Detection performance of PCR for Legionella pneumophila in environmental samples: a systematic review and meta-analysis.

BACKGROUND: Legionellosis remains a public health problem. The most common diagnostic method to detect Legionella pneumophila (L. pneumophila) is culture. Polymerase chain reaction (PCR) is a fast and accurate method for this detection in environmental samples. METHODS: Four databases were searched for studies that evaluated the detection efficiency of PCR in L. pneumophila. The quality evaluation was conducted using Review Manager 5.3. We used Meta-DiSc 1.4 software and the Stata 15.0 software to create forest plots, a meta-regression, a bivariate boxplot and a Deeks' funnel plot. RESULTS: A total of 18 four-fold tables from 16 studies were analysed. The overall pooled sensitivity and specificity of PCR was 94% and 72%, respectively. The positive likelihood ratio (RLR) and negative likelihood ratio (NLR) was 2.73 and 0.12, respectively. The result of the diagnostic odds ratio (DOR) was 22.85 and the area under the curve (AUC) was 0.7884. CONCLUSION: Establishing a laboratory diagnostic tool for L. pneumophila detection is important for epidemiological studies. In this work, PCR demonstrated a promising diagnostic accuracy for L. pneumophila.

Strength Development Monitoring of Cemented Paste Backfill Using Guided Waves.

The strength of cemented paste backfill (CPB) directly affects mining safety and progress. At present, in-situ backfill strength is obtained by conducting uniaxial compression tests on backfill core samples. At the same time, it is time-consuming, and the integrity of samples cannot be guaranteed. Therefore guided wave technique as a nondestructive inspection method is proposed for the strength development monitoring of cemented paste backfill. In this paper, the acoustic parameters of guided wave propagation in the different cement-tailings ratios (1:4, 1:8) and different curing times (within 42 d) of CPBs were measured. Combined with the uniaxial compression strength of CPB, relationships between CPB strength and the guided wave acoustic parameters were established. Results indicate that with the increase of backfill curing time, the guided wave velocity decreases sharply at first; on the contrary, attenuation of guided waves increases dramatically. Finally, both velocity and attenuation tend to be stable. When the CPB strength increases with curing time, guided wave velocity shows an exponentially decreasing trend, while the guided wave attenuation shows an exponentially increasing trend with the increase of the CPB strength. Based on the relationship curves between CPB strength and guided wave velocity and attenuation, the guided wave technique in monitoring the strength development of CPB proves feasible.

Influence of Nanopore Structure Deformation on Gas Migration in Coal.

To better understand the influence and control of nanopore characteristics on gas migration, three kinds of coal samples with different metamorphic degrees were selected for the experiments including high-pressure isothermal gas adsorption, low-pressure CO2 adsorption, and low-pressure Ar adsorption. The changes of the pore volume (PV) and specific surface area (SSA) of coal samples before and after adsorption-desorption were compared and analyzed. The adsorption data of all coal samples at a low pressure stage (<8 MPa) conformed to the Langmuir equation, and the adsorption capacity of powdered coal samples was higher than that of columnar coal samples. Some adsorption data deviated from the original fitting curve at a high pressure stage (>8 MPa), and this was the most remarkable in columnar coal samples. There was a positive correlation between the cumulative SSA of pores and adsorption capacity of coal samples. When the adsorption time was more than 10 min, the adsorption efficiency of 200 mesh coal samples from YJL was lower than those of 200 mesh coal samples from CZ and WY, which was due to the good development and connectivity of micro-fissures and nanopores in YJL coal samples. The pore size distribution of coal samples had changed after adsorption-desorption, and the cumulative deformation of the nanopore structure was anisotropic. As a result of the swelling or shrinkage deformation of the coal matrix, the PV and SSA with the same pore size presented many forms, such as almost unchanged, increased, or decreased. There are two types of deformation mechanisms: the whole collaborative deformation and partial deformation. Both gas adsorption and desorption can lead to the shrinkage or swell deformation of nanopores and fissures. In brief, the research provides theoretical and technical support for reservoir evaluation, fine drainage, and efficient development of coalbed methane.

The effect of environmental factors on the differential expression of miRNAs in patients with chronic obstructive pulmonary disease: a pilot clinical study.

