Effect Of Recombinant Interferon-gamma Research Articles

Immune responses to live and inactivated Nocardia seriolae and protective effect of recombinant interferon gamma (rIFN γ) against nocardiosis in ginbuna crucian carp, Carassius auratus langsdorfii

Looking into the fact that substantial mortality and morbidity is associated with intracellular Gram +ve bacterium, Nocardia seriolae infection, an effective vaccine against this pathogen is necessary to control the significant losses in aquaculture practices. Therefore, an attempt was made to evaluate the effect of live (sub-lethal) and inactivated (antigenic form) N. seriolae on cellular and humoral immunity in ginbuna crucian carp, Carassius auratus langsdorfii as well as the therapeutic potency of recombinant interferon gamma (rIFN γ) against N. seriolae infection. Effect of live and inactivated N. seriolae immunisation on the proliferation of CD4+ T cells, CD8α+ T cells and surface Ig M+ cells in peripheral blood leucocytes, spleen, head kidney and trunk kidney of ginbuna was studied after 1st, 3rd, 7th, 15th and 30th day post immunisation. The percentage of CD8α+ T cells in spleen and head kidney of ginbuna was significantly higher at 3rd day post immunisation. Similarly, surface Ig M+ cells level was found to increase in both live and inactivated N. seriolae immunised groups. On the contrary, high percentage of CD4+ T cells was observed in live N. seriolae immunised group in both the head and trunk kidneys at 30th day post immunisation. The humoral immune response to live and inactivated N. seriolae immunised ginbuna showed high antibody titre at 15th day post immunisation but the level declined subsequently in both the immunised groups. On challenge with virulent N. seriolae (1.2 × 108 CFU/ml), the relative percent survival was 62.5 and 75 in live and inactivated N. seriolae immunised groups, respectively. Furthermore, we have also studied the therapeutic potency of rIFN γ and found the possible involvement of IFN γ in resistance mechanism in fish. Administration of rIFN γ into ginbuna (at 10 μg/fish) one day before challenge study was found to protect ginbuna. The relative percent survival of ginbuna was 43.75 and 60 when challenged with 2 different doses of N. seriolae i.e., 1.2 × 108 CFU/ml and 5 × 107 CFU/ml, respectively. In summary, this study indicates that both forms of N. seriolae immunisation as well as rIFN γ indeed elicit an effective protective immunity which will help in designing suitable vaccine and/or adjunct therapy against N. seriolae infection in fish.

Production of large numbers of plasmacytoid dendritic cells with functional activities from CD34+ hematopoietic progenitor cells: Use of interleukin-3

Plasmacytoid dendritic cells (pDC), a subset of dendritic cells characterized by a rapid and massive type-I interferon secretion through the Toll-like receptor pathway in response to viral infection, play important roles in the pathogenesis of several diseases, such as chronic viral infections (e.g., hepatitis C virus, human immunodeficiency virus), autoimmunity (e.g., psoriasis, systemic lupus erythematosus), and cancer. As pDC represent a rare cell type in the peripheral blood, the goal of this study was to develop a new method to efficiently generate large numbers of cells from a limited number of CD34(+) cord blood progenitors to provide a tool to resolve important questions about how pDC mediate tolerance, autoimmunity, and cancer. Human CD34(+) hematopoietic progenitor cells isolated from cord blood were cultured with a combination of Flt3-ligand (Flt3L), thrombopoietin (TPO), and one of the following cytokine: interleukin (IL)-3, interferon-β(IFN-β), or prostaglandin E2(PGE(2)). Cells obtained in the different culture conditions were analyzed for their phenotype and functional characteristics. The addition of IL-3 cooperates with Flt3L and TPO in the induction of pDC from CD34(+) hematopoietic progenitor cells. Indeed, Flt3L/TPO alone or supplemented with prostaglandin E2 or interferon-β produced smaller amounts of pDC from hematopoietic progenitor cells. In addition, pDC generated in Flt3L/TPO/IL-3 cultures exhibited morphological, immunohistochemical, and functional features of peripheral blood pDC. We showed that IL-3, in association with Flt3L and TPO, provides an advantageous tool for large-scale generation of pDC. This culture condition generated, starting from 2 × 10(5) CD34(+) cells, up to 2.6 × 10(6) pDC presenting features of blood pDC.

