced OPC loss and thereby enhance remyelination in MS patients.

Myeloid type I interferon signaling promotes atherosclerosis by stimulating macrophage recruitment to lesions.

Inflammatory cytokines are well-recognized mediators of atherosclerosis. Depending on the pathological context, type I interferons (IFNs; IFNalpha and IFNbeta) exert either pro- or anti-inflammatory immune functions, but their exact role in atherogenesis has not been clarified. Here, we demonstrate that IFNbeta enhances macrophage-endothelial cell adhesion and promotes leukocyte attraction to atherosclerosis-prone sites in mice in a chemokine-dependent manner. Moreover, IFNbeta treatment accelerates lesion formation in two different mouse models of atherosclerosis and increases macrophage accumulation in the plaques. Concomitantly, absence of endogenous type I IFN signaling in myeloid cells inhibits lesion development, protects against lesional accumulation of macrophages, and prevents necrotic core formation. Finally, we show that type I IFN signaling is upregulated in ruptured human atherosclerotic plaques. Hereby, we identify type I IFNs as proatherosclerotic cytokines that may serve as additional targets for prevention or treatment.

IFN-beta increases listeriolysin O-induced membrane permeabilization and death of macrophages.

Type I IFN (IFN-I) signaling is detrimental to cells and mice infected with Listeria monocytogenes. In this study, we investigate the impact of IFN-I on the activity of listeriolysin O (LLO), a pore-forming toxin and virulence protein released by L. monocytogenes. Treatment of macrophages with IFN-beta increased the ability of sublytic LLO concentrations to cause transient permeability of the plasma membrane. At higher LLO concentrations, IFN-beta enhanced the complete breakdown of membrane integrity and cell death. This activity of IFN-beta required Stat1. Perturbation of the plasma membrane by LLO resulted in activation of the p38MAPK pathway. IFN-beta pretreatment enhanced LLO-mediated signaling through this pathway, consistent with its ability to increase membrane damage. p38MAPK activation in response to LLO was independent of TLR4, a putative LLO receptor, and inhibition of p38MAPK neither enhanced nor prevented LLO-induced death. IFN-beta caused cells to express increased amounts of caspase 1 and to produce a detectable caspase 1 cleavage product after LLO treatment. Contrasting recent reports with another pore-forming toxin, this pathway did not aid cell survival as caspase 1-deficient cells were equally sensitive to lysis by LLO. Key lipogenesis enzymes were suppressed in IFN-beta-treated cells, which may exacerbate the membrane damage caused by LLO.

[Panniculitis induced by interferon beta-1a vascular toxicity]. / Panniculites secondaires à la toxicité vasculaire de l'interféron béta-1a.

BACKGROUND: Interferon-beta-1b is a valuable first-line therapy for patients with relapsing-remitting multiple sclerosis. Many non-severe cutaneous reactions to recombinant interferon beta are described at injection sites. Panniculitis after subcutaneous injection of beta interferon is a rare adverse event; we describe two such cases at beta interferon injection sites. CASE-REPORTS: Two women aged 22 years and 45 years with severe multiple sclerosis receiving immunotherapy with beta interferon were admitted to an emergency department following the appearance of extremely painful induration at injection sites rendering walking impossible after several months of interferon injections. One of the patients had fever. Histology tests showed vasculitis and capillary thrombosis in one-woman and dermal oedema in the other. MRI scanners showed extensive avascular necrosis of soft tissue without fasciitis in both patients. Interferon withdrawal and surgical debridement was carried out in one case and beta interferon was successfully reintroduced in both cases. DISCUSSION: Only two cases have been reported of panniculitis induced by subcutaneous beta interferon injection. Clinically, such cases may mimic infectious processes. The present cases show that MRI may be useful in diagnosis and that the vascular toxicity of interferon beta probably plays a role in panniculitis. Temporary withdrawal of treatment, rotation of several injection sites and alternative routes of administration may all be proposed.

Comparative tolerance of IFN beta-1a regimens in patients with relapsing multiple sclerosis. The EVIDENCE study.