Objective: The objective of the study was to analyze the effect of environmental factors on the differential expression of microRNAs in the peripheral blood of migratory and local patients in northern People's Republic of China and on clinical symptoms of local patients in northern People's Republic of China with COPD. Methods: A total of 118 patients in the northern region and 8 migratory patients were enrolled in this prospective study. We collected general information. Blood samples were collected from 9 patients in the Beijing group, from 8 patients in the migratory group and from 9 healthy control subjects. After extracting the total RNA from these 3 groups, serum miRNA was identified by Solexa sequencing. We collected COPD assessment test (CAT) and Modified British Medical Research Council (mMRC) scores at different levels of air pollution and also collected the number of exacerbations over the year prior to the baseline and in the year preceding the follow-up. Results: In total 9 miRNAs were differentially expressed. When air quality index (AQI) >100, the CAT and mMRC scores at baseline were significantly higher than those when the AQI ≤100 (P<0.001). When AQI >100, the follow-up CAT and mMRC scores were significantly higher than those when AQI ≤100 (P<0.001). Follow-up mMRC scores were significantly higher than baseline scores (P=0.04). When AQI ≤100, the baseline CAT score of the group with fewer symptoms was 6.50 (4.00-8.75). However, when AQI >100, the baseline CAT score of this fewer symptoms group was 10.00 (6.25-12.00). The median CAT score was close to 10. When AQI ≤100, the follow-up CAT score of the fewer symptoms group was 8.00 (4.25-12.00). However, when AQI >100, the follow-up CAT score of the fewer symptoms group was 9.50 (6.00-16.75). The median CAT score was close to 10. Conclusion: Environmental factors may cause differential expression of miRNAs in the peripheral blood of migratory and local patients in northern People's Republic of China. Air pollution may aggravate clinical symptoms of patients with COPD.

Pulmonary function tests findings and their diagnostic value in patients with IgG4-related disease.

BACKGROUND: IgG4-related disease (IgG4-RD) is an immune-mediated fibroinflammatory disorder that can affect most organs. To date, there have been no detailed assessments of pulmonary function in patients with IgG4-RD. In this study, we investigated pulmonary function in IgG4-RD patients and evaluated the value of pulmonary function tests (PFTs) in diagnosing IgG4-related respiratory disease (IgG4-RRD). METHODS: This was a retrospective study of 17 patients with IgG4-RD. The patients were divided into two groups: IgG4-RRD group and IgG4-related disease extrapulmonary involvement (IgG4-RDEI) group. The PFT results were compared between the two groups. RESULTS: All patients in the IgG4-RRD group had pulmonary dysfunction. Five of 8 (62.5%) patients in the IgG4-RDEI group had pulmonary dysfunction, despite having normal thoracic computed tomography scans and no respiratory symptoms. Patients in both groups showed restrictive ventilatory dysfunction and abnormal diffusing capacity, and two patients in the IgG4-RRD group had obstructive ventilatory dysfunction. The incidence of diffusing capacity of the lung for carbon monoxide per liter of alveolar volume (DLCO/VA) decrease were significantly higher in the IgG4-RRD group than in the IgG4-RDEI group (P=0.029). DLCO/VA were significantly higher in the IgG4-RDEI than in the IgG4-RRD group (P=0.044), but otherwise, there were no significant differences. We report the first finding of a negative correlation between pulmonary diffusing capacity and total serum concentrations of IgG and IgG subclasses (IgG4, IgG3 and IgG2). CONCLUSIONS: DLCO/VA plays an important role for detecting lung involvement in IgG4-RD patients. The patient with high serum IgG may be more prone to respiratory involvement.

Pulmonary function test findings in patients with acute inhalation injury caused by smoke bombs.

