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1.
Ying Yong Sheng Tai Xue Bao ; 35(3): 622-630, 2024 Mar 18.
Article in English | MEDLINE | ID: mdl-38646749

ABSTRACT

Soil nitrogen and phosphorus are two key elements limiting tree growth in subtropical areas. Understanding the regulation of soil microorganisms on nitrogen and phosphorus nutrition is beneficial to reveal maintenance mechanism of soil fertility in plantations. We analyzed the characteristics of soil nitrogen and phosphorus fractions, soil microbial community composition and function, and their relationship across three stands of two-layered Cunninghumia lanceolata + Phoebe bournei with different ages (4, 7 and 11 a) and the pure C. lanceolata plantation. The results showed that the contents of most soil phosphorus fractions increased with increasing two-layered stand age. The increase in active phosphorus fractions with increasing stand age was dominated by the inorganic phosphorus (9.9%-159.0%), while the stable phosphorus was dominated by the organic phosphorus (7.1%-328.4%). The content of soil inorganic and organic nitrogen also increased with increasing two-layered stand age, with NH4+-N and acid hydrolyzed ammonium N contents showing the strongest enhancement, by 152.9% and 80.2%, respectively. With the increase of stand age, the composition and functional groups of bacterial and fungal communities were significantly different, and the relative abundance of some dominant microbial genera (such as Acidothermus, Saitozyma and Mortierella) increased. The relative abundance of phosphorus solubilization and mineralization function genes, nitrogen nitrification function and aerobic ammonia oxidation function genes tended to increase. The functional taxa of fungi explained 48.9% variation of different phosphorus fractions. The conversion of pure plantations to two-layered mixed plantation affected soil phosphorus fractions transformation via changing the functional groups of saprophytes (litter saprophytes and soil saprophytes). Changes in fungal community composition explained 45.0% variation of different nitrogen fractions. Some key genera (e.g., Saitozyma and Mortierella) play a key role in promoting soil nitrogen transformation and accumulation. Therefore, the conversion of pure C. lanceolata plantation to two-layered C. lanceolata + P. bournei plantation was conducive to improving soil nitrogen and phosphorus availability. Bacteria and fungi played important roles in the transformation process of soil nitrogen and phosphorus forms, with greater contribution of soil fungi.


Subject(s)
Nitrogen , Phosphorus , Soil Microbiology , Soil , Phosphorus/analysis , Nitrogen/analysis , Nitrogen/metabolism , Soil/chemistry , Cunninghamia/growth & development , China , Bacteria/classification , Bacteria/growth & development , Bacteria/metabolism
2.
Cancer Lett ; 482: 126-135, 2020 07 10.
Article in English | MEDLINE | ID: mdl-31954770

ABSTRACT

Glioblastoma (GBM) has limited therapeutic options. DNA repair mechanisms contribute GBM cells to escape therapies and re-establish tumor growth. Multiple studies have shown that POLD2 plays a critical role in DNA replication, DNA repair and genomic stability. We demonstrate for the first time that POLD2 is highly expressed in human glioma specimens and that expression correlates with poor patient survival. siRNA or shRNA POLD2 inhibited GBM cell proliferation, cell cycle progression, invasiveness, sensitized GBM cells to chemo/radiation-induced cell death and reversed the cytoprotective effects of EGFR signaling. Conversely, forced POLD2 expression was found to induce GBM cell proliferation, colony formation, invasiveness and chemo/radiation resistance. POLD2 expression associated with stem-like cell subsets (CD133+ and SSEA-1+ cells) and positively correlated with Sox2 expression in clinical specimens. Its expression was induced by Sox2 and inhibited by the forced differentiation of GBM neurospheres. shRNA-POLD2 modestly inhibited GBM neurosphere-derived orthotopic xenografts growth, when combined with radiation, dramatically inhibited xenograft growth in a cooperative fashion. These novel findings identify POLD2 as a new potential therapeutic target for enhancing GBM response to current standard of care therapeutics.


Subject(s)
Brain Neoplasms/therapy , DNA Polymerase III/genetics , DNA Polymerase III/metabolism , Glioblastoma/therapy , RNA, Small Interfering/administration & dosage , Temozolomide/administration & dosage , Up-Regulation , Animals , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/radiation effects , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , DNA Polymerase III/antagonists & inhibitors , Drug Synergism , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/radiation effects , Glioblastoma/genetics , Glioblastoma/metabolism , Humans , Mice , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/radiation effects , RNA, Small Interfering/pharmacology , Radiotherapy , Survival Analysis , Temozolomide/therapeutic use , Up-Regulation/drug effects , Up-Regulation/radiation effects , Xenograft Model Antitumor Assays
3.
Cancer Commun (Lond) ; 39(1): 81, 2019 12 03.
Article in English | MEDLINE | ID: mdl-31796105

