Human Γ-interferon Research Articles

LEVELS OF IL-4 AND Γ- INTERFERON IN CHILDREN’S BLOOD SERUM AS PREDICTORS OF ASTHMA FORMATION

Introduction. The prevalence of asthma continues to increase every year. Asthma is the leading chronic disease. Infl ammation,both acute and chronic, is closely related to the action of cytokines (pro- and anti-infl ammatory). T-helper 2 lymphocytesrelease IL-4, which triggers the production of immunoglobulin E (IgE) antibodies, which set the stage for allergy and activateinfl ammatory cells. The regulators of this process are T helper-1 lymphocytes, which produce cytokines (IFN-gamma) thatsuppress Th-2 activity. Disturbances in the ratio of Th-1/Th-2 lymphocyte clones are a subject of debate in the early detectionof asthma and the prediction of the development of this disease.The aim of the study was to evaluate the levels of IL-4 and γ-interferon in the blood serum of children with transient wheezeand asthma..Material and methods. 121 children were included in the study. The patients were divided into groups: group 1 – childrenwith transient wheezing (TW) (n = 81), group 2 – children with doctor- diagnosed asthma (DDA) (n = 15), group 3 – healthychildren (n = 25).IL-4 and γ-interferon in blood serum were analyzed by ELISA using commercial kits (human IL-4 and human γ-interferon“Vector Best- Ukraine”). Data were analyzed using Statsoft Statistica version 8 (Tulsa, OK) and the MedCalc version 17.2statistical program.The planned clinical trials were conducted after obtaining the approval of the local ethics committee (date: February 1, 2015;number: 2015/01) and were conducted in accordance with the principles of the Declaration of Helsinki, as amended in October 2013.This article was conducted within the framework of the Department of Pediatrics No. 2 of Kharkiv National MedicalUniversity «Medical and biological aspects of adaptation of children with somatic pathology to modern conditions. Predictionof asthma control in children taking into account infl ammatory markers and the state of the airway barrier of the lungs» (stateregistration number 0120U102471).Results. It was found that in children of the fi rst and second groups, in the midst of clinical manifestations of wheezing, IL-4levels signifi cantly increased and serum γ-interferon levels decreased. The highest levels of IL-4 and the lowest levels of serumγ-interferon were found in patients with DDA.Serum levels of IL-4 above 15.33 pg/mL and γ-interferon below 98.62 pg/mL in children with wheezing episodes may beconsidered as a possible indicator of asthma development.Conclusions. Serum IL-4 and γ-interferon are involved in the regulation of allergic infl ammatory processes in the body.A statistically signifi cant increase in IL-4 levels and a decrease in γ-interferon levels in the blood serum of patients with asthmais likely to indicate the development of a chronic form of infl ammation.

The recombinant fusion protein CFP10-ESAT6-dIFN has protective effect against tuberculosis in guinea pigs.

Development of effective vaccine candidates against tuberculosis is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein ESAT6-CFP10-dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute tuberculosis. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of animals with the tested fusion protein (2 × 0.5μg) induces a protective effect against subsequent Mtb infection. The immunized animals do not develop the symptoms of acute tuberculosis and their body weight gain was five times more as compared with the non-immunized-infected animals. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages.

The recombinant fusion protein CFP10-ESAT6-dIFN has protective effect against tuberculosis in guinea pigs.

Development of effective vaccine candidates against tuberculosis (TB) is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein CFP10-ESAT6-dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute TB. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of guinea pigs with the tested fusion protein (2×0.5µg) induces a protective effect against subsequent Mtb infection. The immunized guinea pigs do not develop the symptoms of acute TB and their body weight gain was five times more as compared with the non-immunized infected guinea pigs. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages.

