Kyeong Cheon Jung

Therapeutic effects of lysophosphatidylcholine in experimental sepsis.

Sepsis represents a major cause of death in intensive care units. Here we show that administration of lysophosphatidylcholine (LPC), an endogenous lysophospholipid, protected mice against lethality after cecal ligation and puncture (CLP) or intraperitoneal injection of Escherichia coli. In vivo treatment with LPC markedly enhanced clearance of intraperitoneal bacteria and blocked CLP-induced deactivation of neutrophils. In vitro, LPC increased bactericidal activity of neutrophils, but not macrophages, by enhancing H2O2 production in neutrophils that ingested E. coli. Incubation with an antibody to the LPC receptor, G2A, inhibited LPC-induced protection from CLP lethality and inhibited the effects of LPC in neutrophils. G2A-specific antibody also blocked the inhibitory effects of LPC on certain actions of lipopolysaccharides (LPS), including lethality and the release of tumor necrosis factor-α (TNF-α) from neutrophils. These results suggest that LPC can effectively prevent and treat sepsis and microbial infections.

Core-needle biopsy is more useful than repeat fine-needle aspiration in thyroid nodules read as nondiagnostic or atypia of undetermined significance by the Bethesda system for reporting thyroid cytopathology.

BACKGROUND Thyroid nodules with fine-needle aspiration (FNA) readings of nondiagnostic or atypia of undetermined significance (AUS), also referred to as follicular lesion of undetermined significance (FLUS) are problematic for their optimal management. The usefulness of performing a core-needle biopsy (CNB) to clarify whether these nodules are benign or malignant has not been established. The purpose of the present study was to determine whether CNB provides better diagnostic information than repeat FNA (rFNA) in thyroid nodules having nondiagnostic or AUS/FLUS readings. MATERIALS AND METHODS The Bethesda System for Reporting Thyroid Cytopathology was used for FNA readings and for CNB readings. The study included 225 thyroid nodules from 220 consecutive patients who previously had nondiagnostic (Group N-DIAG, n=64) or AUS/FLUS (Group AF, n=161) FNA readings. All patients simultaneously underwent rFNA and CNB of each nodule. The nondiagnostic and AUS/FLUS readings by rFNA and by CNB were compared. The diagnostic sensitivities of rFNA and CNB for malignancy in thyroid nodules were also assessed. Statistical analysis was performed using a McNemars test. RESULTS In N-DIAG Group, the nondiagnostic readings for the CNBs were lower than that those for rFNAs (1.6% vs. 28.1%, p<0.001). In the AF Group, the AUS/FLUS readings for the CNBs were lower than those for the rFNAs (23.6% vs. 39.8%, p<0.001). The inconclusive diagnoses (nondiagnostic or AUS/FLUS) for the CNBs were lower than those for the rFNAs in Group N-DIAG (12.5% vs. 45.3%, p<0.001) and Group AF (26.7% vs. 49.1%, p<0.001). The sensitivity of CNB for thyroid malignancy was higher than that of rFNA in Group N-DIAG (100% vs. 71.4%, p=0.125) and Group AF (78.5% vs. 55.4%, p<0.001). CONCLUSION After patients have had one FNA of a thyroid nodule yielding inconclusive diagnostic results (nondiagnostic or AUS/FLUS), CNB is more useful than rFNA for reducing the frequency of inconclusive diagnostic results. CNB will improve the diagnostic performance for malignancy more than rFNA in thyroid nodules that on the first FNA had nondiagnostic or AUS/FLUS readings.

Long-term control of diabetes in immunosuppressed nonhuman primates (NHP) by the transplantation of adult porcine islets.

Pig islets are an alternative source for islet transplantation to treat type 1 diabetes (T1D), but reproducible curative potential in the pig‐to‐nonhuman primate (NHP) model has not been demonstrated. Here, we report that pig islet grafts survived and maintained normoglycemia for >6 months in four of five consecutive immunosuppressed NHPs. Pig islets were isolated from designated pathogen‐free (DPF) miniature pigs and infused intraportally into streptozotocin‐induced diabetic rhesus monkeys under pretreatment with cobra venom factor (CVF), anti‐thymocyte globulin (ATG) induction and maintenance with anti‐CD154 monoclonal antibody and low‐dose sirolimus. Ex vivo expanded autologous regulatory T cells were adoptively transferred in three recipients. Blood glucose levels were promptly normalized in all five monkeys and normoglycemia (90–110 mg/dL) was maintained for >6 months in four cases, the longest currently up to 603 days. Intravenous glucose tolerance tests during the follow‐up period showed excellent glucose disposal capacity and porcine C‐peptide responses. Adoptive transfer of autologous regulatory T cells was likely to be associated with more stable and durable normoglycemia. Importantly, the recipients showed no serious adverse effects. Taken together, our results confirm the clinical feasibility of pig islet transplantation to treat T1D patients without the need for excessive immunosuppressive therapy.

