Enhancement of defective monocyte function during immunotherapy with recombinant interferon

Summary The influence of immunotherapy with high dose (50×10⁶ units/m²) recombinant leukocyte A interferon on blood monocyte functions was studied in eight patients with bronchogenic carcinoma. Monocyte chemotactic responsiveness (MCR) was initially depressed (9.8±1.6 cells/field) compared to healthy controls (17.6±5.1 cells/field), P<0.01. Recombinant interferon was administered three times weekly, and after 7 days a significant improvement in chemotaxis was observed (16.6±3.0 cells/field), P<0.05. The MCR remained normal until cessation of interferon therapy (>1 month). Phagocytic and candidacidal activities were normal in the patients and were not influenced by treatment with interferon. In conclusion, high dose recombinant interferon given to cancer patients caused a normalization of defective blood monocyte chemotaxis, which persisted for >1 month.     

The Mode of Production of Endotoxin-Induced Interferon in Rabbit Tissue Cells

In vitro production of endotoxin-induced interferon in rabbit tissue cell cultures could be enhanced by pretreatment with interferon. The enhancible state developed from the first hr of incubation at 37 C and a maximal priming effect was attained at 6 hr of incubation. Yields of interferon from unprimed cultures were usually 20–200 units/ml. In contrast, the primed cultures constantly yielded 1,000–2,500 units/ml of interferon. The pretreatment with interferon seemed to cause an earlier appearance of detectable interferon and the primed cells became more sensitive to endotoxin. It turned out that 10–30 units/ml of rabbit interferon were enough to develop the maximal priming. Even when cells were pretreated with higher doses of rabbit interferon such as 1.0 × 10⁴–1.0 × 10⁵ units/ml, the same level of priming effect was always observed without diminution. Various types of homologous (rabbit) and heterologous (human and mouse) interferon preparations showed similar dose-dependent enhancement of interferon production in proportion to the antiviral titers of these preparations as tested with RK-13 cells of rabbit origin.     

High-level expression of human interferon alpha-2b in transgenic carrot (Daucus carota L.) plants

In this study, we report the obtaining of carrot plants expressing human interferon alpha-2b via Agrobacterium-mediated transformation using two vector constructs containing the sequence coding for interferon gene fused with Nicotiana plumbagenifolia calreticulin apoplast targeting signal driven by 35S CaMV promoter and root-specific Mll promoter. The human interferon alpha-2b gene was correctly translated in carrot plants according to Western blot analysis. The recombinant protein exhibited antiviral activity in vitro by inhibition of vesicular stomatitis virus replication in established piglet testicular cells. The results demonstrated the higher activity of interferon accumulated in carrot plants for young leaves (up to 50.7 × 10³ IU/g FW) compared to the mature ones probably due to the degradation-susceptible nature of this protein. The taproot-expressing system could have also provided the sufficient protein amounts (up to 16.5 × 10³ IU/g FW) and could possibly be used for generating interferon alpha-2b protein in planta for preventing and curing infectious diseases.     

Super‐infection with Staphylococcus aureus inhibits influenza virus‐induced type I IFN signalling through impaired STAT1‐STAT2 dimerization

Bacterial super‐infections are a major complication in influenza virus‐infected patients. In response to infection with influenza viruses and bacteria, a complex interplay of cellular signalling mechanisms is initiated, regulating the anti‐pathogen response but also pathogen‐supportive functions. Here, we show that influenza viruses replicate to a higher efficiency in cells co‐infected with Staphylococcus aureus (S. aureus). While cells initially respond with increased induction of interferon beta upon super‐infection, subsequent interferon signalling and interferon‐stimulated gene expression are rather impaired due to a block of STAT1‐STAT2 dimerization. Thus, S. aureus interrupts the first line of defence against influenza viruses, resulting in a boost of viral replication, which may lead to enhanced viral pathogenicity.     

