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Production of interferon by human leukocytes
... Human leukocyte suspensions produce interferon after induction with a suitable inducer (4). Optimal yields of interferon are obtained only in the presence of serum (6); lower yields are produced in the absence of serum. Although whole serum can be present in interferon for evaluation of interferon against viral infections, serum antibodies may complicate interpretation of the results (1). ...
... Although whole serum can be present in interferon for evaluation of interferon against viral infections, serum antibodies may complicate interpretation of the results (1). Milk and nonhuman sera can be substituted for human serum in interferon production (3,(5)(6)(7)(8), but foreign proteins in these materials are unacceptable for use in man. This report describes the preparation of human leukocyte interferon by use of an antibody-free human serum fraction. ...
... Buffy coats were used within 24 h of collection at the New York Blood Center. Red blood cells were removed by treatment with 0.83% ammonium chloride (6). ...
An antibody-free human serum fraction has been prepared by ammonium sulfate precipitation. This fraction can be substituted for whole serum in the production of optimal yields of interferon by induced human leukocytes.
... The Sendai strain of parainfluenza 1 virus was grown in chick embryos, and the Indiana strain of vesicular stomatitis virus (VSV) was passed in U cells. Details of the procedures have been described previously (6,7). Cells. ...
... A continuous line, "U," of human amnion cells was grown in Roux bottles. Leukocytes were stored at 4 C in the presence of 0.5% (w/v) ethylenediaminetetraacetate and purified by treatment with NH4Cl by using a procedure which has been described previously in detail (6,7). No serum was used during the purification. ...
... At present, it is not possible to explain how casein can substitute for serum so effectively, especially since so little is known about the active components of serum and the mechanism of serum action is not understood (2, 7). Strander could only conclude that the serum albumin molecule, or something firmnly attached to it, was in all likelihood the most critical of the required serum components but that it alone could not account for all of the activity of serum (7). It is intriguing that a protein like casein, which is chemically and biologically different from all of the proteins present in serum (4), possesses full activity in stimulating human leukocytes to produce interferon. ...
The presence of serum in suspensions of Sendai-induced human leukocytes is necessary for the synthesis of significant amounts of interferon. Very little interferon is obtained from serum-free suspensions. Cow's milk or milk casein can substitute for serum in the production of high yields of human leukocyte interferon.
... Sources of human cells which can be obtained in large quantities for interferon production are: placentae, embryos, foreskins, skin, peripheral blood leukocytes, and established lines of lymphoid cells and heteroploid epithelium-like cells. These cells are known to release interferon upon proper stimulation (1,7,9,11,13,18,19,21,23,26,30,35,37,39,42,43,45). Optimal conditions for maximal release of interferon by foreskin fibroblasts, peripheral leukocytes, amniotic membrane, and lymphoid cells in continuous culture have also been described (9,11,23,26,35,37,39,45). ...
... These cells are known to release interferon upon proper stimulation (1,7,9,11,13,18,19,21,23,26,30,35,37,39,42,43,45). Optimal conditions for maximal release of interferon by foreskin fibroblasts, peripheral leukocytes, amniotic membrane, and lymphoid cells in continuous culture have also been described (9,11,23,26,35,37,39,45). Yet, to the best of our knowledge, there is no publication on a comparative study of the relative efficacy in interferon production by cell types from such a wide variety of sources. ...
... The procedures adopted in this study were based on findings published by others (11,26,37,39,45). We emphasize simplicity. ...
The relative capacity of several types of human cells and tissue to produce interferon was studied. Types of cells and tissue included were fibroblasts from embryos, foreskins, and biopsied skins; amnion cells; peripheral leukocytes; established lymphoid cell lines; established heteroploid cell lines; and chorioamniotic membrane. When Newcastle disease virus was used as the inducer, fibroblasts and amnion cells produced more interferon per 10⁶ cells than leukocytes, lymphoid cells, and heteroploid cells. Only minor variations in interferon-producing capacity were observed among fibroblasts from 36 persons. Culture passage level, cell concentration, and inducer were factors that significantly affected interferon production.
... The data reported demonstrate that the critical macromolecular, age-independent, and speciesunspecific serum principle can be omitted from the suspensions if the medium is supplemented with a combination of crystalline serum albumin and high concentrations of any one of five studied dipolar ionic buffers [ Considerable purification of a few interferons has been achieved during the recent years (5), but no procedure has succeeded in avoiding great losses of the antiviral activity. Previous studies on interferon production by purified human leukocytes have been directed toward the development of preparations suitable for extensive purification (15,16), and a convenient source of large amounts of interferon is now available (1). Unfortunately, suspensions of human leukocytes require high serum concentrations to yield substantial quantities of interferon (3,16). ...
... The Sendai strain of parainfluenza 1 virus was grown in chick embryos, and the Indiana strain of vesicular stomatitis virus (VSV) was passed in U cells (see below). Similar preparations of both viruses have been characterized previously (15). 1 Cells. A continuous line, "U," of human amnion cells was grown in Roux bottles (15). ...