Evaluation of E1B gene-attenuated replicating adenoviruses for cancer gene therapy

Gene-attenuated replication-competent adenoviruses are emerging as a promising new modality for the treatment of cancer. For the aim of improving adenoviral vectors for cancer gene therapy, we have constructed genetically attenuated adenoviral vectors with different combinations of E1B genes and investigated the possibility of enhanced oncolytic and replication effects of these engineered replication-competent adenoviruses. We show here that the cytolytic potency of each gene-attenuated replicating adenovirus differed significantly depending on the presence or deletion of E1B 55 kDa and E1B 19 kDa function. More specifically, among the constructed vectors (Ad-ΔE1B19, Ad-ΔE1B55, Ad-ΔE1B19/55, and Ad-wt), E1B 19 kDa–inactivated adenovirus (Ad-ΔE1B19) was the most potent against all tumor cells tested, inducing the largest-sized plaques and marked CPE. Further, cells infected with either Ad-ΔE1B19 or E1B19/55 kDa–deleted adenovirus (Ad-ΔE1B19/55) showed complete cell lysis with disintegrated cellular structure, whereas cells infected with Ad-wt maintained intact cellular and nuclear membrane with properly structured organelles. TUNEL and DNA fragmentation assay also revealed that the Ad-ΔE1B19 or Ad-ΔE1B19/55 adenovirus-infected cells showed more profound induction of apoptosis in comparison to wild-type adenovirus-infected cells. The presence of E1B 55 kDa gene was required for efficient viral replication and deletion of E1B 19 kDa function in replicating adenovirus-induced apoptosis, leading to increased cytopathic effects. Moreover, Ad-ΔE1B19 adenovirus showed a better antitumor effect than other E1B-attenuated adenoviruses. Taken together, the replicating adenoviruses deleted in E1B 19 kDa function may serve as an improved vector for anticancer gene therapy in combination with apoptosis-inducing modalities such as chemotherapeutic agents and radiation therapy.

Generation of PLZF+ CD4+ T cells via MHC class II–dependent thymocyte–thymocyte interaction is a physiological process in humans

Human thymocytes, unlike mouse thymocytes, express major histocompatibility complex (MHC) class II molecules on their surface, especially during the fetal and perinatal stages. Based on this observation, we previously identified a novel developmental pathway for the generation of CD4+ T cells via interactions between MHC class II–expressing thymocytes (thymocyte–thymocyte [T–T] interactions) with a transgenic mouse system. However, the developmental dissection of this T–T interaction in humans has not been possible because of the lack of known cellular molecules specific for T–T CD4+ T cells. We show that promyelocytic leukemia zinc finger protein (PLZF) is a useful marker for the identification of T–T CD4+ T cells. With this analysis, we determined that a substantial number of fetal thymocytes and splenocytes express PLZF and acquire innate characteristics during their development in humans. Although these characteristics are quite similar to invariant NKT (iNKT) cells, they clearly differ from iNKT cells in that they have a diverse T cell receptor repertoire and are restricted by MHC class II molecules. These findings define a novel human CD4+ T cell subset that develops via an MHC class II–dependent T–T interaction.

MHC class II-restricted interaction between thymocytes plays an essential role in the production of innate CD8+ T cells.

We have recently shown that MHC class II-dependent thymocyte–thymocyte (T–T) interaction successfully generates CD4+ T cells (T–T CD4+ T cells), and that T–T CD4+ T cells expressing promyelocytic leukemia zinc finger protein (PLZF) show an innate property both in mice and humans. In this article, we report that the thymic T–T interaction is essential for the conversion of CD8+ T cells into innate phenotype in the physiological condition. CD8+ T cells developed in the presence of PLZF+ CD4+ T cells showed marked upregulation of eomesodermin (Eomes), activation/memory phenotype, and rapid production of IFN-γ on ex vivo stimulation. Their development was highly dependent on the PLZF expression in T–T CD4+ T cells and the IL-4 secreted by PLZF+ T–T CD4+ T cells. The same events may take place in humans, as a substantial number of Eomes expressing innate CD8+ T cells were found in human fetal thymi and spleens. It suggests that PLZF+ T–T CD4+ T cells in combination with Eomes+ CD8+ T cells might actively participate in the innate immune response against various pathogens, particularly in human perinatal period.

