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Induction of a TRAIL Mediated Suicide Program by Interferon Alpha in Primary E€Usion Lymphoma

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Oncogene (2001) 20, 7029 ± 7040

2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00

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Induction of a TRAIL mediated suicide program by interferon alpha in primary e€usion lymphoma

Ngoc L Toomey1,4, Vadim V Deyev2,4, Charles Wood3, Lawrence H Boise2, Duncan Scott1, Lei Hua Liu1, Lisa Cabral1, Eckhard R Podack2, Glen N Barber2 and William J Harrington Jr*,1

2

1Department of Medicine University of Miami School of Medicine, Miami, Florida, FL 33136, USA; Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida, FL 33136, USA; School of Biological Sciences, University of Nebraska, Lincoln, Nebraska, NE 68588, USA

3

Gammaherpes viruses are often detected in lymphomas arising in immunocompromised patients. We have found that Azidothymidine (AZT) alone induces apoptosis in Epstein Barr Virus (EBV) positive Burkitt's lymphoma (BL) cells but requires interferon alpha (IFN-a) to induce apoptosis in Human Herpes Virus Type 8 (HHV-8) positive Primary E€usion Lymphomas (PEL). Our analysis of a series of AIDS lymphomas revealed that IFN-a selectively induced very high levels of the Death Receptor (DR) tumor necrosis factor-related apoptosisinducing ligand (TRAIL) in HHV-8 positive PEL lines and primary tumor cells whereas little or no induction was observed in primary EBV+ AIDS lymphomas and EBV7Burkitt's lines. AZT and IFN-a mediated apoptosis in PEL was blocked by stable overexpression of dominant negative Fas Associated Death Domain (FADD), decoy receptor 2 (DcR2), soluble TRAIL receptor fusion proteins (DR-4 and DR-5) and thymidine. Trimeric TRAIL (in place of IFN-a) similarly synergized with AZT to induce apoptosis in HHV-8 positive PEL cells. This is the ®rst demonstration that IFN-a induces functional TRAIL in a malignancy that can be exploited to e€ect a suicide program. This novel antiviral approach to Primary E€usion lymphomas is targeted and may represent a highly e€ective and relatively non-toxic therapy. Oncogene (2001) 20,

7029 ± 7040.
Virus (EBV) or Human Herpes Virus Type 8 (HHV-8) have been isolated from lymphomas found in immunosuppressed organ transplant recipients, children with hereditary immunode®ciencies and patients with acquired immunode®ciency (AIDS) (Swinnen, 1999; Goldsby and Carroll, 1998; Knowles, 1999). Many of these tumors can be categorized into distinct subtypes based on a variety of morphologic and molecular criteria. For example, AIDS associated large cell di€use or immunoblastic lymphomas (DLCL, IBL) are often EBV positive while AIDS associated Burkitt's lymphomas (BL) less frequently contain EBV (Gaidano et al., 1994). A recently de®ned subtype, AIDS related HHV-8 associated Primary E€usion Lymphoma (PEL) (Nador et al., 1996), usually occurs in the setting of severe immunode®ciency of advanced HIV infection. PELs di€er from most AIDS lymphomas by their absence of a discernable primary tumor mass, infre-

  • quent expression of
  • B
  • lymphocyte di€erentiation

antigens, and lack of c-myc gene rearrangement (Mullaney et al., 2000; Gaidano et al., 2000; Demario and Liebowitz, 1998). In general, immunode®ciency related herpesvirus associated lymphomas are aggressive and poorly responsive to conventional chemotherapy (Swinnen, 2000; Levine, 2000).
Although AZT was originally developed as an anticancer agent, this thymidine nucleoside analog has demonstrated relatively little activity in solid tumors (Findenig et al., 1996). Interest in AZT was revived only when it was found to inhibit HIV reverse transcriptase (De Clercq, 1992). To exert this antiviral activity, AZT must be phosphorylated by cellular thymidine kinase (TK) (Arner et al., 1992). Our initial studies indicated that there were two distinctly di€erent pro-apoptotic e€ects of AZT and Interferon alpha (IFN-a) in primary lymphoma cell lines derived from AIDS patients. EBV+ BL cells underwent apoptosis in the presence of AZT alone while HHV-8+ PELs required the addition of IFN-a to undergo signi®cant programmed cell death (Lee et al., 1999).
Keywords: human Herpes Virus Type 8; Epstein Barr virus; TRAIL; apoptosis; lymphoma; FADD

Introduction

Gammaherpes viruses are frequently associated with lymphoproliferative disease in immunocompromised individuals (Okano and Gross, 2000). Epstein-Barr

*Correspondence: WJ Harrington Jr, University of Miami School of Medicine/Sylvester Comprehensive Cancer Center, Room 3400 (D8- 4), 1475 NW 12th Avenue, Miami, Florida, FL 33136, USA; E-mail: [email protected]

Interferons have multiple activities involved in host defense including anti-proliferative and antiviral e€ects. It is known that the interferons, which are potently upregulated by viruses and double stranded RNA (dsRNA), can synthesize e€ectors of apoptosis such as

4These authors contributed equally to this work Received 14 May 2001; revised 17 July 2001; accepted 2 August 2001

