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Background. Memory T cells specific for donor antigens are currently recognized as a significant barrier for maintaining a successful transplant. Furthermore, it has been shown that commonly used immunosuppressive drugs do not alleviate this memory response. Here, we report that rapamycin allows significant proliferation of memory T cells and bortezomib can abrogate the proliferation of rapamycin-resistant memory T cells when preserving the survival of regulatory T cells. Methods. Peripheral blood mononuclear cells freshly isolated from non-human primates were stimulated with anti-CD3/ CD28 antibodies, and inhibitory and apoptotic effects of rapamycin and bortezomib on memory T-cell proliferation were investigated. The CD95 marker in CD3T cells was used for the separate enrichment of memory T cells and naive T cells. Results. Rapamycin at the level even higher than therapeutic concentration could not suppress the proliferation of a significant proportion of memory T cells. However, the combined administration of bortezomib and rapamycin abrogatedtheproliferationofrapamycin-resistantmemoryTcells.Furthermore,bortezomibpreservedthesurvivalof preexisting CD4FoxP3 regulatory T cells, while inducing apoptosis of CD4FoxP3 conventional T cells. The combined administration of low doses of rapamycin and bortezomib also exerted an additive effect on suppressing T-cell proliferation. Cytokine analysis demonstrated that bortezomib could not only suppress rapamycin-permissive interleukin (IL)-6 production, but also production of interferon (IFN)-, IL-4, and IL-10. Conclusions. This article provides in vitro data from which immunosuppressive regimens for the effective control of memory T cells in non-human preclinical experiments and in clinical trials are selected.

Glycosphingolipid (GSL) is a major component of the plasma membrane in eukaryotic cells that is involved directly in a variety of immunological events via cell-to-cell or cell-to-protein interactions. In this study, qualitative and quantitative analyses of GSL-derived glycans on endothelial cells and islets from a miniature pig were performed and their glycosylation patterns were compared. A total of 60 and 47 sialylated and neutral GSL-derived glycans from the endothelial cells and islets, respectively, were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and collision-induced fragmentation using positive-ion electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS). In accordance with previous immunohistochemistry studies, the alpha-Gal-terminated GSL was not detected but NeuGc-terminated GSLs were newly detected from miniature pig islets. In addition, the neutral GSL-derived glycans were relatively quantified by derivatization with carboxymethyl trimethylammonium hydrazide (so called Girard's T reagent) and MALDI-TOF MS. The structural information of the GSL-derived glycans from pig endothelial cells and islets suggests that special attention should be paid to all types of glycoconjugates expressed on pig tissues or cells for successful clinical xenotransplantation.

In addition to CD4+CD25+Foxp3+ regulatory T (T(reg)) cells which protect against autoimmune tissue injury, IL-17-producing CD4+ T (T(h)17) cells have been recently described and shown to play a crucial role in autoimmune injury. It appears that there is a reciprocal developmental pathway between T(h)17 and T(reg) cells. Although IL-17 is known to be associated with allograft rejection, the cellular source of IL-17 and the nature of T(h)17 in the context of allograft rejection remain unknown. In the current study, the dynamics of T(reg) and IL-17-producing cells after syngeneic and allogeneic transplantation were examined using a wild-type murine cardiac transplantation model. Ly6G+ cells were found to produce IL-17 during the early postoperative period and CD8+ as well as CD4+ T cells were also found to produce IL-17 during alloimmune response. Graft-infiltrating Ly6G+, CD4+, and even CD8+ cells were found to express IL-17 highly compared to those in spleen. Although the frequencies of T(h)17 and T(reg) were found to gradually increase in both syngeneic and allogeneic recipients, T(h)17/T(reg) ratios were significantly higher in recipients with allograft rejection than in syngeneic recipients. In conclusion, IL-17 is produced by neutrophils during the early postoperative period and subsequently by T(h)17 and CD8+ T cells during allograft rejection. T(h)17/T(reg) imbalance is associated with the development of allograft rejection. This study would provide basic information on T(h)17 biology for future investigation in the field of transplantation.

Human neural stem cells (hNSCs) can control inflammation in the central nervous system, although the underlying mechanisms are not understood fully. We investigated the immunomodulatory effect of hNSCs on human T cells and the underlying mechanisms. Culture supernatant from an immortalized hNSC cell line, HB1.F3, which has a therapeutic effect on acute stroke and intracerebral hemorrhage, suppressed the proliferation of allogeneically or mitogenically stimulated human peripheral T cells, including the CD3(+)CD103(+) subpopulation. CFSE labeling and flow cytometry showed that the suppression of proliferation was caused by cell cycle arrest and induction of apoptosis. The lack of significant change in caspase-8 levels and the significant reduction in Bcl-2 expression in the affected T cells suggest that the intrinsic pathway plays a major role in soluble-factor-mediated T-cell apoptosis. The addition of culture supernatant from hNSCs to activated T cells reduced the expression of the activation markers CD69 and CD25 at 24 hr after activation, but at 48 hr only CD69 was down-regulated. A cytometry bead assay showed that the secretion of interleukin (IL)-2 decreased significantly, whereas that of IL-4, IL-10, tumor necrosis factor-alpha, and interferon-gamma increased. These results show that hNSCs can negatively affect human peripheral T cells by suppressing their activation and proliferation through soluble mediators, suggesting that hNSCs have a bystander immunomodulatory effect on T cells.

To evaluate the differences in graft survival and histopathological characteristics between full-thickness and lamellar orthotopic corneal xenotransplantation in a pig-to-rabbit model, we orthotopically transplanted a full-thickness or the anterior half of a pig's cornea onto the OD of 16 rabbits. As a result, the median survival were 16.83 and 29.07 days for the full-thickness and lamellar xenografts, respectively (P = 0.0005). Histologically, the full-thickness corneal xenografts had massive infiltration by eosinophils, whereas the lamellar xenografts showed predominantly mononuclear infiltrates (P < 0.05). Given these preliminary findings, lamellar corneal xenografts in rabbits survived longer than the full-thickness xenografts and each type of graft demonstrated different rejection mechanisms.

