Interactions between NCR + ILC3s and the Microbiome in the Airways Shape Asthma Severity

Asthma is a heterogeneous disease whose development is shaped by a variety of environmental and genetic factors. While several recent studies suggest that microbial dysbiosis in the gut may promote asthma, little is known about the relationship between the recently discovered lung microbiome and asthma. Innate lymphoid cells (ILCs) have also been shown recently to participate in asthma. To investigate the relationship between the lung microbiome, ILCs, and asthma, we recruited 23 healthy controls (HC), 42 patients with non-severe asthma, and 32 patients with severe asthma. Flow cytometry analysis showed severe asthma associated with fewer natural cytotoxicity receptor (NCR) + ILC3s in the lung.Similar changes in other ILC subsets, macrophages, and monocytes were not observed. The asthma patients did not differ from the HC in terms of the alpha and beta-diversity of the lung and gut microbiomes. However, lung function correlated positively with both NCR + ILC3 frequencies and microbial diversity in the lung. Sputum NCR + ILC3 frequencies correlated positively with lung microbiome diversity in the HC, but this relationship was inversed in severe asthma. Together, these data suggest that airway NCR + ILC3s may contribute to a healthy commensal diversity and normal lung function.

SH2 domain–containing adaptor protein B expressed in dendritic cells is involved in T-cell homeostasis by regulating dendritic cell–mediated Th2 immunity

PURPOSE: The Src homology 2 domain–containing adaptor protein B (SHB) is widely expressed in immune cells and acts as an important regulator for hematopoietic cell function. SHB silencing induces Th2 immunity in mice. SHB is also involved in T-cell homeostasis in vivo. However, SHB has not yet been studied and addressed in association with dendritic cells (DCs). MATERIALS AND METHODS: The effects of SHB expression on the immunogenicity of DCs were assessed by Shb gene silencing in mouse bone marrow–derived DCs (BMDCs). After silencing, surface phenotype, cytokine expression profile, and T-cell stimulation capacity of BMDCs were examined. We investigated the signaling pathways involved in SHB expression during BMDC development. We also examined the immunogenicity of SHB-knockdown (SHB(KD)) BMDCs in a mouse atopic dermatitis model. RESULTS: SHB was steadily expressed in mouse splenic DCs and in in vitro–generated BMDCs in both immature and mature stages. SHB expression was contingent on activation of the mitogen- activated protein kinase/Foxa2 signaling pathway during DC development. SHB(KD) increased the expression of MHC class II and costimulatory molecules without affecting the cytokine expression of BMDCs. When co-cultured with T cells, SHB(KD) in BMDCs significantly induced CD4+ T-cell proliferation and the expression of Th2 cytokines, while the regulatory T cell (Treg) population was downregulated. In mouse atopic dermatitis model, mice inoculated with SHB(KD) DCs developed more severe symptoms of atopic dermatitis compared with mice injected with control DCs. CONCLUSION: SHB expression in DCs plays an important role in T-cell homeostasis in vivo by regulating DC-mediated Th2 polarization.

Dendritic Cell-based Immunotherapy for Rheumatoid Arthritis: from Bench to Bedside

Dendritic cells (DCs) are professional antigen presenting cells, and play an important role in the induction of antigen-specific adaptive immunity. However, some DC populations are involved in immune regulation and immune tolerance. These DC populations are believed to take part in the control of immune exaggeration and immune disorder, and maintain immune homeostasis in the body. Tolerogenic DCs (tolDCs) can be generated in vitro by genetic or pharmacological modification or by controlling the maturation stages of cytokine-derived DCs. These tolDCs have been investigated for the treatment of rheumatoid arthritis (RA) in experimental animal models. In the last decade, several in vitro and in vivo approaches have been translated into clinical trials. As of 2015, three tolDC trials for RA are on the list of ClinicalTrial.gov (www.clinicaltrials.gov). Other trials for RA are in progress and will be listed soon. In this review, we discuss the evolution of tolDC-based immunotherapy for RA and its limitations and future prospects.

Dendritic cell-based therapeutic cancer vaccines: past, present and future

No abstract available.