Neisseria gonorrhoeae effectively blocks HIV‐1 replication by eliciting a potent TLR9‐dependent interferon‐α response from plasmacytoid dendritic cells

Clinical and epidemiological research provides evidence for a positive correlation between Neisseria gonorrhoeae infection and HIV transmission; however, mechanistic studies examining this relationship have yielded conflicting results. To explore this interaction, we exposed ex vivo cultured peripheral blood cells from acute HIV⁺ individuals to N. gonorrhoeae. Unexpectedly, we observed a profound inhibition in HIV‐1 replication in the ex vivo cultures, and this was recapitulated when peripheral blood mononuclear cells (PBMCs) from healthy donors were co‐infected with HIV‐1 and N. gonorrhoeae. Next, we established that gonococcal‐infected PBMCs liberated a soluble factor that effectively blocked HIV‐1 replication. Cytokine analyses and antibody blocking experiments revealed that the type I interferon, interferon‐α (IFNα), was expressed upon exposure to N. gonorrhoeae and was responsible for the inhibition of HIV‐1. Intracellular staining, TLR9‐blocking and cell depletion‐based studies demonstrated that the IFNα was elicited by plasmacytoid dendritic cells (pDCs) in a TLR9‐dependent manner. The pDC response to N. gonorrhoeae was unexpected given pDCs more established role in innate defence against intracellular pathogens, suggesting this may be a bacterial immune evasion strategy. In the context of HIV, this overcomes the virus's otherwise effective avoidance of the interferon response and represents a previously unrecognized intersection between these two sexually transmitted pathogens.     

Tryptophan reversal of recombinant human gamma-interferon inhibition ofChlamydia trachomatis growth

Recombinant human gamma-interferon was shown to inhibit the growth ofChlamydia trachomatis (L₂/434/Bu) in HEp-2 cells. This inhibition could be reversed by the addition of tryptophan. The effect of tryptophan was dose dependent and determined by the interferon concentration. At low concentrations of interferon, the addition of tryptophan completely restoredC. trachomatis infectivity, whereas at high concentrations (100–1000 IU/ml) the effect of interferon could not be totally reversed. The reversal effect of tryptophan could be achieved even when the addition was 48 h after infection. The probability that tryptophan degradation induced by gamma-interferon might be the mechanism involved in its antichlamydial activity is discussed.     

Effect of mouse genotype on interferon production

Upon induction with Newcastle disease virus, peritoneal macrophages derived from C57BL/6 mice produced ten times as much interferon as macrophages derived from BALB/c mice. This suggested that the alleles of theIf-1 locus are expressed in vitro by these cells. Further evidence for this was obtained by studying interferon production by peritoneal macrophages derived from seven recombinant inbred and one congenic line: in each case there was complete correlation between in vivo and in vitro phenotype: macrophages fromIf-1^l mice were low producers in vitro, and macrophages fromIf-1 ^h mice were high producers in vitro.     

The use of the chemostat to study the relationship between cell growth rate, viability, and the effect of interferon on L 1210 cells

Mouse leukemia L 1210 cells were cultivated in the chemostat at growth rates ranging from 0.1 day⁻¹ (population doubling time (T_d) 166.3 h) to 2.0 day⁻¹ (T_d 8.3 h). At growth rates of 1.0 day⁻¹ and above, the viability of the steady-state culture was greater than 99%. However, below 1.0 day⁻¹ there was a progressive decrease in the viability of the culture with decreasing growth rate until a minimum growth rate (0.1 day⁻¹) was reached below which steady-state cultures of L 1210 cells could not be established. Interferon treatment had no effect on the viability (>99%) of L 1210 cells cultivated at fast growth rates in the chemostat, whereas at slow growth rates (0.35 day⁻¹) interferon treatment markedly reduced the viability of the culture, even though the percentage increase in the doubling time of interferon-treated cultures was the same for cells cultivated at both fast and slow growth rates. Thus, although interferon is not directly cytotoxic, it can cause cell death by reducing the rate of cell multiplication below the minimum value compatible with viability.     