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2'5' oligoadenylate synthetase (2' ± 5'A) which activates RNAseL and degrades viral mRNA (Player and Torrence, 1998). Interferon also induces synthesis of dsRNA activated protein kinase (PKR), which phosphorylates the translation initiator eIF-2-a and inhibits protein synthesis in the cell (Zamanian-Daryoush et al., 2000). Recently, interferons were also found to induce expression of the pro-apoptotic protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in human dendritic cells and T lymphocytes which is capable of inducing apoptosis in TRAIL receptor expressing targets (Fanger et al., 1999; Grith et al., 1999; Balachandran et al., 2000).
These data demonstrate that IFN-a upregulates
TRAIL in Primary E€usion Lymphomas while AZT sensitizes these cells to TRAIL mediated apoptosis resulting in the activation of a suicide program in this cancer. The unique tumorcidal e€ect of AZT and IFN-a may have important applications for therapy of herpesvirus associated lymphoproliferative disease.

Results

AZT and IFN-a act synergistically to induce apoptosis in HHV-8+ PEL cells

IFN also potentiates the adaptive immune response through upregulation of major histocompatibility complexes (MHC) and activation of cytotoxic T cells (CTLs) and natural killer cells (NKs) (Mingari et al., 2000). CTLs can recognize and kill cells that present foreign peptides, in association with MHC, through the perforin/granzyme pathway (Barry et al., 2000). Another important mechanism of immune e€ector clearance of virally infected or cancerous cells is by transmission of an apoptotic signal through ligand binding to members of the tumor necrosis (TNF) family of receptors (Peter et al., 1997). The binding of ligands such as Fas-L or TRAIL to extracellular receptor domains (APO-1 (Fas/CD95), DR-5) results in recruitment of components of the death domain containing the adaptor molecule Fas associated death domain (FADD) and subsequent activation of FLICE (caspase 8) by autoproteolysis. Following recruitment and cleavage of procaspase 8, downstream caspases are activated, including caspase 3, resulting in apoptosis. TRAIL mediated signaling and apoptosis is blocked upon its binding to decoy receptors (DcR1, DcR2) which do not recruit FADD (Walczak and Krammer, 2000; Ashkenazi and Dixit, 1999; Bodmer et al., 2000). The role of DR-4 in apoptosis is unclear since its expression in FADD7/7 ®broblasts still results in cell death (Yeh et al., 1998). Signaling through other members of the TNF receptor family also may activate non-apoptotic processes such as in¯ammatory responses and lymphoid organogenesis (Magnusson and Vaux, 1999).
We have demonstrated that AZT and IFN-a have clinical activity against malignant herpesvirus associated lymphomas (Lee et al., 1999). To further investigate the apoptotic e€ects, we studied the HHV-8+ PEL lines, BC-3 and BCBL-1, as well as the EBV+ AIDS BL primary lines, BL-7 and BL-5. All lines were cultured for 48 h in the presence of medium, AZT (10 mg/ml) or IFN-a (1000 u/ml) alone, or AZT and IFN-a together. As shown in Figure 1a, AZT alone induced marked apoptosis in the EBV+Burkitt's lymphoma lines (BL-7, BL-5) while IFN-a did not induce apoptosis nor add to the cytopathic e€ect of AZT. In contrast, HHV-8+ PEL cells (BC-3, BCBL-1) only underwent marked apoptosis in the presence of both AZT and IFN-a (Figure 1a). Similar experiments revealed that other HHV-8+ PEL lines, BC-1 and BC-5, also required both AZT and IFN-a to undergo signi®cant apoptosis (data not shown). Therefore, a general property of HHV-8+ PEL cells was that AZT and IFN-a induced substantial apoptosis together but not separately. This cytotoxic e€ect was seen only in virus infected lymphomas as both agents had no e€ect on herpes virus negative BL cells (Ramos) (Figure 1b). Further experiments on another herpesvirus negative lymphoma cell line (BJAB) demonstrated that AZT, with or without IFN-a, induced little or no cytotoxic activity (data not shown).

To further investigate the mechanism of AZT and
IFN-a mediated apoptosis, we studied a series of lymphoma cell lines and primary lymphoma cells derived from AIDS patients. We report here that HHV-8+ PEL cell lines and primary tumor cells from a PEL patient express high levels of TRAIL when cultured with IFN-a. Despite the induction of TRAIL in PEL cells by IFN-a, apoptosis was only potentiated upon the addition of AZT. In PEL cells, AZT and IFN-a mediated apoptosis was blocked by expression of dominant negative FADD (FADD DN), decoy receptor 2 (DcR2), soluble TRAIL receptor fusion proteins and by the addition of thymidine. In contrast to HHV-8+ PEL, EBV+ AIDS BL lines and EBV7 BL lines did not express signi®cant amounts of TRAIL, nor undergo increased apoptosis in response to IFN-a.

