BPI23-Fcγ alleviates lethal multi-drug-resistant Acinetobacter baumannii infection by enhancing bactericidal activity and orchestrating neutrophil function.

Antibiotic resistance has become a major threat, contributing significantly to morbidity and mortality globally. Administering non-antibiotic therapy, such as antimicrobial peptides, is one potential strategy for effective treatment of multi-drug-resistant Gram-negative bacterial infections. Bactericidal/permeability-increasing protein (BPI) derived from neutrophils has bactericidal and endotoxin-neutralizing activity. However, the protective roles and mechanisms of BPI in multi-drug-resistant bacterial infections have not been fully elucidated. In this study, a chimeric BPI23-Fcγ recombined protein comprising the functional N terminus of BPI and Fcγ was constructed and expressed by adenovirus vector 5 (Ad5). Ad5-BPI23-Fcγ or recombinant BPI23-Fcγ protein significantly improved the survival of mice with pneumonia induced by a minimal lethal dose of multi-drug-resistant Acinetobacter baumannii or Klebsiella pneumoniae by ameliorating lung pathology and reducing pro-inflammatory cytokines. Transfection with Ad5-BPI23-Fcγ significantly decreased the bacterial load and endotoxaemia, which was associated with enhanced bactericidal ability and elevated the phagocytic activity of neutrophils in vitro and in vivo. In addition, Ad5-BPI23-Fcγ transfection significantly increased the recruitment of neutrophils to lung, increased the proportion and number of neutrophils in peripheral blood, and promoted the maturation of bone marrow (BM) neutrophils after drug-resistant A. baumannii infection. BPI23-Fcγ and neutrophils synergistically enhanced bactericidal activity and decreased pro-inflammatory cytokines. These results demonstrated that the chimeric BPI23-Fcγ protein protected mice from pneumonia induced by multi-drug-resistant A. baumannii infection by direct bactericidal effects and promotion of neutrophil recruitment, phagocytosis and maturation. Chimeric BPI23-Fcγ may be a promising candidate as a non-antibiotic biological agent for multi-drug-resistant A. baumannii infection.

Ficolin A derived from local macrophages and neutrophils protects against lipopolysaccharide-induced acute lung injury by activating complement.

Ficolins are important and widely distributed pattern recognition molecules that can induce lectin complement pathway activation and initiate the innate immune response. Although ficolins can bind lipopolysaccharide (LPS) in vitro, the sources, dynamic changes and roles of local ficolins in LPS-induced pulmonary inflammation and injury remain poorly understood. In this study, we established a ficolin knockout mouse model by clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology, and used flow cytometry and hematoxylin and eosin staining to study the expressions and roles of local ficolins in LPS-induced pulmonary inflammation and injury. Our results show that besides ficolin B (FcnB), ficolin A (FcnA) is also expressed in leukocytes from the bone marrow, peripheral blood, lung and spleen. Further analyses showed that macrophages and neutrophils are the main sources of FcnA and FcnB, and T and B cells also express a small amount of FcnB. The intranasal administration of LPS induced local pulmonary inflammation with the increased recruitment of macrophages and neutrophils. LPS stimulation induced increased expression of FcnA and FcnB in neutrophils at the acute stage and in macrophages at the late stage. The severity of the lung injury and local inflammation of Fcna-/- mice was increased by the induction of extracellular complement activation. The recovery of LPS-induced local lung inflammation and injury was delayed in Fcnb-/- mice. Hence, these findings suggested that the local macrophage- and neutrophil-derived FcnA protects against LPS-induced acute lung injury by mediating extracellular complement activation.

Bactericidal Permeability Increasing Protein Deficiency Aggravates Acute Colitis in Mice by Increasing the Serum Levels of Lipopolysaccharide.