Small ubiquitin-like modifiers (SUMOs) are structurally related to ubiquitin and are ligated to lysine residues within sumoylation target proteins. Currently, a growing body of evidence shows that the SUMO family has evolved as an important modifier of many proteins in a variety of cellular pathways. In this study, we have cloned cDNA encoding for three different pig SUMO isoforms. The full-length cDNA encoding for pig Sumo1, Sumo2 and Sumo4 consists of 306, 288 and 288 nucleotide base pairs of the open reading frame, respectively, and the putative amino acid sequences of pig Sumo1, Sumo2 and Sumo4 are composed of 101, 95 and 95 peptides, respectively. The structures of pig SUMOs are evolutionally well conserved, and their expression has been detected in a broad range of tissues. We also determined that all pig SUMOs are localized within the nucleus. However, the different tissue expression observed in individual pig SUMOs may show the divergent or specialized role of each of the pig SUMO isoforms. Future studies will focus on the identification of targets for sumoylation by different pig SUMO isoforms and the analysis of the functional consequences of sumoylation during the course of infectious diseases in pigs.

The ability to induce and maintain antigen-specific immunologic tolerance is the ultimate goal in allo-transplantation. Here, we report that the transplantation of unmanipulated murine pancreatic islets across the HY disparity induced transplantation tolerance that prevented HY-mismatched skin grafts being rejected.Three hundred islet equivalent of freshly isolated islets from male C57BL/6 donor mice was transplanted underneath the kidney capsule of syngeneic female recipients rendered diabetic by streptozotocin. Nephrectomy was carried out to remove the islet graft and retransplantation was performed using the contralateral kidney. For skin transplantation, donor tail skin was transplanted onto the lateral thorax.Islets from male C57BL/6 donors transplanted to syngeneic female recipients cured diabetes and the mice survived indefinitely. The acceptance of second grafts and rejection of third party islet grafts indicated antigen-specific transplantation tolerance. However, flow cytometry and ELISPOT analysis demonstrated that the HY-specific T cells were not deleted or anergized. A 2-fold increase of CD4+Foxp3+ regulatory T cells (Tregs) was observed in spleen and lymph nodes. Notably, CD25- Tregs increased threefold over levels in naïve mice. Adoptive transfer of CD4+ T cells to neonatal mice could transfer tolerance. At the graft site in long-term tolerant mice, CD4+ T cells, 40% of which were CD4+Foxp3+ Tregs (43% CD25-, 57% CD25+) infiltrated the peri-islet spaces.Unmanipulated pancreatic islets can induce immunologic tolerance associated with peripherally induced CD4+Foxp3+ Tregs, a significant proportion of which notably are CD25-.

As alkylating agents, cyclophosphamides (CTX) are used to treat various cancers and, ironically, to boost immune responses. In the present study, we attempted to elucidate the mechanism responsible for the immunomodulatory effect of high-dose CTX in an established tumor model. A single injection of high-dose CTX increased the survival rate of immunocompetent, but not immunodeficient, mice. Notably, 10 days after CTX injection, the number of CD44(high) memory T cells significantly increased, without a selective decrease in the actual number and percentage of CD4+CD25+Foxp3+ regulatory T cells (Tregs). However, the proportion of Tregs among CD4+ T cells decreased due to expansion of memory and other CD4+ T cell subtypes. This outcome was accompanied by an increase in IL-15 mRNA and up-regulation of IL-15 receptors in the CD44+CD8+ T cell compartment. We postulate that the CTX-induced change in T cell balance may increase anti-tumor immunity.

Genotype-phenotype relationships between genetic polymorphisms of DNA repair genes and DNA repair capacity were evaluated in a case-control study of breast cancer. Selected DNA repair genes included were those involved in double-strand break repair (ATM, XRCC2, XRCC4, XRCC6, LIG4, RAD51, RAD52), base excision repair (LIG1), nucleotide excision repair (ERCC1), and mismatch repair (hMLH1). The subjects consisted of histologically confirmed breast cancer cases (n=132) and controls (n=75) with no present or previous history of cancer. Seventeen single nucleotide polymorphisms of 10 genes (ATM -5144AT, IVS21+1049TC, IVS33-55TC, IVS34+60GA, and 3393TG, XRCC2 31479G/A, XRCC4 921G/T, XRCC6 1796G/T, LIG4 1977T/C, RAD51 135G/C, 172G/T, RAD52 2259C/T, LIG1 583A/C, ERCC1 8092A/C, 354C/T, hMLH1 5' region -93G/A, 655A/G) were determined by TaqMan assay (ATM) or MALDI-TOF (all other genes). DNA repair capacity was measured by a host cell reactivation assay of repair of ultraviolet damage. The DNA repair capacity (%) did not differ between cases (median 37.2, interquartile range: 23.6-59.6) and controls (median 32.7, interquartile range: 26.7-53.2). However, DNA repair capacity significantly differed by the genotypes of ATM and RAD51 genes among cancer-free controls. Our findings suggest that DNA repair capacity might be influenced by genetic polymorphisms of DNA damage response genes and DNA repair genes.

The majority of newly acquired nonhuman primates encounter serious problems adapting themselves to new environments or facilities. In particular, loss of appetite and abnormal behavior can occur in response to environmental stresses. These adaptation abnormalities can ultimately have an affect on the animal's growth and well-being. In this study, we evaluated the affects of a puzzle feeder on the food intake and abnormal behavior of newly acquired rhesus monkeys for a short period. The puzzle feeder was applied to 47- to 58-month-old animals that had never previously encountered one. We found that there was no difference in the change of food intake between the bucket condition and the puzzle feeder condition. In contrast, the time spent for consumption of food was three times longer in the puzzle feeder condition than in the bucket condition. Two monkeys initially exhibited stereotypic behavior. One showed a decreasing, and the other an increasing pattern of abnormal behavior after introduction of the puzzle feeder. In conclusion, this result suggests that over a short period, the puzzle feeder can only affect the time for food consumption since it failed to affect the food intake and did not consistently influence stereotypic behaviors in newly acquired rhesus monkeys.