IFN-a induces TRAIL in PEL cells

It has recently been shown that IFN-a mediates the expression of several pro-apoptotic genes and can

  • induce cell death through
  • a
  • FADD dependent

mechanism (Balachandran et al., 2000). Since IFN-a potentiated apoptosis in PEL, we investigated its e€ect on the regulation of pro-apoptotic factors in IFN-a sensitive and resistant B cell lymphomas. Accordingly, BC-3 and BL-7 cells were treated for 8 h with AZT (10 mg/ml), IFN-a (1000 m/ml) or the chemotherapeutic agent etoposide (10 mg/ml). Total RNA was extracted and analysed for pro-apoptotic gene expression by ribonuclease protection assay (RPA). RPA analysis revealed that IFN-a induced markedly higher levels of TRAIL mRNA in the IFN- a sensitive PEL cells (BC-3) compared to the resistant

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EBV+ BL cells (BL-5) (Figure 2a, lanes 2 and 4 compared to lanes 7 and 9). In contrast, AZT and etoposide, at doses sucient to kill these cells, had no e€ect on TRAIL expression in either type of

ab

Figure 1 (a) AZT and IFN-a synergize to induce apoptosis in HHV-8+ PEL (BC-3, BCBL-1) while EBV+ BL cells (BL-5, BL-7) undergo apoptosis with AZT alone. BC-3 and BCBL-1 (HHV-8+ PEL's) and BL-5 and BL-7 (EBV+ BL's) were treated in medium (Panels A), AZT 10 mg/ml (Panels B), IFN-a 1000 u/ml (Panels C), or AZT 10 mg/ml plus IFN-a 1000 u/ml (Panels D) for 48 h. Cells were then analysed for apoptosis by PI annexin staining and FACs analysis. Upper and lower right quadrants are the apoptotic populations. (b) AZT and IFN-a are synergistically cytotoxic in HHV-8+ PEL but not EBV7 BL. PEL cells BCBL-1 and BC-3, EBV+ BL cells BL-5 and BL-7, and EBV7 Ramos cells were cultured for 48 h in medium (control), IFN-a 1000 u/ml, AZT 10 mg/ml, and IFN-a 1000 u/ml plus AZT 10 mg/ml. Cell viability was then determined by Trypan blue exclusion. Bars represent per cent viable cells

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lymphoma (Figure 2a, lanes 3 and 5 and lanes 8 and 10). We then investigated the e€ect of IFN-a on a larger panel of lymphoma lines including HHV-8+ PELs, EBV+ BLs (derived from HIV positive patients) and EBV7 BL lines. We found that IFN- a signi®cantly induced TRAIL mRNA only in the HHV-8+ PEL lines and primary tumor cells (BCBL- LN) derived from a patient with PEL, while little or no induction was noted either in EBV positive or negative BL lines. IFN-a also had no e€ect on the expression of two death receptors, DR-4 and DR-5, which are known to bind to TRAIL (Figure 2b). Further RPA experiments demonstrated that neither AZT nor IFN-a a€ected mRNA or protein surface expression levels of DR-4 or DR-5 in PEL cells (data not shown). FACS analysis demonstrated that IFN-a induced TRAIL protein surface expression in PEL cells but not in EBV+ BL cells (Figure 2c, panels 3 and 4 versus panels 1 and 2). Therefore, the ability of IFN-a to potentiate AZT mediated apoptosis in PEL correlated with its ability to induce the proapoptotic ligand TRAIL in these tumors.

ab

AZT and IFN-a mediated apoptosis in PEL is FADD dependent

TRAIL has been reported to induce apoptosis by binding its cognate receptors. Furthermore, TRAIL mediated apoptosis has recently been shown to be dependent upon signaling through the death adaptor protein FADD (Bodmer et al., 2000). To investigate the mechanism of IFN-a induced apoptosis we ®rst con®rmed that HHV-8+ PEL cells (BCBL-1) did express TRAIL receptors on their surface (DR-4 was more highly expressed than DR-5) (Figure 3a). BC-3 cells also expressed similar levels of DR-4 and DR-5 (data not shown). To study whether apoptosis induced by AZT and IFN-a in PEL involved a FADD dependent mechanism, we transfected the BCBL-1 line with a dominant negative construct of FADD that lacks a death e€ector domain (Bala-

c

AZT 10 mg/ml. Lane 4=BC-3 in IFN-a 1000 u/ml and AZT 10 mg/ml. Lane 5=BC-3 in etoposide 10 mg/ml. Lane 6=BL-5 in medium. Lane 7=BL-5 in IFN-a 1000 u/ml. Lane 8=BL-5 in AZT 10 mg/ml. Lane 9=BL-5 in IFN-a 1000 u/ml and AZT 10 mg/ml. Lane 10=BL-5 in etoposide 10 mg/ml. (b) IFN-a induces a high level of expression of TRAIL mRNA in HHV- 8+ PEL but not EBV+ BL and EBV7 BL. PEL lines and primary tumor isolates (BCBL-1, BC-3, BC-1, BCBL-LN, BC-2, BC-5), EBV+ BL (P3HR-1), EBV+AIDS BL lines (BL-8, SM-1, BL-5, BL-7), and EBV7 BL lines (Ramos, BJAB) were treated with media (C) or IFN-a 1000 u/ml (I) for 8 h. mRNA was extracted and assayed by ribonuclease protection assay for expression of DR-4, DR-5 and TRAIL. GAPDH expression was used as an internal control. (c) IFN-a induces TRAIL surface expression in HHV-8+ PEL but not EBV+ BL. EBV+ BL: BL- 5 and BL-7 cells (Panels 1 and 2) and HHV-8+ PEL cells: BCBL- 1 and BC-3 (Panels 3 and 4) were treated for 24 h with IFN-a 1000 u/ml or media and assayed for TRAIL expression by FACs analysis. Shaded area equals isotype control (mouse IgG1). Dotted line are cells treated with media and solid line are cells treated with IFN-a
Figure 2 (a) TRAIL mRNA is induced by IFN-a but not by AZT or Etoposide in HHV-8+ PEL. PEL cells (BC-3, Lanes 1 ± 5) and EBV+cells (BL-5, Lanes 6 ± 10) were treated in the following manner for 8 h and examined for TRAIL mRNA expression by ribonuclease protection assay. Lane 1=BC-3 in medium. Lane 2=BC-3 in IFN-a 1000 u/ml. Lane 3=BC-3 in