Objective: The objective of this study was to understand the role of bactericidal permeability increasing protein (BPI) in the pathogenesis of experimental murine colitis. Methods: We used the Cre-LoxP system to generate BPI knockout (BPI KO) mice. Acute colitis was induced in BPI KO mice and wild-type (WT) mice by subjecting the mice to 5% dextran sulfate sodium (DSS). Mice were observed for symptoms of experimental colitis. The survival of BPI KO mice to infection with Acinetobacter baumannii, a gram-negative bacterium, was also assessed. Results: Southern blot, RT-PCR, and western blot results showed that the 2nd and 3rd exons of the murine Bpi gene were knocked out systemically, confirming successful construction of the BPI KO mouse. BPI KO mice subjected to DSS showed increased symptoms of experimental colitis, increased colonic mucosal damage, increased epithelial permeability, elevated levels of serum LPS, and a disrupted fecal microbiome as compared with WT mice. Furthermore, BPI KO mice challenged intraperitoneally with A. baumannii died sooner than WT mice, and the total number of bacteria in the abdominal cavity, spleen, and liver was increased in BPI KO mice as compared to WT mice. Conclusions: We successfully generated BPI KO mice. The BPI KO mice developed worse colitis than WT mice by increased colitis symptoms and colonic mucosal damage, elevated levels of serum LPS, and a disrupted microbiome. BPI could be a potential target for treatment of ulcerative colitis in humans.

Rapamycin ameliorates lipopolysaccharide-induced acute lung injury by inhibiting IL-1ß and IL-18 production.

Interleukin (IL)-1ß and IL-18 play central and detrimental roles in the development of acute lung injury (ALI), and mammalian target of rapamycin (mTOR) is involved in regulating IL-1ß and IL-18 production. However, it is not clear whether the mTOR specific inhibitor rapamycin can attenuate lipopolysaccharide (LPS)-induced ALI by modulating IL-1ß and IL-18 production. In this study, we found that rapamycin ameliorated LPS-induced ALI by inhibiting NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated IL-1ß and IL-18 secretion. Mechanistically, elevated autophagy and decreased nuclear factor (NF)-κB activation were associated with downregulated IL-1ß and IL-18. Moreover, rapamycin reduced leukocyte infiltration in the lung tissue and bronchoalveolar lavage fluid (BALF), and contributed to the alleviation of LPS-induced ALI. Consistently, rapamycin also significantly inhibited IL-1ß and IL-18 production by RAW264.7 cells via increased autophagy and decreased NF-κB signaling in vitro. Our results demonstrated that rapamycin protects mice against LPS-induced ALI partly by inhibiting the production and secretion of IL-1ß and IL-18. mTOR and rapamycin might represent an appropriate therapeutic target and strategy for preventing ALI induced by LPS.

Delayed oseltamivir plus sirolimus treatment attenuates H1N1 virus-induced severe lung injury correlated with repressed NLRP3 inflammasome activation and inflammatory cell infiltration.

Severe influenza A virus infection causes high mortality and morbidity worldwide due to delayed antiviral treatment and inducing overwhelming immune responses, which contribute to immunopathological lung injury. Sirolimus, an inhibitor of mammalian target of rapamycin (mTOR), was effective in improving clinical outcomes in patients with severe H1N1 infection; however, the mechanisms by which it attenuates acute lung injury have not been elucidated. Here, delayed oseltamivir treatment was used to mimic clinical settings on lethal influenza A (H1N1) pdm09 virus (pH1N1) infection mice model. We revealed that delayed oseltamivir plus sirolimus treatment protects mice against lethal pH1N1 infection by attenuating severe lung damage. Mechanistically, the combined treatment reduced viral titer and pH1N1-induced mTOR activation. Subsequently, it suppressed the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated secretion of interleukin (IL)-1ß and IL-18. It was noted that decreased NLRP3 inflammasome activation was associated with inhibited nuclear factor (NF)-κB activation, reduced reactive oxygen species production and increased autophagy. Additionally, the combined treatment reduced the expression of other proinflammatory cytokines and chemokines, and decreased inflammatory cell infiltration in lung tissue and bronchioalveolar lavage fluid. Consistently, it inhibited the mTOR-NF-κB-NLRP3 inflammasome-IL-1ß axis in a lung epithelial cell line. These results demonstrated that combined treatment with sirolimus and oseltamivir attenuates pH1N1-induced severe lung injury, which is correlated with suppressed mTOR-NLRP3-IL-1ß axis and reduced viral titer. Therefore, treatment with sirolimus as an adjuvant along with oseltamivir may be a promising immunomodulatory strategy for managing severe influenza.

