Recombinant Human Tumor Necrosis Factor-alpha Research Articles

Pigment epithelium-derived factor is an interleukin-6 antagonist in the RPE: Insight of structure-function relationships.

Retinal and choroidal inflammatory lesions increase the levels of the pro-inflammatory cytokine interleukin-6 (IL-6). Pigment epithelium-derived factor (PEDF) has anti-inflammatory properties, but it is not known if it can prevent the production of IL-6 by the retinal pigment epithelium. To investigate the anti-inflammatory effects of PEDF in the RPE, we used human ARPE-19 cells stimulated with human recombinant tumor necrosis factor-alpha (TNF-α) to induce overexpression of the IL6 gene. We found that the viability of ARPE-19 cells decreased by 22% with TNF-α at 10ng/ml, being drastically decreased at ≥50ng/ml. TNF-α at 5-100ng/ml elevated the production and secretion of IL-6 protein, as measured by ELISA. To challenge the TNF-α-mediated stimulation of IL-6, we used recombinant human PEDF protein. PEDF at 100nM recovered the TNF-α-mediated loss of cell viability and repressed IL-6 gene expression as determined by RT-PCR. PEDF at 10-100nM attenuated the IL-6 protein secretion in a dose dependent fashion (IC50 = 65nM), being abolished with 100nM PEDF. To map the region that confers the IL-6 blocking effect to the PEDF polypeptide, we used chemically synthesized peptides designed from its biologically active domains, pro-death 34-mer, and pro-survival 44-mer and 17-mer (H105A), to challenge the IL-6 overproduction. The pro-survival peptides recovered the TNF-α-mediated cell viability loss, and inhibited IL-6 secretion, while the 34-mer did not have an effect, suggesting a role for the pro-survival domain in blocking TNF-α-mediated cell death and IL-6 stimulation. Our findings position PEDF as a novel antagonistic agent of IL-6 production in RPE cells, underscoring its use for the management of retinal disease-related inflammation.

Calcitonin Gene-Related Peptide in Charcot Foot Neuroarthropathy

Category:Basic Sciences/Biologics; DiabetesIntroduction/Purpose:Charcot neuroarthropathy (CNA) is a destructive joint condition secondary to neuropathic deficiency. For more than a century, the pathogenesis of CNA has been interpreted as unprotected injury and/or uncontrollable inflammation in a neuropathic joint, with little understanding its cellular and molecular pathology. Histologically, a hallmark of CNA is bone and cartilage fragments engulfed by hyperplastic synovium. Synovium is richly innervated. Neuropeptides, such as calcitonin gene- related peptide (CGRP), are secreted by neuronal and non-neuronal cells of the nervous, endocrine, and immune systems, and regulate inflammation. This study investigated the expression of CGRP in the synovial samples collected from CNA and non-CNA joint conditions, and the effects of CGRP on the proliferation and collagenolysis of fibroblast-like synoviocytes (FLS) isolated from CNA synovium in vitro.Methods:For this study, six CNA and 14 non-CNA synovial samples were collected during foot and ankle surgery. The donors included 10 male and 10 female, age from 14 to 79 years (mean 48). The non-CNA conditions consisted of osteoarthritis, ankle instability, Os Trigonum and osteochondritis dissecans.Western blot and immunohistochemistry of CGRP were performed to detect and localize CGRP expression in the CNA and non- CNA synovium.FLS was isolated from CNA synovium and cultured with, or without, human CGRP (10nM) in the culture media, for comparison of cell proliferation. Additionally, FLS were seeded on the collagen-coated 24-well plate and simulated with CGRP (10nM) and recombinant human tumor necrosis factor-alpha (TNF-α). After 7 days, the residual collagen on the bottom of the culture plate were stained and imaged for area measurements.Data were analyzed with t test or one-way Analysis of Variance, followed with post hoc Tukey's test.Results:By western blot, there was significant CGRP expression in the CNA synovium (5/6; Fig1). Except of an intense CGRP band in one of the osteoarthritis samples, only recognizable CGRP bands were shown in the samples of other non-CNA conditions. The average density of CGRP (in greyscale) in the CNA group was about 2-fold of that in the non-CNA group (10126 +- 5346 vs. 5377+-3734; p < 0.05). Immunohistochemistry demonstrated intense CGRP staining in the intimal layer of the CNA synovium (arrows) but not in the non-CNA synovium (Fig1). Treated with CGRP, the number of FLS in tissue culture increased. Cell number doubling time was 1.0 day (+-0.4) for the CGRP treated FLS and 2.2 days (+-0.5) for the control (p < 0.05). When the culture media were supplemented with CGRP and TNF-α, FLS eroded a larger area of collagen coating comparing with TNF-α alone in the media (p < 0.05).Conclusion:A knowledge gap in understanding the molecular and cellular pathology of CNA hampers the development of disease-modifying therapies. This study showed an increased CGRP, with a concentration in the intimal layer, in the CNA synovium as compared with the synovium in the non-CNA foot conditions. The increased CGRP expression in CNA synovium may be part of the unbalanced neuropeptide signaling in the neuropathic pathology. Functionally, CGRP stimulated FLS proliferation and degrading collagen in vitro. These effects suggest that CGRP may play a role in bone and joint destruction in the Charcot foot.

