Interferon Species Research Articles

The role of the interferon/JAK-STAT axis in driving islet HLA-I hyperexpression in type 1 diabetes.

The hyperexpression of human leukocyte antigen class I (HLA-I) molecules on pancreatic beta-cells is widely accepted as a hallmark feature of type 1 diabetes pathogenesis. This response is important clinically since it may increase the visibility of beta-cells to autoreactive CD8+ T-cells, thereby accelerating disease progression. In this review, key factors which drive HLA-I hyperexpression will be explored, and their clinical significance examined. It is established that the presence of residual beta-cells is essential for HLA-I hyperexpression by islet cells at all stages of the disease. We suggest that the most likely drivers of this process are interferons released from beta-cells (type I or III interferon; possibly in response to viral infection) or those elaborated from influent, autoreactive immune cells (type II interferon). In both cases, Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathways will be activated to induce the downstream expression of interferon stimulated genes. A variety of models have highlighted that HLA-I expression is enhanced in beta-cells in response to interferons, and that STAT1, STAT2 and interferon regulatory factor 9 (IRF9) play key roles in mediating these effects (depending on the species of interferon involved). Importantly, STAT1 expression is elevated in the beta-cells of donors with recent-onset type I diabetes, and this correlates with HLA-I hyperexpression on an islet-by-islet basis. These responses can be replicated in vitro, and we consider that chronically elevated STAT1 may have a role in maintaining HLA-I hyperexpression. However, other data have highlighted that STAT2-IRF9 may also be critical to this process. Thus, a better understanding of how these factors regulate HLA-I under chronically stimulated conditions needs to be gathered. Finally, JAK inhibitors can target interferon signaling pathways to diminish HLA-I expression in mouse models. It seems probable that these agents may also be effective in patients; diminishing HLA-I hyperexpression on islets, reducing the visibility of beta-cells to the immune system and ultimately slowing disease progression. The first clinical trials of selective JAK inhibitors are underway, and the outcomes should have important implications for type 1 diabetes clinical management.

Cellular and humoral factors of innate antiviral immunity

The study presents new data on the biological effects of well-known cellular and humoral factors that ensure the functioning of innate immunity. Four mechanisms induced by viruses, which lead to the destruction of inhibitory proteins and trigger the transcription of interferon genes, are described. The paper also presented the order of synthesis of species of interferons and other pro-inflammatory cytokines in the development of an antiviral immune response. This is of great importance because viruses have significantly different resistance to the biological effects of interferons. Interferon lambda played a role in the development of innate immune reactions against many viruses, and the effectiveness of the functioning of the mechanisms of the innate and adaptive immunity in viral infections was evaluated, depending on the state of the stat l, 4, 6 genes and genes of interferon regulators. An interferon-independent variant of the innate immune response in viral infections that occurs a few hours after infection and is associated with chemokine CXCL10 has been described. Data on the most important role of the ubiquitin-proteasome cleavage pathway of proteins and the complement system in the implementation of antiviral effects of innate immunity are also presented. The uniqueness of the microbicidal effects of natural killers, which are realized only in cells that reduced the expression of major histocompatibility complex I molecules, has been established. In addition, natural killers can recognize and do not attack target cells carrying antigens of HLA-E, the sublocus of the major histocompatibility complex I molecules. The natural killers were found to acquire the properties of memory cells. This is facilitated by interleukins 12 and 18. The extracellular neutrophil traps of neutrophils showed microbicidal effects against many viruses. The congenital lymphoid cells have multifaceted effects on the development of an antiviral immune response.

Gene expression analysis delineates the potential roles of multiple interferons in systemic lupus erythematosus

A role for interferon (IFN) in systemic lupus erythematosus (SLE) pathogenesis is inferred from the prominent IFN gene signature (IGS), but the major IFN species and its relationship to disease activity are unknown. A bioinformatic approach employing individual IFN species gene signatures to interrogate SLE microarray datasets demonstrates a putative role for numerous IFN species, with prominent expression of IFNB1 and IFNW signatures. In contrast with other SLE-affected organs, the IGS is less prominent in lupus nephritis. SLE patients with active and inactive disease have readily detectable IGS and the IGS changes synchronously with a monocyte signature but not disease activity, and is significantly related to monocyte transcripts. Monocyte over-expression of three times as many IGS transcripts as T and B cells and IGS retention in monocytes, but not T and B cells from inactive SLE patients contribute to the lack of correlation between the IGS and SLE disease activity.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-α single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-α protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-α single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-α subtypes.This new methodology allows the detection and quantification of IFN-α protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-α single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-α protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN. The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-α single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-α subtypes. This new methodology allows the detection and quantification of IFN-α protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Abstract 3021: CCR5 inhibition: macrophage repolarization therapy for colorectal cancer