... Similar preparations of both viruses have been characterized previously (15). 1 Cells. A continuous line, "U," of human amnion cells was grown in Roux bottles (15). The leukocytes were stored at 4 C in the presence of 0.5% (w/v) ethylenediaminetetraacetate and were used within 30 hr after the donors were bled (15). ...
The recovery of interferon from Sendai-infected suspensions of purified human leukocytes is dependent on the serum concentration in the incubation medium. Very little interferon is obtained from serum-free suspensions. The data reported demonstrate that the critical macromolecular, age-independent, and species-unspecific serum principle can be omitted from the suspensions if the medium is supplemented with a combination of crystalline serum albumin and high concentrations of any one of five studied dipolar ionic buffers [N,N-bis(2-hydroxyethyl) glycine (Bicine), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), N-tris(hydroxymethyl)methyl-2-amino-ethanesulfonic acid (TES), and N-tris(hydroxymethyl)methylglycine (Tricine)]. The optimal combination [TES (1.0%, w/v) and bovine serum albumin (0.8%, w/v)] allows the production of potent preparations of serum-free human interferon.
... To test if A3A, A3B and A3G (used as a control) can be induced as a part of interferon response, we infected three bladder (HT-1376, HTB-9 and RT-4) and three breast cancer cell lines (MCF-7, MDA-MB-231 and T-47D) with Sendai virus (SeV), which is a model nonlytic RNA-virus that induces robust interferon response in diverse human cells 32 . These cell lines were chosen because they represent some of the major clinical subtypes of bladder and breast tumors (Materials and Methods). ...
... Stocks of Sendai virus (SeV) strain Cantell were purchased from Charles River Laboratories (Wilmington, MA). SeV is a single-strand RNA murine parainfluenza virus which infects human cells and induces a robust antiviral interferon response that quickly controls infection 32 . Bladder and breast cancer cells were infected in triplicates or quadruplicates with SeV (7.5x10 5 CEID 50 /ml Chicken Embryo Infectious Dose 50%) for 1 hour, washed with PBS and then collected at 0, 3, 6, and 12 hours post-infection. ...
High rates of APOBEC-signature mutations are found in many tumors, but factors affecting this mutation pattern are not well understood. Here we explored the contribution of two common germline variants in the APOBEC3 region. SNP rs1014971 was associated with bladder cancer risk, increased APOBEC3B expression, and enrichment with APOBEC-signature mutations in bladder tumors. In contrast, a 30-kb deletion that eliminates APOBEC3B and creates an APOBEC3A–APOBEC3B chimera was not important in bladder cancer, whereas it was associated with breast cancer risk and enrichment with APOBEC-signature mutations in breast tumors. In vitro, APOBEC3B expression was predominantly induced by treatment with a DNA-damaging drug in bladder cancer cell lines, and APOBEC3A expression was induced as part of the antiviral interferon-stimulated response in breast cancer cell lines. These findings suggest a tissue-specific role of environmental oncogenic triggers, particularly in individuals with germline APOBEC3 risk variants.
... In 1970, a study was initiated at the Karolinska hospital to study the effect of IFN-α preparations, made from semi-purified interferon-α produced by human leukocytes, in patients with viral and tumor diseases, including osteosarcoma (Gresser 1961;Strander and Cantell 1966;Cantell et al. 1981). The preparation was modified in 1979 upon the production of recombinant IFN-α. ...
Osteosarcoma is the most prevalent bone tumor in children, adolescents, and young adults. It is considered a systemic disease with micrometastatic spread present already at diagnosis. Hence, while radical surgery is considered a prerequisite to successful treatment of localized or metastatic osteosarcoma, cure can – with few exceptions – only be achieved in combination with intensive chemotherapy. Despite multimodal therapy, 5-year event-free and overall survival for resectable metastatic and localized osteosarcoma have stagnated for almost four decades at <60% and approximately 70%, respectively. Unresectable osteosarcoma has survival rates below 10%. As several clinical trials have failed to improve outcomes with further intensified cytotoxic regimens, other treatment modalities to improve osteosarcoma survival are urgently needed. As osteosarcoma anecdotally and historically was among the first cancers to respond to immunotherapy and at the same time frequently lacks recurrent targetable genetic lesions and only shows moderate responses to tyrosine-kinase inhibitors with broad specificity, immunotherapeutic strategies appear one of the most promising ways to substantially improve long-term survival in both resectable and unresectable osteosarcoma. However, classical immunotherapy with monotherapeutic immune checkpoint inhibitors has had disappointing results. Several case reports and preclinical evidence suggest that immunotherapeutic agents need to be combined with one another and/or chemotherapeutic agents. At the same time, several biomarkers are emerging that might identify subgroups of osteosarcoma that could be particularly susceptible to immunotherapy. This chapter aims to give a biological, preclinical, and clinical overview on the current knowledge of immunotherapy in osteosarcoma as a basis for future efforts to develop effective combinatorial immunotherapies.