LncRNA MEG3 Reduces Hippocampal Neuron Apoptosis via the PI3K/AKT/mTOR Pathway in a Rat Model of Temporal Lobe Epilepsy.

PurposeTemporal lobe epilepsy (TLE) is a common neurological disorder, which is characterized by recurrent spontaneous seizures. Exploring the mechanisms of epileptogenesis has been considered as a priority. The aim of this study is to investigate the effects of LncRNA MEG3 in spontaneous recurrent epileptiform discharges (SREDs) and rats with TLE.MethodsRat model of TLE was produced by intraperitoneal injection of lithium chloride and pilocarpine. Rat hippocampal neuronal model of SREDs was established by Mg2+-free treatment. MEG3 was overexpressed by transfection of AAV-MEG3 in TLE and SREDs model. The expression of MEG3, interleukin-1β (IL-1β), interleukin-6 (IL-6) and recombinant human tumor necrosis factor-alpha (TNF-α) was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were detected by corresponding kit. The apoptosis of hippocampal neurons was detected by terminal deoxynucleotidyl transferase transfer‑mediated dUTP nick end‑labeling (TUNEL) assay and flow cytometry. The expression of proteins related to apoptosis (Caspase-3, Bax, and Bcl-2) and the PI3K/AKT/mTOR pathway was detected by Western blot.ResultsMEG3 expression was downregulated in SREDs and rats with TLE. Overexpression of MEG3 reduced the expression of IL-1β, IL-6, and TNF-α, MDA content, apoptosis rate of hippocampal neuron, increased SOD activity, and inhibited the PI3K/AKT/mTOR pathway in rats with TLE. In addition, overexpression of MEG3 enhanced cell viability and inhibited apoptosis through the activation of the PI3K/AKT/mTOR pathway in SREDs.ConclusionMEG3 reduced proinflammatory cytokines, oxidative stress, and apoptosis rate of hippocampal neuron and enhanced cell viability through the activation of the PI3K/AKT/mTOR pathway in SREDs and rats with TLE. Our findings may contribute to find a new therapeutic target for the treatment of epilepsy.