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chandran et al., 2000). After G418 selection, we derived several stably transfected subclones which
FADD protein (BCBL FADD DN) (Figure 3b). BCBL FADD DN cells were completely resistant to

  • AZT and IFN-a mediated apoptosis but retained
  • expressed
  • a
  • dominant negative mutation of the

  • a
  • d

bc

Figure 3 (a) HHV-8+ PEL cells express TRAIL receptors. PEL cells (BCBL-1) were grown in IMDM media enriched with 10% FCS and surface expression of DR-5 (dotted line) and DR-4 (solid line) was determined by FACs analysis. Shaded area is mouse isotype control (IgG1). (b) Transfection of BCBL-1 cells with FADD-DN. BCBL-1 cells were transfected with FADD-DN by electroporation. Subclones were derived after G418 selection. Subclones (BCBL-2A4, BCBL-3A3, BCBL-3B2, BCBL-3C2, and BCBL-1A2), BCBL-Neo (transfected with neo-resistance gene only), and BCBL-1 parental cells were assayed for expression of FADD and FADD-DN by Western blot. (c) BCBL-1 FADD-DN cells are resistant to AZT and IFN-a but sensitive to VP-16. BCBL-1, BCBL-1 Neo transfectants and BCBL-1 FADD-DN clones (BCBL-2A4, BCBL-3C2, BCBL-1A2) were treated for 48 h with medium, AZT 10 mg/ml, IFN-a 1000 u/ml, AZT 10 mg/ml plus IFN-a 1000 u/ml, or VP-16 10 mg/ml (for 24 h) and cell death was measured by Trypan blue exclusion. The ®gure is representative of 52 experiments. (d) BCBL-1 FADD-DN cells express TRAIL in response to IFN-a. BCBL-1A2 cells were treated with medium (C) or IFN-a (I) for 8 h and caspase 8, DR-4 and DR-5 expression levels were detected by RPA

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their sensitivity to etoposide (Figure 3c). To determine that resistant cells were not generated by the selection process, clones were also characterized for expression of anti-apoptotic proteins, Bcl-2 and Bcl-x and found to express similar low levels (compared to the parental BCBL-1 cells) of each protein (data not shown). BCBL-1 FADD DN cells also expressed similar levels of DR-4 and DR-5 and TRAIL mRNA when treated with IFN-a (Figure 3d). Therefore, our data indicates that apoptosis induced by AZT and IFN-a in HHV-8+ PEL is a FADD dependent process.

  • a
  • c

b

Figure 4 (a) Activation of caspases 3 and 8 by AZT and IFN-a in PEL cells is blocked by the caspase inhibitor DEVD and expression of FADD DN. PEL cells; BC-3 (top panel), BCBL-1 (middle panel) and BCBL-1A2 (FADD-DN cells) (lower panel) were cultured for 48 h in media (C), IFN-a 1000 u/ml (I), AZT 10 mg/ml (A), AZT 10 mg/ml plus IFN-a 1000 u/ml (A+I), AZT 10 mg/ml and IFN-a 1000 u/ml plus the caspase inhibitor DEVD 10 mM (A+I+D) and assayed by Western blot for caspase 3 and 8. The caspase 8 antibody used only detects the uncleaved pro-caspase form. (b) Soluble TRAIL receptors inhibit AZT and IFN-a mediated cytotoxicity in PEL cells. BCBL-1 (HHV-8+ PEL cells) were treated in triplicate with the following conditions: medium (Control); AZT 10 mg/ml+IFN-a 1000 u/ml; soluble DR-4 2 mg/ml+soluble DR-5 5 ng/ml; pre-treatment for 8 h with soluble DR- 4 2 mg/ml and soluble DR-5 5 ng/ml followed by treatment for 48 h with AZT 10 mg/ml+IFN-a 1000 u/ml; pre-treatment for 8 h with soluble DR-4 2 mg/ml+soluble DR-5 5 ng/ml+ZB4 1 mg/ml followed by treatment for 48 h with AZT 10 mg/ml and IFN-a 1000 u/ml; TRAIL 100 ng/ml; pre-treatment for 8 h with soluble DR-4 2 mg/ml+soluble DR-5 5 ng/ml followed by treatment for 48 h with TRAIL 100 ng/ml. Cytotoxicity was measured after 48 h by Trypan blue exclusion. (c) DcR2 expression in BCBL-1 blocks apoptosis induced by AZT and IFN-a. Top panel: Flow cytometry pro®les of DcR2 expression in transfected BCBL-1 cells (bold line) versus wild type (thin line). Dotted line is isotype control. Bottom panel: Wild type and BCBL-1 cells were treated with IFN-a 1000 u/ml, AZT 10 mg/ml or IFN-a 1000 u/ml plus AZT 10 mg/ml and viability was measured by Trypan blue exclusion

