Application of loop-mediated isothermal amplification with propidium monoazide treatment to detect live Salmonella in chicken carcasses.

Raw chicken products are major causes of human foodborne salmonellosis worldwide. In particular, there is a significant risk of human exposure to Salmonella originating from the chicken slaughtering process. Controlling the contamination of chicken carcasses by Salmonella has been a considerable challenge in chicken-slaughtering facilities and involves routine microbiological monitoring using reliable detection methods. Simple and rapid detection methods, particularly those capable of determining cell viability, will significantly facilitate routine monitoring of Salmonella Here, we report an invA-based loop-mediated isothermal amplification method coupled with a simple propidium monoazide treatment (PMA-LAMP) for simple and rapid detection and quantification of viable Salmonella in rinse water of chicken carcasses. In this study, PMA-LAMP consistently gave negative results for isopropanol-killed Salmonella with concentrations up to 8.0 × 106 CFU/reaction. The detection limit of PMA-LAMP was 8.0 × 101 CFU/reaction with viable Salmonella in both pure culture and rinse water of chicken carcasses, and 10-fold lower than a conventional polymerase chain reaction coupled with PMA (PMA-PCR) targeting invA There was a high correlation (R2 = 0.99 to 0.976) between LAMP time threshold (TT) values and viable Salmonella with a quantification range of 1.0 × 103 to 1.0 × 108 CFU/mL in pure culture and rinse water of chicken carcasses. The PMA-LAMP assay took less than 2 h to detect Salmonella contaminated in test samples. Therefore, this simple and rapid method will be a very useful tool to detect live Salmonella contamination of chicken carcasses without pre-enrichment at the slaughterhouse where sanitizing treatments are commonly used.

Tannerella forsythia GroEL induces inflammatory bone resorption and synergizes with interleukin-17.

Tannerella forsythia is a major periodontal pathogen, and T. forsythia GroEL is a molecular chaperone homologous to human heat-shock protein 60. Interleukin-17 (IL-17) has been implicated in the pathogenesis of periodontitis and several systemic diseases. This study investigated the potential of T. forsythia GroEL to induce inflammatory bone resorption and examined the cooperative effect of IL-17 and T. forsythia GroEL on inflammatory responses. Human gingival fibroblasts (HGFs) and periodontal ligament (PDL) fibroblasts were stimulated with T. forsythia GroEL and/or IL-17. Gene expression of IL-6, IL-8, and cyclooxygenase-2 (COX-2) and concentrations of IL-6, IL-8, and prostaglandin E2 (PGE2 ) were measured by real-time reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assays, respectively. After stimulation of MG63 cells with T. forsythia GroEL and/or IL-17, gene expression of osteoprotegerin (OPG) was examined. After subcutaneous injection of T. forsythia GroEL and/or IL-17 above the calvaria of BALB/c mice, calvarial bone resorption was assessed by micro-computed tomography and histological examination. Tannerella forsythia GroEL induced IL-6 and IL-8 production in HGFs and PDL cells, and IL-17 further promoted IL-6 and IL-8 production. Both T. forsythia GroEL and IL-17 synergistically increased PGE2 production and inhibited OPG gene expression. Calvarial bone resorption was induced by T. forsythia GroEL injection, and simultaneous injection of T. forsythia GroEL and IL-17 further increased bone resorption. These results suggest that T. forsythia GroEL is a novel virulence factor that can contribute to inflammatory bone resorption caused by T. forsythia and synergizes with IL-17 to exacerbate inflammation and bone resorption.

Increased efficacy of inactivated vaccine candidates prepared with Salmonella enterica serovar Typhimurium strains of predominant genotypes in ducks.