MRNA-based approach to monitor recombinant gamma-interferon restoration of LPS-induced endotoxin tolerance

IntroductionIt is now well accepted that sepsis is associated with the development of a pronounced immunosuppressive state, characterized by severe immune alterations (e.g. reduced proliferative capacity, endotoxin tolerance, apoptosis) participating in increased mortality and susceptibility to nosocomial infections. Efforts are currently aimed at restoring a functional immune response in septic patients. Successful therapy depends on the identification of appropriate immunostimulatory drugs and on the development of suitable biomarkers that could be used to stratify patients and to follow response to treatment.MethodsIn this study, we evaluated the ex vivo effect of recombinant interferon gamma (rIFN-γ) in restoring monocyte functionality (endotoxin-induced Tumor Necrosis Factor-α production) in a two-hit model of endotoxin tolerance (ET) with peripheral blood mononuclear cells from healthy volunteers and in whole blood of septic shock patients. Importantly, we used quantitative-reverse transcription polymerase-chain reaction to monitor the effect of rIFN-γ on the expression of seven genes known to participate in ET (TNF-α, IL-10, HLA-DRA, CIITA, IRAK-M, ABIN-3 and LY64).ResultsExpression analysis of those genes confirmed the presence of an immunosuppression state and the ex vivo restoration of immune functions by rIFN-γ. We show for the first time that rIFN-γ is able to bypass, at the mRNA level, the effect of negative regulators of the LPS signalling pathway such as IRAK-M, ABIN-3 and LY64.ConclusionsOverall, mRNA expressions of a panel of genes could represent promising candidates for the ex vivo evaluation of rIFN-γ effect on monocyte functionality. This ex vivo translational research study demonstrates the potential of a mRNA-based approach to successfully monitor drug efficacy.

924 The effect of recombinant interferon gamma on IgE synthesis in adult asthmatic patients

924 The effect of recombinant interferon gamma on IgE synthesis in adult asthmatic patients

Augmented Procoagulant Activity in Cancer Patients, Treated with Recombinant Interferon-γ in Addition to Recombinant Tumor Necrosis Factor-α and Melphalan

Several investigators have reported that interferon-gamma (IFN gamma) can alter tumor necrosis factor alpha induced effects in vitro. We assessed in vivo effects of recombinant interferon-gamma (rIFN gamma) on recombinant tumor necrosis factor-alpha (rTNF alpha) induced activation of systemic blood coagulation in a non-randomized study in 20 consecutive cancer patients. Eight patients were treated with rIFN gamma prior to and during hyperthermic isolated limb perfusion with rTNF alpha and melphalan (IFN gamma group). They were compared with twelve patients who did not additionally receive rIFN gamma (non-IFN gamma group). Before start of perfusion, higher levels of TNF alpha, F1+2 and TAT levels were found in the IFN gamma group. Fibrinogen and ATIII levels tended to be lower in this group. High TNF alpha levels, due to leakage during perfusion, were associated with activation of coagulation in all patients, that became obvious after the end of perfusion, when heparin treatment had been antagonized. Activation, measured by increased F1+2 and TAT levels, was significantly stronger in the IFN gamma group. Monocytic TF remained low, possibly due to shedding of TF positive vesicles and/or sequestration of TF positive activated monocytes against the vessel wall. In both groups F1+2 and TAT levels declined 24 h after the perfusion, whereas monocytic TF increased to levels that were higher in the IFN gamma group. In conclusion, our data confirm a strong activation of coagulation induced by rTNF alpha in cancer patients. They suggest that rIFN gamma may lead to a slight activation of coagulation and augments TNF alpha induced procoagulant activity. These effects may be due to rIFN gamma induced sustained monocytic TF activity.

In Vivo Activity Of Interferon- In Combination With Amphotericin B In The Treatment Of Experimental Cryptococcosis

The effect of recombinant interferon-gamma (rIFN-gamma) on Cryptococcus neoformans infection was investigated in vivo. BALB/c mice were injected intravenously with 2 x 10(6) C. neoformans. rIFN-gamma alone (10 micrograms intraperitoneally 18 h before, at, 24 h after infection) significantly increased the survival and decreased the colony-forming unit counts in the lungs compared with untreated mice. rIFN-gamma association significantly enhanced the effect of a single dose of amphotericin B (0.25 mg/kg 24 h after infection) to prolong mouse survival and to reduce colony counts in the brain. Tumor necrosis factor-alpha levels, measured in spleen 3, 6, and 24 h after infection, were not increased by rIFN-gamma. These results suggest that exogenous rIFN-gamma might improve the effect of antifungal therapy during cryptococcosis.

