Presence Of IFN Research Articles

Agonistic anti-Fas antibody has the growth inhibitory effect on pancreatic cancer cells independent of DcR3

(BACKGROUND) Agonistic anti-Fas antibody (CH-11) has been reported to have little growth inhibiting potency to almost all pancreatic cancer cell lines (PCCs). DcR3 is antagonistic soluble receptor against Fas ligand, which belongs to a new TNF superfamily. Genomic amplification and mRNA expression of DcR3 have been reported in colon and lung cancers. However no information is available of the DcR3 status in PCCs. Here in the study we examined the effect of CH-11 in the presence of IFN,yand DcR3 genomic amplification and mRNA expression in PCCs. (METHORD) 1) PCCs (AsPC-1, BxPC-3, Capan-2, CFPAC-1, HPAC, MIA PaCa-2) were incubated with various concentrations of CH-11 after pretreatment with IFN~. Attached ceils were harvested and counted using Coulter Counter. 2) Genomic DNA was extracted from PCCs and quantitative PCR was performed using TaqMan system. The ratio of DNA copy number of DcR3 to beta actin was calculated. 3) mRNA was isolated from subconfluent PCCs and converted to cDNA. Quantitications of mRNA of DcR3 and beta actin were carried out with TaqMan RT-PCR. (RESULT) 1) CH-11 suppressed the growth of PCCs except for AsPC-1 to various extents when pretreated with IFN-/. 2) BxPC-3 and HPAC showed DcR3 genomic amplification (2.6 + 0.4, 2.9 + 0.4 times). Genomic amplification of DcR3 and the sensitivity to CH-11 were not correlated in PCCs. 3) Capan-2 and CFPAC-1 strongly expressed DcR3 mRNA. DcR3 mRNA expression and the sensitivity of CH-11 were not correlated in PCCs. Furthermore genomic amplification and mRNA expression of DcR3 were not correlated each other in PCCs. (CONCLUSION) Agonistic anti-Fas antibody inhibited the growth of pancreatic cancer cell lines in the presence of IFN~. Several pancreatic cancer cell lines strongly expressed DcR3 mRNA, while DcR3 genomic amplification and mRNA expression are not related to the sensitivity to CH-11 in pancreatic cancer cells. The expression of DcR3 is not involved in the inhibitory effect of CH-11 on pancreatic cancer cell growth.

Responsiveness of Mouse Corpora Luteal Cells to Fas Antigen (CD95)-Mediated Apoptosis1

Regression of the corpus luteum (CL) occurs by apoptosis. The Fas antigen (Fas) is a cell surface receptor that induces apoptosis in sensitive cells when bound to Fas ligand or agonistic anti-Fas monoclonal antibodies (Fas mAb). A potential role for Fas to induce apoptosis in dispersed CL cell preparations was tested in cells isolated from mice on Days 2-4 of pseudopregnancy. Total CL dispersates, containing steroidogenic luteal cells, fibroblasts, and endothelial cells, were cultured. The effect of pretreatment of cultures with cytokines interferon gamma (IFN) and tumor necrosis factor alpha (TNF) was examined because these cytokines demonstrated effects on Fas-mediated apoptosis in other cell types. Fas mAb had no effect on viability of CL cells cultured in 5% fetal bovine serum (FBS) and pretreated with or without IFN or TNF, but Fas mAb did kill 23% of the cells in cultures pretreated with IFN + TNF. Fas mRNA was detectable in cultured CL cells and was increased 2.1-, 2. 0-, and 11.8-fold by treatment with TNF, IFN, or IFN + TNF, respectively. CL cells treated with the protein synthesis inhibitor cycloheximide (CX) were killed by Fas mAb in the absence of cytokine pretreatment (34%); pretreatment with IFN or IFN + TNF further potentiated killing (62% and 96%, respectively), whereas pretreatment with TNF had no effect (42%). Cells cultured in medium supplemented with insulin, transferrin, and selenium instead of FBS were killed by Fas mAb in the presence of IFN (23%) or IFN + TNF (29%) but not in the presence of TNF. Cells derived from the mouse CL have a functional Fas pathway that is inhibited by FBS and activated by treatment with CX, IFN, and IFN + TNF.