ABSTRACT

BACKGROUND: High-grade glioma (HGG) is a fatal human cancer. Bortezomib, a proteasome inhibitor, has been approved for the treatment of multiple myeloma but its use in glioma awaits further investigation. This study aimed to explore the chemotherapeutic effect and the underlying mechanism of bortezomib on gliomas. METHODS: U251 and U87 cell viability and proliferation were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, tumor cell spheroid growth, and colony formation assay. Cell apoptosis and cell cycle were detected by flow cytometry. Temozolomide (TMZ)-insensitive cell lines were induced by long-term TMZ treatment, and cells with stem cell characteristics were enriched with stem cell culture medium. The mRNA levels of interested genes were measured via reverse transcription-quantitative polymerase chain reaction, and protein levels were determined via Western blotting/immunofluorescent staining in cell lines and immunohistochemical staining in paraffin-embedded sections. Via inoculating U87 cells subcutaneously, glioma xenograft models in nude mice were established for drug experiments. Patient survival data were analyzed using the Kaplan-Meier method. RESULTS: Bortezomib inhibited the viability and proliferation of U251 and U87 cells in a dose- and time-dependent manner by inducing apoptosis and cell cycle arrest. Bortezomib also significantly inhibited the spheroid growth, colony formation, and stem-like cell proliferation of U251 and U87 cells. When administrated in combination, bortezomib showed synergistic effect with TMZ in vitro and sensitized glioma to TMZ treatment both in vitro and in vivo. Bortezomib reduced both the mRNA and protein levels of Forkhead Box M1 (FOXM1) and its target gene Survivin. The FOXM1-Survivin axis was markedly up-regulated in established TMZ-insensitive glioma cell lines and HGG patients. Expression levels of FOXM1 and Survivin were positively correlated with each other and both related to poor prognosis in glioma patients. CONCLUSIONS: Bortezomib was found to inhibit glioma growth and improved TMZ chemotherapy efficacy, probably via down-regulating the FOXM1-Survivin axis. Bortezomib might be a promising agent for treating malignant glioma, alone or in combination with TMZ.


Subject(s)
Antineoplastic Agents/therapeutic use , Bortezomib/therapeutic use , Brain Neoplasms/drug therapy , Forkhead Box Protein M1/genetics , Glioma/drug therapy , Survivin/genetics , Temozolomide/therapeutic use , Adolescent , Adult , Aged , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Bortezomib/pharmacology , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Down-Regulation/drug effects , Female , Forkhead Box Protein M1/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Glioma/genetics , Glioma/metabolism , Humans , Male , Mice, Inbred BALB C , Mice, Nude , Middle Aged , Survivin/metabolism , Temozolomide/pharmacology , Young Adult
4.
Front Genet ; 10: 1176, 2019.
Article in English | MEDLINE | ID: mdl-31803245

ABSTRACT

Acute otitis media (AOM) is the most common pediatric infection for which antibiotics are prescribed in the United States. The role of the respiratory tract microbiome in pathogenesis and immune modulation of AOM remains unexplored. We sought to compare the nasopharyngeal (NP) microbiome of children 1 to 3 weeks prior to onset of AOM vs. at onset of AOM, and the NP microbiome with the microbiome in middle ear (ME). Six children age 6 to 24 months old were studied. Nasal washes (NW) were collected at healthy visits 1 to 3 weeks prior to AOM and at onset of AOM. The middle ear fluids (MEF) were collected by tympanocentesis at onset of AOM. Samples were stored in Trizol reagents or phosphate-buffered saline (PBS) at -80°C until use. The microbiome was characterized by 16S rRNA gene sequencing. Taxonomic designations and relative abundance of bacteria were determined using the RDP classifier tool through QIIME. Cumulative sum scaling normalization was applied before determining bacterial diversity and abundance. Shannon diversity index was calculated in Microsoft excel. The relative abundance of each bacteria species was compared via Mann-Whitney U test. We found that the NW microbiome of children during healthy state or at baseline was more diverse than microbiome during AOM. At AOM, no significant difference in microbiome diversity was found between NW and MEF, although some bacteria species appear to differ in MEF than in NW. The microbiome of samples stored in PBS had significant greater diversity than samples stored in Trizol reagent.

5.
Clin Infect Dis ; 68(9): 1566-1574, 2019 04 24.
Article in English | MEDLINE | ID: mdl-30188973

ABSTRACT

BACKGROUND: Viral upper respiratory infections (URIs) are common and often precipitate acute otitis media (AOM), caused by bacterial otopathogens, in young children. Acute inflammatory responses initiated in the early phase of viral URI contribute to preventing the development of AOM. Stringently-defined otitis-prone (sOP) children are susceptible to recurrent AOM. METHODS: We assessed proinflammatory cytokine and chemokine levels in the nasopharynxes during viral URIs, and examined the different nasopharyngeal responses between viral URI events and the following AOM episodes in both sOP and non-otitis-prone (NOP) children. RESULTS: The sOP children exhibited significantly more AOM episodes per child (8.86-fold higher), viral URIs (P < .0001), and viral URIs followed by AOMs (P < .0001) than the NOP children. The sOP children had lower nasal proinflammatory levels of interleukin (IL)-6 (P = .05), IL-10 (P = .001), tumor necrosis factor (TNF)-α (P = .004), and regulated on activation, normal T-cell-expressed and -secreted (RANTES; P = .002) than NOP children during viral URIs. NOP children had higher levels of IL-6 (P = .02), IL-10 (P = .02), interferon-γ (P = .003), TNF-α (P = .006), IL-1ß (P = .022), monocyte chemoattractant protein 1 (P = .028), RANTES (P = .005), IL-2 (P = .002), and IL-17 (P = .007) during viral URIs versus AOMs following the URIs, when compared to sOP children. CONCLUSIONS: We conclude that sOP children have more frequent viral URIs than NOP children, due to deficient antiviral nasopharyngeal proinflammatory cytokine and chemokine responses.