... Heparinized bone marrow aspirates in 0.5to l-ml amounts were obtained from patients who suffered neither from hematologic nor from malignant diseases. Each sample was immediately treated with eight volumes of 0.83% ammonium chloride for 10 min at 4 C to remove excess erythrocyte contamination (14). The suspension was then centrifuged and the pellet was processed as for peripheral leukocyte preparation. ...
Interferon was optimally produced in human peripheral leukocyte cultures incubated for approximately 19 hr in the presence of Sendai virus at a multiplicity of 10 to 50 EID50/cell. For determining whether deoxyribonucleic acid (DNA) synthesis per se was essential for interferon production, 1-β-D-arabinofuranosylcytosine (Ara-C), a potent DNA inhibitor was studied for its effect on interferon production in leukocytic and bone marrow cell cultures. These cells showed no impaired capacity to produce interferon when treated with 15 μg of Ara-C per ml. Interferon yields were also determined in leukocyte cultures treated with actinomycin D (0.1 μg/ml) and puromycin (10 μg/ml) at various times before and after virus inoculation. The data suggested that sequential transcriptive and translational events were required for the de novo synthesis of interferon by the infected leukocytes, in a manner similar to other known virus-induced interferon-producing systems. The synthesis of macromolecules and the effects of antimetabolites in leukocytes and bone marrow cell cultures were followed by measuring the incorporation of thymidine-2-¹⁴C, uridine-5-³H, and L-phenylalanine-1-¹⁴C. The effect of 0.1 μg of actinomycin per ml on the capacity of leukemic leukocytes to produce interferon was also studied. Preliminary data showed that, in contrast to nonleukemic leukocytes, interferon production by leukemic leukocytes was only partially inhibited by actinomycin.
... Two-fold serial dilutions of compounds 1, 2, and 3 were prepared in nutrient medium supplemented with 2% FBS, starting from 19 µM, 8.24 µM, and 17 µM, respectively, based on the reported cytotoxicity results obtained previously in the Hep-2 cell line (Đorđević et al., 2014). Cells treated with interferon α-2a served as a positive control of antiviral activity (Cantell and Strander, 1966). It was previously established that interferon α-2a at concentrations of up to 1000 IU/mL are not cytotoxic. ...
Metal coordination compounds have an important role in the development of novel drugs. Using the resazurin microtitration assay we assessed the cytotoxicity and antiviral activity of the ligand 2-(diphenylphosphino)benzaldehyde 1-adamantoylhydrazone and its Pd(II) and Pt(II) complexes. Cytotoxicity was tested in A549 human lung adenocarcinoma epithelial cells. We observed that the ligand displayed a more pronounced cytotoxic activity than the platinum-based drug, carboplatin. Morphological evaluation of A549 cells treated with the ligand by acridine orange and ethidium bromide double staining revealed the presence of signs of apoptosis. Antiviral activity against poliovirus type 1 was assessed by examination of the cytopathic effect (CPE) in Hep-2 cells. Cells that were exposed to the 19 μM ligand before infection displayed a maximal significant reduction (by 24.42 ± 1.49%) of the CPE. This was likely due to the inhibition of virus receptors and prevention of viral adsorption. Treatment with 17 μM Pt(II) complex after viral infection caused a maximal significant reduction (by 30.52 ± 3.12%) of the CPE, presumably through an effect on viral replication. The results indicate that the ligand should be viewed as a potential anticancer agent. The ligand and the Pt(II) complex show promising results for further investigation of antiviral activity.
... The lack of availability of sufficient amounts of interferon was the principal hurdle. This problem has been alleviated to some extent by the development of three procedures for large-scale production of human interferon: (a) human leukocytes induced with Newcastle disease virus or Sendai virus (GRESSER 1961;STRANDER and CANTELL 1966;CANTELL and HIRVONEN 1978); (b) human diploid fibroblast cultures induced with poly(I)· poly(C) together with the judicious use of cycloheximide and actinomycin D (TAN et al. 1970;HAVELL andVILCEK 1972, BILLIAU et al. 1973a); and (c) human lymphoblastoid cell lines (e.g., Namalva) induced with Sendai or Newcastle disease virus FINTER and BRIDGEN 1978;ZOON et al. 1979 b). These procedures for large-scale production have provided amounts of human interferon sufficient for the purification of these proteins as well as for preliminary clinical investigations. ...
Interferons are a group of inducible cellular proteins that interact with mammalian and other vertebrate cells and render them resistant to infection by a wide variety of RNA-containing or DNA-containing viruses. In addition to a marked antiviral effect, interferons cause numerous changes in the phenotype of target cells, including a reduction in the rate of cell proliferation and alterations in the structure and function of the cell surface, the distribution of cytoskeletal elements, and the expression of several differentiated cellular functions. The use of interferons and interferon inducers to treat human viral and neoplastic diseases is currently under intensive investigation. Considerable attention is thus focused on the structure and function of human interferons. Furthermore, investigations of the mechanisms which underlie the induction and the actions of interferons have led to novel insights into the regulation of gene expression in eukaryotic cells as well as into the pathophysiology of viral diseases. Thus, interferon research represents an important and rapidly advancing area of biomedical research today.