Vγ4+γδT Cells Aggravate Severe H1N1 Influenza Virus Infection-Induced Acute Pulmonary Immunopathological Injury via Secreting Interleukin-17A.

The influenza A (H1N1) pdm09 virus remains a critical global health concern and causes high levels of morbidity and mortality. Severe acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are the major outcomes among severely infected patients. Our previous study found that interleukin (IL)-17A production by humans or mice infected with influenza A (H1N1) pdm09 substantially contributes to ALI and subsequent morbidity and mortality. However, the cell types responsible for IL-17A production during the early stage of severe influenza A (H1N1) pdm09 infection remained unknown. In this study, a mouse model of severe influenza A (H1N1) pdm09 infection was established. Our results show that, in the lungs of infected mice, the percentage of γδT cells, but not the percentages of CD4+Th and CD8+Tc cells, gradually increased and peaked at 3 days post-infection (dpi). Further analysis revealed that the Vγ4+γδT subset, but not the Vγ1+γδT subset, was significantly increased among the γδT cells. At 3 dpi, the virus induced significant increases in IL-17A in the bronchoalveolar lavage fluid (BALF) and serum. IL-17A was predominantly secreted by γδT cells (especially the Vγ4+γδT subset), but not CD4+Th and CD8+Tc cells at the early stage of infection, and IL-1ß and/or IL-23 were sufficient to induce IL-17A production by γδT cells. In addition to secreting IL-17A, γδT cells secreted interferon (IFN)-γ and expressed both an activation-associated molecule, natural killer group 2, member D (NKG2D), and an apoptosis-associated molecule, FasL. Depletion of γδT cells or the Vγ4+γδT subset significantly rescued the virus-induced weight loss and improved the survival rate by decreasing IL-17A secretion and reducing immunopathological injury. This study demonstrated that, by secreting IL-17A, lung Vγ4+γδT cells, at least, in part mediated influenza A (H1N1) pdm09-induced immunopathological injury. This mechanism might serve as a promising new target for the prevention and treatment of ALI induced by influenza A (H1N1) pdm09.

Long-term anti-endotoxin/E. coli efficacy in mice transfected with AAV2/1-muBPI25 -muFcγ1.

Bactericidal/permeability increasing (BPI) is an antibiotic protein which kills Gram-negative bacteria and neutralizes endotoxin. We have previously developed a recombinant adeno-associated virus which contains human BPI amino acid residues 1-199 and Fc fragment of human IgG1 gene (AAV-hBPI-Fc) and shown that the recombinant virus can protect mice from lethal endotoxemia. However, whether AAV-hBPI-Fc can be used in vivo for the long term remains unclear. To address this, we established an adeno-associated virus-containing mouse BPI and Fc fragment genes (muBPI-Fc) and compared antigenicity of these recombinant proteins in murine models. Immunohistochemistry showed the expression of both fusion proteins at injected sites. ELISA and Western blotting showed that the muBPI-Fc protein was detected in serum up to 8 weeks after injection, without generation of autoantibodies against muBPI-Fc. In contrast, expressed hBPI-Fc protein was only detected on the 2nd week, whereas the autoantibody against hBPI-Fc protein occurred in serum from the 4th week to the end of study. muBPI-Fc also reduced production of proinflammatory cytokines and protected mice from endotoxemia and bacteremia. Our data showed that AAV-muBPI-Fc has potential long-term efficacy as an anti-endotoxin and has anti-bacterial activity in mice, suggesting the potential clinical application of AAV-hBPI-Fc, such as in endotoxin shock.

IL-25 induces airways angiogenesis and expression of multiple angiogenic factors in a murine asthma model.