Kinetic study and optimization of recombinant human tumor necrosis factor-alpha (rhTNF-α) production in Escherichia coli

Tumor necrosis factor-alpha (TNF-α) is an inflammatory cytokine that plays a major role in immune regulation, homeostatic function, and cellular organization. The present study was undertaken to overproduce recombinant human TNF-α (rhTNF-α) in Escherichia coli (E.coli) in high cell density culture. The use of a codon-optimized gene and strong promoter-based (T7) expression system, choice of Terrific Broth (TB) as medium, and subsequent optimization of culture conditions in shake flasks resulted in production of 0.95 g/L insoluble rhTNF-α comprising upto 50% of total cellular protein (TCP) The protein yield further increased upto 1.26 g/L in 1 L TB medium batch culture in bioreactor with the controlled temperature, pH, and dissolved oxygen. In a series of chemostats operated at dilution rates of 0.2 h−1, 0.3 h−1, 0.4 h−1 and 0.5 h−1 the specific growth rate (μ) positively correlated with specific yield (Yp / x ) and a maximum yield of 164 mg/g DCW was obtained at μ = 0.4 h−1 within 4 h post-induction. A fed-batch cultivation in TB with an exponential feeding profile (μ = ∼0.4 h−1) of concentrated feed resulted in an accumulation of 5.5 g/L of rhTNF-α within 14 h of cultivation which accounted for ∼29% of TCP.

In vitro propagation of chia (Salvia hispanica L.) and assessment of genetic fidelity using random amplified polymorphic DNA and intersimple sequence repeat molecular markers

Nonconventional induction strategies for production of recombinant human tumor necrosis factor-alpha in Escherichia coliTapan Kumar Singha, Pooja Gulati, Sanjay Kumar

Interaction between CD177 and platelet endothelial cell adhesion molecule-1 downregulates membrane-bound proteinase-3 (PR3) expression on neutrophils and attenuates neutrophil activation induced by PR3-ANCA

BackgroundA recent study found that CD177 served as a receptor of membrane-bound proteinase-3 (mPR3) in a subset of neutrophils. Furthermore, CD177 has been identified as a high-affinity heterophilic binding partner for the endothelial cell platelet endothelial cell adhesion molecule-1 (PECAM-1). The current study aimed to investigate whether the interaction between PECAM-1 and CD177 could influence mPR3 expression as well as PR3-antineutrophil cytoplasmic antibody (ANCA)-induced neutrophil activation and glomerular endothelial cell (GEnC) injury.MethodsThe effect of interaction between CD177 and PECAM-1 on mPR3 expression was explored by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The effect of PECAM-1 on neutrophil activation and GEnC injury induced by PR3-ANCA-positive immunoglobulin (Ig)Gs was evaluated by dihydrorhodamine (DHR) assay and ELISA. CD177-negative neutrophils were selected by magnetic cell sorting (MACS), and the inhibitory effect of PECAM-1 on CD177-negative and mixed neutrophils was explored by measuring neutrophil degranulation.ResultsThe level of specific interaction between CD177 and PECAM-1 was elevated with increasing CD177 concentration. The expression of mPR3 significantly decreased in neutrophils preincubated with PECAM-1 in a dose-dependent manner. Consistently, the levels of respiratory burst and degranulation induced by PR3-ANCA-positive IgGs in recombinant human tumor necrosis factor-alpha (TNF-α)-primed neutrophils was significantly reduced by preincubation with PECAM-1 (440.6 ± 123.0 vs. 511.4 ± 95.5, p < 0.05; and 3155.0 ± 1733.0 ng/ml vs. 5903.0 ± 717.5 ng/ml, p < 0.05, respectively). In CD177-negative neutrophils incubated with PR3-ANCA-positive IgGs, the level of degranulation was not significantly changed by preincubation with PECAM-1. However, in mixed neutrophils, PECAM-1 significantly decreased the level of degranulation induced by PR3-ANCA-positive IgGs (1015.9 ± 229.2% vs. 1725.2 ± 412.4%, p < 0.01). Furthermore, with preincubation of TNF-α-primed neutrophils with PECAM-1, the level of soluble intercellular cell adhesion molecule-1 (sICAM-1), a marker of endothelial cell activation and injury, in the supernatant of GEnCs treated with primed neutrophils plus PR3-ANCA-positive IgGs was significantly attenuated (112.7 ± 24.2 pg/ml vs. 167.5 ± 27.7 pg/ml, p < 0.05).ConclusionsPECAM-1 can decrease the level of mPR3 expression on neutrophils, resulting in attenuation of neutrophil activation and subsequent GEnC injury induced by PR3-ANCA-positive IgGs.