Subject(s)
Cytokines/immunology , Inflammation/immunology , Nasopharynx/microbiology , Otitis Media/microbiology , Respiratory Tract Infections/virology , Child, Preschool , Female , Humans , Infant , Male , Otitis Media/etiology , Prospective Studies , Respiratory Tract Infections/complications
6.
J Med Microbiol ; 67(10): 1527-1532, 2018 Oct.
Article in English | MEDLINE | ID: mdl-30136923

ABSTRACT

PURPOSE: Nontypeable Haemophilus influenzae (NTHi) is a commensal in the human nasopharynx and the cause of pneumonia, meningitis, sinusitis, acute exacerbations of chronic obstructive pulmonary disease and acute otitis media (AOM). AOM is the most common ailment for which antibiotics are prescribed in the United States. With the emergence of new strains of antibiotic-resistant bacteria, finding an effective and broad coverage vaccine to protect against AOM-causing pathogens has become a priority. Mouse models are a cost-effective and efficient way to help determine vaccine efficacy. Here, we describe an NTHi AOM model in C57BL/6J mice, which also utilizes a mouse-adapted H1N1 influenza virus to mimic human coinfection. METHODOLOGY: We tested our coinfection model using a protein vaccine formulation containing protein D, a well-studied NTHi vaccine candidate that can be found in the 10-valent Streptococcus pneumoniae conjugate vaccine. We verified the usefulness of our mouse model by comparing bacterial loads in the nose and ear between protein D-vaccinated and control mice. RESULTS: While there was no measurable difference in nasal bacterial loads, we did detect significant differences in the bacterial loads of ear washes and ear bullae between vaccinated and control mice. CONCLUSION: The results from this study suggest that our NTHi AOM coinfection model is useful for assessing protein vaccines.


Subject(s)
Bacterial Proteins/immunology , Carrier Proteins/immunology , Haemophilus Infections/prevention & control , Haemophilus Vaccines/immunology , Haemophilus influenzae/immunology , Immunoglobulin D/immunology , Lipoproteins/immunology , Otitis Media/prevention & control , Administration, Intranasal , Animals , Antibodies, Bacterial/immunology , Bacterial Proteins/administration & dosage , Bacterial Proteins/genetics , Carrier Proteins/administration & dosage , Carrier Proteins/genetics , Coinfection/microbiology , Coinfection/prevention & control , Coinfection/virology , Disease Models, Animal , Female , Haemophilus Infections/microbiology , Haemophilus Vaccines/administration & dosage , Haemophilus Vaccines/genetics , Haemophilus influenzae/genetics , Haemophilus influenzae/growth & development , Humans , Immunoglobulin D/administration & dosage , Immunoglobulin D/genetics , Influenza A Virus, H1N1 Subtype/physiology , Lipoproteins/administration & dosage , Lipoproteins/genetics , Male , Mice , Mice, Inbred C57BL , Nose/microbiology , Nose/virology , Otitis Media/immunology , Otitis Media/microbiology , Vaccines, Conjugate/administration & dosage , Vaccines, Conjugate/immunology
7.
Oncogene ; 37(38): 5160-5174, 2018 09.
Article in English | MEDLINE | ID: mdl-29849122

ABSTRACT

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene have been identified in a number of cancer types, including brain cancer. The Cancer Genome Atlas project has revealed that IDH1 mutations occur in 70-80% of grade II and grade III gliomas. Until recently, most of the functional studies of IDH1 mutations in cellular models have been conducted in overexpression systems with the IDH1 wild type background. In this study, we employed a modified CRISPR/Cas9 genome editing technique called "single base editing", and efficiently introduced heterozygous IDH1 R132H mutation (IDH1R132H/WT) in human astroglial cells. Global DNA methylation profiling revealed hypermethylation as well as hypomethylation induced by IDH1R132H/WT. Global gene expression analysis identified molecular targets and pathways altered by IDH1R132H/WT, including cell proliferation, extracellular matrix (ECM), and cell migration. Our phenotype analysis indicated that compared with IDH1 wild type cells, IDH1R132H/WT promoted cell migration by upregulating integrin ß4 (ITGB4); and significantly inhibited cell proliferation. Using our mutated IDH1 models generated by "single base editing", we identified novel molecular targets of IDH1R132H/WT, namely Yes-associated protein (YAP) and its downstream signaling pathway Notch, to mediate the cell growth-inhibiting effect of IDH1R132H/WT. In summary, the "single base editing" strategy has successfully created heterozygous IDH1 R132H mutation that recapitulates the naturally occurring IDH1 mutation. Our isogenic cellular systems that differ in a single nucleotide in one allele of the IDH1 gene provide a valuable model for novel discoveries of IDH1R132H/WT-driven biological events.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Astrocytes/cytology , Down-Regulation/genetics , Heterozygote , Isocitrate Dehydrogenase/genetics , Mutation , Phosphoproteins/metabolism , Astrocytes/metabolism , Cell Movement/genetics , Cell Proliferation/genetics , Epigenesis, Genetic , Humans , Transcription Factors , YAP-Signaling Proteins
8.
Biochem Biophys Res Commun ; 499(4): 882-888, 2018 05 23.
Article in English | MEDLINE | ID: mdl-29625108

ABSTRACT

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene have been identified as one of the earliest events in gliomagenesis, occurring in over 70% of low grade gliomas and are present in the vast majority of secondary glioblastoma (GBM) that develop from these low-grade lesions. The aim of this study was to investigate whether the IDH1 R132H mutation influences the expression of oncogenic miR-20a and shed light on the underlying molecular mechanisms. The findings of the current study demonstrate presence of the IDH1 R132H mutation in primary human glioblastoma cell lines with upregulated HIF-1α expression, downregulating c-MYC activity and resulting in a consequential decrease in miR-20a, which is responsible for cell proliferation and resistance to standard temozolomide treatment. Elucidating the mechanism of oncogenic miR-20a activity introduces its role among well-established signaling pathways (i.e. HIF/c-MYC) and may be a meaningful prognostic biomarker or target for novel therapies among patients with IDH1-mutant glioma.


