Huey-Kang Sytwu

The Roles of Fas/APO-1 (CD95) and TNF in Antigen-Induced Programmed Cell Death in T Cell Receptor Transgenic Mice

The possible involvement of Fas/APO-1 (CD95) and TNF in antigen-specific AICD of thymocytes and mature T cells has been investigated. Antigenic stimulation in vivo of influenza hemagglutinin (HA)-specific TCRtg mice was used to demonstrate that the kinetics of thymocyte and peripheral CD4+ T cell deletion are similar in mice with normal (+/+) or defective Fas (lpr/lpr) background, indicating that a Fas-independent pathway(s) is responsible for the deletion of activated T cells. TCRtg-+/+ or TCRtg-lpr/lpr mice injected with murine TNF-blocking MAb (TN3) showed rapid apoptosis of thymocytes after HA stimulation, indicating that death signaling through Fas and TNF receptors is not essential for HA-induced thymocyte deletion. CDC peripheral T cells in TCRtg-lpr/lpr mice did not undergo apoptosis following injection with HA and TN3, indicating that TNF-mediated apoptosis is involved in the deletion of mature T cells after antigenic stimulation. However, apoptosis still occurred in TCRtg-+/+ mice injected with TN3, indicating that both Fas- and TNF-mediated cell death can contribute to the deletion of activated peripheral T cells.

SPAK-Knockout Mice Manifest Gitelman Syndrome and Impaired Vasoconstriction

Polymorphisms in the gene encoding sterile 20/SPS1-related proline/alanine-rich kinase (SPAK) associate with hypertension susceptibility in humans. SPAK interacts with WNK kinases to regulate the Na(+)-K(+)-2Cl(-) and Na(+)-Cl(-) co-transporters [collectively, N(K)CC]. Mutations in WNK1/4 and N(K)CC can cause changes in BP and dyskalemia in humans, but the physiologic role of SPAK in vivo is unknown. We generated and analyzed SPAK-null mice by targeting disruption of exons 9 and 10 of SPAK. Compared with SPAK(+/+) littermates, SPAK(+/-) mice exhibited hypotension without significant electrolyte abnormalities, and SPAK(-/-) mice not only exhibited hypotension but also recapitulated Gitelman syndrome with hypokalemia, hypomagnesemia, and hypocalciuria. In the kidney tissues of SPAK(-/-) mice, the expression of total and phosphorylated (p-)NCC was markedly decreased, but that of p-OSR1, total NKCC2, and p-NKCC2 was significantly increased. We observed a blunted response to thiazide but normal response to furosemide in SPAK(-/-) mice. In aortic tissues, total NKCC1 expression was increased but p-NKCC1 was decreased in SPAK-deficient mice. Both SPAK(+/-) and SPAK(-/-) mice had impaired responses to the selective α(1)-adrenergic agonist phenylephrine and the NKCC1 inhibitor bumetanide, suggesting that impaired aortic contractility may contribute to the hypotension of SPAK-null mice. In summary, SPAK-null mice have defects of NCC in the kidneys and NKCC1 in the blood vessels, leading to hypotension through renal salt wasting and vasodilation. SPAK may be a promising target for antihypertensive therapy.

Recombinant Adeno-Associated Virus Expressing Human Papillomavirus Type 16 E7 Peptide DNA Fused with Heat Shock Protein DNA as a Potential Vaccine for Cervical Cancer

ABSTRACT In this study, we explore a potential vaccine for human papillomavirus (HPV)-induced tumors, using heat shock protein as an adjuvant, a peptide vaccine for safety, and adeno-associated virus (AAV) as a gene delivery vector. The tumor vaccine was devised by constructing a chimeric gene which contained HPV type 16 E7 cytotoxic T-lymphocyte (CTL) epitope DNA (M. C. Feltkamp, H. L. Smits, M. P. Vierboom, R. P. Minnaar, B. M. de Jongh, J. W. Drijfhout, J. ter Schegget, C. J. Melief, and W. M. Kast, Eur. J. Immunol. 23:2242–2249, 1993) fused with the heat shock protein gene as a tumor vaccine delivered via AAV. Our results demonstrate that this vaccine can eliminate tumor cells in syngeneic animals and induce CD4- and CD8-dependent CTL activity in vitro. Moreover, studies with knockout mice with distinct T-cell deficiencies confirm that CTL-induced tumor protection is CD4 and CD8 dependent. Taken together, the evidence indicates that this chimeric gene delivered by AAV has potential as a cervical cancer vaccine.

