Objective: To explore the relationship between methylation of interferon gamma (IFN-γ) gene and tumorigenesis in cervical cancer tissues, the biopsy specimens of cervical cancer and cervical intraepithelial neoplasia (CIN) (I-III) patients as well as normal controls were collected and analyzed. Methods: The methylation of the IFN-γ gene was verified by using methylation-specific PCR and DNA sequencing analysis, and the expression levels of IFN-γ mRNA were detected using quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). Results: The methylation rates of the IFN-γ gene were significantly higher in cervical cancer tissues (15/43, 34.9%) than those in CIN (3/23, 13.0% of CIN I; 6/39, 15.4% of CIN II/III) and normal cervical tissues (2/43, 4.7%) (P < 0.01). Furthermore, the mRNA expression of IFN-γ in cervical tumors with methylation (0.71 ± 0.13, n = 8) was lower than that in those without methylation (1.58 ± 0.32, n = 27) (P < 0.05). Likewise, the IFN-γ expression levels in CIN II/III tissues with methylation (0.87 ± 0.16, n = 5) were significantly (P < 0.01) lower compared to those without methylation (2.12 ± 0.27, n = 32). Conclusion: The hypermethylation of IFN-γ gene may be related with tumorigenesis of cervical cancer.
The application of active dry yeast (ADY) in bioethanol production simplifies operation processes and reduces the risk of bacterial contamination. In the present study, we constructed a novel ADY strain with improved stress tolerance and ethanol fermentation performances under stressful conditions. The industrial Saccharomyces cerevisiae strain ZTW1 showed excellent properties and thus subjected to a modified whole-genome shuffling (WGS) process to improve its ethanol titer, proliferation capability, and multiple stress tolerance for ADY production. The best-performing mutant, Z3-86, was obtained after three rounds of WGS, producing 4.4% more ethanol and retaining 2.15-fold higher viability than ZTW1 after drying. Proteomics and physiological analyses indicated that the altered expression patterns of genes involved in protein metabolism, plasma membrane composition, trehalose metabolism, and oxidative responses contribute to the trait improvement of Z3-86. This work not only successfully developed a novel S. cerevisiae mutant for application in commercial bioethanol production, but also enriched the current understanding of how WGS improves the complex traits of microbes.
Interleukin-10 (IL-10) has opposing effects as an anti-inflammatory (potentially cancer-promoting) and antiangiogenic (potentially cancer-inhibiting) agent. The role of IL-10 in cervical cancer is also dual. Here, we review the IL-10-mediated tumor-promoting effect and tumor-inhibiting effects in cervical cancer, among which, human papilloma virus (HPV), human leukocyte antigen-G (HLA-G) and IL-10 polymorphisms are associated with the development of cervical cancer. IL-10 is also used for the therapy of cervical cancer through enhancing proliferation, expression of immunologically important surface molecules and increasing Th1 cytokine production and cytotoxic potential in HPV-specific CD8 (+) cytotoxic T lymphocytes.
interleukin-10; cervical cancer; paradox
Mesenchymal stroma cells (MSCs) have a high potential for novel cell therapy approaches in clinical transplantation. Commonly used bone marrow-derived MSCs (BM-MSCs), however, have a restricted proliferative capacity and cultures are difficult to standardize. Recently developed human embryonic stem cell-derived mesenchymal stroma cells (hES-MSCs) might represent an alternative and unlimited source of hMSCs. We therefore compared human ES-cell-derived MSCs (hES-MP002.5 cells) to normal human bone marrow-derived MSCs (BM-MSCs). hES-MP002.5 cells had lower yet reasonable CFU-F capacity compared with BM-MSC (8±3 versus 29±13 CFU-F per 100 cells). Both cell types showed similar immunophenotypic properties, i.e. cells were positive for CD105, CD73, CD166, HLA-ABC, CD44, CD146, CD90, and negative for CD45, CD34, CD14, CD31, CD117, CD19, CD 271, SSEA-4 and HLA-DR. hES-MP002.5 cells, like BM-MSCs, could be differentiated into adipocytes, osteoblasts and chondrocytes in vitro. Neither hES-MP002.5 cells nor BM-MSCs homed to the bone marrow of immune-deficient NSG mice following intravenous transplantation, whereas intra-femoral transplantation into NSG mice resulted in engraftment for both cell types. In vitro long-term culture-initiating cell assays and in vivo co-transplantation experiments with cord blood CD34+ hematopoietic cells demonstrated furthermore that hES-MP002.5 cells, like BM-MSCs, possess potent stroma support function. In contrast to BM-MSCs, however, hES-MP002.5 cells showed no or only little activity in mixed lymphocyte cultures and phytohemagglutinin (PHA) lymphocyte stimulation assays. In summary, ES-cell derived MSCs might be an attractive unlimited source for stroma transplantation approaches without suppressing immune function.