Assessment of goat activin receptor type IIB knockdown by short hairpin RNAs in vitro

Abstract

Background: Targeted knockdown of ACVR2B, a receptor for TGF beta superfamily, has been seen as a potential candidate to enhance the muscle mass through RNAi approach. Methods: We have evaluated the potential short hairpin RNAs targeting goat ACVR2B in human HEK293T cells and goat myoblasts cells by transient transfection and measured their knockdown efficiency and possible undesired interferon response by quantitative real-time PCR. Results: We observed a significant silencing (64–81%) of ACVR2B in 293T cells with all seven shRNAs (sh1 to sh7) constructs and 16–46% silencing with maximum of 46% by sh6 (p?=?0.0318) against endogenous ACVR2B whereas up to 66% (p?=?0.0002) silencing by sh6 against exogenously expressed ACVR2B in goat myoblasts cells. Transient knockdown of ACVR2B in goat myoblasts cells by shRNAs did not show significant correlation with the expression of MyoD (r?=?0.547; p?=?0.102), myogenin (r?=?0.517; p?=?0.126) and Myf5 (r?=?0.262; p?=?0.465). As reported earlier, transfection of plasmid DNA induced potent interferon response in 293T and goat myoblasts cells. Conclusions: The present study demonstrates the targeted knockdown of ACVR2B by shRNAs in HEK293T and goat myoblasts cells in vitro. The transient knockdown of ACVR2B by shRNAs in goat myoblasts did not alter the myogenic gene expression program. However, shRNAs showing significant knockdown efficiency in our study may further be tested for long term and stable knockdown to assess their potential to use for enhancing muscle mass in vivo. As reported earlier, expression of shRNAs through plasmid expression vectors induces potent interferon response raising the concern of safety of its application in vivo.     

Enhancement by interferon of natural killer cell activity in mice.

Injection of mice with several interferon inducers, Newcastle Disease virus, polyinosinic-polycytidylic acid and tilorone resulted in an increase in spleen cell cytotoxicity for ⁵¹chromium-labeled mouse YAC tumor target cells in 4-hr in vitro assays. This increase in spleen cell cytotoxicity was abrogated by injection of mice with potent anti-mouse interferon globulin. Inoculation of mice with mouse interferon (but not human leucocyte or mock interferon preparations) also resulted in a marked enhancement of spleen cell cytotoxicity. The extent of enhancement of spleen cell cytotoxicity was directly proportional to the amount of interferon injected and a significant increase was observed after inoculation of as little as 10³ to 10⁴ units of interferon. An effect could be detected as soon as 1 hr after injection of interferon. The increase of spleen cell cytotoxicity after inoculation of an interferon inducer was not due to a localization and accumulation of cytotoxic cells in the spleen but reflected a general increase in cytotoxic cell activity in various lymphoid tissues (except the thymus). The splenic cytotoxic cells from interferon or interferon-inducer-injected mice had the characteristics of natural killer (NK) cells since (i) interferon enhanced spleen cell cytotoxicity in athymic (nu/nu) nude mice, (ii) classical spleen cell fractionation procedures by nylon wool columns, anti-Thy 1.2 serum plus complement, anti-Ig columns, and depletion of FcR+ rosette-forming cells, failed to remove the effector cells generated in vivo or in vitro. Therefore like NK cells, interferon-induced cytotoxic cells lack the surface markers of mature T and B lymphocytes, are not adherent, and are devoid of avid Fc receptors. Furthermore like NK cells, the spleen cells from interferon-treated mice lysed various target cells (known for their sensitivity to NK cells) without H-2 or species restriction. Incubation in vitro of normal spleen cells with interferon also resulted in an increase in cytotoxicity for YAC tumor cells. We conclude that interferon acts directly on NK cells and enhances the inherent cytotoxic activity of these cells.     