Salmonella enterica serovar Typhimurium has been a major causative agent of food-borne human disease, mainly due to consumption of contaminated food animal products. In particular, ducks serve as a reservoir of serovar Typhimurium, and are one of the common sources of human infection. To prevent infection of ducks, and therefore minimize human infection, it is critical to control the persistent epidemic strains in ducks. Here, we analyzed the genetic diversity and virulence of serovar Typhimurium isolates from ducks in Korea to identify the predominant strains that might be used as efficient vaccine candidates for ducks. Among the isolates, 2 representative isolates (ST26 and ST76) of predominant genotypes were selected as vaccine strains on the basis of genotypic analysis by pulsed-field gel electrophoresis and DNA microarrays. Two-week-old ducks were then injected intramuscularly with inactivated vaccine candidates prepared using ST26 or ST76 (10(8) cfu/0.5 mL/duck or 10(9) cfu/0.5 mL/duck), and oral challenge with a highly virulent serovar Typhimurium strain (10(9) cfu/0.5 mL/duck) was carried out 2 wk later. Shedding of the challenge strain was significantly decreased in group 2 after vaccination. The antibody levels by enzyme-linked immunosorbent assay in all vaccinated groups were enhanced significantly (P < 0.05) compared to the unvaccinated control group. Overall, vaccination with ST26 or ST76 reduced bacterial shedding and colonization in internal organs, and induced elevated antibody response. In particular, serovar Typhimurium ST26 (10(8) cfu/0.5 mL/duck) was the most effective vaccine candidate, which can provide efficient protection against serovar Typhimurium in ducks with higher effectiveness compared to a commercial vaccine currently used worldwide.

Gingipain-dependent augmentation by Porphyromonas gingivalis of phagocytosis of Tannerella forsythia.

In the pathogenesis of periodontitis, Porphyromonas gingivalis plays a role as a keystone pathogen that manipulates host immune responses leading to dysbiotic oral microbial communities. Arg-gingipains (RgpA and RgpB) and Lys-gingipain (Kgp) are responsible for the majority of bacterial proteolytic activity and play essential roles in bacterial virulence. Therefore, gingipains are often considered as therapeutic targets. This study investigated the role of gingipains in the modulation by P. gingivalis of phagocytosis of Tannerella forsythia by macrophages. Phagocytosis of T. forsythia was significantly enhanced by coinfection with P. gingivalis in a multiplicity of infection-dependent and gingipain-dependent manner. Mutation of either Kgp or Rgp in the coinfecting P. gingivalis resulted in attenuated enhancement of T. forsythia phagocytosis. Inhibition of coaggregation between the two bacterial species reduced phagocytosis of T. forsythia in mixed infection, and this coaggregation was dependent on gingipains. Inhibition of gingipain protease activities in coinfecting P. gingivalis abated the coaggregation and the enhancement of T. forsythia phagocytosis. However, the direct effect of protease activities of gingipains on T. forsythia seemed to be minimal. Although most of the phagocytosed T. forsythia were cleared in infected macrophages, more T. forsythia remained in cells coinfected with gingipain-expressing P. gingivalis than in cells coinfected with the gingipain-null mutant or infected only with T. forsythia at 24 and 48 h post-infection. Collectively, these results suggest that P. gingivalis, mainly via its gingipains, alters the clearance of T. forsythia, and provide some insights into the role of P. gingivalis as a keystone pathogen.

Pathogenic potential of Tannerella forsythia enolase.

Although enolases are cytosolic enzymes involved in the glycolytic pathway, they can also be secreted or expressed on the surface of a variety of eukaryotic cells and bacteria. Surface-exposed enolases of eukaryotes and bacteria can function as plasminogen receptors. Furthermore, antibodies raised against bacterial enolases can react with host enolases, suggesting molecular mimicry between bacterial and host enzymes. In this study, we analyzed an enolase of the major periodontopathogen Tannerella forsythia, which is either secreted or present on the cell surface, via matrix-assisted laser desorption ionization time-of-flight mass spectrometry and immunofluorescence, respectively. The T. forsythia enolase retained the enzymatic activity converting 2-phosphoglycerate to phosphoenolpyruvate and showed plasminogen binding and activating ability, which resulted in the degradation of fibronectin secreted from human gingival fibroblasts. In addition, it induced proinflammatory cytokine production, including interleukin-1ß (IL-1ß), IL-6, IL-8, and tumour necrosis factor-α (TNF-a) in the human THP-1 monocytic cell line. Taken together, our results demonstrate that T. forsythia enolase plays a role in pathogenesis in the host by plasminogen activation and proinflammatory cytokine induction, which has the potential to exaggerate inflammation in periodontitis.