Subject(s)
Down-Regulation/genetics , Glioma/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Isocitrate Dehydrogenase/genetics , MicroRNAs/genetics , Mutation/genetics , Oncogenes , Proto-Oncogene Proteins c-myc/metabolism , Binding Sites , Cell Line, Tumor , Cell Proliferation/genetics , Dacarbazine/analogs & derivatives , Dacarbazine/pharmacology , Dacarbazine/therapeutic use , Down-Regulation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Glioma/drug therapy , Glioma/pathology , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Isocitrate Dehydrogenase/metabolism , MicroRNAs/metabolism , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins c-myc/genetics , Signal Transduction/drug effects , Temozolomide , Up-Regulation/drug effects , Up-Regulation/genetics
9.
J Infect ; 75(1): 26-34, 2017 07.
Article in English | MEDLINE | ID: mdl-28412081

ABSTRACT

OBJECTIVES: We sought to understand how polymicrobial colonization varies during health, viral upper respiratory infection (URI) and acute upper respiratory bacterial infection to understand differences in infection-prone vs. non-prone patients. METHODS: Nasopharyngeal (NP) samples were collected from 74 acute otitis media (AOM) infection-prone and 754 non-prone children during 2094 healthy visits, 673 viral URI visits and 631 AOM visits. Three otopathogens Streptococcus pneumoniae (Spn), Nontypeable Haemophilus influenzae (NTHi), and Moraxella catarrhalis (Mcat) were identified by culture. RESULTS: NP colonization rates of multiple otopathogens during health were significantly lower than during viral URI, and during URI they were lower than at onset of upper respiratory bacterial infection in both AOM infection-prone and non-prone children. AOM infection-prone children had higher polymicrobial colonization rates than non-prone children during health, viral URI and AOM. Polymicrobial colonization rates of AOM infection-prone children during health were equivalent to that of non-prone children during viral URI, and during viral URI were equivalent to that of non-prone during AOM infection. Spn colonization was positively associated with NTHi and Mcat colonization during health, but negatively during AOM infection. CONCLUSION: The infection-prone patients more frequently have multiple potential bacterial pathogens in the NP than the non-prone patients. Polymicrobial interaction in the NP differs during health and at onset of infection.


Subject(s)
Coinfection/epidemiology , Coinfection/microbiology , Nasopharynx/microbiology , Respiratory Tract Infections/microbiology , Respiratory Tract Infections/virology , Acute Disease/epidemiology , Bacteria/classification , Bacteria/isolation & purification , Bacterial Infections/epidemiology , Bacterial Infections/immunology , Bacterial Infections/microbiology , Child, Preschool , Coinfection/immunology , Disease Susceptibility/epidemiology , Disease Susceptibility/microbiology , Female , Haemophilus influenzae/isolation & purification , Haemophilus influenzae/physiology , Healthy Volunteers , Humans , Infant , Longitudinal Studies , Male , Moraxella catarrhalis/isolation & purification , Moraxella catarrhalis/physiology , Otitis Media/microbiology , Prospective Studies , Respiratory Tract Infections/epidemiology , Streptococcus pneumoniae/isolation & purification , Streptococcus pneumoniae/physiology
10.
Vaccine ; 35(2): 337-344, 2017 01 05.
Article in English | MEDLINE | ID: mdl-27919628

ABSTRACT

BACKGROUND: Currently licensed serotype-based pneumococcal vaccines are effective in preventing invasive pneumococcal diseases, but less effective in preventing non-bacteremic pneumonia and acute otitis media (AOM). We previously reported that a trivalent pneumococcal protein recombinant vaccine (PPrV) protected against pneumonia in a murine model. Here we evaluated PPrV protection against AOM in an infant murine model. METHODS: C57BL/6J mice were intramuscularly vaccinated at 1-3weeks of age with monovalent pneumococcal histidine triad protein D (PhtD), or pneumococcal choline binding protein A (PcpA), or detoxified pneumolysin (PlyD1), or trivalent vaccine, and transtympanically challenged at 7-8weeks of age with 1×102CFU of pneumococcal strain BG7322 (6A) or 1×104CFU of pneumococcal nontypeable strain 0702064MEF. Serum IgG titers were determined by ELISA. At 24 and 48h post infection (hpi), animals were sacrificed and middle ear fluid (MEF) samples were collected to determine pneumococcal CFUs. RESULTS: We found that vaccination of infant mice with monovalent and trivalent pneumococcal proteins elicited significant serum IgG antibody responses to corresponding component proteins. Vaccination with PhtD reduced BG7322 bacterial burdens in MEF at both 24 (p=0.05) and 48hpi (p=0.16). Vaccination with PcpA significantly reduced the bacterial burdens in MEF at both 24 (p=0.02) and 48hpi (p=0.004), and PlyD1 significantly reduced bacterial burden in MEF at 48hpi (p=0.02). Vaccination with trivalent PPrV (PhtD, PcpA and PlyD1) significantly reduced Spn burdens in MEF at both 24 (p=0.001) and 48hpi (p<0.0001). Similar reductions of bacterial burdens were found when the vaccinated animals were challenged with a non-typeable Spn strain. Vaccinated mice had significantly milder inflammatory cytokine levels (IL-1ß, IL-6, TNF-α, MIP-2 and KC) in middle ears at 24hpi (all p values<0.05). CONCLUSION: Trivalent PPrV confers protection against pneumococcal AOM in an infant murine model.