Herpes simplex virus ICP0 mutants are hypersensitive to interferon.

Interferon (IFN) is an important immune system molecule capable of inducing an antiviral state within cells. Herpes simplex virus type 1 (HSV-1) replication is somewhat reduced in tissue culture in the presence of IFN, presumably due to decreased viral transcription. Here, we show mutations that inactivate immediate-early (IE) gene product ICP0 render HSV-1 exquisitely sensitive to IFN inhibition, resulting in greatly decreased levels of viral mRNA transcripts and the resulting polypeptides and a severe reduction in plaque formation ability. Mutations in other HSV-1 genes, including the genes coding for virion transactivator VP16 and the virion host shutoff protein vhs, IE gene ICP22, and the protein kinase UL13 gene, do not increase the IFN sensitivity of HSV-1. Interestingly, ICP0 mutants demonstrate the same level of sensitivity to IFN as wild-type virus on U2OS cells, an osteosarcoma cell line that is known to complement mutations in ICP0 and VP16. Thus, in some cell types, functional ICP0 is required for HSV-1 to efficiently bypass the inhibitory effects of IFN in order to ensure its replication. The significance of this link between ICP0 and IFN resistance is discussed.

Vaccination of immunocompetent elderly subjects with a live attenuated Oka strain of varicella zoster virus: a randomized, controlled, dose-response trial

After primary infection in childhood, varicella zoster virus (VZV) remains latent in the dorsal route ganglia. Its reactivation later in life can lead to a zoster episode. VZV-specific, T-cell-mediated immunity (VZV-CMI) is likely to be important in preventing symptomatic reactivation. As CMI declines with age, a vaccine enhancing VZV-CMI might be effective in decreasing the incidence or severity of zoster in elderly subjects. A randomized, double blind controlled trial assessing CMI responses of elderly subjects immunized with a live attenuated, VZV-Oka vaccine was conducted. Two hundred healthy volunteers (55–75 years of age) received either a single injection of the VZV vaccine (PMC), containing 3200 (Oka 3200), 8500 (Oka 8500), or 41,650 (Oka 41650) PFU of live VZV, or a pneumococcus vaccine control group (Pneumo 23(®). The immune response to VZV was assessed by measuring the T-cell response to VZV antigens, i.e. proliferation (stimulation index, SI), precursor cell frequency (PCF), cytokine secretion, and antibody titers. Six weeks post-vaccination, VZV-specific SI (adjusted mean values) was significantly greater ( P<0.0001) in the 3 vaccine groups (with SI=5.6 for Oka 3200; SI=5.0 for Oka 8500, and SI=7.2 for Oka 41,650) than in the control group (SI=2.9). The increase in PCF was striking, with 72.4, 91.2 and 85.1 precursors per million cells respectively in these 3 vaccine groups, vs 26.3 in the control group. No significant IL-4 secretion was observed in any subject, whereas the presence of IFN- γ secretion was found to correlate with good responder status. The increase of these CMI parameters did not depend upon the titer of virus injected. Geometric mean titers of VZV antibodies increased in all vaccine groups and remained unchanged in the control group. Nevertheless, no correlation between the antibody response and the cell-mediated response was found. Live attenuated VZV vaccine caused a significant increase in VZV-CMI in a healthy, elderly population. No relationship between vaccine dose and the intensity of the specific response was found.

Synergistic neurotoxic effects of combined treatments with cytokines in murine primary mixed neuron/glia cultures

Activation of brain glial cells with the bacterial endotoxin lipopolysaccharide (LPS), the HIV-1 coat protein gp120, or β-amyloid-derived peptides, stimulates the expression of several cytokines, including tumor necrosis factor- α (TNF α), interleukin-1 (IL-1) and IL-6, and nitric oxide (NO) which have been proposed as causes of neurodegeneration in the brain. In the present study, the neurotoxic effects of several cytokines, alone or in various combinations, and the correlations of the release of lactate dehydrogenase, the loss of neurons, and the secretion of NO in brain neuronal cell injury were investigated in murine primary mixed neuronal/glial cell cultures. A specific combination of cytokines, i.e., IL-1 (1 ng/ml)+TNF α (10 ng/ml)/interferon- γ (IFN γ) (200 u/ml), induced a dramatic neuronal cell injury in the neuron/glia cultures, and its cytotoxic profile was very similar to that seen with the LPS/IFN γ-induced neuron injury. This indicates that among the many toxic immune mediators secreted in response to LPS, IL-1 and TNF α can mimic LPS as the triggering signals and primary mediators for glia-mediated neuron injury in the presence of IFN γ. This study provides new insights about the cytotoxic mechanism(s) for cytokine-mediated neuron injury.