Anomalous diamagnetic shifts in InP-GaP lateral quantum-wires.

Linearly polarized photoluminescence (PL) measurements were carried out on InP-GaP lateral nanowires grown using a lateral composition modulation method in pulsed magnetic fields up to ∼ 50 T. In these structures, the energy band alignment becomes type-I and type-II in In-rich wire and Ga-rich barrier regions, respectively. It is revealed that the polarization of the type-I PL is oriented along the [11̄0] crystal direction, whereas that of the type-II PL is along the [110] direction in the absence of magnetic field. These two different PL peaks exhibit anomalous energy shifts with respect to the direction of the magnetic field due to the variation of the confined energy in the exciton center of mass potential.

The E3 ubiquitin ligase NEDD4 negatively regulates HER3/ErbB3 level and signaling.

HER3/ErbB3, a member of the epidermal growth factor receptor (EGFR) family, has a pivotal role in cancer and is emerging as a therapeutic antibody target. In this study, we identified NEDD4 (neural precursor cell expressed, developmentally downregulated 4) as a novel interaction partner and ubiquitin E3 ligase of human HER3. Using molecular and biochemical approaches, we demonstrated that the C-terminal tail of HER3 interacted with the WW domains of NEDD4 and the interaction was independent of neuregulin-1. Short hairpin RNA knockdown of NEDD4 elevated HER3 levels and resulted in increased HER3 signaling and cancer cell proliferation in vitro and in vivo. A similar inverse relationship between HER3 and NEDD4 levels was observed in prostate cancer tumor tissues. More importantly, the upregulated HER3 expression by NEDD4 knockdown sensitized cancer cells for growth inhibition by an anti-HER3 antibody. Taken together, our results suggest that low NEDD4 levels may predict activation of HER3 signaling and efficacies of anti-HER3 antibody therapies.

Tannerella forsythia BspA increases the risk factors for atherosclerosis in ApoE(-/-) mice.

OBJECTIVE: The aim of this study was to evaluate the effects of Tannerella forsythia and its major surface virulence factor, BspA, on the progression of atherosclerosis in ApoE(-/-) mice and the expression of lipid metabolism-related genes. METHODS: PMA-differentiated THP-1 cells were treated with BspA to detect foam cell formation. The proximal aortas of ApoE(-/-) mice injected with T. forsythia or BspA were stained with oil red O to examine lipid deposition. The serum levels of CRP, HDL, and LDL were detected by ELISA. The liver tissue of T. forsythia- or BspA-injected ApoE(-/-) mice was examined for mRNA expression of lipid metabolism-related genes, such as liver X receptors (LXRα and LXRß) and ATP-binding cassette transporter A1 (ABCA1). RESULTS: Tannerella forsythia and BspA induced foam cell formation in THP-1 cells and accelerated the progression of atherosclerotic lesions in ApoE(-/-) mice. Mouse serum levels of CRP and LDL were increased, and HDL was decreased by T. forsythia and BspA. The expression levels of LXRα and LXRß, and ABCA1 in liver tissue were decreased by T. forsythia and BspA. CONCLUSIONS: Tannerella forsythia and BspA augmented atherosclerotic lesion progression in ApoE(-/-) mice. This process may be associated with downregulation of lipid metabolism-related gene expression.

Photothermal optical modulation of ultra-compact hybrid Si-VO2 ring resonators.

We demonstrate photothermally induced optical switching of ultra-compact hybrid Si-VO2 ring resonators. The devices consist of a sub-micron length ~70 nm thick patch of phase-changing VO2 integrated onto silicon ring resonators as small as 1.5 µm in radius. The semiconductor-to-metal transition (SMT) of VO2 is triggered using a 532 nm pump laser, while optical transmission is probed using a tunable cw laser near 1550 nm. We observe optical modulation greater than 10dB from modest quality-factor (~10³) resonances, as well as a large -1.26 nm change in resonant wavelength Δλ, resulting from the large change in the dielectric function of VO2 in the insulator-to-metal transition achieved by optical pumping.