Continuous production of human leukocyte interferon withEscherichia coli and continuous cell lysis in a two stage chemostat

Summary The continuous production and extrac-tion of human leukocyte interferon (type α2) in a two stage chemostat is described. Interferon con-tainingEscherichia coli cells were produced in the first stage and transferred to the second stage, where the cells were lysed continuously by the ad-dition of ampicillin. The medium used was based on corn steep liquor. Highest interferon titres and the best extraction efficiencies were achieved when running the first and second stages at dilution rates of 0.3 h⁻¹ and 0.1 h⁻¹, and at temperatures of 30° C and 25 °C, respectively. In order to prevent loss of interferon in the second stage, oxygen limitation had to be avoided. For optimal cell lysis there should be excess glucose in the second stage and the ampicillin concentration should be maintained above 100 mg 1⁻¹.     

Inhibition of type I interferon induction and signalling by mosquito‐borne flaviviruses

The Flavivirus genus (Flaviviridae family) contains a number of important human pathogens, including dengue and Zika viruses, which have the potential to cause severe disease. In order to efficiently establish a productive infection in mammalian cells, flaviviruses have developed key strategies to counteract host immune defences, including the type I interferon response. They employ different mechanisms to control interferon signal transduction and effector pathways, and key research generated over the past couple of decades has uncovered new insights into their abilities to actively decrease interferon antiviral activity. Given the lack of antivirals or prophylactic treatments for many flaviviral infections, it is important to fully understand how these viruses affect cellular processes to influence pathogenesis and disease outcome. This review will discuss the strategies mosquito‐borne flaviviruses have evolved to antagonise type I interferon mediated immune responses.     

Porcine interferon-γ inhibits the growth ofLegionella pneumophila in WiREF cellsin vitro

The intracellular growth ofLegionella pneumophila in WiREF (Wistar rat embryonal fibroblast) cells was inhibited by porcine interferon-γ. The effect was compared with that of different human interferons (α and γ). The growth inhibition was dose-dependent and required the pretreatment of WiREF cells with interferon. The development of an antibacterial state of the cells was observed. When interferon was added together with bacteria or 1 d after the infection there was no inhibition. Also, there was no direct antibacterial effect of the interferon. In addition, cell pretreatment with a combination of interferon and antibiotics failed to show a synergistic effect.     

αs1-Casein-specific CD8+ T Cell Clone That Inhibits Its Own Interferon-γ Production by Producing Interleukin-10

A CD8⁺ T cell clone specific to α_s1-casein, one of the major allergens in milk, is shown to inhibit its own production of interferon (IFN)-γ by producing interleukin (IL)-10. Anti-IL-10 antibodies enhanced the production of IFN-γ induced by the antigen plus antigen-presenting cells from 12h onward after initiating the culture. This enhancing effect was observed only when the cells were stimulated in the presence of the antigen-presenting cells. Neither IL-2 nor IL-4 abrogated this enhancing effect. This reveals a new regulating mechanism for IFN-γ production from CD8⁺ T cells.     

Interferon induction by platinum(II)-poly(I)·poly(C) complexes

The effect of the interaction between poly(I)·poly(C) and cis-dichloro-diammineplatinum(II) (cis-Pt), its trans analogue and chloro-diethylene-triamminoplatinum(II) (dien-Pt) on interferon induction activity was investigated. The covalent monodentate fixation of the three compounds on N⁷ of inosine has different effects on the structure and thermostability of poly(I)·poly(C) which is well reflected by the interferon induction activity of the samples. Thus, the sandwich stabilization by dien-Pt at low binding ratios is manifested by an increased interferon induction and a high resistance towards RNAase degradation. The destabilization of the duplex by cis-Pt decreases interferon induction, accompanied by an increase in RNAase sensitivity of the complexes. In the case of trans-Pt the duplex structure is little perturbed and interferon induction is essentially maintained.     

