Seung-Yong Hwang

POTENTIATION OF ANTIGEN-SPECIFIC, TH1 IMMUNE RESPONSES BY MULTIPLE DNA VACCINATION WITH AN OVALBUMIN/INTERFERON-GAMMA HYBRID CONSTRUCT

The preferential differentiation of T helper (Th) cells to Th1 or Th2 subsets is important with respect to susceptibility or resistance to particular infections, or to autoimmune diseases and allergic diseases. To more effectively drive immune responses toward antigen‐specific Th1 responses, we constructed a mammalian expression vector (pOVA/IFN‐γ) carrying a hybrid gene in which the ovalbumin (OVA) (a model antigen) cDNA was covalently linked to murine interferon‐γ (IFN‐γ) cDNA. Intramuscular injection of BALB/c mice with the pOVA/IFN‐γ DNA increased both the production of OVA‐specific IFN‐γ by CD4+ T cells and the ratio of anti‐OVA immunoglobulin G (IgG)2a to IgG1 isotypes, while the injection with the pOVA alone, or with the mixture of the pOVA and pIFN‐γ, caused no or little increase. Furthermore, the OVA‐specific, Th1 immune responses were dramatically augmented by multiple injections with the pOVA/IFN‐γ DNA. These studies indicate that the direct linkage of an OVA gene to an IFN‐γ gene in the expression plasmid is required for efficiently confining the Th1 effects of IFN‐γ to the OVA‐specific cells, and the linkage effect of the OVA/IFN‐γ DNA can be potentiated by multiple vaccination.

Protection of cardiomyocytes from ischemic/hypoxic cell death via Drbp1 and pMe2GlyDH in cardio-specific ARC transgenic mice

The ischemic death of cardiomyocytes is associated in heart disease and heart failure. However, the molecular mechanism underlying ischemic cell death is not well defined. To examine the function of apoptosis repressor with a caspase recruitment domain (ARC) in the ischemic/hypoxic damage of cardiomyocytes, we generated cardio-specific ARC transgenic mice using a mouse α-myosin heavy chain promoter. Compared with the control, the hearts of ARC transgenic mice showed a 3-fold overexpression of ARC. Langendoff preparation showed that the hearts isolated from ARC transgenic mice exhibited improved recovery of contractile performance during reperfusion. The cardiomyocytes cultured from neonatal ARC transgenic mice were significantly resistant to hypoxic cell death. Furthermore, the ARC C-terminal calcium-binding domain was as potent to protect cardiomyocytes from hypoxic cell death as ARC. Genome-wide RNA expression profiling uncovered a list of genes whose expression was changed (>2-fold) in ARC transgenic mice. Among them, expressional regulation of developmentally regulated RNA-binding protein 1 (Drbp1) or the dimethylglycine dehydrogenase precursor (pMe2GlyDH) affected hypoxic death of cardiomyocytes. These results suggest that ARC may protect cardiomyocytes from hypoxic cell death by regulating its downstream, Drbp1 and pMe2GlyDH, shedding new insights into the protection of heart from hypoxic damages.

PDMS–glass serpentine microchannel chip for time domain PCR with bubble suppression in sample injection

This paper reports on the development of a low-cost microreactor (10 µl) biochip for DNA polymerase chain reaction (PCR). The microbiochip (20 mm × 28 mm) is a hybrid type that is composed of a polydimethylsiloxane (PDMS) layer with a serpentine microchannel (360 µm × 100 µm) chamber and glass substrate integrated with a microheater and thermal microsensor. Because of the hydrophobic chip surface, bubbles are usually created during sample loading in the PMDS-based microchip. These bubbles disrupt the stable biochemical reaction. An improved microreactor chamber was designed using microfluidic simulation. The reactor has a rounded-corner serpentine channel architecture, which enables stable injection into the hydrophobic surface using only a micropipette. The reactor temperature needed for the PCR reaction is controlled within ±0.5 °C by the LabVIEW software proportional-integrative-derivative (PID) controller. PCR analyses of the sex-determining Y chromosome (SRY) gene and mouse GAPDH gene were successfully performed in less than 54 min by the fabricated microreactor chip.

Chloromethyl ketones inhibit interleukin-12 production in mouse macrophages stimulated with lipopolysaccharide.