Human mesoangioblasts are currently in a phase I/II clinical trial for the treatment of patients with Duchenne muscular dystrophy. However, limitations associated with the finite life span of these cells combined with the significant numbers of mesoangioblasts required to treat all of the skeletal muscles in these patients restricts their therapeutic potential. Induced pluripotent stem cell (iPSC)-derived mesoangioblasts may provide the solution to this problem. Although, the idea of using iPSC-derived cell therapies has been proposed for quite some time, our understanding of how the immune system interacts with these cells is inadequate. Herein, we show that iPSC-derived mesoangioblasts (HIDEMs) from healthy donors and, importantly, limb-girdle muscular dystrophy 2D patients exert immunosuppressive effects on T cell proliferation. Interferon gamma (IFN-γ) and tumour necrosis factor alpha (TNF-α) play crucial roles in the initial activation of HIDEMs and importantly indoleamine 2,3 dioxygenase (IDO) and prostaglandin E2 (PGE-2) were identified as key mechanisms involved in HIDEM suppression of T cell proliferation. Together with recent studies confirming the myogenic function and regenerative potential of these cells, we suggest that HIDEMs could provide an unlimited alternative source for mesoangioblast-based therapies.
Pelgipeptin, a potent antibacterial and antifungal agent, is a non-ribosomally synthesised lipopeptide antibiotic. This compound consists of a β-hydroxy fatty acid and nine amino acids. To date, there is no information about its biosynthetic pathway.
A potential pelgipeptin synthetase gene cluster (plp) was identified from Paenibacillus elgii B69 through genome analysis. The gene cluster spans 40.8 kb with eight open reading frames. Among the genes in this cluster, three large genes, plpD, plpE, and plpF, were shown to encode non-ribosomal peptide synthetases (NRPSs), with one, seven, and one module(s), respectively. Bioinformatic analysis of the substrate specificity of all nine adenylation domains indicated that the sequence of the NRPS modules is well collinear with the order of amino acids in pelgipeptin. Additional biochemical analysis of four recombinant adenylation domains (PlpD A1, PlpE A1, PlpE A3, and PlpF A1) provided further evidence that the plp gene cluster involved in pelgipeptin biosynthesis.
In this study, a gene cluster (plp) responsible for the biosynthesis of pelgipeptin was identified from the genome sequence of Paenibacillus elgii B69. The identification of the plp gene cluster provides an opportunity to develop novel lipopeptide antibiotics by genetic engineering.
Non-ribosomal peptide; Biosynthesis; Gene cluster; Antimicrobial agent
Hospital-acquired infections caused by drug-resistant bacteria are a significant challenge to patient safety. Numerous clinical isolates resistant to almost all commercially available antibiotics have emerged. Thus, novel antimicrobial agents, specifically those for multidrug-resistant Gram-negative bacteria, are urgently needed. In the current study, we report the isolation, structure elucidation, and preliminary biological characterization of a new cationic lipopeptide antibiotic, battacin or octapeptin B5, produced from a Paenibacillus tianmuensis soil isolate. Battacin kills bacteria in vitro and has potent activity against Gram-negative bacteria, including multidrug-resistant and extremely drug-resistant clinical isolates. Hospital strains of Escherichia coli and Pseudomonas aeruginosa are the pathogens most sensitive to battacin, with MICs of 2 to 4 μg/ml. The ability of battacin to disrupt the outer membrane of Gram-negative bacteria is comparable to that of polymyxin B, the last-line therapy for infections caused by antibiotic-resistant Gram-negative bacteria. However, the capacity of battacin to permeate bacterial plasma membranes is less extensive than that of polymyxin B. The bactericidal kinetics of battacin correlate with the depolarization of the cell membrane, suggesting that battacin kills bacteria by disrupting the cytoplasmic membrane. Other studies indicate that battacin is less acutely toxic than polymyxin B and has potent in vivo biological activity against E. coli. Based on the findings of the current study, battacin may be considered a potential therapeutic agent for the treatment of infections caused by antibiotic-resistant Gram-negative bacteria.