Poliovirus-derived CTL-inducing Hepatitis C Vaccine by Modification of RPS-Vax with Protein Transduction Domain

We have reported RPS-Vax system by introducing multiple cloning site (MCS) and 3C-protease cutting site at the N-terminal end of the poliovirus Sabin 1 cDNA. Potential vaccine genes can be easily introduced into recombinant polioviral genome and expressed during the viral replication as a part of virus polyprotein and subsequently processed from the mature viral protein by the poliovirus-specific 3C-protease. However, these poliovirus vector-mediated chimeric viral vaccine was not efficient to induce the cell-mediated immunity because of its rapid cytolytic capacity. In order to make CTL-inducing vaccine vector, we integrated a protein transduction domain (PTD) into the pRPS-Vax vector system right ahead of the MCS, named RPS-Vax/PTD. We have incorporated the HCV core (N-terminal 100aa) antigen into the MCS of pRPSvax-PTD vector, followed by production of chimeric virus, named RPSvax-PTD/HCVc. The chimeric virus was genetically stable during the serial passages. Replication capacity of the RPSvax-PTD/HCVc was 1~2 log lower than that of RPS-Vax control virus. These chimeric virus was very efficient to inducing antigen-specific IgG2a in the immunized mice, implying that the recombinant virus has a capacity to induce HCV-specific Th1 type immunity in the immunized animals or humans.

Poliovirus-derived CTL-inducing Hepatitis C Vaccine by Modification of RPS-Vax with Protein Transduction Domain

We have reported RPS-Vax system by introducing multiple cloning site (MCS) and 3C-protease cutting site at the N-terminal end of the poliovirus Sabin 1 cDNA. Potential vaccine genes can be easily introduced into recombinant polioviral genome and expressed during the viral replication as a part of virus polyprotein and subsequently processed from the mature viral protein by the poliovirus-specific 3C-protease. However, these poliovirus vector-mediated chimeric viral vaccine was not efficient to induce the cell-mediated immunity because of its rapid cytolytic capacity. In order to make CTL-inducing vaccine vector, we integrated a protein transduction domain (PTD) into the pRPS-Vax vector system right ahead of the MCS, named RPS-Vax/PTD. We have incorporated the HCV core (N-terminal 100aa) antigen into the MCS of pRPSvax-PTD vector, followed by production of chimeric virus, named RPSvax-PTD/HCVc. The chimeric virus was genetically stable during the serial passages. Replication capacity of the RPSvax-PTD/HCVc was 1~2 log lower than that of RPS-Vax control virus. These chimeric virus was very efficient to inducing antigen-specific IgG2a in the immunized mice, implying that the recombinant virus has a capacity to induce HCV-specific Th1 type immunity in the immunized animals or humans.

HIV-1 Infection Causes Intracellular Expression of p53, Which Induces PKR Expression, Followed by Inhibition of HIV-1 Tat Activity

Few papers have reported that the HIV-1 replication was inhibited by p53 in the infected cells. However, the detail mechanism for the p53-medicated HIV-1 suppression has not yet been clearly demonstrated. In our previous report, we addressed that p53-mediated Tat suppression is very likely associated with PKR. In the present study, we found that the amounts of p53 in the HIV-1 infected cells increased over 10 times in the early stages of infection as much as those in normal cells. Particularly noteworthy is that the both exogenous p53 and endogenous p53 enhanced PKR expression in the transformed or treated cells, and the amounts of PKR induced by p53 were almost equivalent to those induced by interferon. In the PKR promoter studies using Ppkr-CAT (CAT reporter system under the control of PKR promoter), CAT activity induced by p53 was stronger than that by interferon, suggesting that the p53-mediated PKR expression might be more efficient than interferon under the control of PKR promoter. Co-immunoprecipitation experiments showed that PKR directly binds to Tat protein. We established eIF-2alpha dominant negative (S51A) Jurkat cells (JK/eIF2alpha-51A) to block the PKR-mediated cell cycle arrest or apoptosis. In the JK/eIF2alpha-51A cells, not only p53 but also PKR inhibited the Tat activity. Taken together, our results demonstrate that the HIV-1 infection induces p53, which enhances PKR expression by promoter activation, followed by the inhibition of the Tat activity, finally resulting in the inhibition of HIV-1 replication. Detail mechanisms for the PKR-mediated Tat inactivation are under investigation.