Nonconventional induction strategies for production of recombinant human tumor necrosis factor-alpha in Escherichia coli

Nonconventional induction strategies for production of recombinant human tumor necrosis factor-alpha in Escherichia coli

Efficacy and safety of recombinant human tumor necrosis factor application for the treatment of malignant pleural effusion caused by lung cancer

Malignant pleural effusion (MPE) signifies a poor prognosis for patients with lung cancer. For treating physicians, the primary goals are to achieve sufficient control of MPE and minimize invasive intervention. Recombinant human mutant tumor necrosis factor-alpha (rhu-TNF) has been used in the treatment of MPE. The aim of our research was to evaluate the efficacy and safety of rhu-TNF application via ultrasound-guided chest tube for the treatment of MPE. rhu-TNF was administered as a single dose to 102 patients with MPE caused by lung cancer, and dexamethasone (Dxm, 5 mg) was administered 30 minutes before rhu-TNF in 35 randomly selected patients in order test its ability to prevent side effects. The primary endpoint was the efficacy of the rhu-TNF treatment (disease response rate) and side effects (pain, fever, and flu-like symptoms), evaluated four weeks after instillation. The disease response rate of rhu-TNF treatment was 81.37%. Side effects included 13 (12.75%) patients complaining of flu-like symptoms, 15 (14.71%) with fever/chill, and 14 (13.73%) with chest pain. A significantly higher efficacy was observed for treatment with 3 MU versus 2 MU of rhu-TNF (P = 0.036), while the adverse effects were similar. There was no significant association between the dose of rhu-TNF and progression-free survival (P = 0.752). In conclusion, our study shows that intra-pleural instillation of rhu-TNF achieves sufficient control of MPE and minimizes invasive intervention.

Juvenile generalized pustular psoriasis treated with etanercept

An 8-year-old boy with general pustular psoriasis (GPP) and iatrogenic secondary Cushing syndrome was treated successfully with etanercept after he had failed on acitretin, methotrexate, and methylprednisolone therapy. GPP is a severe and very rare variant of psoriasis in children often accompanied by life-threatening complications. Retinoids, cyclosporine, methotrexate, or dapsone used in a small number of case series and case reports were effective. Etanercept is a recombinant human tumor necrosis factor-alpha (TNF-alpha) receptor protein fused with Fc portion of IgG1 that binds to TNF-alpha, approved by Food and Drug Administration for the treatment of moderate-to-severe plaque psoriasis in children and teens who have not responded to other psoriasis treatments. In our patient, etanercept demonstrated significant clinical response associated with long-term efficacy without acute exacerbation, excellent tolerability, and good safety profile.

A dendrimer-based immunosensor for improved capture and detection of tumor necrosis factor-α cytokine

A dendrimer-based sandwich type enzyme-linked immunosorbent assay (ELISA) was developed for the improved detection of recombinant human tumor necrosis factor-alpha (TNF-α) for early diagnosis of perinatal diseases. Hydroxyl-terminated generation four poly(amidoamine) dendrimer (G4-OH) was used for the development of a solid phase bio-sensing platform. The surface of the ELISA plate was modified with polyethylene-glycol (PEG) and thiol-functionalized G4-OH was immobilized on the PEG-functionalized plate. A capture antibody was oxidized and covalently immobilized onto the dendrimer-modified ELISA plate, which provides favorable orientation for the antigen binding sites toward the analyte. The dendrimer-modified plate showed enhanced sensitivity, and the detection limit for TNF-α was found to be 0.48pgmL−1, which is significantly better than the commercially available ELISA kit. The selectivity of the dendrimer-modified ELISA plate was further evaluated with a mixture of cytokines, which showed results for similar to that of TNF-α alone. The modified plate provides a greater opportunity for the detection of a wide range of cytokines and biomarkers.