Subject(s)
Otitis Media/prevention & control , Pneumococcal Infections/prevention & control , Pneumococcal Vaccines/immunology , Streptococcus pneumoniae/immunology , Animals , Animals, Newborn , Antibodies, Bacterial/blood , Bacterial Load , Bacterial Proteins/immunology , Colony Count, Microbial , Disease Models, Animal , Ear, Middle/microbiology , Enzyme-Linked Immunosorbent Assay , Exudates and Transudates/microbiology , Female , Immunoglobulin G/blood , Injections, Intramuscular , Male , Mice, Inbred C57BL , Pneumococcal Vaccines/administration & dosage , Treatment Outcome , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunology
11.
Infect Immun ; 85(2)2017 02.
Article in English | MEDLINE | ID: mdl-27895132

ABSTRACT

An increase in Streptococcus pneumoniae nasopharynx (NP) colonization density during a viral coinfection initiates pathogenesis. To mimic natural S. pneumoniae pathogenesis, we commensally colonized the NPs of adult C57BL/6 mice with S. pneumoniae serotype (ST) 6A or 8 and then coinfected them with mouse-adapted H1N1 influenza A virus (PR/8/34). S. pneumoniae established effective commensal colonization, and influenza virus coinfection caused S. pneumoniae NP density to increase, resulting in bacteremia and mortality. We then studied histidine triad protein D (PhtD), an S. pneumoniae adhesin vaccine candidate, for its ability to prevent invasive S. pneumoniae disease in adult and infant mice. In adult mice, the efficacy of PhtD vaccination was compared with that of PCV13. Vaccination with PCV13 led to a greater reduction of S. pneumoniae NP density (>2.5 log units) than PhtD vaccination (∼1-log-unit reduction). However, no significant difference was observed with regard to the prevention of S. pneumoniae bacteremia, and there was no difference in mortality. Depletion of CD4+ T cells in PhtD-vaccinated adult mice, but not PCV13-vaccinated mice, caused a loss of vaccine-induced protection. In infant mice, passive transfer of antisera or CD4+ T cells from PhtD-vaccinated adult mice led to a nonsignificant reduction in NP colonization density, whereas passive transfer of antisera and CD4+ T cells was needed to cause a significant reduction in NP colonization density. For the first time, these data show an outcome with regard to prevention of invasive S. pneumoniae pathogenesis with a protein vaccine similar to that which occurs with a glycoconjugate vaccine despite a less robust reduction in NP bacterial density.


Subject(s)
Bacterial Proteins/immunology , Influenza A virus , Influenza, Human , Nasopharynx/immunology , Nasopharynx/microbiology , Pneumococcal Infections/immunology , Pneumococcal Infections/microbiology , Pneumococcal Vaccines/immunology , Streptococcus pneumoniae/immunology , Animals , Antibodies, Bacterial/immunology , Bacterial Load , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Carrier State , Coinfection , Disease Models, Animal , Humans , Lymphocyte Activation/immunology , Mice , Pneumococcal Infections/prevention & control , Serogroup , Streptococcus pneumoniae/classification
12.
Oncol Rep ; 36(5): 2935-2945, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27632941

ABSTRACT

The substrates and mechanisms of ubiquitin specific peptidase 7 (USP7) in glioma remain unclear. Lysine­specific demethylase 1 (LSD1) may undergo proteasomal degradation; however, a reciprocal mechanism that stabilizes LSD1 in glioma has not been dertermined. Here co-immunoprecipitation and GST pull-down assays revealed that LSD1 is associated with USP7 in vivo and in vitro. USP7 inhibited LSD1 ubiquitination and stabilized LSD1 in A172 and T98G cells. MTT, EdU proliferation, flow cytometry and Transwell assays indicated that LSD1 played a critical role in the proliferation and invasion of glioblastoma (GBM) cells. We defined the mechanism of USP7 in GBM, through counterbalanced LSD1 ubiquitylation. USP7 caused G0/G1 arrest, promoted tumorigenesis and invasion of A172 and T98G cells. We also uncovered the suppression of the p53 signaling pathway that mediated the activity of USP7 and LSD1. Furthermore, USP7 and LSD1 expression levels were higher in the 150 glioma patients than these levels in normal brain tissues and were correlated with glioma progression. LSD1 was increased concurrently with USP7 during glioblastoma progression and both were predictors for worsened prognosis. Collectively, our study suggested that USP7-LSD1 affects GBM cell proliferation and invasion and may be valuable as novel therapeutic targets and prognostic tools for GBM.


Subject(s)
Glioblastoma/genetics , Histone Demethylases/biosynthesis , Tumor Suppressor Protein p53/genetics , Ubiquitin Thiolesterase/biosynthesis , Adult , Aged , Carcinogenesis/genetics , Cell Proliferation/genetics , Glioblastoma/pathology , Histone Demethylases/genetics , Humans , Male , Middle Aged , Neoplasm Metastasis , Prognosis , Signal Transduction , Ubiquitin/genetics , Ubiquitin Thiolesterase/genetics , Ubiquitin-Specific Peptidase 7
13.
Cancer Sci ; 107(5): 583-9, 2016 May.
Article in English | MEDLINE | ID: mdl-27171351

ABSTRACT

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Despite great improvements in the therapeutic regimen, relapse and leptomeningeal dissemination still pose great challenges to the long-term survival of MB patients. Developing more effective strategies has become extremely urgent. In recent years, a number of malignancies, including MB, have been found to contain a subpopulation of cancer cells known as cancer stem cells (CSCs), or tumor initiating/propagating cells. The CSCs are thought to be largely responsible for tumor initiation, maintenance, dissemination, and relapse; therefore, their pivotal roles have revealed them to be promising targets in MB therapy. Our growing understanding of the major medulloblastoma molecular subgroups and the derivation of some of these groups from specific stem or progenitor cells adds additional layers to the CSC knowledge base. Herein we review the current knowledge of MB stem cells, highlight the molecular mechanisms relating to MB relapse and leptomeningeal dissemination, and incorporate these with the need to develop more effective and accurate therapies for MB patients.