BACKGROUND: Th2-promoting cytokine IL-25 might contribute to bronchial mucosal vascular remodelling in asthma through its receptor expressed by vascular endothelial and vascular smooth muscle cells. METHODS: By utilising a newly established chronic asthma murine model induced by direct exposure of the airways to IL-25 alone, we examined effects of IL-25 on angiogenesis, vascular remodelling and expression of angiogenic factors, compared changes with those in a "classical" ovalbumin (OVA)-induced murine asthma model. IL-25 and OVA were intranasally instilled into the airways of BALB/c mice for up to 55 days. Airways vessels and angiogenic factors, including Von Willebrand Factor (vWF), amphiregulin, angiogenin, endothelin-1, transcription factor ERG, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor (IGF-1) and vascular endothelial growth factor (VEGF) in lung sections, homogenates and BAL fluid were detected and quantified by immunostaining or enzyme linked immunosorbent assay (ELISA). An in house assay was also utilised to compare the effects of IL-25 and other Th2-cytokines on angiogenesis by human vascular endothelial cells. RESULTS: Repetitive intranasal challenge with IL-25 alone or OVA alone in OVA-presensitised animals significantly increased peribronchial vWF (+) vessels in the murine airways, which was associated with remarkably elevated expression of amphiregulin, angiogenin, endothelin-1, bFGF, EGF, IGF-1, VEGF and ERG. IL-25, but not Th-2-cytokines induced human angiogenesis in vitro. CONCLUSIONS: The data suggest that chronic exposure of murine airways to IL-25 alone is able to reproduce a local angiogenic milieu. Thus, blocking IL-25 may attenuate vascular remodelling and improve outcomes in asthma patients.

Distinct sustained structural and functional effects of interleukin-33 and interleukin-25 on the airways in a murine asthma surrogate.

Interleukin-25 (IL-25) and IL-33, which belong to distinct cytokine families, induce and promote T helper type 2 airway inflammation. Both cytokines probably play a role in asthma, but there is a lack of direct evidence to clarify distinctions between their functions and how they might contribute to distinct 'endotypes' of disease. To address this, we made a direct comparison of the effects of IL-25 and IL-33 on airway inflammation and physiology in our established murine asthma surrogate, which involves per-nasal, direct airway challenge. Intranasal challenge with IL-33 or IL-25 induced inflammatory cellular infiltration, collagen deposition, airway smooth muscle hypertrophy, angiogenesis and airway hyper-responsiveness, but neither increased systemic production of IgE or IgG1. Compared with that of IL-25, the IL-33-induced response was characterized by more sustained laying down of extracellular matrix protein, neoangiogenesis, T helper type 2 cytokine expression and elevation of tissue damping. Hence, both IL-25 and IL-33 may contribute significantly and independently to asthma 'endotypes' when considering molecular targets for the treatment of human disease.

IL-35 over-expression increases apoptosis sensitivity and suppresses cell growth in human cancer cells.

Interleukin (IL)-35 is a novel heterodimeric cytokine in the IL-12 family and is composed of two subunits: Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 is expressed in T regulatory (Treg) cells and contributes to the immune suppression function of these cells. In contrast, we found that both IL-35 subunits were expressed concurrently in most human cancer cell lines compared to normal cell lines. In addition, we found that TNF-α and IFN-γ stimulation led to increased IL-35 expression in human cancer cells. Furthermore, over-expression of IL-35 in human cancer cells suppressed cell growth in vitro, induced cell cycle arrest at the G1 phase, and mediated robust apoptosis induced by serum starvation, TNF-α, and IFN-γ stimulation through the up-regulation of Fas and concurrent down-regulation of cyclinD1, survivin, and Bcl-2 expression. In conclusion, our results reveal a novel functional role for IL-35 in suppressing cancer activity, inhibiting cancer cell growth, and increasing the apoptosis sensitivity of human cancer cells through the regulation of genes related to the cell cycle and apoptosis. Thus, this research provides new insights into IL-35 function and presents a possible target for the development of novel cancer therapies.

Cytokine-like 1 is involved in the growth and metastasis of neuroblastoma cells.

Cytokine-like 1 (CYTL1) was first identified in CD34+ cells derived from bone marrow and cord blood. The biological functions of CYTL1 remain largely unknown. Here, we reveal a relationship between CYTL1 expression and the biological characteristics of neuroblastoma (NB). The expression of CYTL1 was detected in 10 human tumor cell lines and human NB tissues by RT-PCR and real-time PCR. The inhibitory effect of CYTL1 knockdown on the proliferation, migration and invasion of SH-SY5Y human neuroblastoma cells was studied using the CCK-8 assay and Transwell chamber assays. Among the 10 human tumor cell lines that we examined, CYTL1 was expressed only in SH-SY5Y human neuroblastoma cells. Furthermore, we also observed high levels of CYTL1 expression in human NB tissues. When CYTL1 expression was blocked by siRNA, SH-SY5Y cells showed decreased proliferation, migration and invasion activities. Taken together, our results showed the first evidence of CYTL1 expression in SH-SY5Y neuroblastoma cells and human NB tissues, revealed a possible link between CYTL1 and NB development, and suggested CYTL1 as a potential therapeutic target and diagnosis biomarker for NB.