Porcine to rat corneal xenotransplantation resulted in severe inflammation and rejection of the corneal stroma, whereas an allograft showed mainly endothelial cell-associated rejection. We, therefore, investigated and compared the gene expression between porcine keratocytes and corneal endothelial cells. RNA was isolated from primary cultured porcine or human keratocytes and porcine corneal endothelial cells. Gene expression was comparatively analyzed after normalization with microarray method using Platinum pig 13 K oligo chip (GenoCheck Co., Ltd., Ansan, Korea). Real-time polymerase chain reaction (PCR) was performed for C1R, CCL2, CXCL6, and HLA-A in porcine keratocytes and corneal endothelial cells. As a result, upregulated expression more than 2 folds was observed in 1,162 genes of porcine keratocytes versus porcine endothelial cells. Among the immune-regulatory genes, SEMA3C, CCL2, CXCL6, F3, HLA-A, CD97, IFI30, C1R, and G1P3 were highly expressed in porcine keratocytes, compared to porcine corneal endothelial cells or human keratocytes. When measured by real-time PCR, the expression of C1R, CCL2, and HLA-A was higher in porcine keratocytes compared to that in porcine corneal endothelial cells. In conclusion, the increased expression of C1R, CCL2, and HLA-A genes in porcine keratocytes might be responsible for the stromal rejection observed in a porcine to rat corneal xenotransplantation.

Pig blood group antigens may be present on grafted tissue as 16 isoforms, including the major one, A substrance. Seoul National University (SNU) miniature pigs and domestic pigs were used in this study. Polymerase chain reaction (PCR) was performed for the erythrocyte antigen A (EAA) gene, and reverse transcriptase-PCR for pig A transferase fucosyl transferase (FUT) 1 and FUT-2. The hemagglutination test was performed with murine monoclonal anti-A and anti-B antibodies (mAb), and immunohistochemistry with anti-human blood antigen mAb. SNU miniature and domestic pigs showed blood groups A and O. Blood group A SNU miniature pigs expressed either EAA(AA) or EAA(AO) and either S(SS) or S(SO); blood group O miniature pigs expressed EAA(OO) and S(SS) or S(SO), and there was no A(weak). Additionally, blood group A could be divided into blood group A(clotting) and blood group A(not clotting) in hemagglutination tests. Pig A substance was expressed in the lung and kidney in blood group A pigs, but we could not detect pig A substance expression in the lung, kidney, and heart of blood group O pigs or the heart of blood group A pigs. In conclusion, we suggest that blood typing of SNU miniature pigs can be easily performed using immunohistochemistry, PCR, and/or RT-PCR. Molecular-based AO typing described in this study may be useful to select SNU miniature pigs bearing a specific blood group.

Background Human bocavirus (hBoV) is regarded as one of the possible etiologic agents in lower respiratory tract infection and bronchial asthma exacerbation in children despite frequent co-detection with other respiratory viruses. The immunologic response in children with hBoV infection is still not clear. Objectives To investigate the profiles of T helper-1 (Th1)/T helper-2 (Th2) cytokines in children with hBoV-associated bronchiolitis. Study design This study utilized of 59 nasopharyngeal aspirates from 59 infants aged 24 months or younger, including 29 from children with hBoV-related bronchiolitis and 30 with respiratory syncytial virus (RSV)-related bronchiolitis. Eighteen infants hospitalized for elective surgeries were included as controls. Nasopharyngeal aspirates were tested simultaneously for cytokines interleukin (IL)-2, IL-4, IL-5, IL-10, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha using the Cytometric Bead Array. Results Significantly higher concentrations of IFN-gamma (p = 0.0001), IL-2 (0.006), and IL-4 (p = 0.0002) were observed in hBoV positive specimens than in controls. The concentration of IL-10 (p = 0.04) and TNF-alpha (p = 0.006) in the RSV-positive group was significantly higher than in the hBoV-positive group, while there was no difference in other cytokines concentration between the two groups. Conclusions These results showed that both of Th1 and Th2 cytokines were increased in children with hBoV-related bronchiolitis compared to normal controls, but Th2-polarized responses were not observed.

Co-infection of herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV) is not uncommon in immunocompromised hosts. Importantly, organ transplant recipients concurrently infected with HSV-1 and HCMV have a worse clinical outcome than recipients infected with a single virus. However, factors regulating the pathologic response in HSV-1, HCMV co-infected tissues are unclear. We investigated the potential biologic role of HCMV gene product immediate early 1 (IE1) protein in HSV-1-induced syncytial formation in U373MG cells. We utilized a co-infection model by infecting HSV-1 to U373MG cells constitutively expressing HCMV IE1 protein, UMG1-2. Syncytial formation was assessed by enumerating nuclei number per syncytium and number of syncytia. HSV-1-induced syncytial formation was enhanced after 24 hr in UMG1-2 cells compared with U373MG controls. The amplified phenotype in UMG1-2 cells was effectively suppressed by roscovitine in addition to inhibitors of viral replication. This is the first study to provide histological evidence of the contribution of HCMV IE1 protein to enhanced cytopathogenic responses in active HSV-1 infection.

Neural stem cell (NSC) transplantation has been investigated as a means to reconstitute the damaged brain after stroke. In this study, however, we investigated the effect on acute cerebral and peripheral inflammation after intracerebral haemorrhage (ICH). NSCs (H1 clone) from fetal human brain were injected intravenously (NSCs-iv, 5 million cells) or intracerebrally (NSCs-ic, 1 million cells) at 2 or 24 h after collagenase-induced ICH in a rat model. Only NSCs-iv-2 h resulted in fewer initial neurologic deteriorations and reduced brain oedema formation, inflammatory infiltrations (OX-42, myeloperoxidase) and apoptosis (activated caspase-3, TUNEL) compared to the vehicle-injected control animals. Rat neurosphere-iv-2 h, but not human fibroblast-iv-2 h, also reduced the brain oedema and the initial neurologic deficits. Human NSCs-iv-2 h also attenuated both cerebral and splenic activations of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and nuclear factor-kappa B (NF-kappaB). However, we observed only a few stem cells in brain sections of the NSCs-iv-2 h group; in the main, they were detected in marginal zone of spleens. To investigate whether NSCs interact with spleen to reduce cerebral inflammation, we performed a splenectomy prior to ICH induction, which eliminated the effect of NSCs-iv-2 h transplantation on brain water content and inflammatory infiltrations. NSCs also inhibited in vitro macrophage activations after lipopolysaccharide stimulation in a cell-to-cell contact dependent manner. In summary, early intravenous NSC injection displayed anti-inflammatory functionality that promoted neuroprotection, mainly by interrupting splenic inflammatory responses after ICH.