Abstract In patients with metastatic colorectal cancer (CRC), the local immune response influences the clinical course. An in-depth analysis of the invasive margin of human CRC liver metastases revealed a distinct immunological microenvironment. Within this microenvironment, two distinct subsets of myeloid cells induce an influx of T cells into the invasive margin via CXCL9/CXCL10. CCL5 is produced by these T cells and stimulates pro-tumoral effects via CCR5, creating an exploitive loop. CCR5 was found on macrophages, lymphocytes and on the vast majority of tumor cells. Inhibition of CCR5 in patient-derived functional in vitro organotypic culture models showed a promising macrophage repolarization with anti-tumoral effects. These effects are mediated by activation of an antiviral program in macrophages, leading to interferon and reactive oxygen species production and subsequent selective tumor cell death. These anti-tumoral effects were confirmed in a phase I trial with a CCR5 antagonist in 14 patients with liver metastases of advanced refractory CRC. Treatment with the oral CCR5 Inhibitor was very well tolerated and objective responses were seen, especially in combination with previously ineffective chemotherapy. Biopsies revealed mitigation of tumor-promoting inflammation within the tumor tissue, confirming the validity of the explant model and highlighting the feasbility of this approach. It furthermore shows the proof-of-concept for macrophage repolarization in cancer patients. Citation Format: Niels Halama, Inka Zoernig, Anna Berthel, Christoph Kahlert, Fee Klupp, Meggy Suarez-Carmona, Karsten Brand, Juergen Krauss, Felix Lasitschka, Alexis Ulrich, Juergen Weitz, Martin Schneider, Markus Buechler, Laurence Zitvogel, Thomas Herrmann, Axel Benner, Christina Kunz, Stephan Luecke, Christoph Springfeld, Christine S. Falk, Dirk Jaeger. CCR5 inhibition: macrophage repolarization therapy for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3021. doi:10.1158/1538-7445.AM2017-3021

In Memoriam: Jean Lindenmann (1924-2015): A Circuitous Interfering Power in Neurology.