Infecting mice with recombinant Ad5-BPI23-Fcγ1 virus protects against systemic Escherichia coli challenge.

Infections caused by Gram-negative bacteria (GNB) are increasingly common and can result in significant mortality rates due to the development of sepsis. To examine the potential usage of a recombinant Ad5-BPI(23)-Fcγ1 virus as a biological treatment against systemic infection by GNB, a construct containing the human bactericidal/permeability increasing protein (BPI) gene, encoding the functional N terminus (amino acid residues 1-199) of human BPI, and the Fcγ1 gene, encoding the Fc segment of human immunoglobulin G1, was inserted into an adenovirus serotype 5 (Ad5) chromosome to produce a recombinant Ad5-BPI(23)-Fcγ1 virus. Human A549 cells in culture and BALB/c mice were infected with the recombinant Ad5-BPI(23)-Fcγ1 virus and BPI(23)-Fcγ1 expression was confirmed by Western blot analysis and ELISA. The concentrations of BPI(23)-Fcγ1 protein were 5.59 µg ml(-1) in vitro and 21.37 ng ml(-1) in vivo and it was observed that these concentrations were sufficient to decrease endotoxin concentrations while enhancing bactericidal activity. In addition, mice treated with the recombinant Ad5-BPI(23)-Fcγ1 virus had decreased levels of IL-1ß and TNF-α and were protected from an E. coli O111 : B4 challenge. Our data support the concept that Ad5-mediated BPI(23)-Fcγ1 gene delivery could be used as an ancillary biological treatment in the management of infection caused by GNB.

Allergen-induced expression of IL-25 and IL-25 receptor in atopic asthmatic airways and late-phase cutaneous responses.

BACKGROUND: IL-25 is thought to participate in allergic inflammation by propagating T(h)2-type responses. OBJECTIVE: To address the hypothesis that allergen provocation increases expression of IL-25 and its receptor IL-25R in the asthmatic bronchial mucosa and skin dermis of atopic subjects. METHODS: Sequential single and double immunostaining was used to evaluate the numbers and phenotypes of IL-25 and IL-25R immunoreactive cells in bronchial biopsies from mild atopic subjects with asthma (n = 10) before and 24 hours after allergen inhalation challenge and skin biopsies from atopic subjects (n = 10) up to 72 hours after allergen subepidermal injection. RESULTS: IL-25 immunoreactivity was expressed by a majority of epidermal cells in both organs at baseline and was not further augmented by challenge. IL-25R immunoreactive cells were rare in the epidermis before or after challenge. Allergen challenge was associated with significantly (P < .01) increased expression of IL-25 and IL-25R immunoreactivity in the submucosa of both organs. IL-25 immunoreactivity colocalized with eosinophils, mast cells, and endothelial cells, whereas IL-25R immunoreactivity colocalized with eosinophils, mast cells, endothelial cells, and T lymphocytes. In both organs, correlations were observed between increases in IL-25 expression and the magnitudes of the late-phase allergen-induced clinical responses. CONCLUSION: Allergen provocation induces functionally relevant, increased expression of IL-25 and its receptor in the asthmatic bronchial mucosa and dermis of sensitized atopic subjects. In addition to T cells, eosinophils, mast cells, and endothelial cells are potential sources and targets of IL-25 in the course of allergic inflammation.

T-helper cell type 2 (Th2) memory T cell-potentiating cytokine IL-25 has the potential to promote angiogenesis in asthma.