THE EXPERIENCE OF THERAPY AND FAMILY PROPHYLAXIS OF ACUTE RESPIRATORY VIRAL INFECTIONS

ARVI is the most common childhood pathology. Search of therapy drugs and prevention is one of the urgent problems of Pediatrics now. Objective of the research – to assess efficacy of Oscillococcinum® drug in ARVI treatment and family prevention in children and their parents. 30 families with 2 children, including 27 girls and 33 boys were observed during 18 months. After randomization, and informed consent of parents, children and their parents were divided into 2 groups: 1st group – 40 children (20 families), with ARVI in older child remaining members of the family received 1 prophylactic dose of Oscillococcinum® once a week. Sick child received Oscillococcinum® and symptomatic therapy (antipyretics, decongestants) for 5 days. 2nd group – 20 children (10 families), with ARVI in older child other members of the family had no prophylactic. Sick child received one of the antiviral drugs (interferon alpha-2b (Viferon®), Kagocel® (no INN) or antibodies to human γ-interferon, affinity-purified (Anaferon®), and symptomatic therapy. 3 children received interferon alpha-2b in suppositories in dose of 150 000 IU, one suppository 2 times a day for 5 days. 5 children received Kagocel® according to the scheme: first 2 days 1 tablet 3 times a day, next 2 days 1 tablet 2 times a day, the course of 4 days. 2 children received antibodies to human γ-interferon for 5 days according to the scheme: in the first 2 hours of disease 1 tablet every 30 minutes (4 tablets) and on the first day another 3 doses, after 2 day – 1 tablet 3 times a day for 5 days. It was found that in the 1st group receiving Oscillococcinum® by all family members with older child disease had a significant preventive effect compared with the control 2nd group (p<0,001). The disease in infected children of the main group receiving Oscillococcinum®, proceeded in a relatively milder form and with shorter duration of ARVI main symptoms (p<0,05 for the fever and runny nose).

Transgenic carrot expressing fusion protein comprising M. tuberculosis antigens induces immune response in mice.

Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human γ-interferon designed for expression in plant tissues. This construct was transferred to the carrot (Daucus carota L.) genome by Agrobacterium-mediated transformation. This study demonstrates that the fusion protein CFP10-ESAT6-dIFN is synthesized in the transgenic carrot storage roots. The protein is able to induce both humoral and cell-mediated immune responses in laboratory animals (mice) when administered either orally or by injection. It should be emphasized that M. tuberculosis antigens contained in the fusion protein have no cytotoxic effect on peripheral blood mononuclear cells.

Modulation of Mucin Synthesis by γ-Interferon in Human Colon Adenocarcinoma Cells

Objectives: Recombinant human interferon has been shown to enhance the expression of histocompatibility antigens and certain tumor-associated antigens in a variety of carcinoma cell lines. Since many tumor-associated antigens are mucins, we investigated the effect of recombinant human γ-interferon on mucin production and secretion by colon cancer cell lines. Methods: Control and γ-interferon-treated cells were labeled with [³H]glucosamine in DMEM-H16 medium with 5% FCS for 24 h. Analysis was performed by gel filtration on Sepharose CL-4B columns, and the high-molecular-weight glycoprotein eluted at the void volume from the cytosol and medium was counted (expressed as dpm/4 × 10⁶ cells). In another experiment the void volume was compared to concentration curves of standard mucins (expressed as mg/10⁷ cells). Results: γ-Interferon increased mucin synthesis in HT-29 and LIM-6 cells, but not in LS174T and CACO2 cells. In HT-29 and LIM-6 cells, mucin synthesis was induced by γ-interferon in a dose-dependent manner. The percentage of mucin secreted into the medium was also increased. Conclusion: The heterogeneity of response of human colon cancer cell lines to γ-interferon by mucin production may limit the specific role of γ-interferon as a modulator of mucin-type tumor-associated antigens.

Development of a Receptor Peptide Antagonist to Human γ-Interferon and Characterization of Its Ligand-bound Conformation Using Transferred Nuclear Overhauser Effect Spectroscopy

Polyclonal anti-idiotypic antibody raised to a synthetic discontinuous peptide derived from the human gamma-interferon (huIFN-gamma) sequence recognizes soluble human gamma-interferon receptor (Seelig, G. F., Prosise, W. W., and Taremi, S. S. (1994) J. Biol. Chem. 269, 358-363). We sought to use this reagent to identify a ligand-binding domain within IFN-gamma-receptor. To do this, the neutralizing anti-idiotypic antibody was used to probe overlapping linear peptide octamers of the extracellular domain of the huIFN-gamma receptor. A 22-amino-acid residue receptor segment 120-141 identified by the antibody was synthesized. CD and NMR analysis indicates that peptide 120-141 has no apparent secondary structure in water or in water containing 50% trifluoroethanol. The synthetic receptor peptide inhibited huIFN-gamma induced expression of HLA/DR antigen on Colo 205 cells with an approximate IC50 of 35 microM. Immobilized peptide specifically bound recombinant huIFN-gamma but did not bind human granulocyte-macrophage colony-stimulating factor on a microtiter plate in a direct binding enzyme-linked immunosorbent assay. The binding results are supported by two-dimensional transferred nuclear Overhauser effect (TRNOE) NMR data obtained on the peptide in the presence of recombinant huIFN-gamma. Characterization of the conformation of the bound peptide by TRNOE suggests that this peptide assumes a distinct conformation. Intramolecular interactions within the bound peptide were detected at two non-contiguous regions and at a third region comprising a beta-turn formed by the sequence DIRK. We believe that this represents the structure of the receptor within the ligand-binding domain.