Expression and Characterization of Human Immunodeficiency Virus Type 1 Envelope Mutant Glycoproteins by Using Baculovirus Expression System

Considerable effort has been directed at understanding the structure and function of HIV-1 envelope glycoproteins. It has been difficult to characterize HIV-1 envelope glycoproteins due to the limited availability of these proteins from virus particles or infected cells. To facilitate the structural and functional analysis of HIV-1 envelope glycoproteins, recombinant baculoviruses were generated to express wild type or mutant HIV-1 envelope glycoproteins. The gp160 precursor protein as well as the gp120 glycoprotein were detected in the cell infected with recombinant BacENVw.t containing wild type HIV-1 envelope gene. In the insect cells infected with recombinant BacENVc with mutations at the cleavage site of gp160, a precursor form of envelope glycoprotein was produced, but not secreted into the culture medium. However, the insect cells infected with recombinant BacENVc/t containing both mutations at the cleavage site and membrane spanning region produced mutant envelope glycoproteins that were efficiently secreted into the culture medium in the form of precursor. Therefore the recombinant HIV-1 envelope glycoproteins produced in this system would be useful as immunogens in the development of a vaccine against AIDS.

Azasugar Nucleotide-containing Phosphorothioate Oligonucleotides as an AIDS Therapeutic Drug

A series of modified oligonucleotides containing P=S backbone and a six-membered azasugar (6-AZS) were synthesized and tested for their ability to inhibit human immunodeficiency virus (HIV) in vitro without the aid of any transfecting agents. While P=S oligonucleotides with natural nucleotides had little anti-HIV-1 activity, six-membered azasugar nucleotide (6-AZN)-containing P=S oligonucleotides (AZPSON) potently inhibited the HIV-1/SHIV production and syncytium formation in vitro (EC50 = 0.02~0.2 micro M) without cytotoxicity up to 100 micro M. AZPSONs are enzymatically stable over 6 days in culture supernatant. Phosphodiester (P=O) backbone only or mixed backbone (P=O and P=S) oligonucleotides that contain 6-AZN did not exhibit anti-HIV-1 activity. The anti-HIV-1 capacity of AZPSON seems to depend on the number and/or distribution patterns of 6-AZN in the oligonucleotides. The oligomer 2198, most effective for anti-HIV-1 activity among the AZPSONs, was much more effective than ddI or ddC in anti-HIV activity. Particularly noteworthy is that the anti-HIV-1 activity of AZPSON-2198 was better than AZT in the long-lasting efficacy after a single treatment.

Construction and Immuno - biochemical Studies of Chimeric Polioviruses Expressing Multivalent V3 / PND - concatamers of Human Immunodeficiency Virus Type 1

Poliovirus Sabin 1 strain has its own special features that make it a particularly attractive live recombinant mucosal vaccine vehicle. Sabin 1 cDNA was manipulated to have multiple cloning site and viral specific 3C-protease cutting site at the N-terminal end of the polyprotein, named RPS-vax system HIV-1 V3- and principal neutralizing domain (PND)-concatamers were successfully cloned into the multiple cloning site of the vector system and produced expected chimeric viruses by transfection of their RNA transcripts into HeLa cells. These chimeric viruses have shown to express introduced HIV-1 subgenome concatamers efficiently during their replication in the infected HeLa cells. Expressed proteins were confirmed to retain the wild type structures at least in parts. Replication capacity of the chimeric viruses was slightly lower than that of wild type Sabin 1 likely to be due to delay in processing steps during their replication. Differing from the virulent Mahoney vectors, the rec-Sabin 1 chimeric viruses maintained the foreign gene stably during the serial passages. These chimeric viruses have also shown to be able to induce specific humoral immunity to the introduced vaccine proteins when inoculated into the poliovirus receptor-expressing transgenic (Tg-PVR) mice. Antiserum obtained from the immunized transgenic mice showed to have neutralizing capacity to HIV-1 in vitro. These results strongly suggest that the chimeric viruses expressing HIV-1 vaccine epitopes can be used as a good live mucosal vaccine candidate against AIDS.