Sometimes, the intended consequences of scientific discovery are not the ones that ultimately have the greatest impact on humanity. This is certainly the case of the interferons, which were discovered jointly by Jean Lindenmann and Alick Isaacs in an attempt to elucidate the role of interferons in host innate immune responses against viral infections. Dr Lindenmann, a Swiss virologist who passed away on January 15, 2015, in Zurich, Switzerland, was an outstanding scientist whose contributions to the interferon field were groundbreaking. He joined Dr Isaacs in the mid-1950s as a postdoctoral fellow at the National Institute for Medical Research in Mill Hill in London, England. The purpose of his research was to study the nature of “viral interference.” When he commenced his work it was already established that incubation of a tissue or tissue culture with heatinactivated virus could prevent the replication of a virus that was subsequently added. Drs Lindenmann and Isaacs subsequently demonstrated that fluid conditioned for 24 hours with chick chorio-allantoic membranes exposed to inactivated virus could transfer this “interfering power” to fresh membranes, indicating that additional “interfering activity” had been generated.1 Dr Lindenmann then showed that a certain time lapse was required to obtain interference, suggesting that a new protein had to be generated to achieve this. Furthermore, dilution experiments indicated a linear relationship between the concentration of interferon and its antiviral potency.2 The seminal article reporting the discovery of interferons was published in 1957,3 which ultimately proved to be a family of proteins with a very high specific activity and a broad spectrum of biological effects. Dr Lindenmann returned to Zurich but did not pursue the purification and structural analysis of interferon. He was convinced that this task would be more or less rapidly accomplished by biochemists.4 In retrospect, this decision may appear misguided: Thousands of man-years later, in 1980, Ernest Knight remarked that “the best way to describe the progress in purification and characterization of the interferon proteins is that it has just begun.”5 At that time, the multiplic ity of interferon species and the low levels of production proved to be an almost unsurmountable barrier. Rather than purifying interferon, Dr Lindenmann explored its mechanism of action and identified a dominant autosomal gene, which he called Mx (now called Mx+), that mediated interferon-induced resistance aga ins t in f luenza and some othe r v i ruses . The Mx promoter is a powerful tool to direct controlled expression of a downstream gene and is now widely used in inducible gene expression in experimental mice.6 After 1980, research in the interferon field progressed rapidly as recombinant DNA technology clarified the relationship between the many interferon species and allowed the production of pure interferon on the kilogram scale. Most importantly, the generation of interferons on an industrial scale led to legitimate clinical applications. In 1987, Hillel Panitch, MD, and colleagues conducted a clinical trial with interferon gamma in patients with relapsingremitting multiple sclerosis (MS).7 At that time, many MS experts considered MS to be a viral, postviral, or paraviral disorder, which made interferon gamma a plausible intervention. Unexpectedly, a disproportionate number of patients displayed disease exacerbations, and bioassays detected an increase in circulating HLA-DR–positive monocytes in peripheral blood of recipients. These observations strongly suggested that MS disease exacerbations are immune mediated and not the consequence of viral illness. In contrast, interferon beta preparations have been approved and used for the treatment of MS for more than 20 years. The biological beneficial effects of interferon beta in MS, which include the reduction of disease attacks and brain lesions on magnetic resonance imaging, are pleiotropic and still incompletely understood. The recent approval of pegylated interferon beta-1a will likely ensure that they remain a mainstay of MS therapy for decades to come. Thus, Dr Lindenmann’s discovery not only opened up a new area of basic research, but also led to practical medical benefits. Those who knew Dr Lindenmann describe him as a gifted communicator who time and again delighted laymen and colleagues alike with his philosophical and historical discourses on scientific topics. Modest and unassuming, he kept out of the limelight toward which many another with his accomplishments would have striven. Jean Lindenmann

Biomarkers of inflammatory and auto-immune central nervous system disorders

Inflammatory and auto-immune disorders of the central nervous system are a heterogeneous group of disorders. Many of these disorders are potentially treatable with immune therapies that can reduce disability or prevent death. We review the clinical value of biomarkers which can aid in the diagnosis of paediatric inflammatory and auto-immune central nervous system (CNS) disorders. This review will first describe the clinical usefulness of nonspecific biomarkers of CNS inflammation such as cerebrospinal fluid neopterin and oligoclonal bands. Neopterin is produced by immune and neuronal cells after stimulation by interferon species and is increased in a broad range of inflammatory and auto-immune CNS disorders. Oligoclonal bands represent clonal production of immunoglobulin G in the CNS and are present in demyelinating, auto-immune, and infectious CNS disorders. In addition, we will review new advances in the immunogenetic investigation of familial auto-inflammatory disorders such as Aicardi-Goutières syndrome and Chronic Infantile Neurologic Cutaneous Articular syndrome. Finally, we will review the clinical utility of auto-antibodies in CNS disorders, with specific focus on auto-antibodies that bind to cell surface proteins such as N-methyl-D-asparate receptor, voltage-gated potassium channels, myelin oligodendrocyte glycoprotein, and aquaporin-4. These biomarkers are increasingly important in the recognition and treatment of inflammatory and auto-immune CNS disorders. Like many biomarkers in paediatric practice, it is essential to interpret the findings in the context of the patient history and examination.

Serum-induced suppression of interferon (IFN) activity. Lack of evidence for the presence of specific autoantibodies to IFN-alpha in normal human sera.

IgG antibodies binding to different IFN species have been described in sera of healthy and diseased individuals. Human serum immunoglobulins have also been shown to interfere with IFN bioactivity. To characterize these antibodies, human recombinant IFN-alpha 2A (rIFN-alpha) was radioiodinated, and ligand binding studies were performed in human sera as well as on the human cell line A-549 in the presence of human serum. 125I-rIFN-alpha bound to serum factors of healthy individuals. However, less than 3% of the binding was to IgG and the binding was non-saturable and therefore most likely non-specific. 125I-rIFN-alpha bound to receptors on A-549 cells, and the ligand-receptor complexes appeared to internalize. However, both cell binding and internalization of 125I-rIFN-alpha were independent of the presence of human serum. We conclude that normal human sera do not contain detectable autoantibodies to rIFN-alpha.