SummaryGlycan recognition leading to cell-cell interactions, signaling, and immune responses is mediated by various glycan-binding proteins (GBPs) showing highly diverse ligand specificities. We describe here a rapid glycan immobilization technique via 4-hydrazinobenzoic acid (HBA)-functionalized beads and its application to high-throughput screening of miniature pig kidney N-glycan-binding proteins by using a mass-spectrometric approach. Without any derivatization steps, the purified pig kidney N-glycans were directly immobilized on to HBA-functionalized beads and subsequently used to identify GBPs from human serum. This screening method showed remarkable performance for identifying potential GBPs closely involved in pig-to-human xenograft rejection mediated by human serum, including antibodies, cytokines, complement components, siglec, and CD antigens. Thus, these results demonstrate that the GBP screening method was firmly established by one-step immobilization of the N-glycans on to microsphere and highly sensitive mass-spectrometric analysis.

Dendritic cells (DCs) are proficient antigen-presenting cells that regulate the activation and suppression of the innate and adaptive immunity. There has been a growing interest in the regulation of DC function by tailoring metabolic requirements in DC-based immunotherapy of cancer. Here we report that targeting acetyl-CoA carboxylase, a key regulator of de novo fatty acid synthesis and lipid oxidation, facilitates the down-modulation of DC functions during the maturation process. DCs treated with ACC blockade, CP-640186, dramatically down-regulated the expression of co-stimulatory molecules including CD80, CD86 and CD40 together with MHC II(major histocompatibility complex II) during LPS-mediated maturation by a dose-dependent manner. Coincidently, the secretion of pro-inflammatory cytokines (IFN-r, IL-6, IL-12) was decreased as well. Blocking fatty acid synthesis by targeting ACC down-regulated the expression of CCR7, a homing receptor of DCs, but up-regulated the expression of CCR5, a chemokine receptor largely expressed in immature DCs. Our data elucidate the role of fatty-acid synthesis in DC maturation and have implications for the development of metabolism based-approaches in DC-mediated immunotherapies.

Pancreatic islet transplantation is one of the most attractive treatment modality for select type 1 diabetes patients suffering from frequent hypoglycemia unawareness. However, because of the shortage of human donor organs, xenotransplantation is becoming more highlighted. Under this circumstance, we have recently achieved reproducible and consistent survivals of adult porcine islet for more than 180 days in the liver of five diabetic monkeys under immunosuppression regimen base on anti-CD154. Interestingly, among five monkeys, one has exhibited graft failure after having experienced severe intestinal infections. Because the porcine islets were exposed to direct blood flow from the intestine, intestinal infection would exert detrimental effect on porcine islets. To test this hypothesis, we performed RNA-seq with peripheral blood from graft maintaining monkey or rejecting monkey after intestinal infections. By using novel bioinformatics tool called Time-series RNA-seq analysis package, we demonstrated highly relevant activated immunologic pathway in graft rejecting monkey, but not in well maintaining monkey. Consistent with this notion, we further confirmed that porcine islets were indeed rejected by CD8+ T cells by immunohistochemistry. Taken together, we for the first time showed that a new bioinformatics analysis using peripheral blood RNA-seq data can pinpoint intestinal infection as a cause of graft rejection in intraportal islet xenotransplantation.

CD81, also known as TAPA-1 (target of antiproliferative antibody 1), is a member of the tetraspanin family of proteins and a component of the B cell co-receptor complex. Several studies have shown that CD81 plays significant roles in a variety of immune responses, including activation of B cells and T cells. In this study, we cloned pig Cd81 cDNA using RT-PCR coupled with rapid amplification of cDNA ends (RACE)-PCR and determined the complete cDNA sequence of pig Cd81. Pig Cd81 cDNA contains an open reading frame (711 bp) encoding 236 amino acids. The identity of pig CD81 with those of human, cattle, rat, and mouse are 90.30%, 92.26%, 86.22%, and 86.22%, respectively. Alignment of the CD81 amino acid sequence with those of mammalian species showed that the large extracellular loop (LEL) is the most divergent, whereas other domains are largely conserved. Pig Cd81 mRNA was detected by RT-PCR in a broad range of tissues, including lymphoid tissues as well as nonlymphoid tissues, indicated variety of cellular functions of CD81 in most pig tissues. Flow cytometry analyses demonstrated that human CD81 antibody recognizes a pig CD81 on the cell surface. Further, immunohistochemistry analysis using human CD81 antibody on pig spleen was revealed that CD81 expression is widely diffused in spleen tissue. Future study will be focused on defining the functional role of CD81 during the course of pig infectious diseases.

Upon antigenic stimulation, CD1d-restricted NKT cells quickly secrete large amounts of cytokines. This prompt response demonstrates that CD1d-restricted NKT cells may potentially prove to be useful therapeutic agents for the treatment of many diseases. Despite the clinical importance of CD1d-restricted NKT cells, the regulating mechanisms of this unique T cell population remain to be defined. We found murine LAG-3 is inducible on CD1d-restricted NKT cells as the result of a variety of stimulants such as concanavalin A (con A) and anti-CD3. Also, antigen-specific CD1d stimulation can elicit LAG-3 in CD1d-restricted NKT cells. Moreover, ectopic LAG-3 expression on CD1d-restricted NKT cells results in cell cycle arrest in the S phase. These results show that LAG-3 signaling on activated CD1d-restricted NKT cells may down-modulate NKT cell proliferation.

