Effect of purified human interferon-α on the expression of differentiation antigens and mitogen reactivity of cultured human thymic cells

Human thymic cells were cultured in vitro either alone or with the addition of a highly purified preparation of human interferon-α. Immunofluorescence techniques using a series of monoclonal antibodies showed that 2-day cultured thymocytes express a more mature phenotype (low HTA 1, high T3 and HLA-A,B,C) than normal, uncultured thymocytes. Interferon addition to the cultures results in a strong increment in the number of HLA⁺ cells and in the total amount of HLA expressed by the cultured cells. Experiments with purified cell populations showed that the cortical, immature, thymocyte was the target cell for interferon action. Phytohemagglutinin responses—but not interleukin 2 responses—were diminished after pretreatment of thymic cells with interferon. We suggest that interferon may favor a pathway of intrathymic differentiation phenotypically characterized by a high content of Class I HLA antigens.     

Modeling of the effects of IL-17 and TNF-α on endothelial cells and thrombus growth

Rheumatoid and psoriatic arthritis are chronic inflammatory diseases, with massive increase of cardiovascular events (CVE), and contribution of the cytokines TNF-α and IL-17. Chronic inflammation inside the joint membrane or synovium results from the activation of fibroblasts/synoviocytes, and leads to the release of cytokines from monocytes (Tumor Necrosis Factor or TNF) and from T lymphocytes (Interleukin-17 or IL-17). At the systemic level, the very same cytokines affect endothelial cells and vessel wall. We have previously shown [1], [2] that IL-17 and TNF-α, specifically when combined, increase procoagulation, decrease anticoagulation and increase platelet aggregation, leading to thrombosis. These results are the basis for the models of interactions between IL-17 and TNF, and genes expressed by activated endothelial cells. This work is devoted to mathematical modeling and numerical simulations of blood coagulation and clot growth under the influence of IL-17 and TNF-α. We show that they can provoke thrombosis, leading to the complete or partial occlusion of blood vessels. The regimes of blood coagulation and conditions of occlusion are investigated in numerical simulations and in approximate analytical models. The results of mathematical modeling allow us to predict thrombosis development for an individual patient.     

No effects of intermittent 50 Hz EMF on cytoplasmic free calcium and on the mitochondrial membrane potential in human diploid fibroblasts

The recently described increase in DNA strand breaks of cultured human diploid fibroblasts after intermittent exposure to extremely-low-frequency electromagnetic fields (ELF-EMF) of more than about 70 µT ELF-EMF is difficult to explain by a direct induction of covalent bond disruption. Therefore the hypothesis has been tested that ELF-EMF-induced DNA strand breaks might be mediated by cellular processes that cause alteration of the intracellular concentration of free calcium ([Ca²⁺]_i) and/or the membrane potential (_m). [Ca²⁺]_i was determined by the ratiometric fura-2 technique. Changes in _m were assessed by using the potential-dependent lipophilic cationic probe JC-1. Human fibroblasts were exposed to intermittent ELF-EMF (50 Hz, 1000 µT). Although exposure of fiboblasts to ELF-EMF resulted in a highly significant increase in DNA strand breaks as determined by the comet assay, no effect on JC-1 fluorescence emission or on [Ca²⁺]_i has been observed when comparing exposed with sham-exposed cells. Therefore, it is suggested that ELF-EMF-induced DNA strand breaks are unlikely to be caused by intracellular changes that affect [Ca²⁺]_i and/or _m.     

Effect of interferon-α 2b on cells from human embryonic nerve tissue in the early stages of neurogenesis

We studied the effects of interferon (IFN)- 2b on cells obtained from the brain of human embryos (4 to 12 weeks of gestation). It was demonstrated that IFN exerts modulatory effects on biochemical and physico-chemical properties of cells of embryonic nerve tissue in the early stages of embryonic development (from 4 weeks of gestation). IFN decreased the content of protein, inhibited the activity of Na⁺,K⁺-ATPase, and induced changes in the superficial charge of the plasma membrane. Based on the obtained experimental data, we suppose that IFN- 2b is involved in regulation of neurogenesis.Neirofiziologiya/Neurophysiology, Vol. 36, Nos. 5/6, pp. 363–369, September–December, 2004.This revised version was published online in April 2005 with a corrected cover date and copyright year.     

T-cell receptor gene rearrangement and expression in human natural killer cells: natural killer activity is not dependent on the rearrangement and expression of T-cell receptor α, β, or γ genes

To test the hypothesis that the T-cell receptor (Tcr) gene encodes a natural killer (NK) cell receptor molecule, three human NK clones and fresh peripheral blood lymphocytes with NK activity from two patients with a CD16⁺ lymphocytosis were analyzed for rearrangements and expression of the human Tcr , , and genes. Two of the clones displayed distinct rearrangements of their Tcr and genes and expressed mature Tcr , , and l RNA. However, one of the clones and both patient samples displayed marked NK activity but failed to rearrange or express any of their Tcr genes. These findings demonstrate that human natural killer activity is not dependent on Tcr gene rearrangement and expression. In addition, they confirm previous findings concerning the lack of Tcr and gene expression in some natural killer cells. Thus, they suggest the existence of additional NK-specific recognition molecules.     