Most Sphingomonas species synthesize the yellow carotenoid nostoxanthin. However, the carotenoid biosynthetic pathway of these species remains unclear. In this study, we cloned and characterized a carotenoid biosynthesis gene cluster containing four carotenogenic genes (crtG, crtY, crtI and crtB) and a β-carotene hydroxylase gene (crtZ) located outside the cluster, from the gellan-gum producing bacterium Sphingomonas elodea ATCC 31461. Each of these genes was inactivated, and the biochemical function of each gene was confirmed based on chromatographic and spectroscopic analysis of the intermediates accumulated in the knockout mutants. Moreover, the crtG gene encoding the 2,2′-β-hydroxylase and the crtZ gene encoding the β-carotene hydroxylase, both responsible for hydroxylation of β-carotene, were confirmed by complementation studies using Escherichia coli producing different carotenoids. Expression of crtG in zeaxanthin and β-carotene accumulating E. coli cells resulted in the formation of nostoxanthin and 2,2′-dihydroxy-β-carotene, respectively. Based on these results, a biochemical pathway for synthesis of nostoxanthin in S. elodea ATCC 31461 is proposed.
The recent increase in bacterial resistance to antibiotics has promoted the exploration of novel antibacterial materials. As a result, many researchers are undertaking work to identify new lantibiotics because of their potent antimicrobial activities. The objective of this study was to provide details of a lantibiotic-like gene cluster in Paenibacillus elgii B69 and to produce the antibacterial substances coded by this gene cluster based on culture screening.
Analysis of the P. elgii B69 genome sequence revealed the presence of a lantibiotic-like gene cluster composed of five open reading frames (elgT1, elgC, elgT2, elgB, and elgA). Screening of culture extracts for active substances possessing the predicted properties of the encoded product led to the isolation of four novel peptides (elgicins AI, AII, B, and C) with a broad inhibitory spectrum. The molecular weights of these peptides were 4536, 4593, 4706, and 4820 Da, respectively. The N-terminal sequence of elgicin B was Leu-Gly-Asp-Tyr, which corresponded to the partial sequence of the peptide ElgA encoded by elgA. Edman degradation suggested that the product elgicin B is derived from ElgA. By correlating the results of electrospray ionization-mass spectrometry analyses of elgicins AI, AII, and C, these peptides are deduced to have originated from the same precursor, ElgA.
A novel lantibiotic-like gene cluster was shown to be present in P. elgii B69. Four new lantibiotics with a broad inhibitory spectrum were isolated, and these appear to be promising antibacterial agents.
AIM: To investigate which surgical techniques and perioperative regimens yielded the best survival rates for diabetic rats undergoing gastric bypass.
METHODS: We performed Roux-en-Y gastric bypass with reserved gastric volume, a procedure in which gastrointestinal continuity was reestablished while excluding the entire duodenum and proximal jejunal loop. We observed the procedural success rate, long-term survival, and histopathological sequelae associated with a number of technical modifications. These included: use of anatomical markers to precisely identify Treitz’s ligament; careful dissection along surgical planes; careful attention to the choice of regional transection sites; reconstruction using full-thickness anastomoses; use of a minimally invasive procedure with prohemostatic pretreatment and hemorrhage control; prevention of hypothermic damage; reduction in the length of the procedure; and accelerated surgical recovery using fast-track surgical modalities such as perioperative permissive underfeeding and goal-directed volume therapy.