T helper 1 (Th1)/T helper 2 (Th2)-biased cytokine regulation may be another reason that neonates are much more susceptible to infectious disease than are adults.We attempted to determine the ability of neonatal mice to direct the Th1 phenotype against Listeria monocytogenes (LM), because LM, an intracellular Gram-positive bacterium, induces profound cellular immunity by Th1 cells in vivo.In order to determine whether neonatal mice evidence strong Th1 activity during LM infection, neonatal mice were compared with adult mice with regard to susceptibility to LM, cytotoxic T lymphocyte activity, and cytokine profiles. Neonatal gene profiles relevant to Th1 and Th2 differentiation during LM infection were also compared between neonatal and adult mice, via real-time PCR and RT-PCR.Neonatal mice were found to be far more susceptible to LM infection than adult mice, due to a lack in the induction of cytotoxic T cell activity, coupled with poor IFN-gamma secretion. Further, LM-infected neonatal mice evidenced much lower levels of expression of Th1-type immune components, including IL-12, IFN-gamma, Delta-4 and T-bet, as compared to those features in adult mice. These results may be due to the comparably lower expressions of mannose-bind lectins and some of toll-like receptors (TLRs) such as TLR-5, -6 and -9, necessary mediators to develop Th1 immune responses.Neonatal mice may not mount an adequate Th1 type immune response due to a significantly lower expression of Th1-type immune components as compared to adult mice, even when forced into a Th1-prone environment.

Background: The dynamic pattern of the cellular infiltration and mRNA expression of chemokines in rat-to-mouse skin xenografts were examined to gain an understanding of the possible role of chemokines in the stronger cellular immune responses to xenografts compared with the allo-response. Methods: The mean survival time of the xenografts was approximately 2 days less than that of allografts (10.7 ± 0.9 days vs. 8.9 ± 0.7 days, P

Free 8-hydroxydeoxyguanosine (oh(8)dG), a nucleoside of 8-hydroxyguanine (oh(8)Gua), present in cytosol is not incorporated into DNA. However, nothing is known about its biological function when it presents in cytosol as a free form. We demonstrate here for the first time that oh(8)dG inhibits lipopolysaccharide (LPS)-induced nitric oxide (NO) production and cyclooxygenase-2 (COX-2) activity, and both gene transcriptions in microglia. Furthermore, oh(8)dG reduced mRNA levels of pro-inflammatory cytokine, such as IL-1beta, IL-6, and TNF-alpha, in activated BV2 cells. We also found that oh(8)dG suppressed reactive oxygen species (ROS) production through reduction of NADPH oxidase activity and blocked Rac1/STATs signal cascade. Finally, oh(8)dG suppressed recruitment of STATs and p300 to the iNOS and COX-2 promoters, and inhibited H3 histone acetylation. Taken together, these results provide new aspects of oh(8)dG as an anti-inflammatory agent.

This study was performed to elucidate the possible mechanism of the disturbance of hemopoiesis by HCMV infection. Saos-2 cells constitutively express mRNA of GM-CSF, and its expression was profoundly decreased by HCMV infection, which required full replication of the virus and was mediated by soluble factors released from the HCMV-infected Saos-2 cells. TGF-beta1 production was statistically and significantly increased from one day after HCMV infection. Expression and production of GM-CSF in Saos-2 cells were restored when a culture supernatant of HCMV-infected Saos-2 cells was reacted with neutralizing anti-TGF-beta antibody. Conclusively, HCMV inhibits GM-CSF expression in Saos-2 cells partly by the increased production of TGF-beta1.

Human cytomegalovirus (HCMV) has many strategies to survive the attack of the host. HCMV infection of host cells induces cellular activation and disturbance of the cell cycle. It is possible that HCMV modulates the behavior of certain cancer cells that are susceptible to HCMV infection. This study was performed to identify the possible mechanism of resistance to apoptotic stimuli in some cancer cell lines by HCMV infection. HCMV-infected cancer cells showed resistance to apoptosis induced by the topoisomerase II inhibitor etoposide. UMG1-2, which constitutively expresses HCMV immediate-early protein-1 (IE1), had resistance to apoptosis induced by etoposide as compared with the parental cell line U373MG. Measurement of caspases activity with fluorogenic substrates in etoposide-treated U373MG and UMG1-2 cells and the direct activation of caspase-3 with peptides containing arginine-glycine-aspartate in U373MG and UMG1-2 cells revealed that the inhibition level of apoptosis by HCMV IE1 would be upstream of caspase-3 in the caspase cascade pathway. Cellular expression of Cdk2 was increased in UMG1- 2 after etoposide treatment while the expression of E2F-1 in UMG1-2 was decreased as compared with that in U373MG. The Cdk2 inhibitor, roscovitine, decreased the resistance to apoptosis on etoposide-treated UMG1-2. These results suggest that aberrant HCMV infection confers resistance to anticancer drugs on some cancer cells and protects cells from apoptosis, possibly due to the deregulation of cyclin-dependent kinase by HCMV immediate-early protein.

Human cytomegalovirus UL18, a MHC class I homologue, is known to serve as a natural killer cell (NK) decoy and to ligate NK inhibitory receptors to prevent lysis of an infected target cell. To explore whether the cell surface expression of UL18 represents a potential immune suppressive approach to evade NK-mediated cytotoxicity in the prevention of xenograft rejection, we examined the effect of the UL18 expression in vitro upon human NK-mediated cytotoxicity against swine endothelial cells (SECs). UL18 expression on SECs by a retroviral vector (PLNCX2) significantly suppressed NK-mediated SEC lysis by approximately 25–100%. The protective effect of UL18 could be mediated through ILT-2 inhibitory receptor on NKs. Additionally, the interaction between UL18 and NKs resulted in the significant reduction of IFN-γ production. This study demonstrates that UL18 can serve as an effective tool for the evasion of NK-mediated cytotoxicity and for the inhibition of IFN-γ production during xenograft rejection.