Fusobacterium nucleatum GroEL induces risk factors of atherosclerosis in human microvascular endothelial cells and ApoE(-/-) mice.

Infection and inflammation are risk factors in the initiation and progression of atherosclerosis. Periodontitis is one of the most prevalent chronic inflammations of the oral cavity, and has been reported to be associated with systemic disease. In this study, we evaluated whether the heat-shock protein GroEL of Fusobacterium nucleatum, one of the most prevalent bacteria in periodontitis, induces factors that predispose to atherosclerosis in human microvascular endothelial cells (HMEC-1) and apolipoprotein E-deficient (ApoE(-/-)) mice. GroEL induced the expression of chemokines such as monocyte chemoattractant protein-1 and interleukin-8 as well as cell adhesion molecules, such as intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin. GroEL induced the activity of tissue factor and reduced the activity of the tissue factor pathway inhibitor. Foam cell formation was induced by GroEL. GroEL-injected ApoE(-/-) mice showed significant atherosclerotic lesion progression compared with control mice. Serum levels of risk factors for atherosclerosis such as interleukin-6, C-reactive protein, and low-density lipoprotein were increased in GroEL-injected ApoE(-/-) mice compared with control mice, whereas serum levels of high-density lipoprotein were decreased. We could detect significantly higher levels of anti-F. nucleatum GroEL antibody in serum and F. nucleatum DNA in gingival crevicular fluid from patients with periodontitis than in that from healthy subjects. Our results indicate that the host response to the GroEL of periodontal pathogens like F. nucleatum may be a mechanism involved in atherosclerosis, supporting the association of periodontitis and systemic infection.

H⁺-myo-inositol transporter SLC2A13 as a potential marker for cancer stem cells in an oral squamous cell carcinoma.

Cancer Stem Cells (CSCs) from tumors of different phenotypes possess a marked capacity for proliferation, self-renewal, and differentiation. They also play a critical role in cancer recurrence. Although CSC has been regarded as a new target for cancer therapy, the fundamental questions in the CSC study have not been resolved mainly due to the lack of proper CSC markers. To find new CSC markers for oral squamous cell carcinoma (OSCC), we cultured the primary tumor cells from OSCC patients the regular culture condition and the sphere-forming culture condition to enrich primary tumor cells and potential CSCs. We compared gene expression profiles between sphere-forming and non-forming cells, thus identifying that 23 membrane protein-coding genes were over-expressed in the sphere-forming cells. Among them, 8 belonged to the solute carrier (SLC) protein family. H⁺-myo-inositol transporter SLC2A13 and monocarbohydrate transporter SLC16A6 genes that were consistently increased in the sphere-forming cells in the primary cultures of OSCC samples. Confocal microscopy revealed that SLC2A13-expressing cells were embedded in the limited areas of tumor tissue as a cluster, while SLC16A6 was uniformly detected in hyperplastic epithelium. Moreover, SLC2A13 an expression was induced in human breast adenocarcinoma MCF7 cells after serum starvation. Taken together, our results suggest that SLC2A13 can be a potential markers for CSC in various tumors.

In-vivo-induced antigenic determinants of Fusobacterium nucleatum subsp. nucleatum.

Fusobacterium nucleatum plays a pivotal role in dental plaque biofilm formation and is known to be involved in chronic inflammatory systemic disease. However, limited knowledge of F. nucleatum genes expressed in vivo interferes with our understanding of pathogenesis. In this study, we identified F. nucleatum genes induced in vivo using in-vivo-induced antigen technology (IVIAT). Among 30,000 recombinant clones screened, 87 reacted reproducibly with pooled sera from 10 patients with periodontitis. The clones encoded for 32 different proteins, of which 28 could be assigned to their functions, which were categorized in translation, transcription, transport, energy metabolism, cell envelope, cellular process, fatty acid and phospholipid metabolism, transposition, cofactor biosynthesis, amino acid biosynthesis, and DNA replication. Putative virulence factors detected were ABC transporter, butyrate-acetoacetate CoA-transferase, hemin receptor, hemolysin, hemolysin-related protein, LysR family transcriptional regulator, serine protease, and transposase. Analysis of immune responses to the in-vivo-induced (ivi) antigens in five patients demonstrated that most were reactive to these proteins, confirming results with pooled sera. IVIAT-identified F. nucleatum genes in this study may accelerate the elucidation of F. nucleatum-mediated molecular pathogenesis.