Interferon- γ-dependent induction of manganese superoxide dismutase activity of SV40-transformed human keratinocytes by anti-Fas antibody and by TNF- α

It has been reported that cellular oxidative stress induces apoptosis, that may be inhibited by scavengers of reactive oxygen intermediates (ROIs). Superoxide dismutase (SOD) is among the most active scavengers of ROIs, providing defense against the cellular oxidative stress. Fas antigen and tumor necrosis factor (TNF) receptor are the cell surface proteins, stimulation of which induces apoptosis of keratinocytes. Using SV40-transformed human keratinocytes (SVHK cells), we investigated the effects of anti-Fas antibody and TNF- α on the SOD activity. Treatment of SVHK cells with anti-Fas antibody or TNF- α in the presence of interferon- γ (IFN- γ) resulted in an increase in Mn-SOD activity, Cu,Zn-SOD activity was not affected. In the absence of IFN- γ, no increase in Mn-SOD activity was detected. The induction of IFN- γ-dependent Mn-SOD activity by anti-Fas antibody or TNF- α was concentration-dependent; the maximal effect was observed at 1–10 μg/ml and 5–10 ng/ml, respectively. The increase in Mn-SOD activity was observed at 6 h following the treatment and remained for at least 48 h. Northern blot analyses showed that Mn-SOD mRNA increased within 3 h without a significant change in Cu,Zn-SOD mRNA. The addition of both anti-Fas antibody and TNF- α in the presence of IFN- γ resulted in an additive increase in Mn-SOD activity. Although the addition of 12- o-tetradecanoylphorbol-13-acetate (TPA) singly to the incubation medium had no effect on either Mn-, or Cu,Zn-SOD activity, it significantly augmented the IFN- γ-dependent induction of Mn-SOD activity by anti-Fas antibody or by TNF- α. The protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (H-7), significantly inhibited the TPA-dependent increase in Mn-SOD activity. These results indicate that the stimulation of Fas antigen or TNF receptor increases Mn-SOD activity of SVHK cells in the presence of IFN- γ and that TPA augments the process through the activation of protein kinase C.

4 Prediction of preterm labour in multiple pregnancies

Various methods of predicting preterm labour in both singleton and multiple pregnancies have been studied including risk scoring systems, home uterine activity monitoring, cervical assessment and biochemical methods. In practice, risk scoring systems for preterm delivery perform poorly. Consensus in the literature regarding the usefulness of home uterine activity monitoring is lacking and well designed randomized controlled trials are required. Transvaginal ultrasound assessment of the cervix appears to have a role to play in the prediction of preterm labour. The presence of IFN in cervicovaginal fluid in the late second and early third trimesters is an important risk factor for preterm labour in asymptomatic women with multiple pregnancies. Preterm labour may be mediated in part by inflammatory cytokines. The measurement of these inflammatory cytokines in cervical secretions may also prove helpful in the prediction of preterm labour. It is anticipated that an improved ability to predict preterm labour in both singleton and multiple pregnancies will depend on increasing understanding of the condition's pathophysiology.

Effects of an anti-IL-10 monoclonal antibody on rIFN β-1b-mediated immune modulation. Relevance to multiple sclerosis

The mechanism of action of recombinant IFN β 1b (IFN β-1b), as a therapy for multiple sclerosis (MS), is still unknown but may result from the enhancement of ConA-induced suppressor cell function and the inhibition of IFN γ secretion by lymphocytes. We previously demonstrated that IFN β-1b stimulated modest amounts of IL-10 secretion by monocytes and IL-10 activity, as cytokine synthesis inhibitory factor, was normal in MS. To determine whether IL-10 plays a role in IFN β-1b modulation of immune function in MS, we studied ConA-induced suppressor cell function and IFN γ production in presence of IFN β-1b and an anti-IL-10 monoclonal antibody (mAb). Anti-IL-10 mAb significantly reduced the effect of IFN β-1b on ConA-induced suppressor cell function and IFN γ production in healthy subjects; MS patients showed a trend of inhibition. We hypothesized that IL-10 may play a role in mediating the effects of IFN β-1b on suppressor cell function and IFN γ production but suppressor molecules other than IL-10 could be also involved.