CTLA4Ig, a fusion protein of CTLA-4 and Fc of immunoglobulin (Ig) heavy chain, inhibits the essential costimulatory signal for full T cell activation via blocking the interaction between CD28 and B7 molecules and renders T cell nonresponsiveness. CTLA4Ig has been used to control deleterious T cell activation in many experimental systems. We hypothesized that by conjugating CTLA4Ig to liposomes the efficacy of CTLA4Ig could be enhanced through multivalent ligand effect, superior targetability, and modification of the fate of ligated costimulatory molecules.Consistent with this hypothesis, liposome-conjugated CTLA4Ig bound to B7 and blocked their binding sites more efficiently than free CTLA4Ig, lowering the half maximal dose for B7 blocking by an order of the magnitude. These results were similar both in B7-1 expressing p815 cells and in activated macrophages. Moreover, CTLA4Ig-liposomes underwent rapid internalization upon cell surface binding through B7 molecules. In allogenic mixed lymphocyte reaction assays, the CTLA4Ig-liposomes were tested to show effective inhibition of T cell proliferation. In vivo, however, when CTLA4Ig-liposomes were injected into mice, a significant fraction was localized to the reticuloendothelial system (RES), presumably because of its binding to Fc receptors expressed on tissue macrophages. The Fc receptor-mediated uptake could be alleviated by coinjection of anti-FcR monoclonal antibody. In the mouse engrafted with pancreatic islets of Langerhans underneath the capsule of one kidney, despite the increased localization in RES, enhanced accumulation of CTLA4Ig-conjugated liposome was observed in the engrafted kidney compared to the contralateral kidney.We show that the conjugation of CTLA4Ig to liposome could increase the efficiency of the targeting by increasing the binding avidity at cellular level and by increasing the concentration at the target site in in vivo system. The biodistribution and circulation time data suggested that the CTLA4Ig-liposomes could be improved upon minimizing the FcR-mediated uptake by Fc receptor-bearing cells. Thus, the strategy of conjugating CTLA4Ig to liposomes could be exploited for immune intervention in transplantation and autoimmune diseases for the efficient blocking of costimulation.

Behçet's disease (BD) is a connective tissue disorder characterized by recurrent orogenital ulcer, uveitis, and skin lesions. Recurrent aphthous ulcer is associated with human cytomegalovirus (HCMV). To investigate the possible role of HCMV in BD, we measured the titers of IgG, IgM, and IgA anti-HCMV antibodies in 73 Korean patients with BD, 50 with scleroderma, 70 with systemic lupus erythematosus, and 50 from healthy controls by indirect immunofluorescent staining. The titer of IgG anti-HCMV antibody was significantly lower in patients with BD than in controls (geometric mean 3115.4 vs 9687.6, P = 0.0001 by Wilcoxon's rank sum test), as was the titer of IgA anti-HCMV antibody (geometric mean 1.9 vs 15.7, P = 0.0001, Wilcoxon's rank sum test). In conclusion, we found significantly lower antibody responses to HCMV in patients with BD.

Purpose The purpose of this pilot study was to evaluate the association between adenosine triphosphate-based chemotherapy response assays (ATP-CRAs) and subsets of tumor infiltrating lymphocytes (TILs) in gastric cancer. Materials and Methods In total, 15 gastric cancer tissue samples were obtained from gastrectomies performed between February 2007 and January 2011. Chemotherapy response assays were performed on tumor cells from these samples using 11 chemotherapeutic agents, including etoposide, doxorubicin, epirubicin, mitomycin, 5-fluorouracil (5-FU), oxaliplatin, irinotecan, docetaxel, paclitaxel, methotrexate, and cisplatin. TILs in the tissue samples were evaluated using antibodies specific for CD3, CD4, CD8, Foxp3, and Granzyme B. Results The highest cancer cell death rates were induced by etoposide (44.8%), 5-FU (43.1%), and mitomycin (39.9%). Samples from 10 patients who were treated with 5-FU were divided into 5-FU-sensitive and -insensitive groups according to median cell death rate. No difference was observed in survival between the two groups (P=0.216). Only two patients were treated with a chemotherapeutic agent determined by an ATP-CRA and there was no significant difference in overall survival compared with that of patients treated with their physician's choice of chemotherapeutic agent (P=0.105). However, a high number of CD3 TILs was a favorable prognostic factor (P=0.008). Pearson's correlation analyses showed no association between cancer cell death rates in response to chemotherapeutic agents and subsets of TILs. Conclusions Cancer cell death rates in response to specific chemotherapeutic agents were not significantly associated with the distribution of TIL subsets.

Waste incinerating workers are exposed to various pyrolysis products including polycyclic aromatic hydrocarbons (PAHs). We examined their PAH exposure by assessing urinary 1-hydroxypyrene glucuronide (1-OHPG), as a measure of internal dose, and aromatic DNA adducts in peripheral white blood cells (WBCs), as a measure of biological effect dose. The potential effect of genetic polymorphisms of three enzymes involved in PAH metabolisms (i.e., CYP1A1, GSTM1, and GSTT1) on these exposure markers was also investigated.Twenty-nine employees including workers incinerating industrial wastes and 21 non-exposed on-site controls were recruited from a company handling industrial wastes in South Korea. Sixteen ambient PAHs were determined by GC/MSD (NIOSH method) from personal breathing zone samples of nine subjects working near incinerators. Urinary 1-OHPG was assayed by synchronous fluorescence spectroscopy (SFS) after immunoaffinity purification using monoclonal antibody 8E11. Aromatic DNA adducts in peripheral WBC were measured by the nuclease P1-enhanced post-labelling assay. Genotypes were assessed by PCR-based methods. Information on smoking habits and use of personal protective equipment were collected by self-administered questionnaire. Urinary 1-OHPG levels were significantly higher in workers handling industrial wastes than in those with presumed lower exposure to PAHs (P=0.006, by Kruskal-Wallis test). A statistically significant dose-response increase in 1-OHPG levels was seen with the number of cigarettes consumed per day (r=0.686, P

Purpose: To investigate the effect of systemically administered anti-CD154 antibody(Ab) on the survival of full-thickness porcine corneal grafts. Methods: Seven rhesus macaques were assigned to control (n=3) or immunosuppression (n=4) group and underwent full-thickness corneal transplantation. Systemic steroid was applied to control group while anti-CD154 Ab were administered to immunosuppression group. Rejection sign, central corneal thickness (CCT), and recipients’ immunologic profile including memory T cell, anti-α-Gal and donor-specific Abs, mixed lymphocyte-like reaction and aqueous complement concentration were evaluated. Cellular infiltration into the xenografts was evaluated by immunohistochemical staining. Results: Anti-CD154 Ab-based immunosuppression significantly prolonged the survival of full-thickness xenocorneal grafts. In control group, CCTs, memory T cells, anti-α-Gal IgG and donor pig-specific IgG Abs continuously increased in correspond with the progression of graft rejection in contrast to immunosuppression group. Reactivities to donor-specific stimulators and concentrations of the aqueous complement were also significantly higher in control group than in immunosuppression group. Rejected grafts showed extensive cellular infiltration. Conclusions: Anti-CD154 antibody-based immunosuppression is effective for prolonging the survival of full-thickness xenocorneal grafts and controlling immunologic responses in pig-to-rhesus corneal transplantation model.

