The Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein, a toll-like receptor 4 agonist, enhances dendritic cell-based cancer vaccine potency

In this study, we showed the direct interaction between Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein (FAP) and toll-like receptor4 (TLR4) via co-localization and binding by using confocal microscopy and co-immunoprecipitation assays. FAP triggered the expression of pro- and anti-inflammatory cytokines in a TLR4-dependent manner. In addition, FAP-induced cytokine expression in bone marrow-derived dendritic cells (BMDCs) was modulated in part by glycogen synthase kinase-3 (GSK-3). FAP-induced expression of CD80, CD86, major histocompatibility complex (MHC) class I, and MHC class II in TLR4+/+ BMDCs was not observed in TLR4-/- BMDCs. Furthermore, FAP induced DC-mediated CD8+ T cell proliferation and cytotoxic T lymphocyte (CTL) activity, and suppressed tumor growth with DC-based tumor vaccination in EG7 thymoma murine model. Taken together, these results indicate that the TLR4 agonist, FAP, a potential immunoadjuvant for DC-based cancer vaccination, improves the DC-based immune response via the TLR4 signaling pathway.

[Survival analysis for laryngeal carcinoma patients with no surgery, radiotherapy or chemotherapy].

OBJECTIVE: To investigate the survival rate and prognostic factors of laryngeal carcinoma patients with no surgery, radiotherapy or chemotherapy. METHODS: One hundred and sixty-seven laryngeal carcinoma cases with no surgery, radiotherapy or chemotherapy were analyzed retrospectively. Survival rates were calculated by Kaplan-Meier product-limit method. With univariate analysis, comparisons among/between groups were performed using Log-rank test. Multivariate analysis was carried out using Cox proportional hazard model. RESULTS: Overall survival time was (16.0 ± 1.4) months (x(-) ± s), overall 1- and 2-year survival rates were 56.4% and 26.5%, respectively. No patient survived over 5 years in these cases who had been diagnosed more than 5 years (except for those who lost). Univariate analysis showed that primary site, pathological grade, T-stage, N-stage and clinical stage were significant prognostic factors for the survival of the patients (P < 0.05). The survival rates of laryngeal carcinoma whether with tracheotomy were no statistically significant (P > 0.05). Multivariate analysis showed survival rates statistically correlated with T stage and N stage (hazard ratio were 1.812 and 1.557, P < 0.05). CONCLUSIONS: The development of laryngeal carcinoma course was faster, without treatment to the tumor itself, even if palliative surgical such as tracheostomy would not improve the survival rate. In laryngeal carcinoma patients with no surgery, radiotherapy or chemotherapy, the factors affecting the survival rates include primary site, pathological grade, T-stage, N-stage and clinical stage, and of them, T-stage and N-stage are the independent prognostic factors.

Down-regulation of ATP-binding cassette transporter G1 expression by unmethylated CpG oligodeoxynucleotides in RAW 264.7 macrophages

We have investigated the effect of various forms of phosphodiester cytidine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) on the production of pro-inflammatory cytokines and related genes in RAW 264.7 macrophages. Treatment with the CpG ODNs increased the expression of tumor necrosis factor alpha (TNF-alpha), IL-6, and inducible nitric oxide synthase but not interleukin-1beta (IL-1beta). We also investigated the effect of CpG ODNs on the expression of ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) genes which are known to facilitate cholesterol efflux from macrophages for anti-atherosclerosis. CpG 2006 significantly reduced the levels of ABCG1 mRNA as determined by real-time polymerase chain reaction, whereas ABCA1 mRNA level was not changed. Western blot analysis further confirmed the reduction of ABCG1 protein expression by CpG 2006. In addition, we also determined the protein level of peroxisome proliferator activated receptor gamma (PPARgamma), which is recognized as a transcriptional activator of ABC transporters, was also reduced by CpG 2006. Thus, these results suggest that ABCG1 is specifically down-regulated by CpG 2006 in a PPARgamma-dependent manner in macrophages.

[RNA interference targeting c-Met inhibits proliferation of human laryngeal carcinoma Hep-2 cell line in vitro and in its xenografts in nude mice].