BACKGROUND: This study aimed to determine the effects of smoke bomb-induced acute inhalation injury on pulmonary function at different stages of lung injury. METHODS: We performed pulmonary function tests (PFTs) in 15 patients with acute inhalation injury from days 3 to 180 after smoke inhalation. We measured the trace element zinc in whole blood on days 4 and 17, and correlations of zinc levels with PFTs were performed. RESULTS: In the acute stage of lung injury (day 3), 3 of 11 patients with mild symptoms had normal pulmonary function and 8 patients with restrictive ventilatory dysfunction and reduced diffusing capacity. Some patients also had mild obstructive ventilatory dysfunction (5 patients) and a decline in small airway function (6 patients). For patients with severe symptoms, PFT results showed moderate to severe restrictive ventilatory dysfunction and reduced diffusing capacity. PaCO2 was significantly higher (P=0.047) in patients with reduced small airway function compared with those with normal small airway function. Whole blood zinc levels in the convalescence stage (day 17) were significantly lower than those in the acute stage (day 4). Zinc in the acute stage was negatively correlated with DLCO/VA on days 3, 10, and 46 (r=-0.633, -0.676, and -0.675 respectively, P<0.05). CONCLUSIONS: Smoke inhalation injury mainly causes restrictive ventilatory dysfunction and reduced diffusing capacity, and causes mild obstructive ventilatory dysfunction and small airway function decline in some patients. Zinc is negatively correlated with DLCO/VA. Zinc levels may be able to predict prognosis and indicate the degree of lung injury.

Correlation of cytotoxic effect of transmembrane and secretory TNF-α to cell cycle.

This study was aimed to examine the correlation of the cytotoxic effects induced by two types of TNF-α to cell cycle. Hoechst 33342 and PI were used to detect the morphological changes in the cell death induced by the two types of TNF-α. TdT and PI co-staining was performed to determine the phase of cell cycle of apoptotic cells. L929 cells in different phases of cell cycle were further synchronized and their sensitivity to the two types of TNF-α was observed. Our results showed that the apoptosis of HepG2 cells triggered by tm-TNF-α mainly occurred in G(1) phase while in HL-60, Raji and K562 cell lines it mainly took place in S phase. The apoptosis of L929 cells induced by tm-TNF-α mainly occurred in S phase while the apoptosis induced by s-TNF-α mainly appeared in G(1) phase. L929 cells were sensitive to s-TNF-α when synchronized in G(1) phase (cytotoxicity 49.8%) while their sensitivity to tm-TNF-α was highest in S phase (45.7%) and G(1)/S phase (cytotoxicity 40.6%). It was concluded that tm-TNF-α-induced apoptosis of different target cells took place in different phases of cell cycle. The apoptosis of the specific cell line induced by the two types of TNF-α occurred in different phases of cell cycle. The sensitivity of the specific cell line to the two types of TNF-α was correlated with the phase of cell cycle.

The genome of Prunus mume.

Prunus mume (mei), which was domesticated in China more than 3,000 years ago as ornamental plant and fruit, is one of the first genomes among Prunus subfamilies of Rosaceae been sequenced. Here, we assemble a 280M genome by combining 101-fold next-generation sequencing and optical mapping data. We further anchor 83.9% of scaffolds to eight chromosomes with genetic map constructed by restriction-site-associated DNA sequencing. Combining P. mume genome with available data, we succeed in reconstructing nine ancestral chromosomes of Rosaceae family, as well as depicting chromosome fusion, fission and duplication history in three major subfamilies. We sequence the transcriptome of various tissues and perform genome-wide analysis to reveal the characteristics of P. mume, including its regulation of early blooming in endodormancy, immune response against bacterial infection and biosynthesis of flower scent. The P. mume genome sequence adds to our understanding of Rosaceae evolution and provides important data for improvement of fruit trees.

In vivo MRSI of hyperpolarized [1-(13)C]pyruvate metabolism in rat hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), the primary form of human adult liver malignancy, is a highly aggressive tumor with average survival rates that are currently less than 1 year following diagnosis. Most patients with HCC are diagnosed at an advanced stage, and no efficient marker exists for the prediction of prognosis and/or response(s) to therapy. We have reported previously a high level of [1-(13)C]alanine in an orthotopic HCC using single-voxel hyperpolarized [1-(13)C]pyruvate MRS. In the present study, we implemented a three-dimensional MRSI sequence to investigate this potential hallmark of cellular metabolism in rat livers bearing HCC (n = 7 buffalo rats). In addition, quantitative real-time polymerase chain reaction was used to determine the mRNA levels of lactate dehydrogenase A, nicotinamide adenine (phosphate) dinucleotide dehydrogenase quinone 1 and alanine transaminase. The enzyme levels were significantly higher in tumor than in normal liver tissues within each rat, and were associated with the in vivo MRSI signal of [1-(13)C]alanine and [1-(13)C]lactate after a bolus intravenous injection of [1-(13)C]pyruvate. Histopathological analysis of these tumors confirmed the successful growth of HCC as a nodule in buffalo rat livers, revealing malignancy and hypervascular architecture. More importantly, the results demonstrated that the metabolic fate of [1-(13)C]pyruvate conversion to [1-(13)C]alanine significantly superseded that of [1-(13)C]pyruvate conversion to [1-(13)C]lactate, potentially serving as a marker of HCC tumors.