The EVIDENCE study was a direct comparative study of two dose regimens of interferon (IFN) beta-1a used in the treatment of relapsing-remitting multiple sclerosis (RRMS): 30 mcg intramuscularly once weekly (qw; n=338) and 44 mcg subcutaneously three times weekly (tiw; n=339). The study continued for an average of 64 weeks. The safety population consisted of all patients receiving at least one dose of study drug. Clinical assessments occurred every 4 weeks for 24 weeks and then every 12 weeks. Blood tests for safety were taken at baseline and at weeks 4 and 12, and every 12 weeks thereafter. Overall adverse events were more common with the 44 mcg tiw regimen (p=0.007), and were due predominantly to differences in injection-site reactions. The majority of adverse events were rated mild by investigators. Hepatic and haematological adverse events and asymptomatic laboratory abnormalities were more common with 44 mcg tiw (p<0.001),with no difference seen for severe events. Flu-like symptoms were more common with 30 mcg qw (p=0.031), were more severe and persisted for longer. Serious adverse events were comparable for both groups, as were drug discontinuations. In conclusion, although adverse events were more common with high-dose, high-frequency IFN therapy, differences were primarily for mild events and did not affect treatment adherence. Based on superior clinical and magnetic resonance imaging outcomes over an average of 64 weeks, coupled with modest safety differences, the risk-benefit ratio for IFN therapy in RRMS favours the 44 mcg tiw regimen over this period of time.

Systemic IFN-beta gene therapy results in long-term survival in mice with established colorectal liver metastases.

Most patients succumbing to colorectal cancer fail with liver-predominant metastases. To make a clinical impact in this disease, a systemic or whole-liver therapy may be required, whereas most cancer gene therapy approaches are limited in their ability to treat beyond local disease. As a preclinical model for cancer gene therapy, recombinant adenovirus containing the human IFN-beta (hIFN-beta) cDNA was delivered systemically in nude mouse xenograft models of human colorectal cancer liver metastases. The vector targeted hepatocytes that produced high levels of hIFN-beta in the liver, resulting in a profound apoptotic response in the tumors and significant tumor regression. hIFN-beta gene therapy not only resulted in improved survival and long-term cure in a micrometastatic model, but provided similar benefits in a clinically relevant gross disease model. A similar recombinant adenovirus containing the murine IFN-beta (mIFN-beta) cDNA also resulted in a therapeutic response and improved survival in syngeneic mouse models of colorectal cancer liver metastases. Depletion studies demonstrate a contribution of natural killer cells to this therapeutic response. The toxicity of an adenoviral vector expressing murine IFN-beta in a syngeneic model is also presented. These encouraging results warrant further investigation of the use of cancer gene therapy for targeting metastatic disease.

Simultaneous but not sequential treatment with sodium butyrate improves the antiproliferative effect of alpha- or beta-interferon on a breast cancer cell line.

Clinical evidence indicates that alpha- and beta-interferon (alpha-IFN, beta-IFN) are only partially effective in human breast cancer. To improve their effectiveness, it has been proposed that differentiation inducers, such as sodium butyrate (NaB), be used to increase the IFN sensitivity of tumors. Therefore, we assessed concomitant or sequential combinations of low/intermediate concentrations of alpha-IFN or beta-IFN (10, 50 and 100 IU/ml) with a low concentration (0.1 mM) of NaB on a human breast cancer cell line (MDA-MB231), which exhibited a moderate sensitivity to IFN. Moreover, to verify the capability of NaB to potentiate IFN effectiveness by increasing IFN receptor (IFN-R) concentration, we investigated the effect of NaB on the synthesis of IFN-R. The concomitant presence of NaB and alpha-IFN or beta-IFN significantly improved the antiproliferative effect of the corresponding IFN alone. Conversely, the sequential treatment NaB-IFN did not enhance the inhibitory activity of the cytokine, although NaB was able to induce the expression of IFN-R. More likely, NaB induced the expression of some component of the IFN system, such as Stat1, Stat2 or p48, whose higher availability in the cytoplasmic compartment promotes formation of the multimeric transcription factor ISGF3, which induces the transcription of IFN-stimulated genes.

Synergistic antitumor effects of a combination of interferons and retinoic acid on human tumor cells in vitro and in vivo.