Anti-IFN BAb and NAb antibodies: a minireview.

Interferon (IFN) -α, -β, and -γ have been used for the management of many diseases with varying clinical effects. Like many other proteins, all IFN species are potentially immunogenic, especially those produced by recombinant (r) gene technologies. The reported frequencies and titers of anti-IFN antibodies (Ab) vary considerably depending on diseases, IFN preparations and administration, and the types of assays being used.1–9⇓⇓⇓⇓⇓⇓⇓⇓ However, Ab-mediated decrease in bioactivity of IFN (ADB), a condition in which the clinical effect of continued injection of rIFN is minimized or abrogated, is increasingly being recognized.10 The need for clinically useful, inexpensive, and standardized screening assays is obvious, but attempts in this direction have failed despite the recommendation of the World Health Organization (WHO) to use biologic assays where antiviral neutralization is expressed as a titer defined as the reciprocal of the serum dilution that reduces the IFN potency from 10 LU/mL to 1 LU/mL (1 LU/mL is the level of IFN inducing 50% protection against the challenge virus in a given assay).11,12⇓ A major reason for the lack of standardization is the inconvenience and high cost of carrying out elaborate antiviral neutralization assays in the clinical setting; additionally, some of these assays have not been validated with regard to clinical relevance and therefore may provide little useful information to the clinician. Several years of use of such assays have contributed to the conception of, and distinction between, “non-neutralizing binding antibodies” (BAb) and “neutralizing antibodies” (NAb) occurring in patients receiving IFN therapies,13,14⇓ and this issue has been complicated further by the frequent use of BAb assays that may give false-positive results. Over the years, we have investigated naturally occurring and therapeutically induced antibodies to cytokines, including BAb and NAb to IFN preparations. …

Trophoblastic Interferons: Do they Modulate Uterine Cellular Markers at the Time of Conceptus Attachment in the Pig?

Between days 12 and 20 of pregnancy, the trophectoderm of the porcine conceptus secretes two species of interferons (IFN): IFN-γ (Type II), which is produced in substantial amounts, and IFN-δ (type I), for which secretion peaks at days 15–16 of gestation. The role of these embryonic IFNs is not known. We made the assumption that, in the pig, one possible role of these IFNs may be the remodelling and/or depolarization of the uterine endometrial epithelium, as a prerequisite for implantation and establishment of a functional placenta. A comparative analysis by immunohistochemistry of several cell membrane markers and ECM components of the cyclic and pregnant uterus was performed at day 15 post-oestrus. The markers were those likely to differ between a pregnant and cyclic uterus, or between different stages of pregnancy. A highly specific marker of IFN-γ activity, namely MHC class II antigens in the uterine mucosa, was also examined. This study provides so far unreported data: in the endometrial epithelium of the pregnant uterus, we observed a partial relocalization of ZO-1, a marker of epithelial tight junctions, thus suggesting significant changes to the endometrial polarity. Heparan-Sulphate Proteoglycan (HSPG) expression did not differ significantly between cyclic and pregnant uteri. In contrast with the accepted rodent model of trophoblast-uterus adhesion, the porcine trophoblast and luminal epithelium were negative for HSPG. Finally, MHC class II antigens were absent from the cyclic uterus, but markedly induced in the day 15 pregnant uterus, particularly in endothelial cells, suggesting that IFN-γ may indeed cross the maternal epithelium. This hypothesis was supported by the observation of IFN-γ immunoreactivity associated with clusters of endometrial cells in the pregnant uterus.

Incidence of autoantibodies against type I and type II interferons in a cohort of systemic lupus erythematosus patients in Slovakia.