In vitro effect of recombinant interferon gamma in combination with LPS on amoebicidal activity of murine Kupffer cells

The present study examines the role of liver macrophages (Kupffer cells), of C57BL/6 mice, as effector cells responsible for the killing of Entamoeba histolytica trophozoites in vitro.It was shown that unstimulated Kupffer cells were inefficient in the killing of E.histolytica trophozoites in vitro.Interferon gamma (IFN-γ) alone was not able to activate Kupffer cells to amoebicidal state.However, Interferon gamma and lipopolysaccharide (LPS) acted synergistically in this phenomenon.It seems that the acquisition of amoebicidal activity is associated with the involvement of hydrogen peroxide, because the addition of catalase partially decreases the killing of this parasite by Kupffer cells.In addition, it appears that the amoebicidal activity of IFN-γ-treated Kupffer cells is contact-dependent. Our results indicate that the immunologic production of IFN-γ is important in the activation of Kupffer cells for controlling this parasite and that Kupffer cells are strong effector cells against the amoebae.

Ability of recombinant interferon gamma in vitro to restore the defective polymorphonuclear-cell- but not lymphocyte-mediated cytotoxic activities in patients with myelodysplastic syndromes.

In this study we analysed the in vitro effect of recombinant interferon gamma on cytotoxic activities mediated by both lymphoid and polymorphonuclear cells from 16 patients with myelodysplastic syndromes. Our results indicate the inability of interferon to restore the defective natural killer activity, natural killer cells and lectin-induced cytotoxicity. On the contrary we detected a boosting effect on the depressed polymorphonuclear cell cytotoxic activities. In our view, the ability of interferon to potentiate polymorphonuclear cell lytic efficiency could support an alternative defensive pathway against either neoplastic or infectious agents.

Recombinant interferon gamma for treatment of chronic granulomatous disease and other disorders

The chemistry, biological activity, and pharmacokinetics of gamma-interferon and recombinant interferon gamma are reviewed, and the agent's clinical efficacy, adverse effects, and dosage and administration for the treatment of chronic granulomatous disease (CGD) and other disorders are described. Endogenous gamma-interferon is a 166-amino-acid protein encoded by a single gene on chromosome 12. Recombinant human interferon gamma is purified from Escherichia coli as a monomer containing 139 amino acids. Gamma-interferon has antiviral, immunomodulatory, and antiproliferative activity. Serum concentrations of recombinant interferon gamma increase in proportion to the dose. Clearance after i.m. or s.c. administration fits a two-compartment model. The half-life is 3.5-7.5 hours, and bioavailability is 89%. Evidence that recombinant interferon gamma can enhance phagocytic oxidative metabolism led to its evaluation for use in the treatment of CGD. Clinical studies showed that the agent decreases the frequency of serious infections in patients with CGD. Recombinant interferon gamma has shown only limited success in the treatment of metastatic renal cell carcinoma (RCC), both as a single agent and in combination with recombinant interferon alfa. Similarly, although interferons appear to be able to change cytogenetic abnormalities in some patients with Philadelphia chromosome-positive chronic myelogenous leukemia, therapy with recombinant interferon gamma has led to minimal success. However, the agent has produced some encouraging results in atopic dermatitis. The adverse effects of recombinant interferon gamma in patients with CGD usually consist only of fever, chills, headache, and erythema. The recommended dosage in CGD-afflicted children whose body surface area is greater than 0.5 sq m is 50 micrograms/sq m given by s.c. injection three times a week for life. Recombinant interferon gamma has given new hope to patients with CGD. Although the drug is very expensive, the cost may be offset by fewer hospitalizations to treat infection.

Cytokine effects in a human colonic goblet cell line. Cellular damage and its partial prevention by 5 aminosalicylic acid.

The aim of the present study was to investigate the effects of cytokines on intestinal goblet cells in vitro. For this purpose, we examined the effects of recombinant interferon gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on the human colonic goblet cell line Cl.16E by morphological and kinetic studies, and by the assessment of mucus production during IFN-gamma/TNF-alpha treatment. Control cultures of Cl.16E cells grown on nitrocellulose filters formed monolayers of polarized goblet cells, which had kinetic characteristics similar to those of a differentiated epithelium in steady state. The combined action of IFN-gamma and TNF-alpha caused a dose-related cellular exfoliation, leading to the formation of a mucoid cap made of mucus and cellular debris. The remaining viable cells underlying the mucoid cap were cuboidal and devoid of mucus granules. A dose-related increase in cellular incorporation of [3H]thymidine was reactive to the cytokine-induced cell loss. The synergistic effects of IFN-gamm and TNF-alpha were found to be reversible when the cells were reincubated in a culture medium without cytokines. Furthermore, 5-aminosalicylic acid partially protected Cl.16E cells against cellular injury caused by IFN-gamma and TNF-alpha. On the whole, these morphological and kinetic findings argue that the changes induced in Cl.16E cells by IFN-gamma and TNF-alpha closely parallel those observed during the acute phase of ulcerative colitis, and show that these cytokines can regulate intestinal mucus production by modulating cellular exfoliation, thus leading probably to a reinforced protection of the damaged mucosa.

