Yong-Soo Bae

PKR, a p53 target gene, plays a crucial role in the tumor-suppressor function of p53.

Type I IFN-induced expression of dsRNA-activated protein kinase (PKR) during viral infection is a well-established antiviral mechanism. However, little is known about the expression of PKR in the context of p53 and about PKR involvement in p53-mediated tumor suppression. Here, we report that PKR is a p53 target gene and plays an important role in the tumor-suppressor function of p53. Activation of p53 by genotoxic stress induces a significant level of PKR expression by acting on the newly identified cis-acting element (ISRE), which is separated from the IFN-stimulated responsive element on the PKR promoter, resulting in translational inhibition and cell apoptosis. The genotoxin-mediated inhibition of translation is associated with the p53/PKR/elF2a (eukaryotic initiation factor-2α) pathway. To some extent, p53 activation induced by DNA damage facilitates cell apoptosis by activating PKR. PKR-knockdown human colon cancer cells grew rapidly in nude mice and proved resistant to anti-cancer drugs. These data indicate that p53-mediated tumor suppression can be attributed at least in part to the biological functions of PKR induced by p53 in genotoxic conditions.

The double-strand RNA-dependent protein kinase PKR plays a significant role in a sustained ER stress-induced apoptosis

Sustained ER stress leads to apoptosis. However, the exact mechanism still remains to be elucidated. Here, we demonstrate that the double strand RNA‐dependent protein kinase (PKR) is involved in the ER stress‐mediated signaling pathway. ER stress rapidly activated PKR, inducing the phosphorylation of eIF2α, followed by the activation of the ATF4/CHOP pathway. ER‐stress‐mediated eIF2α/ATF4/CHOP signaling and associated cell death was markedly reduced by PKR knockdown. We also found that PKR activation was mediated by PACT, the expression of which was elevated by ER‐stress. These results indicate that the ER‐stress‐mediated eIF2α/ATF4/CHOP/cell death pathway is, to some degree, dependent on PACT‐mediated PKR activation apart from the PERK pathway.

Phase I/II study of immunotherapy using tumor antigen-pulsed dendritic cells in patients with hepatocellular carcinoma

Dendritic cells (DCs) are increasingly used as adjuvants for vaccination strategies; however, there has been very little development in DC vaccines for patients with hepatocellular carcinoma (HCC). In this study, we assessed the safety, feasibility and efficacy of a multiple tumor-associated antigen (TAA)-pulsed DC vaccine in 5 patients with advanced HCC. DCs were generated by culturing blood monocytes in the presence of granulocyte macrophage-colony stimulating factor and interleukin-4 for 5 days. The DC vaccine was prepared by pulsing DCs with cytoplasmic transduction peptide-attached α-fetoprotein, glypican-3 and MAGE-1 recombinant fusion proteins and cultivating them in the presence of maturation cocktail. DCs were injected subcutaneously near the inguinal lymph nodes, followed by topical application of toll-like receptor-7 agonist around the injection site. We showed that our DC vaccine was safe and well-tolerated over 6 vaccinations in 5 patients. All 5 patients showed T cell responses against TAAs. Clinical benefit was observed in one of the 5 patients. In conclusion, the feasibility, safety and immune activity of DCs pulsed with TAAs were confirmed in HCC patients. However, clinical response was detected only in one patient. Future trials may consider applying this therapy in a less advanced stage to obtain better clinical responses.

Semi-mature DC are immunogenic and not tolerogenic when inoculated at a high dose in collagen-induced arthritis mice

Semi‐mature DC (smDC) have been shown to be tolerogenic and thus applicable to the treatment of autoimmune disease. However, in our repeated experiments, even the same batches of smDC were found to be profoundly immunogenic rather than tolerogenic when inoculated at high doses into arthritic mice. In a cytokine chip assay, smDC were characterized by remarkable production of IL‐2, IL‐3, IL‐5, and IL‐13 together with well‐known Th2 cytokines. Low doses (2×105) of smDC showed excellent anti‐arthritic activity in collagen‐induced arthritis animals, whereas high doses (2×106) of smDC uniformly accelerated arthritic symptoms. SmDC, vaccinated at lower doses, markedly induced forkhead box P3 Treg, Th2 cytokines (IL‐4/IL‐10), and TGF‐â in their immune deviation. Interestingly, however, as the number of smDC increased from 2×105 to 2×106 in the same assay, the Treg population, Th2 cytokines, and TGF‐β were dramatically reduced. Our present study clearly indicates that smDC could induce either T‐cell tolerance or T‐cell activation, depending on the inoculum size. Special attention should be paid to the optimal range of smDC in DC‐mediated immunotherapy for the treatment of rheumatoid arthritis.