RESULTS: The series of modifications we adopted reduced operation time from 110.02 ± 12.34 min to 78.39 ± 7.26 min (P < 0.01), and the procedural success rate increased from 43.3% (13/30) to 90% (18/20) (P < 0.01), with a long-term survival of 83.3% (15/18) (P < 0.01).
CONCLUSION: Using a number of fast-track and damage control surgical techniques, we have successfully established a stable model of gastric bypass in diabetic rats.
Duodenal-jejunal bypass; Type 2 diabetes mellitus; Minimally invasive surgery; Fast-track surgery; Damage control surgery; Permissive underfeeding; Goal-directed volume therapy
Paenibacillus sp. F6‐B70 was selected from several dozens of isolates with activity against methicillin‐resistant Staphylococcus aureus using a 16S rDNA‐based screening method. F6‐B70 contained polyketide synthase (PKS) and non‐ribosomal peptide synthetase (NRPS) clusters in its genome revealed by PCR amplification of conserved adenylation and ketosynthase (KS) domains. Phylogenetic data suggested that the strain hosts trans‐AT PKSs and their product may be a branched molecule. An antibiotic was subsequently isolated from the methanol extract of F6‐B70 cells. The molecular formula of the antibiotic was deduced to be C33H50NaO6 ([M + Na]+, m/z 565.3505) by analysis of electrospray ionization mass spectral data. Elucidation of the structure by nuclear magnetic resonance and infrared spectroscopy revealed that the active compound, paenimacrolidin (PAM), was a novel 22‐membered macrolide with side‐chains. The new antibiotic, mainly as a bacteriostatic agent, inhibits a couple of multidrug‐resistant Staphylococcus sp. strains. The antibiotic capacity of PAM was compromised by its instability, which can be overcome significantly with addition of an anti‐oxidant. To our knowledge, this is the first report of the isolation of an active macrolide from paenibacilli, which may be a promising source of novel antibiotics.
Directed differentiation of human embryonic stem cells (hESCs) into any desired cell type has been hailed as a therapeutic promise to cure many human diseases. However, substantial roadblocks still exist for in vitro differentiation of hESCs into distinct cell types, including T lymphocytes. Here we examined the hematopoietic differentiation potential of six different hESC lines. We compare their ability to develop into CD34+ or CD34+CD45+ hematopoietic precursor populations under several differentiation conditions. Comparison of lymphoid potential of hESC derived- and fetal tissue derived-hematopoietic precursors was also made. We found diverse hematopoietic potential between hESC lines depending on the culture or passage conditions. In contrast to fetal-derived hematopoietic precursors, none of the CD34+ precursors differentiated from hESCs were able to develop further into T cells. These data underscore the difficulties in the current strategy of hESC forward differentiation and highlight distinct differences between CD34+ hematopoietic precursors generated in vitro versus in vivo.
HMGA proteins are not translated in normal human somatic cells, but are present in high copy numbers in pluripotent embryonic stem cells and most neoplasias. Correlations between the degree of malignancy, patient prognostic index and HMGA levels have been firmly established. Intriguingly, HMGA2 is also found in rare tumor-inducing cells which are resistant to chemotherapy. Here, we demonstrate that HMGA1a/b and HMGA2 possess intrinsic dRP and AP site cleavage activities, and that lysines and arginines in the AT-hook DNA-binding domains function as nucleophiles. We also show that HMGA2 can be covalently trapped at genomic abasic sites in cancer cells. By employing a variety of cell-based assays, we provide evidence that the associated lyase activities promote cellular resistance against DNA damage that is targeted by base excision repair (BER) pathways, and that this protection directly correlates with the level of HMGA2 expression. In addition, we demonstrate an interaction between human AP endonuclease 1 and HMGA2 in cancer cells, which supports our conclusion that HMGA2 can be incorporated into the cellular BER machinery. Our study thus identifies an unexpected role for HMGA2 in DNA repair in cancer cells which has important clinical implications for disease diagnosis and therapy.