OBJECTIVE: The proto-oncogene c-Met was found to express on human laryngeal carcinoma Hep-2 cell line in previous research. In the present study, the author further examined whether inhibition of c-Met by RNA interference (RNAi) might inhibit biologic activity of Hep-2 cell line in vitro and proliferation using a murine laryngeal carcinoma model. METHODS: RNAi plasmid that can express small interfering RNA targeting c-Met or siRNA that did not match any known human coding mRNA(control siRNA plasmid)was designed, constructed, and transfected into Hep-2 cell line by using cationic liposome Lipofectamine2000 as transfecting agent. In vitro, the transfection efficacy was tested by RT-PCR and Western Blot method, then elected the most inhibitive c-Met-siRNA sequence. Cell proliferation, movement and invasion were studied using MTT, cell migration assay and cell invasion assay, respectively. The Hep-2 cells were transplanted into nude mice, then the time of tumor formation and growth were observed. After tumor formation, c-Met-siRNA was given as the anti-tumor therapy. Expression of c-Met, MMP-9 and VEGF were detected by Western Blot method. RESULTS: After the pSilencer2.0/c-Met-shRNA recombinant plasmid transfection into laryngeal carcinoma Hep-2 cells, the expression of mRNA and protein of c-Met decreased significantly in Hep-2 cells. On the 35th day after tumor vaccination, the tumor volume was (138 ± 27) mm³ in c-Met-siRNA transfection group, Which was diminished significantly in contrast with control group (P < 0.01). The expression of c-Met, MMP-9 and VEGF in the tumor of experiment group was decreased significantly, respectively (P < 0.05). CONCLUSIONS: The results indicated that c-Met-siRNA can down-regulate the expression of c-Met and markedly inhibit laryngeal carcinoma Hep-2 cell proliferation, movement and invasion and the growth of transplantation tumor of nude mice. The siRNA expressing plasmid mediated gene therapy might be a new strategy in targeting molecular therapy of cancer of larynx.

The interaction between interferon-induced protein with tetratricopeptide repeats-1 and eukaryotic elongation factor-1A.

It has been shown previously that in mammalian cells, interferon-induced protein with tetratricopeptide repeats-1(IFIT1) is rapidly synthesized in response to viral infection, functions as an inhibitor of translation by binding to the eukaryotic initiation factor-3, and consequently assigns resistive activity against viral invasion to cells. It has also been reported that IFIT1 is rapidly produced in response to other cell stress agents with no direct relation to virus such as bacterial lipopolysaccharide and interleukin-1, but its function under these non-viral infection cell stress conditions has yet to be elucidated. Here, we demonstrate an interaction between IFIT1 and eukaryotic elongation factor-1A (eEF1A) both in vitro, using recombinant proteins as bait in pull-down assays, and in vivo, using laser confocal microscopy and immunoprecipitation. In addition, we report the initial determination of the domain of IFIT1 that mediates this interaction. We also display that both IFIT1 and eEF1A protein levels are rapidly elevated, prolonged in tumor necrosis factor alpha pre-treated Raw264.7 cells, and most of those cells are induced to death by the end of investigations. Our results imply that under some stressful stimulations IFIT1 may participate in cell death pathways by interaction with eEF1A.

{N'-[(E)-(5-Bromo-2-oxidophen-yl)(phen-yl)methyl-ene]benzohydrazidato}pyridine-copper(II).

The asymmetric unit of title complex, [Cu(C(20)H(13)BrN(2)O(2))(C(5)H(5)N)], contains two independent mol-ecules. In each mol-ecule, the central Cu(II) atom has a square-planar environment formed by the tridentate hydrazone and the monodentate pyridine ligands, with the N atoms in a trans arrangement about the Cu(II) atom.

(meso-5,7,7,12,14,14-Hexamethyl-1,4,8,11-tetra-azacyclo-tetra-deca-4,11-diene)nickel(II) bis-[O,O'-bis(4-methyl-phen-yl) dithio-phosphate].