Prevention of Virus - induced Diabetes by Single Immunization with Recombinant BCG in SJL/J Male Mice / 대한면역학회지

D variant of encephalomyocarditis (EMC-D) virus causes diabetes in susceptible mice by direct infection and cytolysis of pancreatic beta cells. cDNA covering the major outer capsid protein (VP1) of EMC-D virus was cloned into Mycobacterium bovis bacillus Calmette-Guerin (BCG). None of the SJL/J male mice, immunized with live recombinant BCG-VP1, became diabetic when challenged with highly diabetogenic EMC-D virus. But the control mice inoculated with normal BCG or rBCG transformed with vector alone developed diabetes in the same challenge. VP1-specific antibodies including neutralizing antibodies were markedly increased as time went on and reached to the maximum titer at week 10 after a single immunization. The plateau of the titer lasted longer than following 4 weeks. Guinea pigs immunized with the live rBCG-VP1 showed strong delayed type hypersensitivity (DTH) to the VP1of EMC-D virus. It means that the live rBCG-VP1 elicit efficient humoral and cell-mediated imrnune responses against EMC-D virus, resulting in prevention of virus-induced diabetes in susceptible mice.

p53-mediated HIV-1 Tat Suppression is Likely to be Assaciated with duble-stranded RNA-dependent Protein Kinase, PKR

No abstract available.

p53-mediated Inhibitory Mechanism on HIV-1 Tat is Likely to be Associated with Tat-Phosphorylation

HIV-1 tat, a strong transactivator, is essential for the HIV-1 replication and AIDS progression. The Tat function is markedly inhibited by human anti-oncogene p53. This work was initiated to identify the p53-associated inhibitory mechanism on tat-mediated transactivation. Inhibitory function of P53 was confirmed by co-transfection of tat-expressing Jurkat cells with LTR-CAT plasmid, or H3Tl cells (LTR-CAT integrated HeLa cells) with different ratio of pSV-tat/pCDNA-p53 plasmids. Results from the direct protein-protein .interaction between soluble p53 and tat, and yeast two-hybrid experiments showed that the co-suppression mechanism is unlikely to be due to the direct interaction. CAT activity was not affected by tat in Jurkat cells which were transfected with p53-promoter-CAT or p53-enhancer-CAT, suggesting that the tat-mediated p53 suppression is not directly associated with p53-promoter. Finally, we have tested protein kinase activity in p53-tranfected Jurkat cells, which might phosphorylate HIV-1 tat, resulting in inhibition of tat function. Some of our data lead us to assume that the p53-mediated tat inhibition is likely to be associated with p53-associated, signaling-mediated phosphorylation of tat, resulting in the dysfunction of tat. This study is now under investigation.

HIV - 1 p24 Expression in BCG and the Immunogenicity of Recombinant BCG in Experimental Animals / 대한면역학회지

HIV-1 p24 was cloned into multiple cloning site of pMV261, extrachromosomal expression vectors carrying BCG replication origin and BCG-specific heat-shock promoter, and then introduced into BCG and E. coli. Western blot experiments showed that the p24 efficiently expressed in recombinant BCG (rBCG), but not in E. coli. Recombinant p24 expression induced by a single heat-shock of rBCG was maintained longer than 3 weeks. Immunoblot experiments with intact rBCG did not show any distinctive positive signal, suggesting that the recombinant protein was not secreted or exposed at the surface of BCG. The guinea pigs immunized with live rBCG showed delayed type hypersensitivity (DTH) by the systemic area as well as an effective humoral immunity, suggesting that tbis rBCG is believed to elicit eKcient immune responses against p24, even though the expression is restricted only in the cytoplasm as reported previously with other antigen. These results demonstrate that BCG can be developed as a live recombinant vaccine vector against a broad spectrum of infectious disease.

Cloning of the Form I-antigen Genes from Shigella sonnei KNIH104

Shigella sonnei KNIH104S, which was selected by Korean National Institute of Health, expresses form I-antigen as a somatic antigen. In this study, we cloned the genes responsible for form I-antigen synthesis from S. sonnei KNIH104S. A Sau3AI-generated cosmid library of S. sonnei KNIH104S plasmids were transfected into E. coli LE392 and transfectants were tested for agglutination with antiserum against S. sonnei form I-antigen. A clone, JH222, showing the strongest agglutination activity was chosen for further analysis. A recombinant cosmid, pJH222, was isolated from the strain JH222 and retransfected into E. coli LE392. All of the transfectants agglutinated with antiserum against form I-antigen, indicating that pJH222 carried the genes required for S. sonnei form I-antigen synthesis. Restriction analysis of pJH222 revealed a 38 kb insert, which was confirmed by Southern hybridization analysis to be present on a large plasmid of S. sonnei KNIH104S.