... Nevertheless, a method for production of IFN had been developed by culturing human leukocytes in Gresser's laboratory [3]. In addition, natural IFN was produced and partially purified at the Finland Red Cross by Cantell [4], for use in the first clinical trial in osteosarcoma patients [5], following the observations on type I IFN antitumor effects obtained in experimental models by Gresser et al. [6]. Many attempts were made to demonstrate the activity of natural IFN on other cancer types -those potentially associated with a viral origin such as juvenile laryngeal papillomatosis, human condylomata acuminata, Hodgkin's disease, acute leukemia in children, multiple myeloma and others. ...
... com elevada percentagem de células infectadas com vírus EB, são as que apresentam títulos mais baixos de interferon, observação já feita por Swart e Young 25 . Também em culturas de leucócitos humanos, infectadas por diferentes vírus, tem sido possível evidenciar a produção de interferon 1,3,6,8,11,12,17,18,19,21,23,24,26 . O trabalho de Carver e Seto 5 mostra que culturas de fibroblastos humanos, depois de submetidas a estimulação com so-ros humanos contendo antígeno Austrália, tornam-se refretárias à multiplicação do vírus da doença de Newcastle. ...
Culturas de células EB3, quando inoculadas com soros humanos contendo antígeno Austrália, produzem interferon ou substância semelhante em títulos significativamente mais elevados do que as culturas controle, não inoculadas. As culturas de células EB3 inoculadas com soros humanos negativos para o antígeno Austrália comportam-se de modo idêntico às culturas controle, no que respeita aos títulos de interferon, ou substância semelhante. Sugere-se a possibilidade daquele efeito poder servir de indicador de uma possível interação entre as células EB3 e o antígeno Austrália.
Leukocyte interferon for clinical trials became available in the early 1970s, the bulk of which was provided from Finland by Cantell et al. (1974) and Strander and CAntell (1966). Fresh buffy coat cells from human blood were stimulated to produce interferon by induction with Newcastle disease or Sendai viruses. Since the production process involved fresh, primary human cells suspended in serum-containing tissue culture medium and infected with an inducing virus, many of the tests used in examining live and inactivated tissue culture-derived vaccines were considered appropriate for application to leukocyte interferon. At first, these included only sterility, general safety, pyrogenicity, and potency. Also, the manufacturer was asked to provide data which identified the product as interferon, e.g., low pH stability, sedimentation characteristics, and biologic activity. As procedures became available for hepatitis B surface antigen (HBsAg) testing, it was reasonable to require that buffy coats used in production should be derived only from blood shown to be negative for the presence of HBsAg. It is quite likely that as tests for hepatitis virus A and for the agents of non-A and non-B hepatitis are developed, these would also be required for source leukocytes used in interferon production. With the refinement of interferon production procedures, experimental materials were also tested for protein and moisture content.
The results of animal studies have long suggested that human interferons should be studied as antiviral and antitumour agents in medicine (see reviews by Finter 1973; Oxman 1973), but clinical trials have been hindered by the shortage of suitable material. Primary human leukocytes obtained from transfusion blood and processed as described by Strander and Cantell (1966,1967) have until recently provided nearly all the interferon used in humans. In 1976, Finnish workers made 1011 IU crude interferon from this source (Cantell and Hirvonen 1977), and have since increased their output severalfold (K. Cantell, personal communication). Such preparations have played a key role in the development of clinical studies. Nevertheless, it is obvious that human blood could not provide enough cells to make interferon in really large amounts. Furthermore, only limited quality control procedures can be applied to individual white blood cell lots, and for example there are few countries where the incidence of hepatitis in blood donors is as low as in Finland.
The development of production methods for human leukocyte interferon (IFN-α) is significant not only for basic and clinical needs, but also as a model for production of many other biologically interesting leukocyte products. The therapeutic potential of interferon was well appreciated early. After its discovery by Isaacs and Lindenmann (1957), further recognition of its action in vitro and in vivo (Lindenmann et al. 1957; Isaacs and Burke 1958; Cantell and Tommila 1960), of factors affecting in vitro production (Burke and Isaacs 1958; Isaacs and Burke 1958), and of pharmaceutical possibilities for human IFN-a (Isaacs 1959,1961) soon followed. However, many years passed before amounts sufficient for clinical trials were produced. Progress toward IFN-α production began with the observation in 1961 that leukocyte cultures were capable of producing interferon (Gresser 1961). Soon thereafter, Cantell and his associates reported progress in the development of IFN-α production from Sendai virus-stimulated leukocyte suspension cultures (Strander and Cantell 1966; Strander and Cantell 1967; Tovell and Cantell 1971; Cantell et al. 1974; Kauppinen et al. 1978). In this chapter, our procedures adopted from Cantell will be described to emphasize what has been most suitable in our hands. The importance of certain steps will be discussed as they are presented, as will pertinent variations from Cantell’s methods.