Longitudinal split of the posterior cruciate ligament: description of a new MR finding and evaluation of its potential clinical significance.

AIM: To evaluate the clinical significance of the intra-substance longitudinal split of the posterior cruciate ligament (LS-PCL) and to evaluate its potential clinical significance on MRI. MATERIALS AND METHODS: The databases of two centres were searched for LS-PCL, 6917 knee magnetic resonance imaging (MRI) examinations undertaken were retrospectively reviewed. LS-PCL was defined as increased signal intensity in a PCL in the longitudinal direction, but with an intact ligament outer surface on MRI. Twelve patients were enrolled in this study. Available arthroscopic results, degree of posterior knee instability, and changes in MRI findings, or the degree of instability during follow-up (FU), were reviewed from the patients medical records and via their MRI images. MRI images were reviewed by two musculoskeletal radiologists in consensus for presence and location of LS-PCL and any combined injuries: menisci lesions, ligament injuries, and bone marrow changes. RESULTS: Seven of 12 patients (58.3%) had morphological or functional evidence of PCL injury or insufficiency according to the change of posterior instability on FU stress testing (n=3), insufficiency during arthroscopy (n=2), or decreased extent and altered shape of the PCL split on the FU MRI (n=3). One patient revealed both change of posterior instability on FU stress testing and insufficiency during arthroscopy. Combined injuries were revealed in seven patients. Five patients had isolated LS-PCL: two patients underwent arthroscopic PCL reconstructions; and another three patients revealed knee instability on stress testing. CONCLUSION: Although LS-PCL has not been described before, it can be a type of partial tear of the PCL, which causes PCL insufficiency.

Identification of immunoreactive epitopes of the Porphyromonas gingivalis heat shock protein in periodontitis and atherosclerosis.

BACKGROUND AND OBJECTIVE: Heat shock protein 60 (HSP60) of Porphyromonas gingivalis, a major periodontal pathogen, might be a trigger molecule linking infectious periodontitis and autoimmune atherosclerosis. The aim of this study was to identify the peptide specificity of anti-P. gingivalis HSP60 monoclonal antibodies and their cross-reactivity with bacterial and human HSPs. Their specific immunoreactivity to periodontal or atherosclerotic lesions was also investigated. METHODS: Twenty patients with chronic periodontitis and 20 atherosclerosis patients who had undergone surgical intervention for atheromatous plaques with evidence of ongoing periodontal disease, were selected. Synthetic peptide 19 ((TLVVNRLRGSLKICAVKAPG)-specific T-cell lines were established from inflamed gingiva and atheromatous plaque and the phenotypes and cytokine profiles were characterized. RESULTS: Thirty per cent of periodontitis patients and 100% of atherosclerosis patients reacted positively to cross-reactive peptide 19 from both P. gingivalis and human HSP60. The peptide 19-specific T-cell lines demonstrated the phenotype characteristic of helper T cells (CD4(+)) but did not express CD25 or FOXP3. The interleukin-10 levels were elevated significantly in the peptide 19 T-cell line. CONCLUSION: Synthetic peptide 19 of P. gingivalis HSP60 is an immunoreactive epitope in the periodontitis-atherosclerosis axis.

Glutathione S-transferase M1 (GSTM1) polymorphism is associated with atopic dermatitis susceptibility in a Korean population.