Subject(s)
Drug Resistance, Neoplasm/drug effects , Medulloblastoma/drug therapy , Medulloblastoma/pathology , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/pathology , Animals , Cell Separation , Humans , Meningeal Neoplasms/secondary , Neoplasm Recurrence, Local , Neoplastic Stem Cells/metabolism , Signal Transduction
14.
Exp Cell Res ; 343(2): 148-158, 2016 05 01.
Article in English | MEDLINE | ID: mdl-27090014

ABSTRACT

PURPOSE: The aim of this study was to investigate the effect of downregulation of HIF-1α gene on human U251 glioma cells and examine the consequent changes of TMZ induced effects and explore the molecular mechanisms. METHODS: U251 cell line stably expressing HIF-1α shRNA was acquired via lentiviral vector transfection. The mRNA and protein expression alterations of genes involved in our study were determined respectively by qRT-PCR and Western blot. Cell proliferation was measured by MTT assay and colony formation assay, cell invasion/migration capacity was determined by transwell invasion assay/wound healing assay, and cell apoptosis was detected by flow cytometry. RESULTS: We successfully established a U251 cell line with highly efficient HIF-1α knockdown. HIF-1a downregulation sensitized U251 cells to TMZ treatment and enhanced the proliferation-inhibiting, invasion/migration-suppressing, apoptosis-inducing and differentiation-promoting effects exerted by TMZ. The related molecular mechanisms demonstrated that expression of O(6)-methylguanine DNA methyltransferase gene (MGMT) and genes of Notch1 pathway were significantly upregulated by TMZ treatment. However, this upregulation was abrogated by HIF-1α knockdown. We further confirmed important regulatory roles of HIF-1α in the expression of MGMT and activation of Notch1 pathways. CONCLUSION: HIF-1α downregulation sensitizes U251 glioma cells to the temozolomide treatment via inhibiting MGMT expression and Notch1 pathway activation.


Subject(s)
Brain Neoplasms/drug therapy , Brain Neoplasms/genetics , Dacarbazine/analogs & derivatives , Down-Regulation/drug effects , Glioma/drug therapy , Glioma/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Apoptosis/drug effects , Brain Neoplasms/pathology , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Dacarbazine/pharmacology , Dacarbazine/therapeutic use , Down-Regulation/genetics , Gene Expression Regulation, Neoplastic/drug effects , Glioma/pathology , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Neoplasm Invasiveness , Neoplasm Proteins/metabolism , RNA, Small Interfering/metabolism , Signal Transduction/drug effects , Temozolomide , Transfection
15.
Hum Vaccin Immunother ; 12(1): 194-205, 2016.
Article in English | MEDLINE | ID: mdl-26539741

ABSTRACT

All currently available Streptococcus pneumoniae (Spn) vaccines have limitations due to their capsular serotype composition. Both the 23-valent Spn polysaccharide vaccine (PPV) and 7, 10, or 13-valent Spn conjugate vaccines (PCV-7, 10, -13) are serotype-based vaccines and therefore they elicit only serotype-specific immunity. Emergence of replacement Spn strains expressing other serotypes has consistently occurred following introduction of capsular serotype based Spn vaccines. Furthermore, capsular polysaccharide vaccines are less effective in protection against non-bacteremic pneumonia and acute otitis media (AOM) than against invasive pneumococcal disease (IPD). These shortcomings of capsular polysaccharide-based Spn vaccines have created high interest in development of non-serotype specific protein-based vaccines that could be effective in preventing both IPD and non-IPD infections. This review discusses the progress to date on development of Spn protein vaccine candidates that are highly conserved by all Spn strains, are highly conserved, exhibit maximal antigenicity and minimal reactogenicity to replace or complement the current capsule-based vaccines. Key to development of a protein based Spn vaccine is an understanding of Spn pathogenesis. Based on pathogenesis, a protein-based Spn vaccine should include one or more ingredients that reduce NP colonization below a pathogenic inoculum. Elimination of all Spn colonization may not be achievable or even advisable. The level of expression of a target protein antigen during pathogenesis is another key to the success of protein based vaccines.. As with virtually all currently licensed vaccines, production of a serum antibody response in response to protein based vaccines is anticipated to provide protection from Spn infections. A significant advantage that protein vaccine formulations can offer over capsule based vaccination is their potential benefits associated with natural priming and boosting to all strains of Spn. One of the most universal and comprehensive approaches of identifying novel vaccine candidates is the investigation of human sera from different disease stages of natural infections. Antigens that are robustly reactive in preliminary human serum screening constitute a pathogen-specific antigenome. This strategy has identified a number of Spn protein vaccine candidates that are moving forward in human clinical trials.