Human Alveolar Macrophages of Anergic Patients with Lung Cancer Lack the Responsiveness to Recombinant Interferon Gamma

We studied the effect of recombinant interferon gamma (rIFN-gamma) on the phagocytic and bactericidal actions in human alveolar macrophages (AM) of patients with pulmonary tuberculosis and lung cancer. Treatment with 100 or 1,000 U/ml of rIFN-gamma for 24 hours resulted in an increased percentage of AM ingesting bacillus Calmette-Guérin (BCG) and an increased number of ingested BCG in individual AM in tuberculin-positive patients with lung cancer and pulmonary tuberculosis. The rIFN-gamma treatment also showed increased killing activity of AM in tuberculin-positive patients. However, rIFN-gamma treatment of AM in tuberculin-negative anergic patients with lung cancer did not induce the increase of phagocytic and killing activities against BCG. These results suggest that responsiveness and activation of AM to rIFN-gamma are inhibited in the anergic environment.

Effects of interferon gamma on cultured synovial cells from patients with rheumatoid arthritis: inhibition of cell growth, prostaglandin E2, and collagenase release.

The effects of recombinant interferon gamma (rIFN gamma) on the in vitro growth of adherent synovial fibroblast-like cells from patients with rheumatoid arthritis (RA) and also on the release of prostaglandin E2 and collagenase from these cells stimulated with recombinant interleukin-1 beta (rIL-1 beta) were investigated. The growth of adherent synovial cells from six of nine samples, determined by [3H]thymidine incorporation, was inhibited by rIFN gamma in a manner dependent on dose. The release of prostaglandin E2 and collagenase from adherent synovial cells stimulated with rIL-1 beta was also suppressed by rIFN gamma in all samples tested, though the basal release of these inflammatory mediators was little influenced. No apparent correlation between inhibition of proliferation by rIFN gamma and either inhibition by rIFN gamma of rIL-1 beta stimulated prostaglandin E2 release or the endogenous synthesis of prostaglandins was found.

Immunological effects of recombinant interferon gamma in vivo in normal mice: failure to induce autoantibodies

We have investigated the effects on the immune system, and especially on the induction of autoimmunity, of treatment of mice with recombinant IFN-γ in vivo by several protocols. Neither antichromatin nor Coombs autoantibody was observed. The spleens of the treated animals enlarged two fold, despite a dramatic decrease in numbers of Thy-1 + spleen cells and a smaller decrease in surface Ig + spleen cells. This was correlated with a markedly diminished Con A response and moderately reduced LPS response. On the other hand, the numbers of IgG secreting cells were augmented in the spleens of treated mice. In addition, IFN-γ-injected mice lost weight and became anemic. This study shows that, although IFN-γ-injected in vivo leads to severe changes in the murine immune system, it is not responsible by itself for the induction of autoimmunity.

Effects of Recombinant Interferon-Gamma and Interleukin-2 on the Generation of Lymphokine-Activated Killer Cells in Vitro

In order to determine if recombinant interferon-gamma (rIFN-gamma) can augment the effect of recombinant interleukin-2 (rIL-2) in generating lymphokine-activated killer (LAK) cells, we have incubated normal peripheral blood mononuclear cells (PBMC) with these lymphokines for 3 days and then tested their LAK and natural killer (NK) cell activity. We have found that LAK activity in PBMC from 13 out of 13 normal donors was increased by the combined lymphokines above that due to either lymphokine alone, provided that rIL-2 was present at suboptimal concentration: Optimal levels of rIFN-gamma (100 U/ml) were able to enhance the LAK-inducing activity of suboptimal levels (5 U/ml) but not optimal levels (100 U/ml) of rIL-2. NK activity showed a similar response to these concentrations of lymphokines. Activation of LAK/NK cells was accompanied by increases in the percentages of Leu 19+ (CD56) cells and TAC+ (IL-2-receptor) cells, and in the intensity of TAC antigen expression. These results indicate that combination rIFN-gamma and rIL-2 may be more effective in generating LAK/NK cells than rIL-2 alone, particularly with suboptimal concentrations of rIL-2 such as occur during continuous infusion therapy with this agent.