In 1957, Isaacs and Lindenmann found that pieces of chick chorioallantoic membranes infected with heat inactivated influenza virus released a substance into the surrounding medium which could make other pieces of chick membrane resistant to virus [1]. They called this unique substance interferon. Similar antiviral activities were rapidly discovered in a great many vertebrates, including man [2]. These substances have been classified as interferons because they all are cellular proteins which act by inducing antiviral resistance in responding vertebrate cells. Now, we know that interferon, even within a single species, is not limited to a single molecular type but that several disparate molecules exhibit antiviral activity. For a number of years, the emphasis of interferon reserach was directed primarily toward its antiviral functions and it is only relatively recently that the immunoregulatory and antitumor activities of the interferons have come under intensive study. This paper will review some of the salient features of the antiviral and antitumor activities of the interferon system and will draw attention to the exciting prospects of employing interferons as agents for tumor management.
So unbestritten die Erfolge von „Stahl und Strahl“ in der modernen Krebstherapie sind, so bescheiden erscheinen die Erfolge der Chemotherapie maligner Tumoren. Wie kommt es zu diesem Nachholbedarf?
Assuming that the majority of readers will be clinical investigators we tried to anticipate what such a specialist may expect to find in this chapter. We concluded that a detailed description of current methods of interferon preparation would not be within the context of this book*. Rather than burden the reader with technical details we decided to give an overview with emphasis on advantages and disadvantages of current production methods and present information about the origin, status of supply, and purity of currently available interferon preparations. Aspects of product quality we assumed would be of foremost importance. Such information we believe will help in the planning of various clinical studies and in the interpretation of results.
Human osteosarcoma is a malignant disease which requires multi-modality treatment including surgery and adjuvant therapy. In this article we have summarized various aspects pertinent to IFN therapy of human osteosarcoma. Different experimental approaches are described but we emphasize that IFN treatment should not be used as the primary single treatment in osteosarcoma. Osteosarcoma exerts biological effects which makes it possible to study biological parameters extensively. Such a biological approach might lead to an extensive combination treatment schedule and a diversified programme for the treatment of human osteosarcoma patients.
Despite the considerable achievements in immunology during the past two decades, control of tumour development by effective immunotherapy is still not possible. This situation reflects the complexity of events that occur in response to tumour growth. Other chapters in this book outline the important advances made in the study of macrophage function and the developments in monoclonal antibody production against tumour-associated antigens. In this chapter the role of the lymphocyte in combating tumour development and metastatic spread is considered. Of importance, however, is the understanding that lymphocyte function is influenced by other cell types, hormones, and products released by tumour cells and that much of our current knowledge has stemmed from in vitro experimentation. Caution must be exercised when interpreting the results of laboratory tests and relating these to their in vivo relevance, since it is not possible to mimic the conditions that the in vivo environment imposes on lymphocyte activity.
α-interferon (α-IFN) is a biological response modifier with a dose-dependent activity. The present study on the treatment of patients with multiple myeloma with high doses of natural α-IFN was designed to meet this dose-dependent concept. 50 previously untreated patients with IgA and BJ myelomas and a s-Creatinine ≤ 200 μmol/l entered the study. Various treatment schedules were tested. The initial plan was to give the patients 30 × 106 U α-IFN daily. This dosage, however, gave unacceptable toxicity. Step-by-step decreasing dose schedules were given to the patients, 10 × 106 U of α-IFN daily for 7 consecutive d repeated every 3rd week was found to be the maximal tolerable dose that could be given to most patients. 36% (95% confidence levels: 22%-50%) of the patients responded: 41% of the IgA myelomas and 23% of BJ myelomas. Median time to response was 1.5 months and median response duration was 20 months. Impaired general condition and central nervous system and gastrointestinal-related toxicity were the main adverse reactions. Hematological side-effects were mild.
Summary After active immunisation with virus infected lymphoblasts the production of guinea pig antibodies against antigens of not infected lymphocytes is increased. This immunological adjuvant effect was observed when human and mice lymphocytes were infected with herpes simplex virus, VSV and vacciniavirus. The increased production of antibodies was demonstrated by an in vitro micro-lymphocytotoxicitytest and in vivo by its immunsuppressive effect when allogeneic tumor cells (Mastocytoma P-815-x2 DBA/2→NMRI) were transplanted.
Interferon-a (IFN-a) production was investigated in whole-blood cultures of 66 bladder cancer patients and 65 control subjects. IFN synthesis was induced with Sendai virus, and IFN activity was assayed in FL cells challenged with vesicular stomatitis virus (VSV). The mean levels of the IFN-a produced were 5,724±2,288 IU/ml in the control subjects and 4,800±2,353 IU/ml in the bladder cancer patients. IFN-a production was significantly suppressed in the bladder cancer patients compared with that in the control subjects (P (0.05). The impairment in IFN-a production correlated with the tumor grade, and it was shown that the tendency toward decreased IFN-a production was closely associated with the advancement of the tumor stage. Our results suggested that the decreased IFN-a production may contribute to the disordered immunoregulation in bladder cancer patients.