Autoantibodies against interferon (IFN) can be found in patients with systemic lupus erythematosus (SLE). However, detailed information about the occurrence of type-specific antihuman IFN antibodies is not available. In this study, we investigated the incidence of autoantibodies specifically recognizing various type I IFNs (alpha1, alpha2, beta, omega) and type II IFN (gamma). Sera from 100 SLE patients were screened for the presence of IFN-binding antibodies by ELISA, using various types of recombinant IFNs as antigen. On the whole, autoantibodies against type I or type II or both IFNs were detected in 45% (45 of 100) of the serum samples investigated. More than half (56%) of the positive samples (25 of 45) contained antibodies specific only for type I IFNs, and 36% of positive sera (16 of 45) had autoantibodies only against type II IFN. Antibodies against both type I and type II IFNs were detected in 8% (4 of 45) of the positive samples. Among autoantibodies to type I IFNs, the most abundant were those against the type IFN-omega (15%) and the subtype IFN-alpha2 (11%). Autoantibodies binding subtype IFN-alpha1 and type IFN-beta were detected at a relatively lower incidence of about 3%-4%. The highest occurrence (20%) showed autoantibodies to the proinflammatory cytokine, IFN-gamma. We did not find any correlation between the production of autoantibodies against particular IFN species and an antibody response to other IFN species. We further observed that 84% (38 of 45) of the positive sera bound only one IFN species, and 13% (6 of 45) of positive samples contained antibodies against two IFN species of five different combinations (alpha1/beta, alpha1/omega, alpha2/omega, alpha2/gamma, omega/gamma). One sample uniquely showed reactivity with three IFN species (alpha2/omega/gamma). Our findings suggest that formation of autoantibodies could reflect humoral immune responses to increased spontaneous production of the respective IFN species in SLE patients.

Editorial: A tribute to Sidney Udenfriend

Editorial: A tribute to Sidney Udenfriend

Effects of four species of interferon -& on cultured erythroid progenitors from congenital dyserythropoietic anaemia type I

Summary. The in vitro effects of interferon (IFN)-a on erythroid progenitor cells derived from the peripheral blood of five patients with congenital dyserythropoietic anaemia (CDA) type I and seven healthy adults were studied. Ficolhypaque-separated mononuclear cells were cultured for 14 d in StemGEM-1d medium with 0, 1, 10 and 100 U/ml of recombinant IFN-a2a, recombinant IFN-a2b, the geneticallyengineered hybrid molecule IFN-a1-8 or the laboratorydesigned molecule IFN-aconsensus. Erythroid bursts and colonies were counted, picked and processed for electron microscopy. In the experiments employing IFN-a2a there were no differences in the numbers of erythroid bursts or colonies between four patients with CDA type I and seven healthy adults. All five patients with CDA type I showed the ‘Swiss-cheese’ ultrastructural abnormality of the heterochromatin in a proportion of the erythroblasts when the progenitor cells were cultured in the absence of added IFN-a. A statistically significant reduction in the proportion of erythroblasts showing the ‘Swiss-cheese’ defect was seen when the erythroid progenitors were cultured in the presence of 0·01‐0·5 U/ml IFN-a2a (five patients), or 0·1 U/ml of IFN-a2b (two patients). In contrast, no reduction was seen in cultures containing 0·1‐100 U/ml of either IFN-a1-8 or IFN-aconsensus or 20 mM hydroxyurea (two patients). The partial correction of the ‘Swiss-cheese’ abnormality by low concentrations of IFN-a2a in vitro provides an experimental model with which the mechanisms underlying the haematological response that occurs after the in vivo administration of this species of IFN-a may be investigated.

Demonstration of luteotrophic responses of human recombinant gamma interferon in porcine corpora lutea using an in-vivo microdialysis system