Interleukin-12 (IL-12) plays a pivotal role in the development of T-helper type 1 (Th1) immune response, which may be involved in the pathogenesis of chronic inflammatory autoimmune disorders. In this study, we investigated the effects of N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), serine protease inhibitors, on the production of IL-12 from macrophages stimulated with lipopolysaccharide (LPS). TPCK and TLCK potently inhibited this LPS-induced IL-12 production in a dose-dependent manner. The effect of TPCK and TLCK on the IL-12 p40 promoter activation was analyzed by transfecting monocytic RAW264.7 cells with p40 promoter-reporter constructs. The repressive effect maps to a region in the p40 promoter containing a binding site for NFkappaB (p40-kappaB). A linker scan mutant of the p40-kappaB site abrogates the inhibitory effect on the p40 promoter, confirming the functional relevance of the NFkappaB site. Our results show that TPCK and TLCK inhibit NFkappaB-mediated IL-12 production in macrophages. reserved.

Interleukin-12-secreting fibroblasts are more efficient than free recombinant interleukin-12 in inducing the persistent resistance to Mycobacterium avium complex infection.

To determine whether the paracrine secretion of interleukin‐12 (IL‐12) can efficiently stimulate the resistance to Mycobacterium avium complex (MAC) infection, 3T3 fibroblasts were stably transfected to secrete IL‐12 (480 U/106 cells/48 hr) and their effect on MAC infection was investigated in genetically susceptible BALB/c mice, compared with that of free recombinant IL‐12 (rIL‐12). Injection with IL‐12‐secreting fibroblasts (3T3‐IL‐12) during intranasal infection with MAC resulted in a significant decrease in the bacterial load of the lung during the entire 10‐week observation period, while rIL‐12 reduced the bacterial load initially, at 2 weeks, but not by 10 weeks postinfection. Lung CD4+ T cells in mice injected with the 3T3‐IL‐12 cells showed a persistent T helper type 1 (Th1) response throughout the 10‐week period. Furthermore, immunization with the 3T3‐IL‐12 cells induced and maintained significantly higher levels of cytotoxic activity and nitric oxide production by lung cells than did rIL‐12 immunization. This work suggests that IL‐12‐secreting fibroblasts may serve as a vehicle for paracrine secretion of IL‐12 for immunotherapy of MAC infection.

Efficient induction of an antigen-specific, T helper type 1 immune response by interleukin-12-secreting fibroblasts.

To determine whether the paracrine secretion of interleukin (IL)-12 can efficiently convert immune responses characterized by high levels of synthesis of IL-4 and immunoglobulin E (IgE) into T helper 1 (Th1)-dominated responses, 3T3 fibroblasts were stably transfected to secrete IL-12 (480 units/10(6) cells/48 hr). Their effects on the T helper cell-mediated immune response were investigated in ovalbumin (OVA)-primed mice. Free mouse recombinant IL-12 was included as a control group. IL-12-secreting fibroblasts (3T3/IL-12) were more effective than free recombinant IL-12 at increasing OVA-specific interferon-gamma (IFN-gamma) production and decreasing OVA-specific IL-4 production in CD4+ T cells. In addition, injection with 3T3/IL-12 cells significantly increased anti-OVA immunoglobulin G2a (IgG2a) levels and decreased anti-OVA IgE levels in OVA-primed mice. This work suggests that IL-12-secreting fibroblasts can efficiently induce an antigen-specific Th1 response and may be beneficial in the treatment of diseases caused by undesirable T helper 2 (Th2)-dominated responses, including allergic diseases.

Simultaneous subtyping and pathotyping of the novel reassortant influenza A (H5N8) virus from clinical samples using a diagnostic microarray

Highly pathogenic avian influenza (HPAI) viruses cause economic losses in the poultry industry and pose a severe threat to human health. Rapid and accurate diagnostic methods are important because they can help prevent further spread of the virus and reduce the time required for eradication of the virus. We developed a low-density microarray for the rapid detection and identification of avian influenza virus subtypes H5, H7, and H9 and their pathotypes in a previous study. In the present study, we report the development of updated probe sets and evaluation of the diagnostic microarray using H5N8 clade 2.3.4.4 HPAI viruses including clinical samples, without the need for egg propagation. Cy3-labeled DNA targets were obtained by reverse transcription polymerase chain reaction using Cy3-labeled universal primers, and labeled amplicons were hybridized to the microarray. All positive samples from RT-PCR showed H5-specific and highly pathogenic pattern in the microarray, without purification of PCR products. Furthermore, it allowed for specific detection of the subtype and pathotype from low DNA concentration samples that did not allow direct sequence analysis. Therefore, this diagnostic microarray has enormous potential for the rapid subtyping and pathotyping from clinical samples without the need for culture.