Acetylation of αA-crystallin in the human lens: effects on structure and chaperone function

α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N(ε)-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.     

Combined effects of interleukin-1α and transforming growth factor-β1 on modulation of human cardiac fibroblast function

During cardiac remodeling, cardiac fibroblasts (CF) are influenced by increased levels of interleukin-1α (IL-1α) and transforming growth factor-β1 (TGFβ1). The present study investigated the interaction between these two important cytokines on function of human CF and their differentiation to myofibroblasts (CMF). CF were isolated from human atrial appendage and exposed to IL-1α and/or TGFβ1 (both 0.1 ng/ml). mRNA expression levels of selected genes were determined after 6–24 h by real-time RT-PCR, while protein levels were analyzed at 24–48 h by ELISA or western blot. Activation of canonical signaling pathways (NFκB, Smad3, p38 MAPK) was determined by western blotting. Differentiation to CMF was examined by collagen gel contraction assays. Exposure of CF to IL-1α alone enhanced levels of IL-6, IL-8, matrix metalloproteinase-3 (MMP3) and collagen III (COL3A1), but reduced the CMF markers α-smooth muscle actin (αSMA) and connective tissue growth factor (CTGF/CCN2). By contrast, TGFβ1 alone had minor effects on IL-6, IL-8 and MMP3 levels, but significantly increased levels of the CMF markers αSMA, CTGF, COL1A1 and COL3A1. Co-stimulation with both IL-1α and TGFβ1 increased MMP3 expression synergistically. Furthermore, while TGFβ1 had no effect on IL-1α-induced IL-6 or IL-8 levels, co-stimulation inhibited the TGFβ1-induced increase in αSMA and blocked the gel contraction caused by TGFβ1. Combining IL-1α and TGFβ1 had no apparent effect on their canonical signaling pathways. In conclusion, IL-1α and TGFβ1 act synergistically to stimulate MMP3 expression in CF. Moreover, IL-1α has a dominant inhibitory effect on the phenotypic switch of CF to CMF induced by TGFβ1.     

Influence of interferon-α on the expression of the cancer stem cell markers in pancreatic carcinoma cells

The cytokine interferon-α (IFNα) belongs to the group of type I interferons already used in cancer therapy. This drug possesses radio- and chemo-sensitizing, and shows anti-angiogenic properties. Cancer stem cells (CSC) are a unique population of tumor cells that initiate secondary tumors, and are responsible for metastasis formation. Patients with pancreatic ductal adenocarcinoma (PDAC) have an especially poor prognosis, with 5-year survival rates of only ~1% and median survival of 4–6 months. PDAC is characterized by the presence of CSC. In this work we demonstrate for the first time that IFNα up-regulates the expression of the CSC markers CD24, CD44 and CD133 in in vitro and in vivo models of PDAC. We showed the IFNα effects on the migration and invasion of PDAC cells, which is associated with the level of the CSC marker expression. In vivo, this drug inhibits tumor growth but promotes metastasis formation in the early stage of tumor growth. We propose that IFNα may enhance the enrichment of CSC in PDAC tumors. Additionally we also suggest that in combination therapy of solid tumors with IFNα, this drug should be given to patients prior to chemotherapy to achieve the CSC activation.     

Identification of Ly 5 on the surface of ‘natural killer’ cells in normal and athymic inbred mouse strains

This report that (1) cells mediating NK activity in different inbred mouse strains selectively express one of two allelic products specified by theLy-5 locus (or a locus tightly linked to it) and (2) this surface structure may directly contribute to NK-mediated cytolysis, since Ly 5 antiserum specifically inhibits NK activity in vitro in the absence of complement.     

Overexpression of 27-kDa heat-shock protein in MCF-7 breast cancer cells: effects on lymphocyte-mediated killing by natural killer and γδ T cells

Overexpression of the heat-shock protein hsp27 protein in primary breast cancers has been associated with early relapse in women with breast cancer. This study was designed to determine the role of the hsp27 protein in lymphocyte recognition of estrogen-receptor(ER)-positive breast cancer cells and to assess the effect of hsp27 expression on lymphocyte-mediated lysis. The hsp27 cDNA was inserted into the pHbAPr-1-neo plasmid expression vector and driven by the constitutive actin promoter. The ER-positive MCF-7 human breast cancer cell line was then transfected with this vector and the resulting clonal cell lines were confirmed to overexpress hsp27. hsp27-transfected clonal cell lines stimulated the proliferation of fresh peripheral blood lymphocytes (PBL) significantly better than control cells transfected with the expression vector alone. When clonal T cell lines were utilized as effectors, hsp27-transfected cell lines were significantly better targets for lysis than a control-transfected MCF-7 cell line. In contrast, hsp27-transfected cell lines had no increase in susceptibility to lymphokine-activated-killer- or natural-killer-mediated lysis. These results suggest that overexpression of the hsp27 protein in ER-positive MCF-7 cells stimulated the proliferation of fresh PBL and the lysis of MCF-7 cells by T cell clones.     