Conceptuses from several mammalian species prior to implantation secrete proteins belonging to the family of interferons. The main species of interferons known to be secreted by the pig blastocyst is interferon gamma (IFNγ), the precise role of which is unclear. We decided to explore its effects on corpus luteum (CL) function using the novel microdialysis technique in vivo. Six cycling miniature pigs were monitored for estrus by daily plasma progesterone analysis and visual symptoms. On day nine of the cycle (day zero being the day of ovulation) the animals underwent surgery, and microdialysis tubing (vitafiber, Amicon U.S.A, cut off mol. wt. 1 million) were implanted in 17 corpora lutea. The inlets and outlets of all tubings were exteriorized and the entry and exit points of tubings in the CLs sealed with tissue glue. The afferent extension tubings were connected to a fraction collector and the system was continuously flushed with Ringer at a flow rate of 2.4 ml/h. After an initial flushout phase of 8 h, fractions were collected every half hour over 3 days. On days 10, 11 and 12 post estrus 12 CLs were stimulated for 4 h with 10 −7 M, 2 × 10 −7 M and 4 × 10 −7 M human recombinant IFNγ (Pharma Biotechnologie) respectively. Simultaneously, fractions were also collected from the remaining five unstimulated corpora lutea which served as controls. Progesterone concentrations in the dialysates were estimated by a sensitive enzymeimmunoassay (EIA). A significant increase ( P < 0.01) in progesterone release was observed in all 3 days following stimulation. The progesterone increase was more marked on the first day of stimulation (1 × 10 −7 M) with the hormone levels rising further even after the end of stimulation. The overall increase in progesterone concentration was 2-fold on day 10 in comparison to 15–30% on subsequent days even though IFN concentrations for stimulation were 2- and 4-fold higher. In the unstimulated CLs, a gradual decline ( P < 0.01) in progesterone levels were observed over days. In conclusion, these data provide evidence that the early conceptus signals its presence by way of IFNγ to maintain the CL in pigs.

Treatment of chronic hepatitis C with interferon alfa-n3: A multicenter, randomized, open-label trial

We studied the antiviral effectiveness and safety of interferon alfa-n3, a natural alpha interferon which contains multiple interferon species, in the treatment of previously untreated patients with chronic hepatitis C. Seventy-seven patients were randomized to receive either 1.0, 2.5, 5.0, or 10.0 million units (MU) of interferon alfa-n3 three times a week for 24 weeks and were then followed for an additional 24 weeks. At the end of therapy, 67% of patients in the 10 MU group normalized serum alanine transaminase (ALT) levels and 59% had no hepatitis C virus (HCV) RNA detected by polymerase chain reaction. At the end of the follow-up period, 44% of patients in the 10 MU group maintained normal ALT, and 24% had nondetectable HCV RNA. Lower doses were much less effective. Interferon alfa-n3 was well tolerated and no patient developed neutralizing anti-interferon antibodies during or after the treatment period. Interferon alfa-n3 appears to be effective against hepatitis C virus and deserves further study in larger randomized controlled trials.(Hepatology 1997 Feb;25(2):445-8)

Cytokines in the treatment of virus infections.

The interferon (IFN) system consists of both the formation of the various IFN proteins, and the diverse cellular responses which these induce: these result from the intracellular changes which follow their binding to a specific cell surface receptor. There is only a single human gamma, omega and beta IFN; in contrast, there are 13 closely related chemical species ("subtypes") of human alpha IFN, which are nevertheless chemically and biologically distinct. IFN preparations made from mass cultured human cells or by using recombinant DNA techniques are now readily available for clinical use. IFN have a major role in the defence of the body against virus infections. In acute virus infections, preformed exogenous IFN cannot be given soon enough to be of value. However, IFN-alpha and IFN-beta have proved of considerable value in some chronic virus infections, particularly chronic virus hepatitis and chronic papillomavirus infections. The doses routinely used are associated with both acute and chronic toxic side effects. Also, some patients form specific neutralising antibodies against the particular IFN preparation injected, which may abrogate all the benefits of the treatment. Nevertheless, IFN are now established as agents for use in routine medical practice.

Positive interactions between human interferon and cepharanthin against human cancer cells in vitro and in vivo.

A human tumor microcytotoxicity-viable cell-staining assay was used to test the antiproliferative effect of recombinant human interferon-beta or -gamma alone and in combination with bisbenzylisoquinoline alkaloid cepharanthin against four human tumor cell lines in vitro and in nude mice. Results obtained in the in vitro study indicate that combinations of interferon-beta/-gamma with cepharanthin show synergistic and, occasionally, additive antiproliferative effects in a dose-dependent manner on tumor viable cell-staining assay. Interferon-gamma combined with cepharanthin suppressed the growth of all four human tumor cell lines (RPMI 4788, PC 10, HeLa, ZR-75-1), and this enhanced antiproliferative effect was not dependent on the interferon species involved, including interferon-beta and -gamma. In an experimental model of pulmonary metastasis, in which human colon tumor cells were inoculated i.v. into nude mice, interferon-gamma alone exerted significant inhibitory activity against pulmonary metastasis in a dose-dependent manner, and cepharanthin alone also significantly inhibited metastasis. Furthermore, a combination of interferon-gamma with cepharanthin resulted in a considerable suppression of pulmonary metastasis. These studies indicate that due to their therapeutic potential, combinations of recombinant human interferon-beta or -gamma with cepharanthin might be a promising therapy for pulmonary metastasis of human cancers.