Xenoreactive antibody responses were investigated in non-human primate (NHP) recipients after porcine islet transplantation (pITx). The levels of anti-αGal and anti-nonGal IgG Abs in serial sera of the recipients were measured by ELISA and flow cytometry using GalT-KO porcine endothelial cells (PECs), respectively. We detected an elicited anti-nonGal IgG response in some sera of the recipients. Through a series of work including EP of protein extracts from GalT-KO PECs, Western blot and MALDI-TOF/TOF mass spectrometry, the putative antigen was identified as bovine albumin. The sera positive of anti-nonGal Ab revealed strong IgG binding to both bovine and porcine albumin, but no binding to human albumin by ELISA. When anti-human, bovine and porcine albumin Abs were measured in pre- and post-pITx sera of 29 NHP recipient, the frequencies of the development of anti-porcine albumin IgG Abs were significantly different according to the types of immunosuppression (IS) regimen: 5/5 (100%) receiving conventional IS regimen, 2/2 (100%) receiving anti-CD154 and sirolimus and 2/21 (9.5%) receiving CD40-CD40L blockade, sirolimus and either anti-ICAM-1 or ATG (P

Background: the association between cancer phenotype and host immune response has rarely been evaluated in gastric cancer. We hypothesized that microsatellite instability (MSI) is associated with pattern of tumor infiltrating lymphocytes (TILs). Methods: we evaluated the status of MSI and subset of TILs in gastric cancer patients who received curative resection. The number of MSI-High and non-MSI-High were matched 1:3. The immunohistochemical staining with anti-CD3, CD4, CD8, Foxp3, and granzyme B were used to quantify the subset of TILs. Results: of the 98 carcinomas studied, 27 were MSI-High and 71 were non-MSI-High. Clinicopathologic profiles were comparable for both groups. The absolute number of CD3+ T-cells was not significantly different. MSI-High tumors carried significantly higher numbers of CD8+ T-cells than non-MSI-High tumors, suggesting favorable prognosis. Paradoxically, MSI-High tumors also contained higher numbers Foxp3+ T-cells and higher ratio of Foxp3+/CD4+ T-cells suggesting unfavorable prognosis. In subgroup analysis using T classification, distribution of TILs according to MSI type changed in CD8 and CD4 subsets. Conclusions: Microsatellite instable gastric cancers harbor distinctive distribution of subset of tumor infiltrating lymphocytes according to tumor progression.

Human cytomegalovirus (HCMV)-specific monoclonal antibody, SCMVM34, recognizes the early antigen encoded by UL44 of HCMV. This antigen is confined to the nucleus of HCMV-infected cells. This study was performed to characterize the DNA-binding activity of the protein encoded by UL44 of HCMV. The nuclear and cytoskeletal fraction of HCMV-infected cells was subjected to 0.4 m NaCl extraction, DEAE-Sephacel ion exchange chromatography, DNA-cellulose chromatography and SDS-PAGE analysis with monitoring of the reactive protein using SCMVM34 monoclonal antibody. The molecular weights of the resultant proteins were found to be 34, 40 and 52 kDa. The internal peptide fragments were isolated by tryptic digestion and reverse-phase HPLC. The internal amino acid sequence analysis of the peptides from the HPLC profile revealed that the antigen recognized by SCMVM34 monoclonal antibody was ppUL44. The reactive antigen began to be eluted from 250 mm NaCl (Tris-HCl pH 7.4) in DNA cellulose. The 34 kDa protein seems to bind to DEAE more tightly than the 52 kDa protein. The surface charge of 34 kDa might be more basic. Conclusively, the antigen recognized by SCMVM34 was the protein encoded by HCMV UL44, which was localized in the nuclei after HCMV infection, and was the DNA-binding protein with the characteristic that the surface charge of the molecule was more basic, as the molecular weights of the protein were decreased.

The compound purified from the fruit of Melia azedarach exerted an antiviral effect on herpes simplex virus-1 (HSV-1) in Vero cells. It was identified as 28-deacetylsendanin (28-DAS). The 50% inhibitory concentration (IC 50 ) of 28-DAS was 1.46 μg/ml without cytotoxicity at 400 μg/ml on Vero cells. Electron microscopy showed that low electron-dense cores of newly synthesized nucleocapsids remained in swollen nuclei and no extracellular virus particles were observed at 15 h p.i. Consistent with this result, it was confirmed by a plaque assay that few infectious progeny viruses were released from the 28-DAS-treated virus-infected cells at 24 h p.i. Intracellular viruses in 28-DAS-treated virus-infected cells were 23% of untreated and infected cells. The synthesis of thymidine kinase (TK) was reduced by 28-DAS at early stage. In conclusion, 28-DAS inhibited the replication of HSV-1, reduced the synthesis of HSV-1 TK, and led to the formation of defective nucleocapsids.

A stable clonal cell line of human neural stem cells (NSCs), HB1.F3, has been shown therapeutic effect on acute stroke or intracerebral haemorrhage by the mechanisms of cell replacement and several bystander effects. To investigate the bystander immunomodulatory functions of HB1.F3 cells, we examined immunosuppressive effects of HB1.F3 on T cell effector functions in vitro. We found that heat-labile soluble factors from hNSCs culture supernatant are attributed to the inhibition of peripheral blood lymphocytes (PBLs) proliferation induced by mixed lymphocyte reaction (MLR) or phorbol myristate acetate (PMA)/ionomycin. In contrast to inhibitory effect of HB1.F3, they promoted cytokine secretion from PBLs including IFN-r, TNF-a, IL-10, IL-6 during mixed lymphocyte reaction by soluble factor dependant-manner. HB1.F3 cell line expressed TGF-beta and Trail mRNA, not FasL. we confirmed that the apoptosis induction of PBLs by HB1.F3 is one of the mechanisms of T cell proliferation inhibition during MLRs. In summary, HB1.F3 cells could suppress the T cell proliferation by induction of apoptosis and promote both inflammatory and immune suppressive cytokines. This study suggested that therapeutically effective hNSCs cell line on several stroke models might be due to the bystander immunomodulatory functions by the secretion of immunosuppressive soluble factors.