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combined with AZT in PEL cells. We found that in PEL cells, TRAIL and AZT caused a synergistic proapoptotic e€ect similar to that caused by IFN-a and AZT (Figure 5a, panel E compared to panel C). Similar to AZT and IFN-a, AZT and TRAIL were not cytotoxic to FADD DN BCBL-1 cells (Figure 5b). These data indicate that AZT enhances TRAIL mediated apoptosis in HHV-8+ PEL cells.

AZT and IFN-a induces cleavage of caspases 3 and 8 in PEL which is inhibited by soluble DR-4, DR-5 or DcR2

Apoptosis mediated through death receptor ligand pathways involves the recruitment of proteins forming the death inducing signaling complex (DISC) resulting in cleavage (activation) of procaspase 8 (FLICE) to its active form and subsequent activation of downstream pro-caspases (Kischkel et al., 1995; Medema et al., 1997). We therefore treated HHV-8+ PEL cells (BCBL-1 and BC-3) and BCBL-DN FADD transfectants (BCBL-1A2) with AZT, IFN-a, or AZT plus IFN-a and assayed for procaspase 8 cleavage. Treatment with either agent alone resulted in no cleavage of procaspase 8. However in BC-3 and BCBL-1 cells, AZT and IFN-a together activated procaspase 8 and procaspase 3 but did not in BCBL-1A2 (FADD DN) cells (Figure 4a, lane 4). We did note some cleavage of procaspase 3 in AZT treated BCBL-1 and BC-3 PEL cells which indicates that AZT alone does cause some degree of apoptosis in PEL cells (lane 3). Treatment with the caspase inhibitor DEVD abrogated AZT and IFN-a mediated cleavage of both procaspases in PEL cells (lane 5). Caspase inhibition of AZT and IFN-a mediated apoptosis with IETD, a speci®c caspase 8 inhibitor, was not possible since the inhibitor itself was toxic to PEL cells (data not shown). We then investigated whether inhibition of TRAIL with soluble TRAIL receptors DR-4 and DR-5 could block AZT and IFN-a mediated apoptosis. These experiments were also performed with ZB4, a blocking anti-Fas antibody. As demonstrated in Figure 4b, soluble DR-4 and DR-5 together inhibited AZT and IFN-a mediated apoptosis, albeit incompletely, while the addition of ZB4 had little e€ect on AZT and IFN-a apoptosis. In contrast, soluble receptors DR-4, DR-5 and ZB4 had no inhibitory e€ect on AZT mediated apoptosis in EBV+ BL cells (data not shown). Control experiments demonstrated that soluble DR-4 and DR-5 almost completely inhibited TRAIL mediated cell death (at a highly cytotoxic concentration of TRAIL [100 ng/ml]). To further con®rm that AZT and IFN-a mediated apoptosis occurs principally through TRAIL signaling, we transfected BCBL-1 cells with the decoy receptor for TRAIL (DcR2). Overexpression of DcR2 markedly abrogated the IFN-a component of AZT and IFN-a mediated apoptosis (Figure 4c).

ab

Figure 5 (a) TRAIL substitutes for IFN-a to potentiate apoptosis in AZT treated PEL cells. BCBL-1 cells were treated under the following conditions for 48 h and apoptosis was measured by Annexin/PI staining. Panel A=BCBL-1 cells in medium. Panel B=BCBL-1 cells in AZT 10 mg/ml. Panel C=BCBL-1 cells in AZT 10 mg/ml and IFN-a 1000 u/ml. Panel D=BCBL-1 cells treated with soluble TRAIL 10 ng/ml. Panel E=BCBL-1 cells treated with AZT 10 mg/ml plus soluble TRAIL 10 ng/ml. The per cent of apoptotic cells in each panel is shown in Panel F, (b) AZT potentiates TRAIL mediated cytotoxicity in BCBL-1. BCBL-1 and BCBL-1 FADD-DN (BCBL-1A2) were treated in media, AZT 10 mg/ml, AZT 10 mg/ml plus IFN-a 1000 u/ml, TRAIL 10 ng/ml, or AZT 10 mg/ml plus TRAIL 10 ng/ml for 48 h and per cent viability was determined by Trypan blue exclusion. Bars represent per cent viable cells. The data is representative of at least two experiments

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  • Dysregulated Interferon Response Underlying Severe COVID-19