Solid tumors are relatively resistant to growth inhibition by IFNs. To enhance sensitivity, we assessed combinations of IFNs with all-trans-retinoic acid (RA). Antiproliferative studies in vitro suggested that the growth of three human breast carcinomas (MCF-7, MDA-MB-231, and MDA-MB-468), an ovarian carcinoma (NIH-OVCAR-3), and a malignant melanoma (SK-MEL-1) was inhibited to a greater degree by combination treatment with human IFN-beta and RA compared to single agents. Some of these cell lines were resistant to 10-100 IU/ml human IFN-alpha2b or IFN-beta or to 0.1-1.0 microM RA. Growth was inhibited significantly by combinations of IFNs and RA in all cell lines tested, and in some cases, cytotoxicity was observed. Sequential treatment of MCF-7 cells with RA followed by IFN-beta was more effective at inhibiting growth than treatment with IFN-beta followed by RA, suggesting that RA modulated the anticellular response of IFN-beta rather than the converse. In nude mice, the growth of MCF-7 and NIH-OVCAR-3 tumors was suppressed completely when combination treatment was started 2 days after tumor inoculation. Established, 6-week-old NIH-OVCAR-3 tumors underwent regression when treated with the combination of IFN-beta and RA but not with single-agent therapy. Together with our recent studies that demonstrated enhancement of IFN-stimulated gene expression by RA pretreatment in IFN-resistant cells, these data suggest that combination treatment with RA and IFNs may increase IFN-stimulated gene expression in IFN-resistant tumors, leading to augmented antitumor effects.

In vitro assessment for neurotoxicity of antitumor agents before local administration into central nervous system.

In vitro assays for neurotoxicity with the aid of cultured mouse fetal neurons and glial cells were applied to investigate neurotoxicity of recombinant murine interferon-beta (rMuIFN-beta). These data were compared with those for MTX, ADR, and ACNU. The range of concentrations of the drugs used in these experiments spanned their clinically achievable concentrations in patient serum (IFN-beta: 1 x 10(4) IU/ml, MTX: 100 micrograms/ml, ADR: 20 micrograms/ml, ACNU: 20 micrograms/ml). rMuIFN- beta damaged both neurons and glial cells at concentrations of more than 1 x 10(5) IU/ml but did not damage them at 1 x 10(4) IU/ml or less. Microtubule-associated protein 1A (MAP1A) staining was decreased in rMuIFN-beta-treated (more than 1 x 10(5) IU/ml) neutrons. In conclusion, since IFN-beta may have some neurotoxic effects at concentrations higher than 1 x 10(5) IU/ml, it should be administered carefully, as should other antitumor agents, into the tumor cavity in the CNS following surgery.

Divergent in vivo and in vitro antileukemic activity of recombinant interferon beta in patients with chronic-phase chronic myelogenous leukemia.

It was the aim of this study to investigate the antileukemic activities of recombinant interferon beta (rIFN beta) in chronic-phase CML in vitro and in vivo. Nine patients in the early chronic-phase of CML were treated in a phase-II trial with escalating doses of rIFN beta. In parallel, antiproliferative and immunomodulatory activities of rIFN beta and rIFN alpha 2b were studied in vitro. rIFN beta exhibited a significantly higher antiproliferative activity on hematopoietic progenitor cells of CML patients in vitro than rIFN alpha 2b. In contrast, only very limited clinical antileukemic efficacy of rIFN beta was observed. None of the patients achieved a complete or partial hematologic response (0% response rate, 0-36% 95 C.I.). Primary resistance of CML patients to rIFN beta treatment was caused neither by antibody formation against the recombinant material nor by deficient IFN receptor targeting and/or signaling; Induction of serum levels of beta-2-microglobulin (beta-2-m) and neopterin after administration of rIFN beta was comparable to that seen after administration of rIFN alpha. However, rIFN beta treatment less effectively induced biosynthesis of interleukin-1 receptor antagonist protein (IL-1-Ra) than rIFN alpha 2b. Thus, we conclude that rIFN beta at doses up to 12 MU/day s.c. is ineffective for treatment of chronic-phase CML. Further investigations into divergent biologic responses to various type-I interferons might help to elucidate mechanisms crucial for IFN action in patients with CML.

Cytotoxicity of human beta-interferon produced in human glioma cells transfected with its gene by means of liposomes.

The cytotoxicity of human beta-interferon (HuIFN-beta) produced in human glioma cells was examined by use of our liposomes entrapping two plasmids, pSV2neo and pSVMTV-IFN-beta. After the cells had been transfected with these genes by means of the liposomes, neomycin-resistant cells were selected. When the selected cells were subjected to a single exposure to dexamethasone, all of the cells were found to produce HuIFN-beta and were eliminated by 8 days. Accordingly, the effect of HuIFN-beta produced in human glioma cells is considered to be cytocidal.