TNF-α inhibitor reverse the effects of human umbilical cord-derived stem cells on experimental arthritis by increasing immunosuppression

To demonstrate the therapeutic potential for cartilage repair of mesenchymal stem cells derived from human umbilical cord (HUCSC), we studied the clinical and histopathological effects of intra-articular injection of HUCSCs in a collagen-induced arthritis (CIA) model. In our study, intra-articular injection of HUCSCs had no benefit in CIA mice; and accelerated the progression of arthritis in the presence of TNF-α. To determine the role of TNF-α, we injected the combination with HUCSCs and TNF inhibitor, showed reduced the disease signs in CIA mice. On exposure of TNF-α, stem cells significantly decreased the expression of CD90, HLA-G, and the levels of IL-10 in vitro and in vivo. Our data showed that TNF-α blocks the immunosuppressive effects of HUCSCs and that inhibition of TNF-α decreases cartilage destruction by suppressing the immunogenicity of HUCSCs. Injecting both a TNF inhibitor and HUCSCs may be a potential effective therapy for ameliorating the disease.     

Interleukin-15 and interferon-γ participate in the cross-talk between natural killer and monocytic cells required for tumour necrosis factor production

We have characterized the lymphocyte subset and the receptor molecules involved in inducing the secretion of TNF by monocytic cells in vitro. The TNF secreted by monocytic cells was measured when they were co-cultured with either resting or IL-15-stimulated lymphocytes, T cells, B cells or natural killer (NK) cells isolated from the peripheral blood of healthy subjects and from the synovial fluid from patients with inflammatory arthropathies. Co-culture with IL-15-activated peripheral blood or synovial fluid lymphocytes induced TNF production by monocytic cells within 24 hours, an effect that was mainly mediated by NK cells. In turn, monocytic cells induced CD69 expression and IFN-γ production in NK cells, an effect that was mediated mainly by β₂ integrins and membrane-bound IL-15. Furthermore, IFN-γ increased the production of membrane-bound IL-15 in monocytic cells. Blockade of β₂ integrins and membrane-bound IL-15 inhibited TNF production, whereas TNF synthesis increased in the presence of anti-CD48 and anti-CD244 (2B4) monoclonal antibodies. All these findings suggest that the cross-talk between NK cells and monocytes results in the sustained stimulation of TNF production. This phenomenon might be important in the pathogenesis of conditions such as rheumatoid arthritis in which the synthesis of TNF is enhanced.     

Inhibitory effects of tamoxifen and tumor necrosis factor α on human glioblastoma cells

We reported previously that tumor necrosis factor α (TNFα) inhibited proliferation and invasiveness of human malignant glial cells. Because tamoxifen, an estrogen antagonist, has also been shown to inhibit growth of such cells, we hypothesized that a combination of tamoxifen and TNFα might be more effective than either reagent alone. TNFα (1–100 ng/ml) or tamoxifen (80 ng/ml-2 μg/ml) alone inhibited proliferation of a human glioblastoma cell line (WITG3) in a dose-dependent fashion; in combination, tamoxifen and TNFα yielded additive growth inhibition. Apoptotic cells characterized by nuclear fragmentation were detectable after 48 h of TNFα or tamoxifen exposure and were significantly increased by combination treatment. In non-neoplastic human astroglia and fibroblasts, proliferation was unaffected by tamoxifen, and enhanced by TNFα as previously reported. Staurosporine (2–50 nM), which has been reported to augment the effects of TNFα, was less effective than tamoxifen against WITG3 and, in addition, was markedly inhibitory to non-neoplastic glial cells. Binding studies yielded no evidence of WITG3 estrogen or progesterone receptors, nor of tamoxifen effects on TNFα receptors. Data suggest that TNFα and tamoxifen in combination display growth-regulatory properties, which (a) are more inhibitory to human glioblastoma cells than either agent alone, (b) do not affect non-neoplastic glia, (c) do not require either estrogen/ progesterone receptors or alteration of external TNFα receptors, and (d) may involve apoptosis.     