In response to a lymphopenic cue, T lymphocytes undergo a slow-paced homeostatic proliferation in an attempt to restore T cell cellularity. The molecular interaction that maintains the pace of homeostatic proliferation is unknown. In this study, we report that in lymphopenic CD24-deficient mice, T cells launch a massive proliferation that results in the rapid death of the recipient mice. The dividing T cells have phenotypes similar to those activated by cognate antigens. The rapid homeostatic proliferation is caused by a lack of CD24 on dendritic cells (DCs). Interestingly, although CD24 expression in T cells is required for optimal homeostatic proliferation in the wild-type (WT) host, mice lacking CD24 on all cell types still mount higher homeostatic proliferation than the WT mice. Thus, a lack of CD24 in the non–T host cells bypassed the requirement for T cell expression of CD24 in homeostatic proliferation in the WT host. Our data demonstrate that CD24 expressed on the DCs limits T cell response to homeostatic cue and prevents fatal damage associated with uncontrolled homeostatic proliferation.
It is well established that T lymphocytes undergo homeostatic proliferation in lymphopenic environment. The homeostatic proliferation requires recognition of the major histocompatibility complex on the host. Recent studies have demonstrated that costimulation-mediated CD28, 4-1BB, and CD40 is not required for T cell homeostatic proliferation. It has been suggested that homeostatic proliferation is costimulation independent. Here, we report that T cells from mice with a targeted mutation of CD24 have a remarkably reduced rate of proliferation when adoptively transferred into syngeneic lymphopenic hosts. The reduced proliferation cannot be attributed to abnormal survival and homing properties of the CD24-deficient T cells. T cell proliferation in allogeneic hosts is less affected by this mutation. These results demonstrate a novel function of CD24 expressed on T cells. Thus, although distinct costimulatory molecules are involved in antigen-driven proliferation and homeostatic proliferation, both processes can be modulated by costimulatory molecules.
costimulation; homeostasis; autoimmune diseases; immunological memory; T cell traffic
In the development of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS), autoreactive T cells must be activated and clonally expand in the lymphoid organs, and then migrate into the central nervous system (CNS) where they undergo further activation. It is unclear whether the autoreactive T cells further expand in the CNS and if so, what interactions are required for this process. We have demonstrated previously that expression by the host cells of the heat-stable antigen (CD24), which was recently identified as a genetic modifier for MS, is essential for their susceptibility to EAE. Here we show that CD24 is essential for local clonal expansion and persistence of T cells after their migration into the CNS, and that expression of CD24 on either hematopoietic cells or nonhematopoietic antigen-presenting cells in the recipient is sufficient to confer susceptibility to EAE.
costimulatory molecules; autoimmune diseases; central nervous system; multiple sclerosis; clonal expansion
B7H1 (PDL1) and B7DC (PDL2) are two new members of the B7 family that can interact with PD-1, a putative negative regulator for immune function. Recent studies have provided evidence for inhibitory functions of both members via PD-1. Meanwhile, compelling evidence exists for costimulatory function of both members. Here we demonstrate that expression of B7DC on the tumor cells promotes CD8 T cell–mediated rejection of tumor cells, at both the induction and effector phase of antitumor immunity. Moreover, B7DC binds to PD-1(−/−) cells and enhances T cell killing in a PD-1–independent mechanism. Our results demonstrate a novel pathway for B7DC to promote tumor immunity and may reconcile the apparently contradictory findings on the function of B7DC.
tumor immunity; costimulatory molecules; cytolytic T lymphocytes
It is established that mutations in viral antigenic epitopes, or antigenic drifts, allow viruses to escape recognition by both Ab’s and T lymphocytes. It is unclear, however, whether tumor cells can escape immune recognition via antigenic drift. Here we show that adoptive therapy with both monoclonal and polyclonal transgenic CTLs, specific for a natural tumor antigen, P1A, selects for multiple mutations in the P1A antigenic epitope. These mutations severely diminish T cell recognition of the tumor antigen by a variety of mechanisms, including modulation of MHC:peptide interaction and TCR binding to MHC:peptide complex. These results provide the first evidence for tumor evasion of T cell recognition by antigenic drift, and thus have important implications for the strategy of tumor immunotherapy.