Liposomal transfection of human γ-interferon gene into human glioma cells and adoptive immunotherapy using lymphokine-activated killer cells

The authors evaluated the effect of liposomal transfection of human gamma-interferon (HuIFN-gamma) gene into human glioma cells and lymphokine-activated killer (LAK) cells, alone and in combination. An HuIFN-gamma gene inserted in a eukaryotic expression vector was entrapped in liposomes bearing positive surface charges. Liposomal gene transfection induced production of HuIFN-gamma and its secretion in culture medium of human glioma cell lines (SK-MG-1 and U-251 MG). At 4 days after transfection, the cells produced 10 to 50 U/ml of HuIFN-gamma in the medium, whereby the major histocompatibility complex (MHC) class I and II antigens, as well as intercellular adhesion molecule-1 (ICAM-1), were induced on the glioma cell surface. The growth-inhibiting effect of transfection-induced HuIFN-gamma was much stronger in comparison with control cultures exposed to 500 U/ml of exogenously added HuIFN-gamma. In addition, 20% to 40% growth inhibition was obtained in the glioma cells when they were treated with LAK cells alone at a 5:1 ratio of effector to target cells. Liposomal transfection of HuIFN-gamma gene into human glioma cells combined with immunotherapy using LAK cells was more effective than either technique alone. The reinforcement of growth inhibition in the case of combined therapy was quenched by anti-ICAM-1 monoclonal antibody, but not by anti-MHC class I or II monoclonal antibodies. These results suggest that the combined effect of liposomal transfection of HuIFN-gamma gene plus LAK cells into human glioma cells is a potentially useful therapy for malignant glioma, and that the mechanisms of the reinforcement of growth inhibition are closely related to the expression of ICAM-1 on the glioma cell surface.

Human γ-interferon expression in the mammary gland of transgenic mice

Transgenic mice carrying a hybrid gene consisting of ovine β-lactoglobulin gene sequences and human γ-interferon (hIFN-g) cDNA were produced. hIFN-g expression in the mammary gland of two lactating transgenic founder females was found. The concentration of active hIFN-g in the milk was estimated as being ca. 1800 IU ml . The hIFN-g ability to express in the mammary gland was found in the progeny of transgenic founder male.

Immunogenicity of recombinant Plasmodium falciparum SERA proteins in rodents

We have expressed defined regions of the serine-repeat antigen (SERA) of the Honduras-1 strain of Plasmodium falciparum in the yeast Saccharomyces cerevisiae. Amino-terminal domains of the natural SERA protein have been shown previously to be targets for parasite-inhibitory murine monoclonal antibodies. Two recombinant SERA antigens were selected for purification and immunological analysis. The first (SERA 1), corresponding to amino acids 24–285 of the natural SERA precursor, was expressed by the ubiquitin fusion method. This allowed for in vivo cleavage by endogenous yeast ubiquitin hydrolase, and subsequent isolation of the mature polypeptide. The second, larger protein (SERA N), encompassing amino acids 24–506, was expressed at only low levels using this system, but could be isolated in high yields when fused to human γ-interferon (γ-IFN). Each purified protein was used to immunize mice with either Freund's adjuvant or a muramyl tripeptide adjuvant that has been used in humans. Sera from immunized mice were shown to be capable of in vitro inhibition of invasion of erythrocytes by the Honduras-1 strain of P. falciparum. The results suggest that a recombinant SERA antigen may be an effective component of a candidate malaria vaccine.

Hormonal regulation of complement biosynthesis in human cell lines—I. Androgens and gamma-interferon stimulate the biosynthesis and gene expression of C1 inhibitor in human cell lines U937 and HepG2

Cl inhibitor (Clinh), a member of the serine protease inhibitor gene superfamily, is a glycosylated plasma protein inhibiting the proteolytic activities of Clr and Cls and involved in the regulation of coagulation, fibrinolysis and kinin-releasing systems. In this study, the in vitro effect of androgen hormones, dehydroepiandrosterone (DHEA), testosterone (TEST) and recombinant human γ-interferon (γ-IFN), has been determined on the production of Clinh in human cell lines. In both human monocytoid/histiocytoid cell line U937 and in hepatoma derived cell line HepG2, DHEA and TEST upregulated the gene expression and secretion of Clinh. The most pronounced effect was detected in the concn range 10 −7−10 −9 M of the hormones. Under the same conditions DHEA and TEST had no detectable effect on the biosynthesis of C3, C2 and factor B by these cells, but DHEA at higher concn (10 −4M) slightly increased that of C4 in HepG2 cells. Both in U937 and in HepG2 cells recombinant γ-IFN markedly increased the gene expression and secretion of Clinh. This effect of γ-IFN was abolished by histamine.