Atopic dermatitis (AD) is a chronic pruritic skin condition affecting as much as 15% of children in industrialized countries. While the underlying pathophysiology of AD is not entirely understood, several studies have suggested that AD may mediated by oxidative stress. Glutathione S-transferases (GSTs) are a class of polymorphic enzymes that function to protect against oxidative stress. To identify any possible associations between GSTs polymorphisms and AD susceptibility, the prevalence of two specific polymorphisms -GSTM1 and GSTT1 (homozygous deletion vs. undeleted) - were quantified by multiplex PCR in 145 patients with AD and 267 healthy controls. In individuals with AD, GSTM1/GSTT1 polymorphisms were compared with family history of AD, age of disease onset, disease severity [per SCORing Atopic Dermatitis (SCORAD)], serum IgE level and presence of other allergic diseases. While the GSTM1-null genotype was found to be significantly associated with AD (P = 0.033, OR = 1.579, 95% CI = 1.037-2.403), the correlation between the GSTT1-null genotype and AD did not reach statistical significance (P = 0.577, OR = 1.125, 95% CI = 0.744-1.702). The GSTM1-null genotype was also found to be significantly associated with a childhood onset of AD, the absence of other allergic diseases, and a family history of AD. In combination, these results suggest that GSTM1 is associated with AD susceptibility in Korean subjects.

Cartilage oligomeric matrix protein-angiopoientin-1 enhances angiogenesis of isolated islet and maintains normoglycemia following transplantation.

Islet transplantation (ITx) has potential as a therapy for patients with type 1 diabetes. For successful engraftment and insulin independence, the transplanted islets must establish an adequate, stable blood supply. Angiopoientin-1 (Ang1) is a specific growth factor that induces vascularization via the Tie2 or Tie1 receptor. In this study, we used an in vitro angiogenesis assay to evaluate islet function following transplantation and the effect of the Ang1 variant cartilage oligomeric matrix protein (COMP) Ang1 on isolated islets. The enhanced function of islets transduced with COMP-Ang1 was also confirmed in a streptozotocin (STZ)-induced diabetic mice model. In a three-dimensional collagen-based culture system, the transduction of COMP-Ang1 into islets significantly increased angiogenesis compared with the bacterial-ß-galactosidase (LacZ)-transduced controls and an intact, nontransduced islet negative control group. COMP-Ang1 transduced islets also attenuated hyperglycemia in syngeneic diabetic C57BL/6 mice and enhanced glucose tolerance by areas under the curves of intraperitoneal glucose tolerance tests. These findings demonstrated the capacity of COMP-Ang1 to promote revascularization in cultured islets, which may contribute to successful transplantation in vivo.

Td92, an outer membrane protein of Treponema denticola, induces osteoclastogenesis via prostaglandin E(2)-mediated RANKL/osteoprotegerin regulation.

BACKGROUND AND OBJECTIVE: Periodontitis is a chronic inflammatory disease of the periodontium that causes significant alveolar bone loss. Osteoclasts are bone-resorbing multinucleated cells. Osteoblasts regulate osteoclast differentiation by expression of RANKL and osteoprotegerin (OPG). Td92 is a surface-exposed outer membrane protein of Treponema denticola, a periodontopathogen. Although it has been demonstrated that Td92 acts as a stimulator of various proinflammatory mediators, the role of Td92 in alveolar bone resorption remains unclear. Therefore, in this study, we investigated the role of Td92 in bone resorption. MATERIAL AND METHODS: Mouse bone marrow cells were co-cultured with calvariae-derived osteoblasts in the presence or absence of Td92. Osteoclast formation was assessed by TRAP staining. Expressions of RANKL, osteoprotegerin (OPG) and prostaglandin E(2) (PGE(2) ) in osteoblasts were estimated by ELISA. RESULTS: Td92 induced osteoclast formation in the co-cultures. In the osteoblasts, RANKL and PGE(2) expressions were up-regulated, whereas OPG expression was down-regulated by Td92. The addition of OPG inhibited Td92-induced osteoclast formation. The prostaglandin synthesis inhibitors NS398 and indomethacin were also shown to inhibit Td92-induced osteoclast formation. The effects of Td92 on the expressions of RANKL, OPG and PGE(2) in osteoblasts were blocked by NS398 or indomethacin. CONCLUSION: These results suggest that Td92 promotes osteoclast formation through the regulation of RANKL and OPG production via a PGE(2) -dependent mechanism.

Influence of human allogenic bone marrow and cord blood-derived mesenchymal stem cell secreting trophic factors on ATP (adenosine-5'-triphosphate)/ADP (adenosine-5'-diphosphate) ratio and insulin secretory function of isolated human islets from cadaveric donor.