The Inhibitory Effect of Staphylococcus epidermidis Slime on the Phagocytosis of Murine Peritoneal Macrophages Is Interferon-Independent

The extracellular slime produced by Staphylococcus epidermidis has been shown to interfere with several human neutrophil functions in vitro, such as chemotaxis, degranulation and phagocytosis. Slime production has been suggested as a useful marker for clinically significant infections with coagulase-negative Staphylococcus. Since the main role of macrophages in defense mechanisms is phagocytosis, the effect of slime on the phagocytic activity of macrophages was investigated. The phagocytic activity of murine peritoneal macrophages treated with slime in vitro decreased in a dose-dependent fashion. A similar decrease was also observed in macrophages isolated from mice that had previously received intraperitoneal injection of slime. To investigate whether interferon also plays a role in this process, mice were treated with interferon or an interferon inducer, polyinosinic-polycytidylic acid (poly I:C), together with slime before macrophage isolation. The slime-suppressed phagocytic activity of macrophages was partially relieved by both agents, and the recovery effect of poly I:C in slime-suppressed phagocytosis of macrophages in vivo might be attributed to the increased interferon level in peritoneal fluid and sera. However, when slime was given to poly I:C-pretreated mice, the phagocytic activity remained suppressed. Thus, it appears that slime is able to suppress the phagocytic activity of macrophages regardless of the state of macrophage activation by poly I:C. The results suggest that the inhibition of phagocytosis by S. epidermidis slime may be independent from the activation of interferon.     

Experiences with the on-line measurement of culture fluorescence during cultivation of Bacillus subtilis, Escherichia coli and Sporotrichum thermophile

Bacillus subtilis and Escherichia coli K12 (both transformed for human leukocyte interferon production) and Escherichia coli B/r and Sporotrichum thermophile (a deuteromycete) were cultivated in submersed culture and the culture fluorescence recorded on-line using a fluorometer. During the cultivation of B. subtilis the signal from the fluorometer correlated with cell density and interferon production and thus could be used for process control (interferon production). However, the culture fluorescence of the other organisms did not increase (S. thermophile), was too weak to be measured with the fluorometer used (E. coli transformed for interferon production), or the signal from the fluorometer was not an accurate measure of the culture fluorescence because of the accumulation of a fluorophor in the culture medium (E. coli B/r).     

Influence of low doses of an oxazaphosphorine on natural killer activity of human lymphocytes

Summary The influence of cis-4-sulfoethylthio-cyclophosphamide (mafosfamide) on natural killer cell activity was examined in vitro in order further to elucidate the possible immunological mechanisms of tumor regressions following low-dose oxazaphosphorine therapy. It was observed that cytotoxicity of human blood lymphoid cells was unchanged or reduced when the lymphocytes were pretreated for 24 h with mafosfamide or when the drug was present during incubation with K562 cells. However, when lymphoid cells were preincubated with human leukocyte interferon plus mafosfamide, natural killer activity was enhanced above the level caused by interferon alone. This enhancement was noted at mafosfamide concentrations of 1 nM-1 M and was only present when the lymphocyte preparation was contaminated with monocytes.     

Suppressive Subtraction Libraries to Identify Interferon-Inducible Genes in Fish

Previous attempts to identify genes in fish that respond to virus infection or interferon induction have not been particularly productive. Since these genes are very important in developing strategies to control disease outbreaks in aquaculture, we began a study of interferon-inducible genes in fish using suppressive subtraction hybridization to construct cDNA libraries enriched for interferon-inducible genes. Subtraction hybridization libraries were constructed with cDNA obtained from the kidney, spleen, and liver of Chinook salmon (Oncorhynchus tshawytscha) and staghorn sculpin (Hemilepidotus spinosus) before and after injection with poly IC, a potent interferon inducer. The ``identified' genes in both cDNA libraries corresponded to previously identified genes of the fish complement system, the interferon-inducible proteins observed in mammalian cells, and the Vig-1 gene, identified in fish cells after infection with fish rhabdoviruses. Received June 19, 2001; accepted July 13, 2001