γ-Interferon-induced monocyte major histocompatibility complex class II antigen expression in individuals with acquired immune deficiency syndrome

Twelve patients with the acquired immune deficiency syndrome (AIDS) and Kaposi's sarcoma were treated with recombinant human γ-interferon (rIFN-γ). A rapid, substantial increase in the fraction of HLA-DQ-positive monocytes was noted after treatment with rIFN-γ. The rIFN-γ-induced increase in monocyte HLA-DQ was seen throughout the course of treatment, with the percentage of HLA-DQ-positive monocytes dropping slightly following each week's treatment with rIFN-γ and then rapidly increasing following the next course of treatment. Although the percentage of HLA-DR-positive monocytes was unchanged (HLA-DR was expressed on > 80% of monocytes prior to treatment), the density of HLA-DR on monocytes also increased following rIFN-γ treatment. Following rIFN-γ treatment, no changes were seen in CD3, CD4, CD8 T cell numbers, in T cell subset ratio (CD4/CD8), in Leu 7 or CD 16 (Leu 11) cell number, in spontaneous Ig secretion, in PHA-induced in vitro proliferation, or in NK activity. These results indicate that exposure to rIFN-γ in vivo led to the increased expression of class II antigens on monocytes in patients with AIDS.

Effective method for obtaining long nucleotide chains on partially complementary templates Processed bovine γ-interferon gene obtained from human γ-interferon gene

The method of obtaining the bovine γ-interferon gene by means of simultaneous multidirected mutagenesis of the human γ-interferon gene is presented. The first strand of the bovine γ-interferon gene was obtained by ligation of synthetic oligonucleotides, using the cDNA of human γ-interferon, cloned in the single-stranded phage M13mpl9 as a template. The second strand was synthesized using a large fragment of E. coli DNA-polymerase 1. The double-stranded gene was then treated by restriction nucleases and cloned in a pUC- 18 derived vector. The primary structure was confirmed by sequencing.

Demonstration of a 1–3 disulfide bond in a synthetic nonapeptide derived from the signal sequence and N-terminus of human γ-interferon

The nonapeptide Cys-Tyr-Cys-Gln-Asp-Pro-Tyr-Val-Lys was prepared by solid-phase peptide synthesis under oxidizing conditions. Fast atom bombardment mass spectrometric analysis of the untreated molecule produced an ion consistent with a structure involving an intramolecular disulfide bond between Cys(1) and Cys(3). Mass spectra of the peptide after treatment with 2-mercaptoethanol gave signals corresponding to the reduced disulfide form of the peptide and to a mixed disulfide of the peptide with 2-mercaptoethanol. Molecular mechanics calculations of the conformation of the 11-membered ring formed by disulfide bond closure predicted a discrete, low-energy structure resembling the locus of a gamma turn. We hypothesize that this structure may be important in the recognition and cleavage of the signal sequence of the parent molecule.

Actions of recombinant human gamma-interferon and tumor necrosis factor alpha on the proliferation and osteoblastic characteristics of human trabecular bone cells in vitro.

Using cultured human osteoblast-like cells, we studied the effects of tumor necrosis factor (TNF) and recombinant human gamma-interferon (gamma-IFN) on osteoblast growth and function, and demonstrated that TNF stimulated bone cell proliferation and prostaglandin production while inhibiting 1,25-(OH)2D3-stimulated alkaline phosphatase activity and osteocalcin release. In contrast, gamma-IFN inhibited proliferation and stimulated alkaline phosphatase activity of the cells, while inhibiting 1,25-(OH)2D3-stimulated osteocalcin production and having variable effects on the release of prostaglandins, depending on the presence of other factors. Our results suggest that TNF and gamma-IFN can act directly on bone-forming cells to affect both their proliferation and their differentiated function, and that changes in the ability of cells to produce these factors in disease states may contribute to alterations in the integrity of connective tissue matrices.