All xenografts from pigs impose infection risk by porcine endogenous retrovirus (PERV). The purpose of this study was to investigate the env constructs with the comparison of the ratio of the competent form to the defective one of env in subtypes, PERV-A, PERV-B and PERV-C in different pig breeds. The results of PCR amplification of env represented that all env subtypes had more than two defective forms which cannot bind to host cells due to the absence of binding regions of env in miniature pigs, SNU and PWG, and farm pig breeds, Duroc, Yorkshire and Landrace. In addition, comparing the full sequences with the defective ones in three subtypes demonstrated that the present percentages of env sequences in defective PERV-A, PERV-B and PERV-C were approximately 50%, 38~45% and 4~11%, respectively, in SNU and PWG pigs whereas PERV-A and PERV-B occupied around 40 to 60% but PERV-C was not detected in farm pigs. Quantitative real-time PCR assays with primers and probes targeted to proline-rich region (PRR) of each env subtype were done to measure the copy numbers of each env subtype. When the reference was set with copy number of PERV-A, the ratio of those of PERV-B and PERV-C to the reference were 1.5 to 6.0 folds high in SNU and PWG pigs while 1.0 or less in farm pigs. These contradictory results of PERV-C constructs and copy numbers in SNU pigs suggests that many truncated or short defective sequences of PERV-C might be present in them.

We found an error in our published article: Application of the Multiplex Cytokine Analysis to Monitor Xenogeneic Immune Responses to the Porcine Islet Graft in Non-Human Primate Yong-Hee Kim,1,2,3,4 Jung-Sik Kim,1,2,3 Il-Hee Yoon,1,2,3 Jun-Seop Shin,1,2,3 Jong-Min Kim,1,2,3 Sang-Joon Kim5 and Chung-Gyu Park1,2,3,4 1Department of Microbiology and Immunology, 2Translational Xenotransplantation Research Center, 3Cancer Research Institute, 4Institute of Endemic Diseases, Seoul National University College of Medicine, Seoul, Korea, 5Organ Transplantation Center, Myong-Ji Hospital, Goyang, Korea J Korean Med Sci. 2013 Dec;28(12):1729-33 Corrected grant number is HI13C0954.

Regulatory T cells (Treg) offer potential for improving long-term outcomes in cell and organ transplantation. The non-human primate (NHP) model has been a valuable resource to bridge Treg therapy to the clinic. Here we address the highly effective Treg expansion protocols and phenotypic and functional characteristics of rhesus monkey. Rhesus monkey CD4+CD25+FoxP3+Treg cells exists 2-5% in CD4+T cells in blood. Around 1-2% of CD4+CD25high population in CD4+T cells were enriched by FACSAria III and cultured, simultaneously stimulated with antiCD3/antiCD28 antibody, in the presence of hIL-2, TGF-beta and irradiated autologous CD4-T cells for 14 days. The CD4+CD25+FoxP3+Treg cells expanded from 400 to 1200 times during two round stimulations. Expansion was far retarded during third round stimulation. Expansion rates were greatly affected by the initial ratio of Treg cell to autologous CD4-T cells. Phenotypically expanded Treg are CD25+CD95+CD27+CD47+FoxP+Treg cells. CCR7, LN-homing receptor, was expressed by 70%. In contrast, inflammatory cytokines including IL-17 and IFN-r were minimally expressed by 0.4% and 1.8%, respectively. Expanded Tregs were able to suppress the monkey CD4+CD25-T cell proliferation at even 1: 32 ratio of T regs to T effector cells. These finding suggests that ex-vivo expanded Treg cells may be promising modalities to overcome cell-mediated graft rejection in preclinical transplantation model.

CTLA-4Ig is regarded as an inhibitory agent of the T cell proliferation via blocking the costimulatory signal which is essential for full T cell activation. To improve applicability, we developed the CTLA-4Ig-CTKC in which the c-terminal lysine had been replaced by cysteine through single amino acid change. The single amino acid mutation of c-terminus of CTLA-4Ig was performed by PCR and was checked by in vitro transcription and translation. DNA construct of mutant form was transfected to Chinese hamster ovary (CHO) cells by electroporation. The purified proteins were confirmed by Western blot and B7-1 binding assay for their binding ability. The suppressive capacity of CTLA-4Ig-CTKC was evaluated by the mixed lymphocyte reaction (MLR) and in the allogeneic pancreatic islet transplantation model. CTLA-4Ig-CTKC maintained binding ability to B7-1 molecule and effectively inhibits T cell proliferation in MLR. In the murine allogeneic pancreatic islet transplantation, short-term treatment of CTLA-4Ig-CTKC prolonged the graft survival over 100 days. CTLA-4Ig-CTKC effectively inhibits immune response both in MLR and in allogeneic islet transplantation model, indicating that single amino acid mutation does not affect the inhibitory function of CTLA-4Ig. CTLA-4Ig-CTKC can be used in vehicle-mediated drug delivery system such as liposome conjugation.

Constructing a bone marrow chimera prior to graft transplantation can induce donor-specific immune tolerance. Mixed chimerism containing hematopoietic cells of both recipient- and donor-origin has advantages attributed from low dose of total body irradiation. In this study, we explored the mechanism of mixed chimerism supplemented with depletion of Natural Killer cells. Mixed chimerism with C57BL/6 bone marrow cells was induced in recipient BALB/c mice which were given 450 cGy of γ-ray irradiation (n = 16). As revealed by reduced proliferation and cytokine production in mixed leukocyte reaction and ELISpot assay (24.6 vs 265.5), the allo-immune response to bone marrow donor was reduced. Furthermore, the induction of transferable immunological tolerance was confirmed by adoptive transfer and subsequent acceptance of C57BL/6 skin graft (n = 4). CD4(+)FoxP3(+) regulatory T cells were increased in the recipient compartment of the mixed chimera (19.2% → 33.8%). This suggests that regulatory T cells may be therapeutically used for the induction of graft-specific tolerance by mixed chimerism.