Subject(s)
Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Drug Discovery/trends , Pneumococcal Vaccines/immunology , Pneumococcal Vaccines/isolation & purification , Streptococcus pneumoniae/immunology , Animals , Clinical Trials as Topic , Drug Evaluation, Preclinical , Humans
16.
Pediatr Infect Dis J ; 35(1): 54-60, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26448450

ABSTRACT

OBJECTIVES: Acute otitis media (AOM) is the most common pediatric bacterial infection, and stringently defined otitis-prone (sOP) children have immunologic deficiencies. We recently found that nasopharyngeal (NP) colonization by Streptococcus pneumoniae (Spn) elicits a NP mucosal antibody response to vaccine candidate pneumococcal proteins that correlate with protection from AOM in non-sOP (NOP) children. Here, we sought to determine if sOP children experience significantly higher colonization rates with Spn than NOP children, develop lower naturally acquired NP mucosal antibody responses to those same pneumococcal proteins after colonization by Spn, and suffer greater frequency of AOM as a consequence. METHODS: NP samples were collected from 130 NOP and 45 sOP children during 270 healthy visits and 201 AOM visits between 6 and 24 months of age. Spn were identified by standard culture. NP mucosal IgG and IgA levels to vaccine candidate proteins pneumococcal histidine triad protein D, pneumococcal choline binding protein A (PcpA) and pneumolysin D1 were measured by quantitative enzyme-linked immunosorbent assay. RESULTS: sOP children had significantly higher colonization frequency by Spn (P < 0.0001) and significantly lower IgG and IgA levels to all 3 vaccine candidate proteins studied compared with NOP children (all P values <0.05) except IgG to Ply D1 (P = 0.31). Spn colonization in NOP children led to 2-fold to 5-fold increase in mucosal IgG and IgA levels to all 3 proteins (all P values <0.01), whereas Spn colonization in sOP children generally failed to elicit antibody responses (all P values >0.05). PcpA was unique in inducing significant increases in mucosal IgA (P = 0.02). When high mucosal IgG levels to all 3 proteins and IgA to PcpA were measured, they correlated with reduced AOM in sOP children. CONCLUSION: sOP children experience significantly higher colonization rates with Spn, develop lower naturally acquired NP mucosal antibody responses to pneumococcal vaccine candidate proteins pneumococcal histidine triad protein D, PcpA and pneumolysin D1 after colonization by Spn, and suffer greater frequency of AOM if they do not generate high mucosal antibody to the studied proteins.


Subject(s)
Adaptive Immunity , Antibodies, Bacterial/immunology , Nasopharynx/immunology , Nasopharynx/microbiology , Otitis Media/immunology , Otitis Media/microbiology , Pneumococcal Infections/immunology , Pneumococcal Infections/microbiology , Respiratory Mucosa/immunology , Respiratory Mucosa/microbiology , Streptococcus pneumoniae/immunology , Antigens, Bacterial/immunology , Case-Control Studies , Child, Preschool , Cohort Studies , Female , Humans , Immunization , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Infant , Male
17.
Oncol Rep ; 34(1): 318-24, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25954994

ABSTRACT

Previous studies have focused on miRNA expression in brain gliomas. However, both the expression pattern of miRNAs in gliomas of different grades and various miRNAs involved in malignant progression of gliomas are poorly understood. In the present study, we used miRNA microarray-based screening to investigate the miRNA expression profile in gliomas, which was further verified by qRT-PCR in selected miRNAs. In total, we found 13 differentially expressed miRNAs between gliomas and their matched surrounding tissues. Among them, 12 miRNAs were upregulated and only one (miR-4489) was downregulated compared with the control. Furthermore, the lower expression level of miR-4489 was confirmed by qRT-PCR in 26 glioma samples. Our microarray result revealed 8, 9 and 15 aberrantly expressed miRNAs in gliomas of World Health Organization (WHO) grade II-IV, respectively. Gene Ontology (GO) and Pathway analysis indicated that target genes of the 13 miRNAs were significantly enriched in central nervous system- and tumor-related biological processes and signaling pathways. The dysregulated miRNAs identified in the present study contribute to the tumorigenesis and malignant progression of gliomas and may serve as useful markers for advanced glioma pathological grading and prognosis.


Subject(s)
Brain Neoplasms/genetics , Gene Regulatory Networks/genetics , Glioma/genetics , MicroRNAs/biosynthesis , Adolescent , Adult , Aged , Brain Neoplasms/pathology , Cell Transformation, Neoplastic/genetics , Child , Child, Preschool , Disease Progression , Female , Gene Expression Regulation, Neoplastic , Glioma/pathology , Humans , Male , MicroRNAs/genetics , Microarray Analysis , Middle Aged , Neoplasm Grading , Prognosis
18.
Vaccine ; 33(8): 993-1000, 2015 Feb 18.
Article in English | MEDLINE | ID: mdl-25597944

ABSTRACT

OBJECTIVE: Due to the fact that current polysaccharide-based pneumococcal vaccines have limited serotype coverage, protein-based vaccine candidates have been sought for over a decade to replace or complement current vaccines. We previously reported that a trivalent Pneumococcal Protein recombinant Vaccine (PPrV), showed protection against pneumonia and sepsis in an infant murine model. Here we investigated immunological correlates of protection of PPrV in the same model. METHODS: C57BL/6J infant mice were intramuscularly vaccinated at age 1-3 weeks with 3 doses of PPrV, containing pneumococcal histidine triad protein D (PhtD), pneumococcal choline binding protein A (PcpA), and detoxified pneumolysin mutant PlyD1. 3-4 weeks after last vaccination, serum and lung antibody levels to PPrV components were measured, and mice were intranasally challenged with a lethal dose of Streptococcus pneumoniae (Spn) serotype 6A. Lung Spn bacterial burden, number of neutrophils and alveolar macrophages, phagocytosed Spn by granulocytes, and levels of cytokines and chemokines were determined at 6, 12, 24, and 48h after challenge. RESULTS: PPrV vaccination conferred 83% protection against Spn challenge. Vaccinated mice had significantly elevated serum and lung antibody levels to three PPrV components. In the first stage of pathogenesis of Spn induced pneumonia (6-24h after challenge), vaccinated mice had lower Spn bacterial lung burdens and more phagocytosed Spn in the granulocytes. PPrV vaccination led to lower levels of pro-inflammatory cytokines IL-6, IL-1ß, and TFN-α, and other cytokines and chemokines (IL-12, IL-17, IFN-γ, MIP-1b, MIP-2 and KC, and G-CSF), presumably due to a lower lung bacterial burden. CONCLUSION: Trivalent PPrV vaccination results in increased serum and lung antibody levels to the vaccine components, a reduction in Spn induced lethality, enhanced early clearance of Spn in lungs due to more rapid and thorough phagocytosis of Spn by neutrophils, and correspondingly a reduction in lung inflammation and tissue damage.