Determination of Diabetogenicity Attributable to a Single Amino Acid, Ala776, on the Polyprotein of Encephalomyocarditis Virus

The best experimental evidence indicating that viruses have an etiological role in the pathogenesis of diabetes comes from studies of mice infected with EMC virus. For this study we generated mutant viruses from stocks of diabetogenic EMC-D and nondiabetogenic EMC-B viruses by serial passages of the viruses in L-cell cultures at high MOI. The genomic sequence information and the biological activities of three different plaque-purified diabetogenic variants of EMC virus (EMC-D, EMC-D1/6A, EMC-D2/4) and six different plaque-purified nondiabetogenic variants (EMC-B, EMC-BS, EMC-B1/G, EMC-DV1, EMC-D4/1B, EMC-D3/1) revealed that only one amino acid, Ala (776th amino acid on the polyprotein), is critical for the diabetogenicity of EMC virus. The G base at the nucleotide position 3155 (Ala[GCC]776 in the polyprotein) is unique to all diabetogenic variants, whereas the A base at the same position (Thr[ACC]776 in the polyprotein) is identical to all nondiabetogenic variants. A single-point mutation (G to A; Ala to Thr) results in the conversion of the diabetogenic variant into a nondiabetogenic variant of EMC virus. On the basis of these observations, we conclude that a single amino acid, Ala776, on the polyprotein of EMC virus appears responsible for the inducement of diabetes in susceptible mice. Conversion of Ala776 into Thr776 on the polyprotein by a point mutation, G to A at the nucleotide position 3155, results in the loss of diabetogenicity.

DC immunotherapy is highly effective for the inhibition of tumor metastasis or recurrence, although it is not efficient for the eradication of established solid tumors.

Dendritic cell (DC)-based immunotherapy has not been as effective as expected in most solid tumors even in the murine model, particularly in renal cell carcinoma (RCC). Our investigation was initiated to identify what causes the limitations of DC-based immunotherapy in solid RCC. We have investigated immunosuppressive factors from tumors and their effects on DC migration, as well as cytotoxic T lymphocyte (CTL) response and lymphocyte infiltration into the tumor mass upon vaccination with mouse renal adenocarcinoma (Renca) cell lysate-pulsed bone marrow (Bm)-derived DC in tumor-bearing mice. We also investigated pulmonary metastasis- and tumor recurrence-inhibitory effects of DC-vaccination in the solid tumor-bearing mice. In these experiments, we found that the limitations of DC-based immunotherapy to solid RCC likely result from tumor-mediated TGF-β hindrance of immune attack rather than insufficient immune induction by DC therapy. In fact, the CTL response induced by DC therapy was quite sufficient and functional for the inhibition of tumor recurrence after surgery or of tumor metastasis induced by additional tumor-challenge to the tumor-bearing mice. Taken together, our present results obtained in mouse model suggest the potential of DC immunotherapy in tumor patients for hindering or blocking disease progression by inhibition of tumor metastasis and/or tumor recurrence after surgery.

Nox2-based NADPH oxidase mediates HIV-1 Tat-induced up-regulation of VCAM-1/ICAM-1 and subsequent monocyte adhesion in human astrocytes

Up-regulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) by the HIV-1 transactivator of transcription (Tat) in activated microglia and astrocytes may play a pivotal role during the development of AIDS-related encephalitis and dementia. Previous studies demonstrated that HIV-1 Tat-induced up-regulation of adhesion molecules was mediated by reactive oxygen species (ROS), although the mechanisms underlying HIV-1 Tat-induced ROS generation are unknown. In this study, we examined the possible role of NADPH oxidase in HIV-1 Tat-induced up-regulation of adhesion molecules in astroglioma cell lines. HIV-1 Tat-induced up-regulation of VCAM-1/ICAM-1 and subsequent increased adhesion of monocytes to astrocytes were blocked by a general NADPH oxidase inhibitor, diphenylene iodonium, and a specific inhibitor of NADPH oxidase assembly, 9R3A-gp91ds. Nox2 knockdown using small interfering RNA (siRNA) inhibited HIV-1 Tat-induced up-regulation of adhesion molecules and subsequent increased adhesion of monocytes to astrocytes. Nox2 siRNA blocked HIV-1 Tat-induced ROS production, increase in NADPH oxidase activity, and Rac1 activation. Furthermore, Nox2 siRNA decreased HIV-1 Tat-induced NF-κB activation as well as activation of MAP kinases including ERK, JNK, and p38. These data indicate that Nox2-based NADPH oxidase is responsible for HIV-1 Tat-induced generation of ROS and plays an important role in the up-regulation of adhesion molecules such as VCAM-1/ICAM-1 and subsequent increased adhesion of monocytes to astrocytes and serves as a novel target for HIV-1 Tat-mediated neurological diseases.