Involvement of β7 integrin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the development of diabetes in nonobese diabetic mice

Nonobese diabetic (NOD) mice develop autoimmune-mediated lymphocytic inflammation of pancreatic islets (insulitis) that leads to β-cell destruction and development of diabetes. Inflamed islets show expression of lymphocyte α4β7 integrin and endothelial mucosal addressin cell adhesion molecule-1 (MAdCAM-1), adhesion molecules involved in tissue-selective migration of lymphocytes to mucosal lymphoid tissues. To elucidate the roles of the mucosal lymphocyte/endothelial adhesion system in the development of diabetes, we treated NOD mice with monoclonal antibody against β7 integrin or MAdCAM-1. Treatment of mice from age 7 to 28 days or 8 to 12 weeks with either antibody led to significant and long-standing protection against the spontaneous development of diabetes and insulitis. In contrast, neither treatment prevented the development of salivary gland inflammation (sialadenitis), indicating that the effect was tissue-selective. Monoclonal antibody treatment had no demonstrable effect on numbers or phenotypes of peripheral lymphocytes or on the immune response to pancreatic islet or exogenous antigens. These data indicate that lymphocyte and endothelial adhesion molecules involved in the migration of lymphocytes into mucosal lymphoid tissues play a role in the development of diabetes in NOD mice. Moreover, the results suggest that treatment of humans with antibodies against tissue-selective lymphocyte or endothelial adhesion molecules may selectively inhibit the development of autoimmune diseases such as diabetes.

SFRP1 and SFRP2 suppress the transformation and invasion abilities of cervical cancer cells through Wnt signal pathway.

OBJECTIVES Aberrant activation of the Wnt/beta-catenin signaling pathway is common in human cancers, including cervical cancer. The secreted frizzled-related proteins (SFRPs) function as Wnt antagonists and play important implications in carcinogenesis. Recently, we have shown that SFRP1 and SFRP2 are frequently downregulated through promoter hypermethylation. However, the function of SFRP1 and SFRP2 in cervical cancer remains unclear. METHODS To improve our understanding of the role of SFRP1 and SFRP2 in cervical cancer cells, we use overexpression or shRNA approach in cervical cancer cell lines. RESULTS Restoration of the expression of SFRP1 and SFRP2 attenuated Wnt signaling in CaSki cells, decreased abnormal accumulation of free beta-catenin in the nucleus, and suppressed cancer cell growth. In addition, different statuses of beta-catenin accumulation in the cytoplasm of CaSki or HeLa3rd cells were observed, suggesting that different Wnt pathways are executed. Furthermore, we demonstrated that SFRP1 and SFRP2 enhance the expression of the epithelial marker E-cadherin, through inhibition of the expression of SLUG, TWIST and SNAIL, three transcription factors involved in the epithelial mesenchymal transition (EMT) program. Finally, in a xenograft animal model, we showed that SFRP1 suppresses tumorigenicity of cancer cells in vivo. CONCLUSIONS Taken together, these data strongly suggest that epigenetic silencing of SFRP genes leads to oncogenic activation of the Wnt pathway and contributes to cervical cancer progression through the EMT program.

Immunomodulatory properties of human adult and fetal multipotent mesenchymal stem cells

In recent years, a large number of studies have contributed to our understanding of the immunomodulatory mechanisms used by multipotent mesenchymal stem cells (MSCs). Initially isolated from the bone marrow (BM), MSCs have been found in many tissues but the strong immunomodulatory properties are best studied in BM MSCs. The immunomodulatory effects of BM MSCs are wide, extending to T lymphocytes and dendritic cells, and are therapeutically useful for treatment of immune-related diseases including graft-versus-host disease as well as possibly autoimmune diseases. However, BM MSCs are very rare cells and require an invasive procedure for procurement. Recently, MSCs have also been found in fetal-stage embryo-proper and extra-embryonic tissues, and these human fetal MSCs (F-MSCs) have a higher proliferative profile, and are capable of multilineage differentiation as well as exert strong immunomodulatory effects. As such, these F-MSCs can be viewed as alternative sources of MSCs. We review here the current understanding of the mechanisms behind the immunomodulatory properties of BM MSCs and F-MSCs. An increase in our understanding of MSC suppressor mechanisms will offer insights for prevalent clinical use of these versatile adult stem cells in the near future.

Single nucleotide polymorphism at Fas promoter is associated with cervical carcinogenesis

The causal role of human papillomavirus (HPV) in cervical carcinogenesis is beyond reasonable questioning. The progression from HPV infection, squamous intraepithelial lesions (SIL) to squamous cell carcinomata (SCC), however, is very uncommon and inefficient. Host genetic factors that may confer the susceptibility of disease progression are largely unknown. Apoptosis is an important fail‐safe check for tumor development, in which Fas/FasL interaction contributes substantially. The purpose of our study is to test the hypothesis that an A/G polymorphism at −670 of Fas promoter with different transcriptional activity is associated with the risk for cervical neoplasia. A hospital‐based case‐control study was conducted, in which 104 patients of low grade SIL (LSIL), 131 high grade SIL (HSIL) and 176 SCC as well as age‐matched, 1:1 controls were tested for Fas polymorphism by PCR‐RFLP. HPV genotypes were determined in case groups by MY PCR‐reverse line blot. The frequency of A allele was significantly (p = 0.006) higher in SCC than in control, conferring an odd ratio of 1.5 (95% CI = 1.1–2.0). The distribution of Fas (−670) genotypes also differed significantly between HSIL, SCC and each of their control (p = 0.017 and 0.03, respectively), with the A/A genotype conferring an OR of 1.3 (95% CI = 1.1–1.6) and 1.6 (95% CI = 1.0–2.5), respectively. Remarkably, the frequency of A allele and A/A genotype increased gradually in accordance with the multi‐step carcinogenesis from LSIL, HSIL to SCC (ptest for trend = 0.0066 and 0.0007, respectively). In addition, there was no difference of Fas genotypes between HPV (+) and HPV (−) cases. Fas genotypes, however, differed in LSIL infected with different HPV types (p = 0.033). The present study demonstrated an association between Fas polymorphism and cervical carcinogenesis. We deduced a possible effect of apoptosis of immune cells in this virus‐induced cancer.