NKT cells play critical roles in the induction of oral tolerance by inducing regulatory T cells producing IL-10 and transforming growth factor β, and by clonally deleting antigen-specific T cells

Oral tolerance is the systemic unresponsiveness induced by orally administered proteins. To explore the roles of natural killer T (NKT) cells in oral tolerance, we induced oral tolerance to ovalbumin (OVA) in NKT cell‐deficient mice. In CD1d–/– mice, the induction of tolerance to orally administered high‐ or low‐dose OVA was impaired. Dendritic cells (DCs) in the Peyers patches (PPs) of CD1d–/– mice fed OVA showed high expression of major histocompatibility complex (MHC) class II and B7 molecules, whereas DCs of control mice fed OVA expressed low levels of these molecules. The adoptive transfer of NKT cells restored oral tolerance and induction of tolerogenic DCs in the PPs and spleens of CD1d–/– mice. Moreover, interleukin (IL)‐10 and transforming growth factor (TGF)‐β1 production in vitro were reduced in cells from the spleen and PPs of CD1d–/– mice compared with those of control mice fed OVA. The numbers of OVA‐specific CD4+ KJ1‐26+ T cells were significantly reduced in the PPs and spleens of DO11·10 mice fed OVA. In contrast, OVA‐specific CD4+ KJ1‐26+ T cells were not deleted in the PPs or spleens of DO11·10 CD1d–/– mice. In conclusion, NKT cells were found to play an indispensable role in oral tolerance by inducing regulatory T cells, and clonally deleting antigen‐specific CD4+ T cells.

TTF-1 mRNA-positive circulating tumor cells in the peripheral blood predict poor prognosis in surgically resected non-small cell lung cancer patients

Circulating tumor cells (CTCs) have been identified in peripheral blood of cancer patients, and reproducible detection of CTCs has demonstrated the potential as useful diagnostic and prognostic tools in several cancers. Present study aimed to determine the clinical relevance of CTCs in surgically resected non-small cell lung cancer (NSCLC) patients. CTC status in presurgery and postsurgery peripheral blood samples from 79 surgically resected NSCLC patients was investigated using thyroid transcription factor-1 (TTF-1) and cytokeratin19 (CK19) mRNA markers by nested real-time RT (reverse transcription)-PCR assay. Detection of TTF-1((+))CTCs was found to be specific to NSCLC patients. TTF-1((+))CTCs were detected in 36.1% (22/61) of patients before surgery and in 37.5% (18/48) after surgery. For CK19 mRNA-expressing CTCs (CK19((+))CTCs), the detection rate was 42.6% (26/61) before surgery, and 25.0% (12/48) after surgery. Cases with postsurgery TTF-1((+)) and/or CK19((+))TCs was more associated with disease progression (P=0.004) and shorter disease progression-free survival (P=0.006) as compared to those without postsurgery CTCs. As an individual marker, postsurgery TTF-1((+))CTCs-positive status was more associated with disease progression (P=0.004) and shorter disease progression-free survival (P=0.004) as compared to postsurgery TTF-1((+))CTCs-negative status. Particularly, patients with postsurgery TTF-1((+))CTCs, but not presurgery (Pre((-))Post((+)) cases) showed marked shorter disease progression-free survival than other patients (P<0.001). On the other hand, a CK19((+))CTC status individually did not show significant clinical relevance, and presurgery CK19((+))CTC status did not either. Present study suggests that TTF-1 mRNA-expressing CTCs might be a useful surrogate predictor of disease progression before clinical manifestations are apparent, and that monitoring of TTF-1((+))CTCs status after surgery may be useful for identifying high-risk patients among surgically resected NSCLC cases.

CD99 Regulates the Transport of MHC Class I Molecules from the Golgi Complex to the Cell Surface

The down-regulation of surface expression of MHC class I molecules has recently been reported in the CD99-deficient lymphoblastoid B cell line displaying the characteristics of Hodgkin’s and Reed-Sternberg phenotype. Here, we demonstrate that the reduction of MHC class I molecules on the cell surface is primarily due to a defect in the transport from the Golgi complex to the plasma membrane. Loss of CD99 did not affect the steady-state expression levels of mRNA and protein of MHC class I molecules. In addition, the assembly of MHC class I molecules and the transport from the endoplasmic reticulum to the cis-Golgi occurred normally in the CD99-deficient cells, and no difference was detected between the CD99-deficient and the control cells in the pattern and degree of endocytosis. Instead, the CD99-deficient cells displayed the delayed transport of newly synthesized MHC class I molecules to the plasma membrane, thus causing accumulation of the molecules within the cells. The accumulated MHC class I molecules in the CD99-deficient cells were colocalized with α-mannosidase II and γ-adaptin in the Golgi compartment. These results suggest that CD99 may be associated with the post-Golgi trafficking machinery by regulating the transport to the plasma membrane rather than the endocytosis of surface MHC class I molecules, providing a novel mechanism of MHC class I down-regulation for immune escape.

In situ induction of dendritic cell–based T cell tolerance in humanized mice and nonhuman primates

Administration of an ICAM-1–specific antibody arrests dendritic cells in a semi-immature state and facilitates antigen-specific T cell tolerance to islet allografts in humanized mice and Rhesus monkeys.