A model of the role of natural killer cells in immune surveillance — I

Summary The theory of immune surveillance of Thomas and Burnet stated in part that antigenic differences between neoplastic and normal cells provide the stimulus for their destruction by cells of the immune system. Burnet pointed to the T lymphocyte as the cell which mediated this surveillance. The existence of some form of surveillance in cases of no T lymphocyte functioning presents the possibility that surveillance, if present at all, is mediated by non T cells.Cells identified as naturally cytotoxic killer (NK) cells appear to have properties required of a surveillance effector population. This paper utilizes properties of NK cells and the effects of interferon on this population to construct a mathematical model of the characteristics that an NK cell surveillance would have. A two level theory of immune surveillance is proposed.This research has been supported in part by the National Science Foundation under grant #NSF-Eng. 7904852     

Cytotoxic effects induced by interferon-ω gene lipofection through ROS generation and mitochondrial membrane potential disruption in feline mammary carcinoma cells

Progress in comparative oncology promises advances in clinical cancer treatments for both companion animals and humans. In this context, feline mammary carcinoma (FMC) cells have been proposed as a suitable model to study human breast cancer. Based on our previous data about the advantages of using type I interferon gene therapy over the respective recombinant DNA derived protein, the present work explored the effects of feline interferon-ω gene (fIFNω) transfer on FMC cells. Three different cell variants derived from a single spontaneous highly aggressive FMC tumor were successfully established and characterized. Lipofection of the fIFNω gene displayed a significant cytotoxic effect on the three cell variants. The extent of the response was proportional to ROS generation, mitochondrial membrane potential disruption and calcium uptake. Moreover, a lower sensitivity to the treatment correlated with a higher malignant phenotype. Our results suggest that fIFNω lipofection could offer an alternative approach in veterinary oncology with equal or superior outcome and with less adverse effects than recombinant fIFNω therapy.     

A model of the role of natural killer cells in immune surveillance — II

The functioning of natural killer (NK) cells as immune surveillance effector cells against tumors is explored. In part I (J. Math. Biol. 12, 363-373 (1981], it was predicted that susceptible tumors would be eliminated if they have parameter lambda 0 value negative. They would not be eliminated if lambda 0 greater than 0. As the lambda 0 less than 0 result was local, one expected either that tumors of all sizes with lambda 0 less than 0 will be eliminated (global stability) or that tumor population will go to zero if in a domain of attraction of the critical point which is not all of the positive orthant. In this paper, the second is shown to be true. The general results are illustrated by a specific model.     

Genetic engineering of α2,6-sialyltransferase in recombinant CHO cells and its effects on the sialylation of recombinant interferon-γ

The CHO cell line has achieved considerable commercial importance as a vehicle for the production of human therapeutic proteins, but is known to lack a functional copy of the gene coding for 2,6-sialyltransferase (EC 2.4.99.1). The cDNA for rat 2,6-ST was expressed in a recombinant CHO cell line making interferon-, using a novel in vitro amplification vector. The enzyme was expressed efficiently, and resulted in up to 60% of the total sialic acids on interferon- being linked in the 2,6-conformation. This sialic acid linkage distribution was more akin to that seen in natural human glycoproteins. In the most successful cell clones, expression of 2,6-sialyltransferase improved the overall level of sialylation by up to 56%, and had no adverse effects on cell growth, IFN- productivity or other aspects of IFN- glycosylation. These experiments demonstrate how the glycosylation machinery of rodent cells can be genetically manipulated to replicate human tissues.Abbreviations AT-III antithrombin-III - CHO Chinese hamster ovary - dhfr dihydrofolate reductase - EPO erythropoietin - IFN- human interferon- - NEO neomycin - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - ST sialyltransferase - tPA tissue plasminogen activator     

Activation of natural killer T cells inhibits the development of induced regulatory T cells via IFNγ

Recent reports have provided evidence for cross-talk between regulatory T (Treg) cells and natural killer T (NKT) cells. However, it is unclear whether NKT cells play a role in the differentiation of Treg cells. By employing NKT cell-abundant Vα14 TCR transgenic (Tg) and NKT cell-deficient CD1d knock-out (KO) mice, we examined the effects of NKT cells on the in vitro differentiation of induced Treg (iTreg) cells with IL2 and TGFβ. We found that iTreg induction from CD1d KO mice was significantly increased compared to the control. Also, the addition of isolated NKT cells from Vα14 TCR Tg mice to naïve CD4⁺ T cells from CD1d KO mice during iTreg differentiation caused a remarkable reduction of iTreg cells. Through IFNγ neutralization, we showed that this reduction was mediated by IFNγ. Furthermore, the main source of IFNγ during iTreg differentiation was NK1.1⁻CD4⁺Foxp3⁻ T cells. This finding implied that early-activated NKT cells induced Th1-type cells and subsequently underwent apoptosis. Taken together, our results suggest that NKT cells inhibit the in vitro development of iTreg cells by increasing IFNγ.