Immunologic patterns associated with cure in human American cutaneous leishmaniasis.

Patients with American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-gamma) and interleukin 4 (IL-4) produced were also determined in the culture supernatants. Two different patterns of Lb-induced T cell responses were observed: a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-gamma and IL-4) during the active disease, and b) similar proportions of responding CD4+ and CD8+ cells, and type 1 cytokine production (presence of IFN-gamma and very low IL-4) at the end of therapy (healed lesions). This last pattern is probably associated with a beneficial T cell response.

Induction of MHC class II antigen expression on human HMC-1 mast cells

Mast cells are not only the primary effector cells of immediate type immune reactions, but they have recently also been considered to contribute to the induction of an immune response. Data on the ability of the cells to express major histocompatibility complex (MHC) antigens and the mechanisms involved are however controversial or unclear. We have therefore studied the expression of MHCI and the induction of MHCII molecules on leukemic HMC-1 mast cells by immunohistochemistry. Cells were incubated for up to 72 h in the presence of IFN γ, TNF α and IL-4, and immunohistochemical staining was done with monoclonal antibodies with specificity for HLA class I heavy chain, HLA-DQ (Tü22), HLA-DR (Tü36) and HLA-DQ, -R and -P (Tü35). All unstimulated mast cells expressed MHC class I, but almost no class II antigens. Incubation with IFN γ caused a rapid, dose-dependent induction of MHC class II molecules, with Tü35 staining maximally one third of the cells within 24 h at the highest dose tested (100 IU/ml), with decline on extended culture. TNF α (2 ng/ml) was less effective but caused more persistent induction with time. IL-4 (200 ng/ml) had hardly any effects at all. Staining with Tü22 and Tü36 was always lower than with Tü35, and additive or even synergistic results were obtained when cells were stimulated with a combination of IFN γ and TNF α. These findings support the concept that mast cells can facultatively participate in immune recognition processes depending on the type of pathological conditions in their microenvironment which allow expression of MHC class II molecules.

The distribution and abundance of MHC and ICAM-1 on Schwann cells in vitro

Schwann cells, the myelin forming glial cells of peripheral nerves, have been implicated as having an immunoregulatory role in inflammatory demyelinating neuropathies (IDNs) such as Guillain Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP). We employed rat IFN- γ, a cytokine released by macrophages and CD4 + T-cells during inflammatory demyelination of the peripheral nervous system, to examine the distribution and abundance of MHC class I, MHC class II and ICAM-1 on Lewis rat Schwann cells and fibroblasts in vitro. MHC class I, class II and ICAM-1 molecules were immunolabelled with 30 nm colloidal gold and observed by scanning electron microscopy. Incubation with IFN- γ for 24 and 72 h, resulted in the clustering of MHC class I and ICAM-1 on Schwann cells and fibroblasts with MHC class II randomly distributed as single particles. MHC class I and ICAM-1 were upregulated after 24 h incubation in the presence of IFN- γ, whereas MHC class II was upregulated after 72 h. The difference in the rate of upregulation may indicate differences in the recycling and/or synthesis of these molecules. Changes in distribution such as clustering, in conjunction with the upregulation of these molecules, suggest a role for Schwann cells in the restimulation of specifically primed CD4 + T-cells in IDNs.

Modulation of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) by interferon-gamma in a human salivary gland cell line.