Temperature sensitivity and drug encapsulation of star-shaped amphiphilic block copolymer based on dendritic poly(ether-amide).

The star-shaped amphiphilic block copolymer (DPEA-PCL-PNIPAAms) with different PCL block lengths was prepared through ring opening polymerization of epsilon-caprolactone (CL) initiated by hydroxyl end-capped dendritic poly(ether-amide) (DPEA-OH), and then coupling with carboxyl end-capped linear poly(N-isopropylacrylamide) (PNIPAAm-COOH) via an esterification process. The molecular structure was characterized by FT-IR, (1)H NMR, and GPC analysis. As the copolymer dissolved in water, the core-shell structural nanoparticle was formed as a micelle. The fluorescence, (1)H NMR, and dynamic light scattering (DLS) techniques were utilized to confirm the formation of micelles. The optical transmittance and US-DSC measurements demonstrated that the micelles performed the reversible dispersion/aggregation behavior in response to temperature through the outer PNIPAAm polymer shell. The micelles loaded with daidzein showed a very rapid drug release speed at temperatures above the lower critical solution temperature (LCST) due to the temperature-induced structural change of the polymeric micelles.

Influence of reverse signaling via membrane TNF-alpha on cytotoxicity of NK92 cells.

Membrane tumor necrosis factor-alpha (mTNF-alpha) serves as a receptor transducing signals into mTNF-alpha-bearing cells. Among human peripheral blood mononuclear cells, natural killer (NK) cells have been reported to be the only cell type constitutively expressing mTNF-alpha, which is involved in the cytotoxicity of resting NK cells. Using an IL-2-dependent human NK cell line, NK92, which constitutively expresses mTNF-alpha, we examined the effect of reverse signaling via mTNF-alpha on cellular activities. When the cells were prestimulated with soluble TNFR1 (sTNFR1) which activated mTNF-alpha-mediated reverse signaling, the cytotoxicity of NK92 cells was significantly increased. Further investigation demonstrated that prestimulation with sTNFR1 augmented exocytosis and mRNA transcription of two cytotoxic molecules, perforin and granzyme B, which could serve as underlying molecular mechanisms by which mTNF-alpha-mediated reverse signaling promoted cytotoxicity of NK cells toward K562 cells. On the other hand, pretreatment of NK92 with sTNFR1 boosted the expression of FasL and TNF-alpha, including both the secretory and membrane forms. These molecules also contribute to the NK-mediated cytotoxicity, although K562 cells are Fas-negative and sTNF-alpha-resistant. Interestingly, the mTNF-alpha reverse signaling was found to act synergistically with IL-2 on NK-mediated cytotoxicity. This synergy markedly promoted the production of secretory as well as membrane cytotoxic molecules which may be responsible for the enhanced NK92-mediated cytotoxicity. Our observations suggest that, via reverse signaling, constitutively expressed mTNF-alpha may sensitize NK cells to activating stimuli, such as IL-2, resulting in increased NK-mediated cytotoxicity through promoting the production of multiple cytotoxic effector molecules.

Estrogen plays a critical role in AAV2-mediated gene transfer in ovarian cancer.