The C Proteins of Human Parainfluenza Virus Type 1 Limit Double-Stranded RNA Accumulation That Would Otherwise Trigger Activation of MDA5 and Protein Kinase R

ABSTRACT Human parainfluenza virus type 1 (HPIV1) is an important respiratory pathogen in young children, the immunocompromised, and the elderly. We found that infection with wild-type (WT) HPIV1 suppressed the innate immune response in human airway epithelial cells by preventing not only phosphorylation of interferon regulatory factor 3 (IRF3) but also degradation of IκBβ, thereby inhibiting IRF3 and NF-κB activation, respectively. Both of these effects were ablated by a F170S substitution in the HPIV1 C proteins (F170S) or by silencing the C open reading frame [P(C−)], resulting in a potent beta interferon (IFN-β) response. Using murine knockout cells, we found that IFN-β induction following infection with either mutant relied mainly on melanoma-associated differentiation gene 5 (MDA5) rather than retinoic acid-inducible gene I (RIG-I). Infection with either mutant, but not WT HPIV1, induced a significant accumulation of intracellular double-stranded RNA (dsRNA). These mutant viruses directed a marked increase in the accumulation of viral genome, antigenome, and mRNA that was coincident with the accumulation of dsRNA. In addition, the amount of viral proteins was reduced compared to that of WT HPIV1. Thus, the accumulation of dsRNA might be a result of an imbalance in the N protein/genomic RNA ratio leading to incomplete encapsidation. Protein kinase R (PKR) activation and IFN-β induction followed the kinetics of dsRNA accumulation. Interestingly, the C proteins did not appear to directly inhibit intracellular signaling involved in IFN-β induction; instead, their role in preventing IFN-β induction appeared to be in suppressing the formation of dsRNA. PKR activation contributed to IFN-β induction and also was associated with the reduction in the amount of viral proteins. Thus, the HPIV1 C proteins normally limit the accumulation of dsRNA and thereby limit activation of IRF3, NF-κB, and PKR. If C protein function is compromised, as in the case of F170S HPIV1, the resulting PKR activation and reduction in viral protein levels enable the host to further reduce C protein levels and to mount a potent antiviral type I IFN response.

Novel Design Architecture for Genetic Stability of Recombinant Poliovirus: the Manipulation of G/C Contents and Their Distribution Patterns Increases the Genetic Stability of Inserts in a Poliovirus-Based RPS-Vax Vector System

ABSTRACT Poliovirus has been studied as a live recombinant vaccine vector because of its attractive characteristics. The genetic instability, however, has hampered recombinant polioviruses (PVs) from being developed as an appropriate vaccine. A variety of different foreign inserts were cloned directly into our poliovirus Sabin 1-based RPS-Vax vector system, resulting in the production of recombinant PVs. The genetic stability of each recombinant PV was examined during 12 rounds of consecutive passage. It was found that the genetic stability of the recombinants was not well correlated with their insert size. Instead, elevated stability was frequently observed in recombinants with inserts of high G/C contents. Furthermore, a comparative study using different constructs of the human immunodeficiency virus env gene revealed that the internal deletion of the unstable insert was seemingly caused by the presence of the adjacent A/T-rich region. The instability of these inserts was completely remedied by (i) increasing the G/C contents and (ii) replacing the local A/T-rich region with the G/C-rich codon without a change of the amino acid. This means that stability is closely associated with the G/C content and the G/C distribution pattern. To see whether these findings can be applied to the design of genetically stable recombinant PV, we have reconstructed the heteromultimeric insert based on our design architecture, including the above-mentioned G/C rules and the template/ligation-free PCR protocol. The heteromultimeric insert was very unstable, as expected, but the manipulated insert with the same amino acid sequence showed complete genetic stability, not only in vitro, but also in vivo. Even though this guideline was established with our RPS-Vax vector system, to some extent, it can also be applied to other live viral vaccine vectors.

A phase I/IIa study of adjuvant immunotherapy with tumour antigen-pulsed dendritic cells in patients with hepatocellular carcinoma.

Background:To date, no adjuvant treatment has been shown to have a clear benefit in patients with hepatocellular carcinoma (HCC). In this prospective phase I/IIa study, we evaluated the safety and efficacy of adjuvant dendritic cell (DC) therapy in HCC patients who received primary treatment for HCC.Methods:Twelve HCC patients who had no viable tumour after primary treatments were included. Dendritic cell vaccines pulsed with cytoplasmic transduction peptide-attached alpha-fetoprotein, glypican-3 and melanoma-associated antigen 1 recombinant fusion proteins were injected subcutaneously near to inguinal lymph nodes. Adverse effects, time to progression (TTP), and associated immune responses were evaluated after DC vaccination.Results:Nine of 12 patients had no tumour recurrence up to 24 weeks after DC vaccination. Among a total of 144 adverse events, 129 events (89.6%) were regarded as adverse drug reactions, all of which were grade 1 or 2. The majority of patients showed enhanced anti-tumour immune responses after DC vaccination. Recurrence-free patients exhibited relatively stronger anti-tumour immune responses than patients who developed recurrence after DC vaccination, as evidenced by lymphocyte proliferation and IFN-γ ELISPOT assays. The median time of TTP was 36.6 months in the DC-vaccination group and 11.8 months in the control group (hazard ratio, 0.41; 95% confidence interval, 0.18–0.95; P=0.0031 by log-rank test).Conclusions:Adjuvant DC vaccine for HCC was safe and well tolerated in phase I/IIa study, and preliminary efficacy data are encouraging to warrant further clinical study in patients with HCC after primary treatments.