IL-25 (IL-17E) is a T-helper cell type 2 (Th2) cytokine best described as a potentiator of Th2 memory responses. Reports of expression of its receptor, IL-25R, on airways structural cells suggest a wider role for IL-25 in remodeling. We hypothesized that IL-25 stimulates local angiogenesis in the asthmatic bronchial mucosa. Immunoreactive IL-25(+), IL-25R(+), and CD31(+) (endothelial) cells in sections of bronchial biopsies from asthmatics and controls were detected by immunohistochemistry. The effect of IL-25 on angiogenesis was examined using an in vitro assay. Real-time PCR was used to detect expression of IL-25R and VEGF mRNA in cultured human vascular endothelial cells (HUVEC), and a cell proliferation kit (WST-8) was used to measure the effect of IL-25 on HUVEC proliferation. Immunostaining showed that IL-25(+), IL-25R(+), and CD31(+)/IL-25R(+) cells were significantly elevated in the bronchial mucosa of asthmatics compared with controls (P < 0.003). In asthmatics, the numbers of IL-25(+) cells correlated inversely with the forced expiratory volume in 1 s (r = -0.639; P = 0.01). In vitro, HUVEC constitutively expressed IL-25R, which was up-regulated further by TNF-α. IL-25 and TNF-α also increased expression of VEGF and VEGF receptors. IL-25 increased HUVEC proliferation and the number, length, and area of microvessel structures in a concentration-dependent manner in vitro. VEGF blockade, the PI3K-specific inhibitor LY294002, and the MAPK/ERK1/2 (MEK1/2)-specific inhibitor U0126 all markedly attenuated IL-25-induced angiogenesis, and the inhibitors also reduced IL-25-induced proliferation and VEGF expression. Our findings suggest that IL-25 is elevated in asthma and contributes to angiogenesis, at least partly by increasing endothelial cell VEGF/VEGF receptor expression through PI3K/Akt and Erk/MAPK pathways.

Allogeneic periodontal ligament stem cell therapy for periodontitis in swine.

Periodontitis is one of the most widespread infectious diseases in humans. It is the main cause of tooth loss and associated with a number of systemic diseases. Until now, there is no appropriate method for functional periodontal tissue regeneration. Here, we establish a novel approach of using allogeneic periodontal ligament stem cells (PDLSCs) sheet to curing periodontitis in a miniature pig periodontitis model. Significant periodontal tissue regeneration was achieved in both the autologous and the allogeneic PDLSCs transplantation group at 12 weeks post-PDLSCs transplantation. Based on clinical assessments, computed tomography (CT) scanning, and histological examination, there was no marked difference between the autologous and allogeneic PDLSCs transplantation groups. In addition, lack of immunological rejections in the animals that received the allogeneic PDLSCs transplantation was observed. Interestingly, we found that human PDLSCs fail to express human leukocyte antigen (HLA)-II DR and costimulatory molecules. PDLSCs were not able to elicit T-cell proliferation and inhibit T-cell proliferation when stimulated with mismatched major histocompatibility complex molecules. Furthermore, we found that prostaglandin E2 (PGE2) plays a crucial role in PDLSCs-mediated immunomodulation and periodontal tissue regeneration in vitro and in vivo. Our study demonstrated that PDLSCs possess low immunogenicity and marked immunosuppression via PGE2-induced T-cell anergy. We developed a standard technological procedure of using allogeneic PDLSCs to cure periodontitis in swine.

Suppression of T cell proliferation by root apical papilla stem cells in vitro.

The use of allogeneic stem cells strongly extends the range of stem cell applications in dentistry; however, immunological rejection remains a major concern. There is little information about the immunological features of dental-related stem cells in the literature. Therefore, we investigated the immunological characteristics of stem cells from the root apical papilla (SCAP) of swine in vitro by measuring T cell immunomodulation and apoptosis. We found that SCAP expressed a low level of swine leukocyte antigen (SLA) class I molecules and were negative for SLA class II DR molecules. Moreover, SCAP could inhibit autologous T cell proliferation stimulated by phytohemagglutinin (PHA) and a one-way mixed lymphocyte reaction in a dose-dependent manner. In addition, SCAP could suppress proliferation of allogeneic T cells in a dose-dependent manner, with or without mitomycin C pretreatment. Moreover, soluble factor(s) may be involved in the SCAP-mediated immune suppression. After a 5-day coculture of SCAP, allogeneic T cells, and PHA, only 1.22% of T cells were apoptotic. These data indicated that SCAP were weakly immunogenic and suppressed T cell proliferation in vitro through an apoptosis-independent mechanism.