    Dysregulated Interferon Response Underlying Severe COVID-19

    viruses Review Dysregulated Interferon Response Underlying Severe COVID-19 LeAnn Lopez y, Peter C. Sang y, Yun Tian y and Yongming Sang * Department of Agricultural and Environmental Sciences, College of Agriculture, Tennessee State University, 3500 John A. Merritt Boulevard, Nashville, TN 37209, USA; [email protected] (L.L.); [email protected] (P.C.S.); [email protected] (Y.T.) * Correspondence: [email protected]; Tel.: +1-615-963-5183 These authors contributed equally. y Academic Editor: Andrew Davidson Received: 27 October 2020; Accepted: 9 December 2020; Published: 13 December 2020 Abstract: Innate immune interferons (IFNs), including type I and III IFNs, constitute critical antiviral mechanisms. Recent studies reveal that IFN dysregulation is key to determine COVID-19 pathogenesis. Effective IFN stimulation or prophylactic administration of IFNs at the early stage prior to severe COVID-19 may elicit an autonomous antiviral state, restrict the virus infection, and prevent COVID-19 progression. Inborn genetic flaws and autoreactive antibodies that block IFN response have been significantly associated with about 14% of patients with life-threatening COVID-19 pneumonia. In most severe COVID-19 patients without genetic errors in IFN-relevant gene loci, IFN dysregulation is progressively worsened and associated with the situation of pro-inflammation and immunopathy, which is prone to autoimmunity. In addition, the high correlation of severe COVID-19 with seniority, males, and individuals with pre-existing comorbidities will be plausibly explained by the coincidence of IFN aberrance in these situations. Collectively, current studies call for a better understanding of the IFN response regarding the spatiotemporal determination and subtype-specificity against SARS-CoV-2 infections, which are warranted to devise IFN-related prophylactics and therapies.
  • Pegasys, INN-Peginterferon Alfa-2A

    Pegasys, INN-Peginterferon Alfa-2A

    SCIENTIFIC DISCUSSION This module reflects the initial scientific discussion and scientific discussion on procedures, which have been finalised before 1 April 2005. For scientific information on procedures after this date please refer to module 8B. 1. Introduction Peginterferon alfa-2a is a polyethylene glycol (PEG)-modified form of human recombinant interferon alfa-2a intended for the treatment of adult patients with chronic hepatitis C (CHC) or chronic hepatitis B (CHB). Chronic hepatitis C is a major public health problem: hepatitis C virus (HCV) is responsible for a large proportion of chronic liver disease, accounting for 70% of cases of chronic hepatitis in industrialised countries. Globally there are an estimated 150 million chronic carriers of the virus, including 5 million in Western Europe. Without treatment approximately 30% of those infected with HCV will develop cirrhosis over a time frame of 30 years or more. For those with HCV-related cirrhosis, the prognosis is poor – a significant proportion will develop a life-threatening complication (either decompensated liver disease or an hepatocellular carcinoma) within a few years. The only therapy for those with advanced cirrhosis is liver transplantation, which carries a high mortality. In those who survive transplantation, viral recurrence in the new liver is almost inevitable and a significant proportion of infected liver grafts develop a progressive fibrosis that leads to recurrence of cirrhosis within 5 years. Interferon alfa monotherapy has been shown to be effective for the treatment of chronic hepatitis although sustained response rates occurred in approximately 15 to 30 % of patients treated for long duration (12-18 months). The current reference therapy is interferon alpha in combination with ribavirin, which resulted in an increase in biochemical and virological sustained response rates to approximately 40 % in naïve patients.
  • Induces Antigen Presentation in B Cells Cell-Activating Factor of The

    Induces Antigen Presentation in B Cells Cell-Activating Factor of The

    B Cell Maturation Antigen, the Receptor for a Proliferation-Inducing Ligand and B Cell-Activating Factor of the TNF Family, Induces Antigen Presentation in B Cells This information is current as of September 27, 2021. Min Yang, Hidenori Hase, Diana Legarda-Addison, Leena Varughese, Brian Seed and Adrian T. Ting J Immunol 2005; 175:2814-2824; ; doi: 10.4049/jimmunol.175.5.2814 http://www.jimmunol.org/content/175/5/2814 Downloaded from References This article cites 54 articles, 36 of which you can access for free at: http://www.jimmunol.org/content/175/5/2814.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology B Cell Maturation Antigen, the Receptor for a Proliferation-Inducing Ligand and B Cell-Activating Factor of the TNF Family, Induces Antigen Presentation in B Cells1 Min Yang,* Hidenori Hase,* Diana Legarda-Addison,* Leena Varughese,* Brian Seed,† and Adrian T.
  • Interferon-Based Biopharmaceuticals: Overview on the Production, Purification, and Formulation

    Interferon-Based Biopharmaceuticals: Overview on the Production, Purification, and Formulation