A Rapid Fluorometric Assay for the Determination of Keratinocyte Proliferation In Vitro

In the present study we describe a simple technique for the determination of keratinocyte proliferation in vitro, based on the hydrolysis of a fluorogenic substrate by cell esterases. Normal and transformed human keratinocytes were grown in microtiter plates and were incubated with 4-methylumbelliferyl heptanoate after 3, 5, and 7 days. The fluorescence was quantified using an automatic fluorescence detection unit. The fluorescence showed a strong correlation with the cell number at various growth phases. In addition, the method reliably detected the growth inhibitory effect of recombinant interferon gamma on human keratinocytes. The fluorometric assay is a simple, fast and reliable method to assess cell number in keratinocyte cultures.

The effect of recombinant interferon-gamma on human monocyte-derived macrophages.

The effect of recombinant interferon-gamma (rIFN-gamma) on human macrophage functions was studied, using monocytes which had matured to macrophages within hydrophobic containers. Following exposure to rIFN-gamma, the number of surface-expressed specific IgG-binding sites was increased. This increase was restricted to high-affinity Fc receptors (FcR), however; low-affinity FcR were not increased in number. Exposure to rIFN-gamma led to an enhanced chemiluminescence (CL) signal in the presence of luminol and a variety of respiratory burst stimuli, such as zymosan, phorbol 12-myristate 13-acetate or IgG-sensitized sheep erythrocytes (EA). In contrast, phagocytosis of EA was markedly depressed in rIFN-gamma-treated cells. Both increase in CL response and decrease in phagocytic activity were manifest after 1 day of treatment and were more pronounced after 2 days. While 5 U/ml of rIFN-gamma was an insufficient dose, 50 to 5000 U/ml yielded significant dose-dependent changes in both functional assays. Thus, using rIFN-gamma as a biological response-modifier, FcR expression and FcR-mediated CL can be dissociated from FcR-mediated phagocytosis.

Effect of recombinant interferon-gamma on hydrogen peroxide-releasing capacity of monocyte-derived macrophages from patients with lepromatous leprosy.

Monocyte-derived macrophages from 14 patients with lepromatous leprosy respond to rIFN-gamma with an enhanced secretion of H2O2 in a fashion similar to that of cells obtained from normal donors. The activation is not dependent on the cutaneous bacterial index, the length of treatment, or the stage and activity of the disease. H2O2 release can be triggered in these cells both by phorbol myristate acetate and by intact irradiated Mycobacterium leprae. Uptake of M. leprae by both normal donors' and patients' macrophages is proportional to the number of bacilli added. Prior ingestion of M. leprae does not interfere with the ability of macrophages to respond to IFN-gamma by the production of oxygen intermediates. We conclude that the immune defect in lepromatous leprosy probably results from a lack of response to M. leprae by the patients' T cells rather than an inability of mononuclear phagocytes to respond to IFN-gamma.

Induction of HLA class-II antigen expression on human carcinoma cell lines by IFN-Gamma.

The effects of recombinant interferon gamma (IFN-gamma) on HLA antigen expression were examined in various human carcinoma cell lines: carcinomas of the lung (ChaGo, Oat 75, SK-LC-LL), colon (HT-29, WiDr), larynx (HEp-2), cervix (ME-180) and mammary gland (AlAb). Surface expression of HLA antigens was determined using monoclonal antibodies directed against monomorphic determinants of HLA class II (HD II, 2.06) and HLA class I (W6/32) by several techniques including indirect immunofluorescence, immunoperoxidase assay, radioimmunoassay of cells, and immunoprecipitation of radioiodinated cells. Without previous exposure to IFN-gamma, HLA class-II antigen was surface-expressed on a small subpopulation of HEp-2 only. All cell lines examined, except AlAb, could be induced to cell-surface expression of HLA class-II antigen after 48 hr incubation in the presence of IFN-gamma (10 units/ml). HLA class-I antigen was present on all cell lines even without treatment, and it was increased after IFN-exposure. Incorporation of 3H-thymidine and 3H-leucine and expression of the transferrin receptor were not significantly altered by IFN-gamma application, indicating a specific regulatory effect of IFN-gamma on HLA antigen expression. Our data demonstrate that HLA class-II surface expression is inducible by IFN-gamma in a variety of epithelial tumor lines. This may have implications for the host immune system defense mechanisms against tumors.