In the title compound, [Ni(C(16)H(32)N(4))](C(14)H(14)O(2)PS(2))(2) or [Ni(trans[14]dien)][S(2)P(OC(6)H(4)Me-4)(2)](2), where trans[14]dien is meso-5,7,7,12,14,14-hexa-methyl-1,4,8,11-tetra-azacyclo-tetra-deca-4,11-diene, the Ni(II) ion lies across a centre of inversion and is four-coordinated in a relatively undistorted square-planar arrangement by the four N atoms of the macrocyclic ligand trans[14]dien. The two O,O'-di(4-methyl-phen-yl)dithio-phos-phates act as counter-ions to balance the charge. Important geometric data include Ni-N = 1.9135 (16) and 1.9364 (15) Å.

{N'-[(E)-(5-Bromo-2-oxidophen-yl)(phenyl)-methyl-ene]-4-chloro-benzo-hydrazidato}pyridine-nickel(II).

The asymmetric unit of title complex, [Ni(C(20)H(12)BrClN(2)O(2))(C(5)H(5)N)], contains one complex with the central Ni atom in a slightly distorted square-planar environment, formed by the tridentate hydrazone and the monodentate pyridine ligands, with N atoms in a trans arrangement about the Ni atom.

N'-[(E)-(5-Bromo-2-hydroxy-phen-yl)(phen-yl)methyl-ene]benzohydrazide.

In the title compound, C(20)H(15)BrN(2)O(2), the C=N double bond displays a trans configuration. The crystal structure features an intra-molecular O-H⋯N hydrogen bond.

{N'-[(E)-1-(5-Bromo-2-oxidophen-yl)ethyl-idene]-4-chloro-benzohydrazidato}pyridinenickel(II).

The title complex, [Ni(C(15)H(10)BrClN(2)O(2))(C(5)H(5)N)], displays a square-planar coordination geometry around the Ni(II) ion, formed by the tridentate hydrazone and monodentate pyridine ligands, with the N atoms in a trans arrangement about the Ni center.

{N'-[(E)-(5-Bromo-2-oxidophen-yl)(phen-yl)methyl-ene]benzohydrazidato}pyridine-nickel(II).

The asymmetric unit of title complex, [Ni(C(20)H(13)BrN(2)O(2))(C(5)H(5)N)], contains two independent mol-ecules. In each mol-ecule, the central Ni(II) atom has a square-planar environment, formed by the tridentate hydrazone and the monodentate pyridine ligands, with the N atoms in a trans arrangement about the Ni(II) atom.

N'-[(E)-(5-Bromo-2-hydroxy-phen-yl)(phen-yl)methyl-idene]-4-chloro-benzo-hydrazide.

The Schiff base, C(20)H(14)BrClN(2)O(2), displays a trans conformation with respect to the C=N double bond. The aromatic rings at either end of the -C(=O)-NH-N=C- fragment are nearly parallel [dihedral angle = 3.4 (5)°]. The hydr-oxy group forms an intra-molecular hydrogen bond to the imino N atom.

(5-Bromo-2-hydroxy-phen-yl)(phen-yl)methanone.

In the title compound, C(13)H(9)BrO(2), the dihedral angle between the aromatic ring planes is 53.6 (1)°. The crystal structure is stabilized by intra-molecular O-H⋯O and inter-molecular C-H⋯O hydrogen bonding and C-H⋯π inter-actions.

N'-[(E)-1-(5-Bromo-2-hydroxy-phen-yl)ethyl-idene]benzohydrazide.

The C=N double bond in the title compound, C(15)H(13)BrN(2)O(2), is transE configured and the dihedral angle between the aromatic ring planes is 22.3 (1)°. The crystal structure is stabilized by intra-molecular O-H⋯O and inter-molecular N-H⋯O hydrogen bonds.