Gelatinases have been shown to be regulated by many cytokines and growth factors, and have been implicated in the pathogenesis of certain autoimmune diseases via tissue destruction. High levels of several cytokines, including IFN-gamma and TNF-alpha, have been demonstrated in the salivary gland microenvironment of patients with Sjogren's syndrome (SS). How these cytokines may be contributing to the pathogenesis of this disease is not well understood. We hypothesized that IFN-gamma with or without (+/-) TNF-alpha could be playing a role in the pathogenesis of SS via the regulation of matrix metalloproteinase (MMP) levels. This study examined the role of IFN-gamma and (+) TNF-alpha in the regulation of the matrix metalloproteinases, MMP-2 (72 kD gelatinase A) and MMP-9 (92 kD gelatinase B). A human salivary gland cell line (HSG) has been used as a possible in vitro model to study the role of IFN-gamma + TNF-alpha in the pathogenesis of SS. The HSG cell line, in the presence of IFN +/- TNF-alpha, displays increased MMP-2 and MMP-9 gelatinolytic activity, protein and RNA levels. The increase in MMP activity was partially blocked with an antibody against the IFN-gamma receptor, and this was associated with a complete inhibition of the previously described IFN-gamma +/- TNF-alpha antiproliferative effect. However, incubation of IFN-gamma treated HSG cells with the synthetic MMP inhibitor BB94 did not alleviate this antiproliferative effect. In addition, we demonstrate that there are very high levels of MMP-9 in the saliva of patients with SS when compared to healthy control subjects. These data suggest that cytokines could be regulating MMP production by salivary epithelial cells and thus indicate a potential role for these cells in the pathogenesis of SS.

Interferon- γ induced increases in intracellular cathepsin B activity in THP-1 cells are dependent on RNA transcription

Interferon- γ (IFN- γ) treatment of human macrophages induces increased intracellular levels of cathepsin B (CB), a lysosomal cysteine proteinase which is implicated in inflammatory tissue injury. To determine the mechanism of the increase, we studied the macrophage-like cell line, THP-1. Dose and time dependent increases in intracellular CB were seen when cells primed with phorbol ester (PMA) were cultured with IFN- γ. To determine whether protein synthesis was required for the increase, PMA primed cells were cultured in the presence of IFN- γ and cycloheximide: The expected increase was inhibited. To determine whether RNA synthesis was required for the IFN- γ induced increases, PMA primed cells were cultured in the presence of IFN- γ and actinomycin D. Again the expected increases were not seen. Direct measurement of CB mRNA levels showed increases in cells not treated with inhibitors. These results suggest that the IFN- γ induced increases in THP-1 cell CB are dependent on RNA and protein synthesis.

Clinical Observations with Topical Liposome-Encapsulated Interferon Alpha for the Treatment of Genital Papillomavirus Infections

This study reports the formulation and preliminary clinical testing of liposomes as a topical drug delivery system for interferon a (IFN α) in the treatment of genital human papillomavirus (HPV) infections. Intron A (IFN α-2b) was encapsulated into multilamellar liposomes, composed of soya phosphatidylcholine: cholesterol: stearic acid 2:1:1.4 molar ratio, by a modified solvent evaporation method (protein to phospholipid ratio 7±103IU/mg). The liposomes were in the size range of 0.5-2 μm as determined by electron microscopy. The incorporation of IFN α into liposomes was 91.7±2.2% (n=4). In an open trial, one female patient with exophytic warts and cervical HPV (type 6/11), responded to a treatment with 2.1 ±106 IU liposomal IFN α-2b per week for 12 weeks and 7±106 IU liposomal IFN α-2b per week for an additional 6 weeks externally administered in divided doses twice a day, and simultaneously 0.7±106 IU liposomal IFN α-2b per week intravaginally. Clinically the cervical lesion completely resolved at the end of the therapy, and the exophytic lesions decreased in size. In situ hybridization showed decreased focal staining for HPV DNA in biopsy tissue after treatment. The presence of IFN α was detected in the biopsy tissue by immunostaining. The male patient, treated with 2.1±106 IU liposomal IFN α-2b per week for 8 weeks showed decreased number of lesions at the end of the observation period.