Bovine respiratory syncytial virus-specific immune responses in cattle following immunization with modified-live and inactivated vaccines. Analysis of proliferation and secretion of lymphokines by leukocytes in vitro

Cattle were immunized with vaccines containing modified-live or inactivated bovine respiratory syncytial virus (BRSV) and lymphocyte proliferative responses and cytokine secretion were monitored sequentially. Compared to pre-inoculated values, significant increases in proliferative responses to modified-live BRSV were detectable by Day 7 after the primary immunization with the vaccine containing inactivated BRSV, and by 7 days after the second immunization with modified-live virus. After a third immunization with the respective vaccines, proliferative responses to live BRSV were significantly higher in the group that received modified-live vaccine compared to the group that received inactivated vaccine. Proliferative responses to live BRSV corresponded with the presence of interleukin-2 (IL-2) in the supernatants from BRSV-stimulated leukocyte cultures and there were significantly higher levels of IL-2 in cultures from the group that received modified-live BRSV. An interferon species with the characteristics of interferon-α was also present in the supernatants from leukocyte cultures and there were no significant differences between the groups of vaccines. The predominant phenotype of proliferating cells in BRSV-stimulated leukocyte cultures derived from both groups of bovine vaccines was a BoCD4+ T-lymphocyte. These in vitro data suggest that both types of vaccines are capable of stimulating cell-mediated immune responses to BRSV in cattle.

Antiviral actions of interferon interferon-regulated cellular proteins and their surprisingly selective antiviral activities

Considerable progress has been made in the understanding of the molecular biology of the human interferon system. The genes encoding the interferons, their receptors, and the proteins that mediate many of their biological effects have been molecularly cloned and characterized. The availability of complete cDNA clones of components of the interferon systems has contributed significantly to our understanding of both the biology and the biochemistry of the antiviral actions of interferons.At the biological level, the antiviral effects of interferon may be viewed to be virus-type nonspecific. That is, treatment of cells with one type or even subspecies of interferon often leads to the generation of an antiviral state effective against a wide array of different RNA and DNA animal viruses. However, at the biochemical level, the antiviral action of interferon is often virus-type selective. That is, the apparent molecular mechanism which is primarily responsible for the inhibition of virus replication may differ considerably between virus types, and even host cells. For example, the IFN-regulated Mx protein selectively inhibits influenza virus but not other viruses when constitutively expressed in mouse cells. The IFN-regulated 2′,5′-oligoadenylate synthetase selectively inhibits EMC and mengo viruses, two picornaviruses, but not viruses of other families when constitutively expressed in transfected cells. Some viruses are typically insensitive to the antiviral effects of interferon, both in cell culture and in intact animals. This lack of sensitivity to IFN may result from a virus-mediated direct antagonism of the interferon system. For example, in the case of adenovirus, the activation of the IFN-regulated RNA-dependent P1/elF-2 protein kinase is blocked by the virus associated VA RNA.The relative sensitivity to interferon of different animal viruses varies appreciably. All three of the basic components required to measure an antiviral response may play a role in determining the relative effectiveness of the antiviral response: the species of interferon administered; the kind of cell treated; and, the type of virus used to challenge the interferon-treated host cell. Thus, the relative sensitivity to interferon observed for a particular interferon-cell-virus combination is likely the result of the equilibrium between the many agonists and antagonists which contribute to the overall response. That is, the relative sensitivity of a virus to the inhibitory action of IFN is governed by the qualitative nature and quantitative amount of the individual IFN-regulated cell proteins that may collectively contribute to the inhibition of virus replication. Furthermore, virus gene products or intermediates that serve as activators of IFN-regulated protein function must be balanced against those products that serve as inhibitors of IFN-regulated protein function.