Synergism of hydroxyapatite nanoparticles and recombinant mutant human tumour necrosis factor-α in chemotherapy of multidrug-resistant hepatocellular carcinoma

Locoregional chemotherapy continues to be the mainstay for the treatment of unresectable hepatocellular carcinoma (HCC). One of the principal obstacles implicated in its unsuccessful therapy is multidrug resistance (MDR). Former studies have identified the multidrug-resistant nature and possible mechanisms of hepatoma cells both in vitro and in vivo. This work aimed to develop an effective strategy for the treatment of HCC with MDR. The treatment was exploited to inhibit the MDR cells by co-administration of the recombinant mutant human tumour necrosis factor-alpha (rmhTNF-alpha), a sublethal dose of chemicals [adriamycin (ADM), mitomycin and 5-FU] and hydroxyapatite nanoparticles (nHAPs). Real-time quantitative reverse transcriptase-polymerase chain reaction and electrochemiluminescence Western blot were used to detect the expression of several related genes. The chemicals acted synergistically with rmhTNF-alpha and nHAP in suppressing the growth of hepatoma cells and inducing apoptosis of the cells, with the MDR phenotype reversed, as measured by intracellular ADM retention. Analysis of mRNA and protein revealed that rmhTNF-alpha inhibited the gene expression of XIAP, survivin, Ki67, PCNA, MDR1 and BCRP to some extent. Moreover, the inhibitory effects mentioned above could be as good or better than when nHAP is incorporated into the regimens. rmhTNF-alpha was not only able to restore the chemotherapeutic sensitivity to HepG2/ADM, its xenograft model and clinical samples but also further inhibited the growth of these tumours by a combination of nHAP. These results strongly suggested that chemicals in combination with rmhTNF-alpha and nHAP may be beneficial for the local treatment of advanced HCC.

Effect of Etanercept on Steroid Refractory Graft-versus-host Disease in Children

Background: Etanercept is a recombinant human soluble tumor necrosis factor-alpha (TNF-) receptor fusion protein that inhibits TNF-, a major mediator in the pathogenesis of graft-versus-host disease (GVHD). The purpose of our study was to evaluate the safety and efficacy of etanercept therapy in children with steroid-refractory acute GVHD (aGVHD) (n=5) and chronic GVHD (cGVHD) (n=3). Methods: Five males and 3 females were enrolled and their median age was 14.4 years (range, 2.1-18.8). Etanercept 0.4 mg/kg per dose (maximum dose, 25 mg) was given subcutaneously twice weekly for 4 weeks followed by 0.4 mg/kg per dose (maximum dose, 25 mg) weekly for 4 weeks. At the time of initiation of etanercept, 5 patients had aGVHD grade III to IV (III=4, IV=1) and 3 patients had moderate to severe cGVHD (moderate=1, severe=2). Results: Overall, 6 of 8 patients (75%) responded to the treatment with etanercept, including 5 patients with aGVHD [n=3 complete response (CR), n=2 partial response (PR)] and 1 patient with cGVHD [n=1 PR, n=2 no response (NR)]. Clinical responses were most commonly seen in patients with refractory gut aGVHD. CMV reactivation occurred in 2 patients, bacterial infection in 1 patient, and fungal infection in 1 patient. Conclusion: Our preliminary data indicate that etanercept is well tolerated and can induce a high response rate in patients with steroid refractory aGVHD, particularly in the setting of intestinal involvement.