β-Amyloid protein-dependent nitric oxide production from microglial cells and neurotoxicity

β-Amyloid protein (A β) is the major component of the senile plaques in Alzheimer's disease (AD), and microglial cells have been shown to be closely associated with these plaques. However, the roles of A β and microglial cells in pathogenesis of AD remain unclear. Incubation of rat microglial cells with A β(1–40) caused a significant increase in nitrite, a stable metabolite of nitric oxide (NO), in culture media, while there was no detectable increase in nitrite in astrocyte-rich glial cells or cortical neurons after incubation with A β(1–40). Nitrite production by microglial cells was also induced by A β(1–42), but not A β(25–35). An inhibitor of NO synthase, N G-monomethyl- l-arginine (NMMA), as well as dexamethasone and actinomycin D, dose-dependently inhibited this nitrite production. Among the various cytokines investigated such as interleukin-1, interleukin-6, tumor necrosis factor- α and interferon- γ (IFN- γ), only IFN- γ markedly enhanced A β-dependent nitrite production. Cultured cortical neurons were injured by microglial cells stimulated with A β in a dose-dependent manner in the presence of IFN- γ. Neurotoxicity caused by the A β plus IFN- γ-stimulated microglial cells was significantly attenuated by NMMA. Thus, although further investigations into the effect of A β on human microglial cells are needed, it is likely that A β-induced NO production by microglial cells is one mechanism of the neuronal death in AD.

INDUCTION BY HUMAN INTERFERON AND MURINE H-2L(D) GENE OF THE SURFACE EXPRESSION OF ENDOGENOUS HLA CLASS-I FREE HEAVY-CHAINS IN HCT HUMAN COLON-CARCINOMA CELLS, DEFICIENT IN BETA-2-MICROGLOBULIN

The colon cancer cell line HCT fails to express HLA class I antigens on the cell surface as a consequence of two independent mutations occurring in both copies of the beta2-microglobulin (beta2-mu) gene. Restoration of HLA class I antigen expression in HCT cells transfected with the wild-type human beta2-mu gene is accompanied by the expression of beta2-mu-free HLA class I heavy chains on the cell surface. HCT cells expressing a transfected H-2L(d) gene (HCT-L(d) transfectants) exhibited high levels of H-2L(d) heavy chains on the cell surface, following treatment with human interferon a (IFN-alpha). IFN-treated HCT-L(d) transfectants also expressed high levels of endogenous beta2-g-free HLA class I heavy chains on the cell surface. Incubation of HCT-L(d) transfectants at 26-degrees-C for 24 hours is associated with a striking increase in the surface expression of H-2L(d) heavy chains (comparable to that observed at 37-degrees-C in the presence of IFN), without a concomitant increase in the surface expression of beta2-mu-free HLA class I heavy chains, suggesting that IFN and low temperature act by different mechanisms. These results also indicate that human IFN and H-2L(d) heavy chain can act synergistically in inducing the surface expression of endogenous beta2-mu-free HLA class I heavy chains by HCT cells. The combination of 26-degrees-C incubation and IFN treatment results in the surface expression of beta2-mu-free HLA class I heavy chains in HCT-neo transfectants, suggesting that IFN plays a crucial role in allowing the surface expression of endogenous HLA class I heavy chains in beta2-mu-negative HCT cells.

Localization of the human indoleamine 2,3-dioxygenase (IDO) gene to the pericentromeric region of human chromosome 8.

Indoleamine 2,3-dioxygenase (IDO) is the first enzyme in the catabolic pathway for tryptophan. This extrahepatic enzyme differs from the hepatic enzyme, tryptophan 2,3-dioxygenase (TDO), in molecular as well as enzymatic characteristics, although both enzymes catalyze the same reaction: cleavage of tryptophan into N-formylkynurenine. The induction of IDO by IFN-[gamma] plays a role in the antigrowth effect of IFN-[gamma] in cell cultures and in the inhibition of intracellular pathogens, e.g., Toxoplasma gondii and Chlamydia psittaci. Tryptophan is also the precursor for the synthesis of serotonin, and reduced levels of tryptophan and serotonin found in AIDS patients have been correlated with the presence of IFN-[gamma] and consequent elevation of IDO activity. The IDO enzyme has been purified and characterized, and its cDNA and genomic DNA clones have been isolated and analyzed. DNA from hybrid cells containing fragments of human chromosome 8 was used to determine the regional localization of the IDO gene on chromosome 8. The hybrids R30-5B and R30-2A contain 8p11 [yields] qter and 8q13 [yields] qter, respectively. Hybrid 229-3A contains the 8pter [yields] q11. The hybrid R30-2A was negative for the IDO gene, whereas R30-5B and 229-3A were positive as analyzed by PCR and verified by Southern blotting. Only themore » region close to the centromere is shared by R30-5B and 229-3A hybrids. The results indicate that the IDO gene is located on chromosome 8p11 [yields] q11.« less