Effect of cryopreservation on biological and immunological properties of stem cells from apical papilla.

Stem cells from apical papilla (SCAP) are a novel population of multipotent stem cells that, although similar to dental pulp stem cells, are a discrete source of dental stem cells. SCAP have potential roles in root development, apexogenesis, pulp/dentin regeneration, and bioroot engineering. However, procedures to store and preserve SCAP for future clinical applications have not been explored. In this study, we compared human freshly isolated SCAP (fSCAP) with cryopreserved SCAP (cSCAP) in terms of cell viability, colony-forming efficiency, cell proliferation rate, multilineage differentiation potential, profiles of mesenchymal stem cell (MSC) markers, karyotype analysis, and immunological assays. cSCAP showed a similar viable cell ratio, colony-forming efficiency, cell proliferation rate, multilineage differentiation potential, MSC surface markers, apoptotic rate, and G-banded karyotype when compared to fSCAP. There was no significant difference between fSCAP and cSCAP with regard to immune properties. In addition, cSCAP of miniature pig possessed the similar proliferation rate, differentiation potential, and immunomodulatory function as seen in fSCAP. This study demonstrates that cryopreservation does not affect the biological and immunological properties of SCAP, supporting the feasibility of SCAP cryopreservation in nitrogen.

Structural immunology and crystallography help immunologists see the immune system in action: how T and NK cells touch their ligands.

Host immune system is an important and sophisticated system, maintaining the balance of host response to "foreign" antigens and ignorance to the normal-self. To fulfill this achievement the system manipulates a cell-cell interaction through appropriate interactions between cell-surface receptors and cell-surface ligands, or cell-secreted soluble effector molecules to their ligands/receptors/counter-receptors on the cell surface, triggering further downstream signaling for response effects. T cells and NK cells are important components of the immune system for defending the infections and malignancies and maintaining the proper response against over-reaction to the host. Receptors on the surface of T cells and NK cells include a number of important protein molecules, for example, T cell receptor (TCR), co-receptor CD8 or CD4, co-stimulator CD28, CTLA4, KIR, CD94/NKG2, LILR (ILT/LIR/CD85), Ly49, and so forth. These receptor molecules interact with their ligands on the target cells, including major histocompatibility complex (MHC) (or human leukocyte antigen, HLA), CD80, CD86, and so forth. Detailed understanding of these receptor-ligand pair interactions is crucial for our full knowledge of the immune system, ultimately for us to manipulate the T cell and NK cell functions. Accumulations of the receptor-ligand complex crystal structures in the recent years have provided us a unique angel to see how the immune cells interacting with their partner cells. In this review, we discussed binding specificity, plasticity, and flexibility of the T cell and NK cell receptor/ligand interaction, fitting the structural data with their functions. Structural immunology indeed helps us see how T and NK cells "touch" their target cells in our immune system.

High-throughput functional screening for autophagy-related genes and identification of TM9SF1 as an autophagosome-inducing gene.

Autophagy, a tightly regulated process responsible for the bulk degradation of most long-lived proteins and some organelles, is associated with several forms of human diseases including cancer, neurodegenerative disease and cardiomyopathies. However, the molecular machinery involved in autophagy in mammalian cells remains poorly understood. Here, we describe a high-throughput, cell-based functional screening platform, based on an automated fluorescence microscopy system, which enables acquiring and quantitatively analyzing images of GFP-LC3 dots in cotransfected cells. From a library of 1,050 human cDNA clones, we identified three genes (TM9SF1, TMEM166 and TMEM74) whose overexpression induced high levels of autophagosome formation. In particular, overexpression of TM9SF1, which colocalized with LC3 according to the confocal assay, led to a significant increase in the number of GFP-LC3 dots. The results of transmission electron microscopy and immunoblotting to examine LC3-II levels further confirmed the ability of TM9SF1 to induce autophagy. Furthermore, knockdown of TM9SF1 expression by RNA interference could hamper starvation-induced autophagy. The functional screening platform therefore can be applied to high-throughput genomic screening candidate autophagy-related genes, which would provide new insights into underlying molecular mechanisms that may regulate autophagy in mammalian cells.