SUMMARY Suspensions of human leucocytes purified by NHaC1 produced interferon in the same time after induction by Sendai virus whether the medium con- tained serum or not. In the absence of serum the interferon titre levelled off after as little as 5 hr, and the yield was less than IO ~o of that obtained when serum was present. The production of interferon was completely inhibited by 2/zg./ml. of actinomycin D added simultaneously with the virus. Inter- feron synthesis gradually became insensitive to actinomycin during the first 4 to 5 hr after induction in both the presence and absence of serum. Large amounts of interferon rapidly appeared after addition of serum to cultures induced and incubated in serum-free medium. The cells responded to added serum by interferon production up to I6 to 20 hr after induction. The rate of interferon production after addition of serum was not affected by actinomycin. Removal of serum from the medium resulted in a reduced yield of interferon; which would not be prevented by addition of actinomycin. The low yields of interferon in a medium devoid of serum were not due to reduced synthesis of the DNA-dependent RNA needed for interferon production.
Several polynucleotides were tested for interferon induction in comparison with the standard Poly (IC) in human diploid fibroblasts and in human leukocyte suspensions.
We received high IFN-titre following superinduction of the polynucleotides in human fibroblasts.
These results show that under superinduction conditions partly 3 times more interferon is induced in comparison with the standard inductor Poly (IC).
The tested concentration of 10 μg/ml polynucleotides did not result in any IFN yields or only in very low ones in human leukocytes, which were only within the range of detectability.
Attempts have been made by in vitro methods to demonstrate the presence of a cell-mediated immune response to the gonococcus in human peripheral leukocytes. Leukocyte-migration tests in the presence of gonococcal antigen were performed in 34 patients with uro-arthritis (Reiter's disease), in 18 patients with newly developed gonorrhoea without joint manifestations, and in 34 controls (blood donors). In the patients with uncomplicated gonorrhoea the mean migration index (MI) was generally the same as in the healthy controls. A MI below 0.75 was noted in 21 of 34 cases of uro-arthritis. The number of cases with inhibited migration rose parallel with the duration of the disease and the presence of prostatitis. Antigen-induced lymphocyte transformation was also studied by quantitation of the 14C-thymidine uptake in cell cultures. Studies were made in three groups of patients, one group with uro-arthritis, one with gonococcal urethritis, and one with gonococcal septicaemia, and also in healthy controls. The lymphocyte stimulation induced by gonococcal antigen was significantly greater in uro-arthritis patients with known gonococcal infection (0.010.05) than in healthy controls. No difference in DNA synthesis was noted between patients with uncomplicated gonorrhoea and healthy controls, while the gonococcal septicaemia patients demonstrated a poor cellular immune response.
Zusammenfassung Die Autoren berichten über eine Steigerung der Interferonproduktion der Leukocyten von chronischer myeloischer Leukämie.
The procedures generally used for the in vitro induction of interferon exploit fibroblast or leucocyte culture suspensions. Several factors and conditions influencing the capacity for IFN induction were studied, with the following results.
In contrast to previous findings, optimum IFN induction was obtained at 37 °C, rather than 30 °C, when human fibroblasts were induced with 100 μg/ml of Poly (IC). There was a direct correlation between the dose of the inducing agent applied and the amount of IFN produced. Also, the cells were excellently superinducible with metabolic inhibitors like actinomycin D and cycloheximine. In addition, priming procedures enhanced IFN induction in human fibroblasts.
Comparing different cell types with respect to their ability to be induced for IFN production, we found that different cell types show great differences in their inducer sensitivity. Finally, the response of different cell culture systems was highly variable when induced with different compounds.
Patients with chronic myelogenous leukemia (CML) and elevated white cell counts afford a large number of leukocytes for production of interferon. Primary leukocyte cultures are established in a synthetic medium with casein instead of the serum requirement and are induced with Newcastle disease virus to produce interferon. Procedures involved in the establishment of the primary culture are performed with aseptic techniques. Equipment is steam-sterilized, heat treated at 230 ° or rinsed with denatured ethyl alcohol (3A) depending on the applicability. Whenever possible, procedures are performed in a laminar-flow hood. Cells are obtained in blood collection bags after leukapheresis of chronic myelogenous leukemia patients. Titers of interferon from leukemic leukocytes can be as high as 200,000 units/ml. A quantitative recovery of interferon activity is obtained for the procedures outlined.