Successful islet transplantation (ITx) is not only dependent on the number of islets, but also their quality, including viability, metabolic activity, and function. Islet quality decreases during cultivation after the isolation procedure. To overcome this obstacle, we established the practice of islet and mesenchymal stem cells (MSCs) coculture. This coculture condition improved the ATP (adenosine-5'-triphosphate)/ADP (adenosine-5'-diphosphate) ratio and insulin secretory function in vitro. It is believed that the enhancement of islet quality in islet-MSCs cocultures may be caused by the secretion of active agents by MSCs. Herein we have shown that interleukin-6 (IL-6), vascular endothelial growth factor-A (VEGF-A), hepatocyte growth factor (HGF), and transforming growth factor-beta (TGF-beta) were significantly increased as measured by enzyme-linked immunosorbent assay (ELISA) in MSCs-cultured medium, factors that have been shown to improve the survival, function, and angiogenesis/revascularization of islets. These results indicated that the quality of human islets was enhanced by trophic molecules secreted by MSCs, which influence the intracellular islet ATP content and insulin secretory function.

Use of epigenetic modification to induce FOXP3 expression in naïve T cells.

We investigated whether epigenetic modification agents can convert naïve T cells to regulatory T cells (T(regs)) which are responsible for limiting immune responses and maintaining self-tolerance. We treated splenic CD4(+)/CD25(-) naïve T cells from BALB/c mice with the DNA-methyltransferase inhibitor 5-aza-2'-deoxycytidine (5AzaD) or the histone protein deacetylase (HDAC) inhibitor Trichostatin A (TSA), and measured the effects on the expression of FOXP3, which encodes a transcription factor (FOXP3) that regulates T(reg) development. FOXP3 expression in naïve T cells was increased by 5AzaD or TSA treatment, administered 72 hours after T-cell receptor (TCR) stimulation with anti-CD3 plus anti-CD28. The T(regs) induced by 5AzaD or TSA expressed greater amounts of the FOXP3 protein than the control and the natural T(regs). The analysis of T(reg)-associated markers also showed T(reg) phenotypes (CD25(+)/CTLA4(+)/GITR(+)/CD127(-)). Finally, the induced T(reg) population also displayed T-cell suppression. These data suggested that epigenetic modification agents can induce FOXP3 expression, promoting the conversion of naïve T cells to T(regs).

Development of functional human immune system with the transplantations of human fetal liver/thymus tissues and expanded hematopoietic stem cells in RAG2-/-gamma(c)-/- MICE.

BACKGROUND: There is an increasing need for suitable animal models for the study of the human immune system and disease. The purpose of this study was to develop a practical in vivo model of human immune cell repopulation using ex vivo expanded human fetal liver-derived CD34(+) hematopoietic stem cells and subrenally coimplanted fetal liver/thymus tissues. METHODS: Freshly isolated fetal liver-derived CD34(+) hematopoietic stem cells were frozen until injected and ex vivo expanded with various cytokines for 7 days. After fetal liver/thymus tissues were subrenally coimplanted into preirradiated Rag2(-/-)gamma(c)(-/-) mice, frozen and ex vivo expanded CD34(+) cells were injected intravenously. The peripheral blood of the mice was monitored for the detection of human cell engraftment using flow cytometry. Then we confirmed human T-cell function by in vitro function assays. RESULTS: After fetal liver/thymus tissues were coimplanted into the irradiated Rag2(-/-)gamma(c)(-/-) mice, with frozen and ex vivo expanded CD34(+) hematopoietic stem cells, human cell engraftments were determined using hCD45 and multilineage markers. The cultured cells with the cytokine combination of stem cell factor, thrombopoietin, Flk2/Flk3 ligand (FL), and interleukin-3 showed stable and long-term engraftment compared to other combinations. The ex vivo expanded human fetal liver-derived CD34(+) hematopoietic stem cells, under our culture conditions, accomplished a large volume of expanded cells that were sustained, demonstrating self-renewal of the evaluated markers, which may have indicated long- term repopulation activity. CONCLUSION: The results of this study demonstrated a practical mouse model of expanded human immune cells especially T cells in Rag2(-/-)gamma(c)(-/-) mice.