Augmentation of the human monocyte/macrophage chemiluminescence response during short-term exposure to interferon-gamma and tumour necrosis factor-alpha

The effects of short-term (30 min) pre-incubation of human monocytes and macrophages (3-day cultured monocytes) with leucocyte-derived human interferon-gamma (IFN-gamma) and recombinant human tumour necrosis factor-alpha (rTNF-alpha) were examined. Pre-incubation of either monocytes or macrophages with rTNF-alpha or IFN-gamma (100 U/5 x 10(5) cells) augmented their respiratory burst to formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), measured by the luminol- and lucigenin-dependent chemiluminescence assay. In addition, both cell types showed a burst of respiratory activity in the presence of rTNF-alpha or IFN-gamma only. The effects of IFN-gamma were removed by adsorption with an anti-IFN-gamma monoclonal antibody and those of rTNF-alpha were abolished by heating at 100 degrees C, or by the addition of anti-TNF-alpha monoclonal antibody. The results demonstrate that both IFN-gamma and rTNF-alpha are stimulators of monocytes and macrophages, and rapidly alter the capacity of the cells to respond to fMLP, which binds to cell surface receptors.

Production of serum amyloid A and C-reactive protein by HepG2 cells stimulated with combinations of cytokines or monocyte conditioned media: the effects of prednisolone.

The hepatic production of the acute phase proteins in response to inflammatory cytokines, and the interaction of corticosteroids within this response, has been the subject of considerable recent research. In this study we have examined the effects of the corticosteroid prednisolone on the production of IL-1 alpha and IL-1 beta by lipopolysaccharide (LPS)-stimulated monocytes, and the ability of the monocyte conditioned media (MOCM) obtained under these conditions to induce human hepatoma HepG2 cells to produce serum amyloid A (SAA) and C-reactive protein (CRP). We also examined the production of SAA and CRP by HepG2 cells exposed to different combinations and concentrations of recombinant human (rh) IL-1 alpha, rhIL-1 beta, rhIL-6, recombinant human tumour necrosis factor-alpha (rhTNF-alpha) and prednisolone. The findings indicate: (i) prednisolone substantially inhibits the production of both IL-1 alpha and IL-1 beta by LPS-stimulated monocytes. The MOCM from prednisolone-treated monocytes induced less SAA and CRP production by HepG2 cells; (ii) IL-1 alpha and IL-1 beta both induced CRP and SAA synthesis by HepG2 cells, but only in the presence of IL-6. IL-1 beta was the more potent inducer for SAA production, but for CRP production IL-1 alpha and IL-1 beta were equivalent; (iii) prednisolone enhances the production of SAA by HepG2 cells, but does not enhance the production of CRP; (iv) TNF-alpha in the presence or absence of IL-6 and/or prednisolone did not induce the production of SAA or CRP by HepG2 cells. These findings offer a tenable solution to a disparate production of SAA compared with CRP in corticosteroid-treated cystic fibrosis (CF) patients.

Modulation of adhesion molecule expression on endothelial cells during the late asthmatic reaction: role of macrophage-derived tumour necrosis factor-alpha.