As the first DNA recombinant technology products effective in cancer treatment, interferons (IFNs) opened an era of biologic therapies for cancer, multiple sclerosis, and infectious diseases. Although more than 40 years have passed since IFNs were first described, scientific and clinical interest in these multifunctional cytokines has not diminished. The mechanisms of antitumor effects, optimal dose, schedule, and type of IFN for specific clinical indications have yet to be fully described. Progress in cloning IFN receptors and other signaling components has allowed further identification and description of molecular pathways by which type 1 and type 2 IFNs mediate their effects. Further characterization and identification of interferon-stimulated genes or factors that mediate IFN-dependent antiproliferative, apoptotic, and immunomodulatory effects will lead to better utilization of IFNs in the treatment of cancer. Anti-infective property of IFNs has provided efficacy against hepatitis B and hepatitis C infection. Although the use of α-IFNs in hepatitis B infection has largely been replaced by oral antiviral drug therapies, IFNs remain the standard of care for treatment of patients with chronic hepatitis C infections. The anti-inflammatory effects of β-IFNs have demonstrated benefits in the treatement of patients with multiple sclerosis. Monographs of IFN therapeutics are included in a later part of this chapter. Innovative approaches such as the introduction of second-generation IFNs with extended biological effects and pharmacokinetic profiles may broaden the therapeutic applications of IFNs over the next decade.
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virus infections;clinical medicine;synthetic polyanions;newcastle disease virus;Semliki Forest virus
We analyzed whether normal human hepatocytes, which normally do not display Class II major histocompatibility complex antigens, can be induced to express them in vitro, and whether this induction has an in vivo counterpart in chronic liver diseases. While both α- and γ-interferon induced expression of Class I antigens, only γ-interferon induced expression of Class II antigens on hepatocytes in vitro. Recombinant interleukin 2 had no effect on major histocompatibility complex antigen expression. Both Class I and Class II antigens could be detected by indirect immunofluorescence on hepatocytes from patients with various forms of chronic liver disease, regardless of etiology. These findings suggest that γ-interferon produced by T lymphocytes that infiltrate the liver during the course of chronic hepatitis induces Class II major histocompatibility complex antigen expression and may endow the hepatocytes with the capacity to perform accessory (antigen-presenting) cell functions.
MATERIALS AND METHODSRESULTSDiscussionACKNOWLEDGEMENTSReferencesDiscussionReferences
Daily subcutaneous injections of 5 to 10 million units of partially purified human leukocyte interferon were given to newborn rabbits for 2 weeks or 1 month. The control groups received mock interferon, saline or nothing. The interferon treatment had no overt effect on the development of the animals during the period of treatment. The rabbits treated with interferon had leukocytosis, splenomegaly and prolonged postnatal extramedullary hematopoiesis in the liver and spleen. Certain immune responses were also demonstrated in the rabbits treated with interferon and mock interferon preparations. Platelet counts and the serum-ASAT, -ALAT, -LD and alkaline phosphatase values were normal.
After treatment with neuraminidase, human leukocyte interferon (HLI) retained its characteristic heterogeneity when subjected to isoelectric focusing. The clearance of HLI from the circulation of rabbits, injected intravenously, was unaffected by treatment with neuraminidase or subsequent treatment with galactose oxidase. Complementary chemical treatments were similarly without effect. None of the treatments resulted in destruction of biological activity. Sialic acid and galactose or its derivatives do not appear to contribute to either the antiviral activity or the pharmaco-kinetics of HLI.
Nine osteosarcoma cell lines, originally developed from six osteosarcoma tumours in five patients, and two cell lines of non-tumour origin (glia and fibro-blast) were grown in vitro in the presence of human leukocyte interferon (L-IF). L-IF exerted a dose-dependent inhibition of growth in all these lines. The inhibitory activity displayed characteristics typical of interferons. Inhibition of cell growth occurred at a much lower L-IF concentration for the osteosarcoma than for the non-tumour-derived lines. Inhibition of tumour cell growth was observed at concentrations obtained in the serum of osteosarcoma patients treated with interferon.
The spontaneous cytotoxicity of peripheral lymphoid cells from five tumor patients was measured before and at various times after the first injection of human leukocyte interferon (IF). Four of the patients' lymphocytes exhibited cytotoxicity before the IF injection. After injection of IF there was an initial decrease in cytotoxicity, followed by an increase to 1.5-5 times above the preinjection level, the peak being reached at 12 hours. Thereafter the spontaneous cytotoxicity decreased but usually remained elevated for 24 hours after the injection. The lymphocytes of the fifth patient had very low spontaneous cytotoxicity before the injection of IF, and this did not markedly change afterwards. The proportion of E-rosette forming cells seemed to decrease slightly in all patients after the injection, followed by a normalization at 24–48 hours.
Lymphotoxin, a glycoprotein lymphokine, possesses direct cytostatic and cytolytic activity for a wide variety of tumor cells. Lymphotoxin has been detected in the plasma from patients with neoplasia as well as individuals with various other diseases. Lymphokine preparations containing lymphotoxin activity can inhibit tumor growth in vivo, and recently lymphotoxin has been shown to irreversibly prevent carcinogen-induced morphological transformation during carcinogenesis. The anticarcinogenic activity of lymphotoxin is more potent than its tumor growth-inhibitory activity; the latter activity, furthermore, is frequently cytostatic rather than cytolytic. Lymphotoxin can also induce an increased susceptibility of tumor cells to cytolytic destruction by natural killer cells. The anticarcinogenic and tumor growth-inhibitory activities of lymphotoxin may be effected through alterations in cellular glycoproteins as the lymphokine stimulates incorporation of glucosamine into larger-molecular-weight glycoproteins in normal cells but inhibits incorporation of glucosamine into the glycoproteins in tumor cells.