    Review Interferon-Based Biopharmaceuticals: Overview on the Production, Purification, and Formulation Leonor S. Castro 1,†, Guilherme S. Lobo 1,†, Patrícia Pereira 2 , Mara G. Freire 1 ,Márcia C. Neves 1,* and Augusto Q. Pedro 1,* 1 CICECO–Aveiro Institute of Materials, Chemistry Department, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal; [email protected] (L.S.C.); [email protected] (G.S.L.); [email protected] (M.G.F.) 2 Centre for Mechanical Engineering, Materials and Processes, Department of Chemical Engineering, University of Coimbra, Rua Sílvio Lima-Polo II, 3030-790 Coimbra, Portugal; [email protected] * Correspondence: [email protected] (M.C.N.); [email protected] (A.Q.P.) † These authors contributed equally to this work. Abstract: The advent of biopharmaceuticals in modern medicine brought enormous benefits to the treatment of numerous human diseases and improved the well-being of many people worldwide. First introduced in the market in the early 1980s, the number of approved biopharmaceutical products has been steadily increasing, with therapeutic proteins, antibodies, and their derivatives accounting for most of the generated revenues. The success of pharmaceutical biotechnology is closely linked with remarkable developments in DNA recombinant technology, which has enabled the production of proteins with high specificity. Among promising biopharmaceuticals are interferons, first described by Isaacs and Lindenmann in 1957 and approved for clinical use in humans nearly thirty years later. Interferons are secreted autocrine and paracrine proteins, which by regulating several biochemical Citation: Castro, L.S.; Lobo, G.S.; pathways have a spectrum of clinical effectiveness against viral infections, malignant diseases, Pereira, P.; Freire, M.G.; Neves, M.C.; and multiple sclerosis.
  • Pegasys®) Peginterferon Alfa-2B (Peg-Intron®

    Pegasys®) Peginterferon Alfa-2B (Peg-Intron®

    Peginterferon alfa-2a (Pegasys®) Peginterferon alfa-2b (Peg-Intron®) UTILIZATION MANAGEMENT CRITERIA DRUG CLASS: Pegylated Interferons BRAND (generic) NAME: Pegasys (peginterferon alfa-2a) Single-use vial 180 mcg/1.0 mL; Prefilled syringe 180 mcg/0.5 mL Peg-Intron (peginterferon alfa-2b) Single-use vial (with 1.25 mL diluent) and REDIPEN®: 50 mcg, 80 mcg, 120 mcg, 150 mcg per 0.5 mL. FDA-APPROVED INDICATIONS PEG-Intron (peginterferon alfa-2b): Combination therapy with ribavirin: • Chronic Hepatitis C (CHC) in patients 3 years of age and older with compensated liver disease. • Patients with the following characteristics are less likely to benefit from re-treatment after failing a course of therapy: previous nonresponse, previous pegylated interferon treatment, significant bridging fibrosis or cirrhosis, and genotype 1 infection. Monotherapy: CHC in patients (18 years of age and older) with compensated liver disease previously untreated with interferon alpha. Pegasys (Peginterferon alfa-2a): • Treatment of Chronic Hepatitis C (CHC) in adults with compensated liver disease not previously treated with interferon alpha, in patients with histological evidence of cirrhosis and compensated liver disease, and in adults with CHC/HIV coinfection and CD4 count > 100 cells/mm3. o Combination therapy with ribavirin is recommended unless patient has contraindication to or significant intolerance to ribavirin. Pegasys monotherapy is indicated for: • Treatment of adult patients with HBeAg positive and HBeAg negative chronic hepatitis B who have compensated
  • Gut Microbiota and Regulation of Myokine-Adipokine Function

    Gut Microbiota and Regulation of Myokine-Adipokine Function

    Available online at www.sciencedirect.com ScienceDirect Gut microbiota and regulation of myokine-adipokine function 1 1 Francesco Suriano , Matthias Van Hul and Patrice D Cani Both skeletal muscle and adipose tissue are considered as particular interest on how they affect metabolic homeosta- endocrine organs due to their ability to produce and secrete sis of the whole body. several bioactive peptides (e.g. myokines and adipokines). Those bioactive molecules are well known for their capacity to Myokines influence whole-body homeostasis and alterations in their In the body, there are different type of muscles (skeletal, production/secretion are contributing to the development of cardiac,smooth),whichperform different functions based on various metabolic disorders. While it is well accepted that their location. They are mainly responsible for maintaining changes in the composition and functionality of the gut and changing body posture, producing force and motion, microbiota are associated with the onset of several generating heat (both through shivering and non-shivering), pathological disorders (e.g. obesity, diabetes, and cancer), its as well as facilitating movement of internal organs, such as contribution to the regulation of the myokine-adipokine profile the heart, digestive organs, and blood vessels [2,3]. Skeletal and function remains largely unknown. This review will focus on muscle is the largest organ in the human body, accounting for myokines and adipokines with a special interest on their about 30% of body mass in women and 40% in men, though interaction with the gut microbiota. muscle mass is affected by several conditions such as fasting, physical inactivity, cancer, obesity, untreated diabetes, hor- Address monal changes, heart failure, AIDS, chronic obstructive UCLouvain, Universite´ catholique de Louvain, WELBIO - Walloon Excellence in Life Sciences and BIOtechnology, Louvain Drug Research pulmonary disease (COPD), or aging [4].
  • Innate and Adaptive Signals Enhance Differentiation and Expansion of Dual-Antibody Autoreactive B Cells in Lupus

    Innate and Adaptive Signals Enhance Differentiation and Expansion of Dual-Antibody Autoreactive B Cells in Lupus