The upstream sequence of Mycobacterium leprae 18-kDa gene confers transcription repression activity in orientation-independent manner

In order to understand the role of the upstream region of the Mycobacterium leprae 18-kDa gene on the gene regulation, the region was divided into two at the -50 position from the first start codon of the gene and their effect on transcription was examined by using a LacZ transcriptional reporter gene assay. The presence of each of these two regions conferred transcription repression not only on its cognate M. lepraerae 18-kDa gene promoter, but also on a heterologous promoter such as the Mycobacterium bovis BCG hsp65 gene promoter. Moreover, it was found that these regions could confer transcription repression activity in both cases in an orientation-independent manner. Thus, these results indicate that the upstream region of the M. leprae 18-kDa gene harbors transcription repression responsive element(s) acting as an operator and can be further divided into two separately functional regions, suggesting a bipartite structure of the element(s). The identification of transcription repression activity of the upstream region in the M. leprae 18-kDa gene will contribute greatly for the understanding of the 18-kDa gene regulation mechanism, and provide also useful information for the manipulation of mycobacterium gene expression.

[Tuberculosis of pharynx and larynx: a report of 32 cases].

OBJECTIVE: To evaluate the trends and the clinical changes in tuberculosis of pharynx and larynx. METHODS: The clinical data of 32 patients with tuberculosis of pharynx and larynx from Jan. 1982 to Dec. 2000 in Daping hospital were studied retrospectively. RESULTS: (1) The local manifestations were mainly single lesion that commonly involved the vocal cord (10 cases). (2) The lesions appearances were mainly the proliferation such as mass (11 cases) or granulation(8 cases). (3) anti-tuberculosis is the main treatment, the operation is the second. Twelve patients cured in clinic, six patients received operation and cured without any complications. Fourteen patients condition controlled. CONCLUSION: The classical manifestations with tuberculosis of pharynx and larynx were not exited, the new clinical manifestations were associated with local lesion in nowadays.

Heterologous Regulation of BCG hsp65 Promoter by M. leprae 18 kDa Transcription Repression Responsive Element

Among a number of antigens characterized in M. leprae, an etiological agent of Leprosy, the 18 kDa antigen, is unique to M. leprae. We have previously determined a sequence specific element in the 18 kDa gene of M. leprae, which confers transcriptional repression. In this report, we have examined if the element could be applied to genes other than the 18 kDa gene of M. leprae. To identify the roles of the regulatory sequence in heterologous promoter, we have constructed pB3 vector series, which contains BCG hsp65 promoter and the M. leprae 18 kDa transcription repression responsive element in tandem using LacZ gene as a reporter gene. Cloning of hsp65 promoters of M. bovis BCG or M. smegmatis in front of LacZ gene resulted in normal beta- galactosidase activity as expected. However, when the sequence element was placed between the promoter and the LacZ gene, beta-galactosidase activity was reduced 10-fold less. Also we have examined with pB3(-) vector, that harbors the transcription repression responsive element in a reversed orientation, the beta-galactosidase activity was found to be similar to pB3(+) vector. Thus, these results further confirm that M. leprae 18 kDa transcription repression responsive element could regulate BCG hsp65 heterologous promoter and that the element could act as an operator for the transcription of mycobacteria.

Pseudotype HIV-1 Particles Carrying CD4

A defective HIV-1 helper virus DNA, pHyPC, was assembled by deleting the RNA packaging signal, env, nef and the 3'LTR sequences. HIV-1 like virus particles that carry the HIV-1 receptor, CD4 were generated by coexpression of pHyPC and plasmid DNAs encoding different chimeric CD4 proteins. The CD4 particles, sharing the CD4 ectodomain, precisely fused to different membrane anchors. CD4(+) particles specifically bound to HIV-1 Env expressing cells, but any signs of infection into these cells were not detected. Binding was only partially blocked by either polyclonal anti-CD4 antibodies or by high concentrations of soluble CD4. Suprisingly, CD4(+) particles also adsorbed to HeLa, CHO, NIH3T3 and COS-7 cells in the absence of HIV-1 Env expression. Adsorption was comparable in strength and speed to the highly specific CD4-Env interaction. CD4(-) particles exhibited only background levels of binding. Cell binding was CD4- dependent, but it was independent of the cell type from which the CD4(+) particles originated. Interestingly, CD4-dependent/Env-independent binding was only found when CD4 was present on virus particles. This suggests that the micro-environment of CD4 on virus particles uniquely expose this new cell binding activity. Its high affinity could explain in part why infection of Env(+) cells by CD4(+) particles was not detected. Further experiments will be required to evlauate whether this strong membrane interaction could represent one step in the multiple-step viral entry process.