Subject(s)
Neutrophils/immunology , Phagocytosis/immunology , Pneumococcal Vaccines/immunology , Pneumonia, Pneumococcal/immunology , Pneumonia, Pneumococcal/prevention & control , Streptococcus pneumoniae/immunology , Animals , Chemotaxis, Leukocyte/immunology , Cytokines/biosynthesis , Disease Models, Animal , Female , Granulocytes/immunology , Humans , Lung/immunology , Lung/metabolism , Lung/microbiology , Lung/pathology , Macrophages, Alveolar/immunology , Male , Mice , Pneumonia, Pneumococcal/metabolism , Pneumonia, Pneumococcal/mortality , Vaccination
19.
Clin Infect Dis ; 59(3): 376-83, 2014 Aug 01.
Article in English | MEDLINE | ID: mdl-24785236

ABSTRACT

BACKGROUND: Acute otitis media (AOM) is a leading cause of bacterial pediatric infections associated with viral upper respiratory infections (URIs). We examined the differential impact of respiratory syncytial virus (RSV) and parainfluenza virus URIs on the frequency of AOM caused by Streptococcus pneumoniae (Spn) and nontypeable Haemophilus influenzae (NTHi) in stringently defined otitis-prone (sOP) and non-otitis-prone (NOP) children as a potential mechanism to explain increased susceptibility to AOM. METHODS: Peripheral blood and nasal washes were obtained from sOP and NOP children (n = 309). Colonization events and antiviral responses consisting of total specific immunoglobulin G (IgG) responses, neutralizing antibody responses, and T-cell responses were determined. Isolated neutrophils were infected with varying multiplicities of infection of both viruses, and opsonophagocytosis potential was measured. RESULTS: A significant increase was found in frequency of AOM events caused by Spn and NTHi, with a concurrent RSV infection in sOP children. These results correlated with diminished total RSV-specific IgG, higher viral nasal burdens, and lower IgG neutralizing capacity. The sOP children had diminished T-cell responses to RSV that correlated with lower Toll-like receptor 3/7 transcript and decreased expression of HLA-DR on antigen-presenting cells. RSV interfered with the Spn phagocytic capacity of neutrophils in a dose-dependent manner. Parainfluenza virus infections did not differentially affect AOM events in sOP and NOP children. CONCLUSIONS: Lower innate and adaptive immune responses to RSV in sOP children may slow the kinetics of viral clearance from the nasopharynx and allow for viral interference with antibacterial immune responses, thus contributing to increased frequency of AOMs.


Subject(s)
Adaptive Immunity , Haemophilus influenzae/isolation & purification , Immunity, Innate , Otitis Media/immunology , Paramyxoviridae/isolation & purification , Respiratory Syncytial Viruses/isolation & purification , Streptococcus pneumoniae/isolation & purification , Acute Disease , Child, Preschool , Female , HLA-DR Antigens/immunology , Humans , Infant , Male , Nasopharynx/microbiology , Otitis Media/microbiology , Prospective Studies , Toll-Like Receptor 3/immunology , Toll-Like Receptor 7/immunology
20.
Vaccine ; 32(26): 3205-10, 2014 May 30.
Article in English | MEDLINE | ID: mdl-24731814

ABSTRACT

Streptococcus pneumoniae infections continue to cause significant worldwide morbidity and mortality despite the availability of efficacious serotype-dependent vaccines. The need to incorporate emergent strains expressing additional serotypes into pneumococcal polysaccharide conjugate vaccines has led to an identified need for a pneumococcal protein-based vaccine effective against a broad scope of serotypes. A vaccine consisting of several conserved proteins with different functions during pathogenesis would be preferred. Here, we investigated the efficacy of a trivalent recombinant protein vaccine containing pneumococcal choline-binding protein A (PcpA), pneumococcal histidine triad D (PhtD), and genetically detoxified pneumolysin (PlyD1) in an infant mouse model. We found the trivalent vaccine conferred protection from lethal pneumonia challenges using serotypes 6A and 3. The observed protection with trivalent PcpA, PhtD, and PlyD1 vaccine in infant mice supports the ongoing study of this candidate vaccine in human infant clinical trials.


Subject(s)
Bacterial Proteins/immunology , Carrier Proteins/immunology , Hydrolases/immunology , Pneumococcal Infections/prevention & control , Pneumococcal Vaccines/immunology , Streptolysins/immunology , Animals , Animals, Newborn , Antibodies, Bacterial/blood , Female , Immunoglobulin G/blood , Intracellular Signaling Peptides and Proteins , Lung/pathology , Male , Mice , Mice, Inbred C57BL , Recombinant Proteins/immunology , Sepsis/prevention & control , Vaccines, Conjugate/immunology
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