    ARTICLE DOI: 10.1038/s41467-018-06293-z OPEN Innate and adaptive signals enhance differentiation and expansion of dual-antibody autoreactive B cells in lupus Allison Sang1, Thomas Danhorn 2, Jacob N. Peterson1, Andrew L. Rankin3,4, Brian P. O’Connor1,2,5,6, Sonia M. Leach2,5, Raul M. Torres1,5 & Roberta Pelanda1,5 1234567890():,; Autoreactive B cells have a major function in autoimmunity. A small subset of B cells expressing two distinct B-cell-antigen-receptors (B2R cells) is elevated in many patients with systematic lupus erythematosus (SLE) and in the MRL(/lpr) mouse model of lupus, and is often autoreactive. Here we show, using RNAseq and in vitro and in vivo analyses, signals that are required for promoting B2R cell numbers and effector function in autoimmune mice. Compared with conventional B cells, B2R cells are more responsive to Toll-like receptor 7/9 and type I/II interferon treatment, display higher levels of MHCII and co-receptors, and depend on IL-21 for their homeostasis; moreover they expand better upon T cell-dependent antigen stimulation, and mount a more robust memory response, which are characteristics essential for enhanced (auto)immune responses. Our findings thus provide insights on the stimuli for the expansion of an autoreactive B cell subset that may contribute to the etiology of SLE. 1 Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO 80045, USA. 2 Center for Genes, Environment and Health, National Jewish Health, Denver, CO 80206, USA. 3 Inflammation and Immunology, Pfizer Research, Cambridge, MA 02140, USA. 4 Immuno- Oncology Discovery, FivePrime Therapeutics, South San Francisco, CA 94080, USA.
  • Introna, INN-Interferon Alfa-2B

    Introna, INN-Interferon Alfa-2B

    SCIENTIFIC DISCUSSION This module reflects the initial scientific discussion and scientific discussion on procedures, which have been finalised before 1 August 2003. For scientific information on procedures after this date please refer to module 8B. 1. Introduction This was a full application to obtain a single European Marketing Authorisation via the centralised procedure for interferon alfa-2b. National Marketing Authorisations exist in all Member States for the use of interferon alfa–2b (IntronA). IntronA is indicated in the treatment of Chronic Hepatitis B, Chronic Hepatitis C, Hairy Cell Leukemia, Chronic Myelogenous Leukemia, Multiple Myeloma, Follicular Lymphoma, Carcinoid tumor, Malignant Melanoma and AIDS-related Kaposi’s Sarcoma. The harmonization of the SPC for the listed indications was achieved in June 1997 through a referral under Article 11 of Council directive 75/319/EEC as amended. The new data filed with the centralised application concerned an extension of Chronic Hepatitis B approval in adults to children (1 to 17 years of age), the combination of interferon alfa-2b with ribavirin as first line treatment for patients with Chronic Hepatitis C and an update of the other indications including acknowledgement that cytarabine is commonly used with interferon alfa-2b in Chronic Myelogenous Leukemia. The indication AIDS-related Kaposi’s Sarcoma was deleted. The CPMP focused the evaluation on the new information provided in support of the centralised application. 2. Chemical, pharmaceutical and biologicals aspects The qualitative and quantitative composition of the medicinal product centrally authorised IntronA and of the nationally licensed IntronA, are strictly identical. The manufacturers and the manufacturing process are those already approved for the manufacture of the nationally authorized Intron-A.
  • Interferon-Á Sensitises Human Osteosarcoma Cells to Trail-Mediated Apoptosis

    Interferon-Á Sensitises Human Osteosarcoma Cells to Trail-Mediated Apoptosis

    CANCER GENOMICS & PROTEOMICS 1: 95-104 (2004) Interferon-Á Sensitises Human Osteosarcoma Cells to Trail-mediated Apoptosis IDUN M. MIKKELSEN1, CECILIE LØKKE1, TROND FLAGSTAD1,3 and ØYVIND S. BRULAND2,4 1Department of Paediatrics and 2Department of Oncology, Institute of Clinical Medicine, University of Tromsø, N-9037 Tromsø;. 3Department of Paediatrics, University Hospital of North Norway, N-9037 Tromsø; 4The Norwegian Radium Hospital, Montebello, Oslo, Norway Abstract. Background: Nearly half of all patients with efficiently induces cell death in osteosarcoma cell lines and osteosarcoma are still not cured by the currently employed should be further investigated as a potential future therapy. multimodal treatment. New strategies are therefore needed to further improve the outcome. Tumour necrosis factor-related Osteosarcoma (OS) is the most common primary malignant apoptosis-inducing ligand (TRAIL/Apo2) is able to induce tumour of the bone in children and adolescents and is programmed cell death in transformed cells, while normal characterised by its high propensity to metastasise to the cells remain unaffected. Materials and Methods: We have lungs and the skeleton (1,2). Currently, only half of the investigated the effect of TRAIL in combination with IFN-Á patients with OS are cured after multimodal therapy with on four human osteosarcoma cell lines; Saos-2, U2OS, surgery, high-dose combinatory chemotherapy and KPDXM and OHS, and on one normal human fibroblast cell irradiation (3,4). The acquisition of resistant phenotypes of line, MRC-5. Results: One of the four osteosarcoma cell lines OS is also a major problem during and after the use of was TRAIL-resistant, but was sensitised to TRAIL-mediated chemotherapy and radiation therapy (4-6).