The sensitivity of primary human ovarian cancer cells to interferon (IFN) was studiedin vitro by the use of a tumor cloning system in semi-solid agar. Tumor colonies were found in 37% (34/93) of the experiments with
tumor cells from ascites and in 35% (6/17) of the experiments with solid tumors. The relative colony-forming ability could
not be correlated to prior treatment.
In 15 out of 18 patients the ascitic tumor cells were sensitive to IFN-α. Sensitivity was found in one test out of three with
solid tumors. The sensitivity was dependent on the dose of IFN but could vary for the natural IFNs α and γ. In seven patients
the relation betweenin vitro andin vivo sensitivity of the tumor cells to IFN could be studied. Any correlation betweenin vitro andin vivo sensitivity could not be revealed in this small group of patients.
Die in vitro-Interferonproduktionsfhigkeit der Leukocyten von Patienten mit Lupus Erythematodes disseminatus und von Leukocyten gesunder Personen ergab keinen wesentlichen Unterschied.The authors made a comparison between in vitro interferon producing capacity of leucocytes originated from SLE syndromic patients and healthy persons, and found no significant differences.
Bei 2 Patienten mit M.S. und einem Patienten mit Dermatomyositis wurde whrend und nach einer ALG-Therapie in Lymphocytenkulturen die Mitoseaktivitt untersucht und Chromosomenanalysen durchgefhrt. Trotz manifester Lymphopenien war whrend der Therapie bis auf 2 Untersuchungen stets eine Antwort auf die PHA-Stimulation erkennbar. Nach Absetzen des ALG lagen die Mitoseindices stets niedriger als in den Kontrollen. Auf Grund dieser Beobachtungen wird das Vorhandensein verschiedener Lymphocytenpopulationen mit unterschiedlicher ALG-Empfindlichkeit diskutiert. Bei der Chromosomenanalyse konnten weder numerische noch strukturelle Chromosomenvernderungen beobachtet werden.The action of ALG-therapy in two patients with M.S. and one patient with dermatomyositis was studied by means of the mitotic activity and by chromosome analysis in lymphocyte cultures. Although persistent lymphopenia was present during therapy in all but two experiments, there was always a response to the PHA stimulation. After the termination of the ALG-therapy the mitotic indices were always lower than in the controls. Because of these observations the presence of different lymphocyte populations with different sensibility to ALG is discussed. Neither numerical nor structural chromosome aberrations could be demonstrated by chromosome analysis.
Interferon (IFN) was induced in suspensions of fresh human leukocytes by incubation with concanavalin A (Con A). The crude supernatant was concentrated and partially purified by adsorption to silicic acid and elution with an ethylene glycol (EG)-containing buffer. Th EG eluate was shown to contain two antivirally active components (tentatively called 22K), separable from each other by gel filtration. The 45K component was strictly species specific, relatively sensitive to acid (pH 2) and poorly neutralized by anti-human IFN-beta (HuIFN-beta) antibody. In all probability, it predominantly contained (a) molecular variant(s) of human gamma-type IFN (HuIFN-gamma ). The 22K component was acid resistant and serologically indistinguishable from HuIFN-beta prepared from fibroblasts. However, in contrast to the latter, it had little activity on bovine kidney cells and no activity on human HEp-2 and monkey Vero cells. A preparation with the same properties was obtained by substituting glycine buffer (pH 2) for EG in the elution of Con A-induced IFN from silicic acid. This IFN failed to adsorb to a zinc chelate column, while HuIFN-beta from fibroblasts can easily be purified by affinity chromatography on this type of column. It was concluded that the Con A-induced IFN contained an acid-resistant component of apparent molecular weight 22000 serologically related to classical HuIFN-beta but differing by certain physicochemical and biological criteria.
In this article the evidence pertaining to the perspectives for clinical applicability of interferons as antitumor drugs is critically reviewed. The mechanisms by which interferons can influence tumor growth and host defense against tumor proliferation are: a direct inhibitory effect on cell growth, alteration of cellular surfaces and immunomodulatory effects. In a particular situation several of these mechanisms may act syngergistically. In experiments on tumor-bearing animals the effect of interferon therapy consists of inhibition of tumor progression rather than induction of regression. Furthermore, the studies with animal models suggest that doses higher than those given in current clinical trials will be necessary to obtain clearly beneficial effects in man. Clinical trials with leukocyte interferon are well advanced, especially with regard to breast cancer, lymphoma and myeloma. They suggest that fairly small doses can cause tumor regression in some but not all cases. Regressions were also seen in all treated cases of laryngeal papilloma. Leukocyte interferon therapy might be useful to prevent metastasis after surgical removal of the primary tumor in osteosarcoma patients. Clinical trials with fibroblast interferon are less well advanced and the available evidence is rather fragmentary. No clinical trials have so far been done with immune type interferon.
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