AIM: The aim of our study was to develop an effective gene delivery system for ovarian cancer gene therapy. METHODS: The expression of heparin sulfate proteoglycan (HSPG) and integrins alpha(upsilon)beta(3) and alpha(upsilon)beta(5) were analyzed with flow cytometry on 2 human ovarian cancer cell lines (OVCAR-3 and SKOV-3ip). The gene transduction efficiencies were evaluated with recombinant adeno-associated viral vector (rAAV)2-green fluorescent protein or rAAV2-lactase Z followed by flow cytometry or cytohistochemistry staining. The effect of 17beta-estradiol on ovarian cancer cell proliferation, HSPG, the expressions of integrins alpha(upsilon)beta(3) and alpha(upsilon)beta(5), and adeno-associated viral vector (AAV)2-mediated gene transduction were determined. RESULTS: In the present study, we found: (1) a variation in HSPG and the expressions of integrins alpha(upsilon)beta(3) and alpha(upsilon)beta(5) between OVCAR-3 and SKOV-3ip; (2) that 17beta-estradiol was shown to significantly stimulate cell proliferation and integrin beta(5) expression in certain ovarian cancer cell lines; and (3) integrintargeted A520/N584RGD-rAAV2, which has alternative interactivity with integrins and abrogates the binding capacity HSPG, showed much higher gene transduction efficiency in ovarian cancer cells than rAAV2 in the presence/absence of 17beta-estradiol. Moreover, this RGD-modified rAAV2 exerted more efficient transduction in ovarian cancer cells in response to 17beta-estradiol. CONCLUSION: Our findings implied that A520/N584RGD-rAAV2 may offer great potential for ovarian cancer treatment in vivo.

Generation of adenovirus-mediated anti-CD20 antibody and its effect on B-cell deletion in mice and nonhuman primate cynomolgus monkey.

Therapeutic monoclonal anti-CD20 antibody (Rituxan) is increasingly applied to treat B-cell-related hematologic malignancies and autoimmune disorders with great clinical success, whereas its widespread application is limited by antibody manufacturing capability. Here, we explored a quick and economical adenovirus-mediated anti-CD20 antibody generating system to directly produce anti-CD20 antibody in vivo. We generated a recombinant adenovirus encoding the anti-CD20 antibody gene and found that infection of cells with this recombinant adenovirus led to the generation of anti-CD20 antibody in cells with a similar CD20 binding affinity and specificity as commercial product Rituxan. After one single administration of the anti-CD20-expressing adenoviruses through tail vein at a dose of 1 x 10(9) plaque-forming units/mouse in nude mice, anti-CD20 antibody in the serum was detectable at day 3, reached to the peak value of 246.34 microg/mL at day 14, and maintained a high serum concentration of >40 microg/mL for 56 days. Furthermore, the in vivo generation of anti-CD20 antibody led a complete elimination of preestablished B-cell lymphoma Raji cells in nude mice, and a single administration of the anti-CD20-expressing adenovirus at a dose of 2.0 x 10(9) plaque-forming units/kg in cynomolgus monkey led a continuous B-cell deletion in circulation blood and bone marrow. These observations thus suggest that adenovirus-mediated in vivo generation of anti-CD20 antibody may serve as a new strategy to combat B-cell-related hematologic disorders.

Effect of human WEE1 and stem cell factor on human CD34+ umbilical cord blood cell damage induced by chemotherapeutic agents.

Myelosuppression is one of the major side-effects of most anticancer drugs. To achieve myeloprotection, one bicistronic vector encoding anti-apoptotic protein human WEE1 (WEE1Hu) and proliferation-stimulating stem cell factor (SCF) was generated. In this study, we selected human umbilical cord blood CD34+ cells as the in vitro model in an attempt to investigate whether WEE1Hu, rather than conventional drug-resistant genes, can be introduced to rescue cells from the damage by chemotherapeutic agents such as cisplatin, adriamycin, mitomycin-c and 5-fluorouracil. Cell viability and cytotoxicity assay, colony-forming units in culture assay and externalization of phospholipid phosphatidylserine analysis showed that the expression of WEE1Hu and SCF in CD34+ cells provided the cells with some protection. These findings suggest that the expression of WEE1Hu and SCF might rescue CD34+ cells from chemotherapy-induced myelosuppression.

Comparison of cell deaths induced by transmembrane and secretory TNF-alpha.