Evaluation of protective efficacy and immune mechanisms of using a non-structural protein NS1 in DNA vaccine against dengue 2 virus in mice.

To evaluate the potential of DNA vaccine against dengue (DEN) infection, we characterize the protective efficacy and immune responses of mice intramuscularly injected with plasmid encoding DEN-2 non-structural protein 1 (NS1). Intravenously challenged by lethal DEN-2, mice vaccinated with NS1-DNA exhibited a delay onset of paralysis, a marked decrease of morbidity, and a significant enhancement of survival. In addition to a moderate increase of NS1-specific antibody titer from immunized mice measured by ELISA, a strong priming effect on anti-NS1 response was also noticed in plasmid NS1-vaccinated mice by radioimmunoprecipitation (RIP) or immunoblot analysis. Interestingly, newborn mice from NS1-DNA-immunized dam showed stronger resistance to viral challenge, as compared to those from vector DNA or PBS-immunized dams, indicating the protective role of NS1-specific antibody. In contrast to humoral immune response, DNA immunization can elicit strong cellular immune responses, including NS1-specific T cell proliferation and cytolytic activity. The NS1-DNA-induced protection can be further augmented by co-injection of plasmid encoding interleukin 12 (IL-12), suggesting an effector role of Th1 immunity against DEN infection. In summary, our results suggest the potential of NS1-DNA vaccine against DEN infection, and indicate both NS1-specific humoral and cellular immune responses contribute to the protection.

Impaired Phosphorylation of Na(+)-K(+)-2cl(-) Cotransporter by Oxidative Stress-Responsive Kinase-1 Deficiency Manifests Hypotension and Bartter- Like Syndrome

Na+-K+-2Cl− cotransporters (NKCCs), including NKCC1 and renal-specific NKCC2, and the Na+-Cl− cotransporter (NCC) play pivotal roles in the regulation of blood pressure (BP) and renal NaCl reabsorption. Oxidative stress-responsive kinase-1 (OSR1) is a known upstream regulator of N(K)CCs. We generated and analyzed global and kidney tubule-specific (KSP) OSR1 KO mice to elucidate the physiological role of OSR1 in vivo, particularly on BP and kidney function. Although global OSR1−/− mice were embryonically lethal, OSR1+/− mice had low BP associated with reduced phosphorylated (p) STE20 (sterile 20)/SPS1-related proline/alanine-rich kinase (SPAK) and p-NKCC1 abundance in aortic tissue and attenuated p-NKCC2 abundance with increased total and p-NCC expression in the kidney. KSP-OSR1−/− mice had normal BP and hypercalciuria and maintained significant hypokalemia on a low-K+ diet. KSP-OSR1−/− mice exhibited impaired Na+ reabsorption in the thick ascending loop on a low-Na+ diet accompanied by remarkably decreased expression of p-NKCC2 and a blunted response to furosemide, an NKCC2 inhibitor. The expression of total SPAK and p-SPAK was significantly increased in parallel to that of total NCC and p-NCC despite unchanged total NKCC2 expression. These results suggest that, globally, OSR1 is involved in the regulation of BP and renal tubular Na+ reabsorption mainly via the activation of NKCC1 and NKCC2. In the kidneys, NKCC2 but not NCC is the main target of OSR1 and the reduced p-NKCC2 in KSP-OSR1−/− mice may lead to a Bartter-like syndrome.

Transgenic expression of decoy receptor 3 protects islets from spontaneous and chemical-induced autoimmune destruction in nonobese diabetic mice.

Decoy receptor 3 (DCR3) halts both Fas ligand– and LIGHT-induced cell deaths, which are required for pancreatic β cell damage in autoimmune diabetes. To directly investigate the therapeutic potential of DCR3 in preventing this disease, we generated transgenic nonobese diabetic mice, which overexpressed DCR3 in β cells. Transgenic DCR3 protected mice from autoimmune and cyclophosphamide-induced diabetes in a dose-dependent manner and significantly reduced the severity of insulitis. Local expression of the transgene did not alter the diabetogenic properties of systemic lymphocytes or the development of T helper 1 or T regulatory cells. The transgenic islets had a higher transplantation success rate and survived for longer than wild-type islets. We have demonstrated for the first time that the immune-evasion function of DCR3 inhibits autoimmunity and that genetic manipulation of grafts may improve the success and survival of islet transplants.