Interferon Stimulates the Expression of 2′,5′ -Oligoadenylate Synthetase and MHC Class I Antigens in Insulin-Producing Cells

The pathogenesis of type 1 diabetes involves autoimmune processes directed against the pancreatic beta-cells. The etiology is not known, but circumstantial evidence suggests a connection between virus infection and development of the disease. Therefore, because the interferon-(IFN) dependent 2',5'-oligoadenylate (2-5A) synthetase system constitutes an important part of the nonspecific immune defense against viral infections, the activity of the enzyme was examined in islets of Langerhans, RIN cells, and GH3 cells. First, the 2-5A synthetase was expressed constitutively in all cell types and, second, all cells were sensitive to stimulation with IFN-alpha. The 2-5A synthetase activity induced by 1,000 U/ml of IFN-alpha increased by 400% in pancreatic islets and by more than 1000% in GH3 and RIN cells. However, the IFN-alpha concentration needed to induce half-maximal 2-5A synthetase activity was nearly the same in the three cell types (i.e., ranging from 59 to 66 U/ml IFN-alpha). The 2-5A synthetase present in islets and RIN cells was highly sensitive to poly (I:C). In pancreatic islets and RIN cells, the 2-5A synthetase enzyme generated dimers and trimers of 2',5'-oligoadenylates. Furthermore, exposure of RIN cells to IFN-alpha showed an increase in MHC class I expression already at 5 U/ml and maximal expression at about 200 U/ml IFN-alpha. The examined endocrine cells express the 2-5A synthetase enzyme as well as MHC class I antigen constitutively, but also by stimulation with IFN in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct induction of Ia antigen on murine thyroid-derived epithelial cells by reovirus.

The ability of thyroid follicular epithelial cells (TFEC) to act as APC is linked to the expression of class II (Ia) molecules of the MHC. The cloned murine thyroid-derived epithelial cell line M.5 was used to demonstrate the potential effects of virus in the direct induction of Ia molecules on TFEC. Membrane binding and replication of reovirus type 1 in TFEC was demonstrated using fluorescein-labeled antireovirus antibody and fluorescence microscopy. One consequence of the interaction between reovirus and M.5 cells was the induction of Ia Ag and augmented class I molecule expression in M.5 cells. The levels of Ia expression at three days after reovirus binding were amplified 17.3-fold over controls and were 2-fold less than that seen upon treatment of M.5 cells with IFN-gamma. Supernatant transfer experiments showed that the induction of Ia expression was directly linked to the binding of virus to M.5 cells, and was not dependent upon virus replication or the presence of IFN. These results indicate that early events of reovirus binding or receptor internalization on TFEC initiate a signaling process which results in the induction of class II and augmentation of class I MHC protein levels on the cell surface.

Enhancer-like interferon responsive sequences of the human and murine (2′-5′) oligoadenylate synthetase gene promoters.

The human (2'-5') oligo(A) synthetase gene contains two independent cis-acting DNA elements, A and B, which act as transcriptional enhancers. Element A alone is not activated by IFN treatment. Element B alone confers IFN-inducibility to the herpes tk promoter. Two murine (2'-5') oligo(A) synthetase genes were isolated and their promoter sequences show high conservation of element A and B. A synthetic oligonucleotide, containing 16 bp of the human element B, or 14 bp of the homologue murine element B, was linked to a TK-CAT construct. These oligonucleotides were shown to be sufficient to activate the TK promoter in the presence of IFN. When multiple repeats of the interferon-responsive sequence (E-IRS) were cloned in 5' of the TK promoter, the activation ratio was increased. In vitro, specific binding of nuclear protein(s) is observed to the radiolabelled synthetic human E-IRS. This binding is competed by the addition of cold synthetic mouse E-IRS or fragments of genomic DNA containing the E-IRS.