Our previous study showed that transmembrane TNF-alpha (TM-TNF-alpha) had broader tumoricidal spectrum than secretory TNF-alpha (s-TNF-alpha). This study examined the difference between the two kinds of TNF-alpha in inducing cells and the relationship between the apoptosis induced by TM-TNF-alpha and the cell cycle. Bioassay was employed to compare the cytotoxic effect of two kinds of TNF-alpha on cell lines L-929 and HepG2. TUNEL was used to detect apoptosis and the TdT and PI co-staining were used for determining the phase of apoptotic cells. Our results showed that TM-TNF-alpha could kill not only s-TNF-sensitive L929 cells but also s-TNF-tolerant HepG2 cells. TM-TNF-alpha predominantly induced apoptosis while s-TNF could induce both apoptosis and necrosis. The apoptosis of L-929 cells induced by TM-TNF-alpha mainly occurred in S phase and the apoptosis of HepG2 predominantly took place in G(1) phase. It is concluded that the cytotoxic effects of the two TNF differ substantially. Since TM-TNF-alpha works locally, mainly induces apoptosis and has broader anti-tumor spectrum, it may be more effective for the treatment of tumor than s-TNF.

Synthesis of amphiphilic poly(ether-amide) dendrimer endcapped with poly(ethylene glycol) grafts and its solubilization to salicylic acid.

An amphiphilic dendrimer (DPEA-PEG) grafting polyethylene glycol at the terminals was prepared by endcapping of dendritic poly(ether-amide) (DPEA) with isocyanate terminated linear polyethylene glycol (PEG-NCO). The molecular structure was verified by gel permeation chromatography (GPC), (1)H NMR and FT-IR. The micelle characteristic of DPEA-PEG in water was investigated. The critical micelle concentration (CMC) was determined by a fluorescence technique to be 55.5 mg/L. The hydrodynamic radius of micelles was measured by dynamic light scattering (DLS) to be 76.2 nm. The UV-vis spectrum showed that the solubility of salicylic acid increased from 1.91 to 2.78 mg/L when the concentration of DPEA-PEG attained 5 mg/mL in an aqueous solution.

Trends in therapeutic monoclonal antibodies of cancer.

Therapeutic monoclonal antibodies are increasingly applied in clinical application with great success. A variety of antibody products have been approved by the FDA since 1997. Furthermore, the industries have been paying more attention to and efforts in the field of antibody development than ever, suggesting the grand potential of the market and benefits. At present, many monoclonal antibodies have proven their therapeutic value in combination with established treatment for many diseases, as shown in FDA approved expanded indications. This old-fashioned immunotherapy exerts profound effects in many refractory and formidable diseases, especially cancers. With further understanding of the interaction between immune system and cancer, more target molecules were discovered and more promising therapeutic antibodies with improved effects will be feasible in the future. Regardless of initial development or ultimate approved drug, therapeutic monoclonal antibodies have always been associated with numerous patent applications. This review mainly focuses on potential therapeutic monoclonal antibodies in oncology and related antibody patents, and discusses the trend for antibody development and therapeutic applications in humans.

Increased safety with preserved antitumoral efficacy on hepatocellular carcinoma with dual-regulated oncolytic adenovirus.

PURPOSE: A dual-regulated adenovirus variant CNHK500, in which human telomerase reverse transcriptase promoter drove the adenovirus 5 (Ad5) E1a gene and hypoxia-response promoter controlled the E1b gene, was engineered. This virus has broad anticancer spectrum and higher specificity compared with mono-regulated adenovirus CNHK300. The objective of the current study is to show its antitumor selectivity and therapeutic potential. EXPERIMENTAL DESIGN: The antitumor specificity of human telomerase reverse transcriptase and hypoxia response promoters was evaluated in a panel of tumor and normal cells. Under the control of these promoters, the tumor-selective expression of E1a and E1b genes was evaluated. Further in vitro antitumor specificity and potency of this virus were characterized by viral replication and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Subsequently, hepatocellular carcinoma xenografts were established to evaluate CNHK500 antitumor efficacy in vivo by different routes of virus administration and different dosages. RESULTS: Human telomerase reverse transcriptase and hypoxia response promoters were activated in a tumor-selective manner or under hypoxia treatment in a broad panel of cells. Selective adenoviral early gene expression, efficient viral replication, and oncolysis were observed in all tested cancer cells with more attenuated replication capacity in normal cells. Significant regression of hepatocellular carcinoma xenografts and prolonged survival were observed by either i.t. or i.v. administration. CONCLUSIONS: CNHK500 greatly reduced side effects in normal cells via dual control of adenoviral essential genes while still preserving potent antitumor efficacy on broad-spectrum cancer cells in vitro and in vivo. It can be used as a powerful therapeutic agent not only for liver cancers but also for other solid tumors.