Induction of human leukocyte antigen-A,B,C and -DR on cultured human oligodendrocytes and astrocytes by human γ-interferon

γ-Interferon (IFN-γ) is known to induce expression of major histocompatibility complex (MHC) class II antigens on murine astrocytes and MHC class I antigens on murine oligodendrocytes. We studied whether the human IFN-γ could induce the expression of Human Leukocyte Antigen (HLA)-A, B, C and -DR antigens on cultured human glia from autopsied brain white matter tissue. HLA-A, B, C antigens were induced on both human astrocytes and oligodendrocytes, whereas HLA-DR antigens were induced only on some astrocytes. From these results, it is suggested that IFN-γ affects the expression of MHC class I and class II antigens on astrocytes and oligodendrocytes derived from human brain. The relationship between the induction of MHC class I and class II antigens by IFN-γ and the pathogenesis of multiple sclerosis is discussed.

Determination of human T cell activity in response to allogeneic cells and mitogens: An immunochemical assay for γ-interferon is more sensitive and specific than a proliferation assay

T lymphocytes proliferate and secrete lymphokines in response to allogeneic cells, mitogens and other stimuli. Cell proliferation as measured by [ 3H]thymidine ([ 3H]Tdr) incorporation into DNA has been routinely used to determine T cell responses in research and clinical laboratories. We have compared the sensitivity of an immunoradiometric assay (IRMA) for human γ-interferon (IFN-γ) (Chang et al., 1984), with that of the conventional [ 3H]Tdr incorporation assay in the measurement of T cell responses to antigens and mitogens in culture. Peripheral blood mononuclear cells (PBMs) were incubated in the presence and absence of phytohemagglutinin (PHA) or mononuclear cells from another individual for various periods of time. The culture fluids were collected for determining IFN-γ and the cells were assayed for [ 3H]Tdr incorporation. Results of measurements were expressed in terms of stimulation indices. Both IFN-γ secretion and thymidine incorporation were measurable in mixed lymphocyte cultures after incubation for 3 days, and in PHA stimulated culture after 24 h of incubation. The stimulation indices reflecting increased γ-interferon were found to be more pronounced and more consistent than those of [ 3H]Tdr incorporation.

The response of cultured human kidney capillary endothelium to immunologic stimuli

We have developed in culture for the first time one of the actual target cells in kidney rejection by establishing long-term cultures of human kidney capillary endothelium and have begun to explore their response to immunologic stimuli. Microvascular endothelial cells were obtained from the human kidney cortex by mechanical separation, enzyme digestion, and density gradient centrifugation. The cells were exposed to 1% PHA, a PHA-stimulated PBL supernatant, human and mouse IL-1 and human γ-interferon. They were stained with FITC-conjugated monoclonalantibodies specific for β 2-microglobulin, HLA-DR, and DQ and analyzed by flow cytometry (FACS-II). HKCE cells express HLA-ABC antigens during growth and confluent phases but do not express DR or DQ antigens. Only the PHA-stimulated PBL supernatant and γ-interferon induced class II antigen expression. A change in cellular morphology was noted after 72 hr of incubation in the presence of lectin-stimulated PBL supernatant. Our findings show that microvascular endothelial cells express selected Ia antigens only after immunologic induction. This is an agreement with studies using umbilical and aortic endothelial cells. These data suggest that a dynamic interaction takes place between activated lymphocytes whose products induce expression of class II antigens and human kidney endothelial cells.

Monoclonal antibody against human IFN-gamma.

Monoclonal antibodies have proved extremely useful for the unambiguous identification, quantification and large-scale purification of a given antigen or a specific epitope. As they are highly specific for a given antigenic determinant, these antibodies are superior to conventional polyclonal antibodies. Since the discovery1 that mouse spleen B lymphocytes can be fused with mouse B-myeloma cells to yield hybrid cells (hybridomas) secreting specific antibodies derived from the spleen cell fusion partner, several modifications of this method have been used to improve the yield of surviving antibody-secreting hybridomas. We have now included, 5 days after the fusion proceedure, an additional step which stabilizes the hybrids against gene loss and improves growth conditions. We report here that, using this method, we have established several stable mouse hybridoma lines secreting monoclonal antibodies to human γ-interferon (HuIFN-γ).

Messenger RNA of human immune interferon: Isolation and partial characterization

Suspension cultures of total and lymphocyte-enriched peripheral white blood cells were used as a source for the preparation of mRNA for human γ-interferon. The cells were lysed and total RNA was extracted with the phenol method. A poly(A)-rich RNA fraction was isolated by affinity chromatography on oligo(dT)-cellulose and further purified on a preparative sucrose gradient. The RNA preparations were translated by oocytes into (a) protein(s) that showed biological activity in the interferon-assay. On sucrose gradient centrifugation the translatable fractions of the RNA migrated with a sedimentation coefficient of approximately 15 S, a value compatible with the molecular weight of human γ-interferon. The translation product was further characterized by serological cross reactivity with purified γ-interferon in neutralization reactions.