Gene therapy using adenovirus-mediated full-length anti-HER-2 antibody for HER-2 overexpression cancers.

PURPOSE: Therapeutic monoclonal antibody is increasingly applied in many clinical applications, although complicated technologies and high cost still limit their wide applications. To obtain the sustained serum antibody concentration with one single injection and lower the cost of antibody protein therapy, an adenovirus-mediated full-length antibody gene therapy was developed. EXPERIMENTAL DESIGN: Full-length antibody light-chain and heavy-chain sequences were linked with internal ribosome entry site and constructed into adenoviral vector under the control of cytomegalovirus promoter. Antibody expression in vitro and in vivo were tested with ELISA, and its antitumor efficacy was evaluated in SKOV-3-inoculated nude mice. RESULTS: Ad5-TAb-generated anti-HER-2 antibody presented the similar binding specificity with commercial trastuzumab. A single i.v. injection of 2 x 10(9) plaque-forming units of Ad5-TAb per mouse resulted in not only a sustained over 40 microg/mL serum antibody level for at least 4 weeks but also significant tumor elimination in the ovarian cancer SKOV-3-inoculated nude mice. CONCLUSIONS: An in vivo full-length antibody gene delivery system allows continuous production of a full-length antibody at high concentration after a single administration. Bioactive antibody macromolecules can be generated via gene transfer in vivo. All the data suggest that this novel adenovirus-mediated antibody gene delivery can be used for the exploitation of antibodies, without being hampered by the sophisticated antibody manufacture techniques and high cost, and, furthermore, can shorten the duration and reduce the expense of antibody developments.

Dual regulation of soluble tumor necrosis factor-alpha induced activation of human monocytic cells via modulating transmembrane TNF-alpha-mediated 'reverse signaling'.

Transmembrane tumor necrosis factor-alpha (mTNF-alpha) is known to be the precursor of soluble TNF-alpha (sTNF-alpha). mTNF-alpha can act as a ligand on the TNF receptor- (TNFR)- bearing cell through 'forward signaling' or as a receptor on the TNF producing cell through 'reverse signaling'. In the current study, we investigated the role of mTNF-alpha-mediated reverse signaling in regulating sTNF-alpha-induced activation of human monocytic U937 cells. We demonstrated that pretreatment with sTNFRI, for inducing reverse signaling through mTNF-alpha, sensitized U937 cells to sTNF-alpha stimulation, as evidenced by an increase in reactive oxygen production and mRNA levels of proinflammatory cytokines (TNF-alpha, IL-1beta, and IL-8) in these cells. Further experiments revealed that IkappaB-alpha degradation was increased in the monocytic cells primed with sTNFRI, implying that reverse signaling of mTNF-alpha sensitizes U937 cells via an NF-kappaB-dependent mechanism. On the other hand, binding of sTNFRI to mTNF-alpha after sTNF-alpha-induced activation of U937 cells reduced mRNA stability (half-life) of IL-1beta and IL-8. The involvement of reverse signaling in the process was verified by using a mutated form of mTNF-alpha lacking the majority of the cytoplasmic domain. Our results clearly showed that enhanced mRNA degradation of the cytokines occurred only in U937 cells transfected with a wild-type mTNF-alpha, but not in those cells transfected with the mutant mTNF-alpha. Taken together, these data suggest that reverse signaling through mTNF-alpha may exert a double role in modulating sTNF-alpha bioactivity. It is positive when reverse signaling occurs prior to sTNF-alpha stimulation, while it is negative when reverse signaling occurs after the sTNF-alpha signal. Thus, our findings strengthen a role of mTNF-alpha-mediated reverse signaling in the regulation of immune-inflammatory response and control of inflammatory reaction.