In a previous work we have demonstrated that in patients exhibiting a late allergic reaction (LAR), alveolar macrophages (AM) collected 18 h after bronchial allergen challenge produced high levels of IL-6 and tumour necrosis factor-alpha (TNF) which is known to up-regulate the endothelial cell expression of adhesion molecules participating in the development of the inflammatory reaction in bronchial asthma. For these reasons, we evaluated the effect of AM supernatants from asthmatic patients developing an LAR on intercellular adhesion molecule-1 (ICAM-1) and endothelial leucocyte adhesion molecule-1 (ELAM-1) expression by human endothelial cells. The expression of adhesion molecules was assessed by an ELISA method and compared with the effect of an optimal dose of human recombinant (hr) TNF. Results showed that AM supernatants, from challenged asthmatics developing an LAR, increased significantly the ICAM-1 and ELAM-1 expression on endothelial cells to a level similar to that obtained in the presence of hrTNF (500 U/ml) (P < 0.001 in both cases, respectively 90.4% and 75.2% of the level obtained with hrTNF). In contrast, AM supernatants from asthmatics at baseline or exhibiting, after challenge, a single early reaction had no significant effect on these parameters (P = NS in both cases, respectively 23.5% and 24.7% of the ICAM-1 expression, 22.7% and 15.3% of the ELAM-1 expression obtained with hrTNF). AM-derived TNF present in these supernatants was thought to play a key role in endothelial cell stimulation, since: (i) TNF concentration in AM supernatants correlated with its effect on ICAM-1 (r = 0.80, P < 10(-4)) and ELAM-1 expression (r = 0.88, P < 10(-5)); and (ii) a neutralizing anti-TNF antibody decreased their effect (68% and 80% respectively on ICAM-1 and ELAM-1 expression). Moreover, the role of IL-6 was excluded on the basis both of the hrIL-6 inefficiency to induce ICAM-1 and ELAM-1 synthesis, even in costimulation with hrTNF, and of anti-IL-6 antibody to neutralize the effect of AM supernatants. Our results suggest that, beside mast cells and lymphocytes, macrophages might participate in the induction of the local inflammatory reaction observed in bronchial asthma. During the LAR, cytokines and especially TNF are able, through an enhanced adhesion molecule expression on endothelial cells, to facilitate the bronchial cellular influx.

Antitumor Effect of Lung Cancer Vaccine with Umbilical Blood Dendritic Cells in Reconstituted SCID Mice

Dendritic cells (DCs) are important cells in initiating an immune response. A generation of functional DCs has potential clinical use in treating cancer. However, the source of DCs and patient immunodeficiency with cancer have been hindrances in clinical therapy. We generated DCs from human umbilical cord blood mononuclear cells (UBMCs) with recombinant human granulocyte-macrophage colony stimulating factor, recombinant human interleukin-4, and recombinant human tumor necrosis factor-alpha. The mature DC-A549 lung cancer vaccine (AgL-DC) was prepared through loading A549 lysate, treating with lipopolysaccharide (LPS) and positive selecting with CD83 magnetic beads. AgL-DC can secrete interleukin (IL)-12 and IL-1. Further in vitro analysis showed that AgL-DC notably induced human UBMC lymphocyte proliferation (p < 0.01) by 3-(4,5-dimethylthiazol-z-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, increased the cytotoxic T-lymphocyte (CTL) activity of UBMC lymphocytes against A549 cells (p < 0.05, at effector cells:target cells ratios of 50:1 and 100:1) by lactate dehydrogenase (LDH) cytotoxic assay, and improved production of IL-6 and tumor necrosis factor-beta (p < 0.01, p < 0.05) by enzyme-linked immunosorbent assay. Subsequently, the reconstitute immunity model in severe combined immunodeficiencies (SCID) mice has been established using human UBMC transplantation, and similar trends to results of UBMC in vitro experiments have been shown in lymphocyte proliferation, CTL activity, and IL-6 and tumor necrosis factor-beta secretion levels in these models. AgL-DC also significantly (p < 0.01) increased the antitumor effect in vivo. The tumor infiltrating immunocytes were positively expressed human CD83 and CD3 molecules, and they were negatively expressed in tumor tissue treated with control. These results have demonstrated that umbilical cord DCs are a useful source of vaccine cells for augmenting CTL-mediated cytotoxicity and have potential usefulness in cellular therapy for human cancer in a new vaccination strategy.

Protection of human islets from induction of apoptosis and improved islet function with HO-1 gene transduction

Islet transplantation represents an ideal therapeutic approach for treatment of type 1 diabetes but islet function and regeneration may be influenced by necrosis or apoptosis induced by oxidative stress and other insults. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in the catabolism of heme into biliverdin, releasing free iron and carbon monoxide. It has also been reported to be an antioxidant enzyme which can improve the function of grafted islets by cytoprotection via free radical scavenging and apoptosis prevention. In the present study, we investigated whether transduction of HO-1 genes into human islets with an adenovirus vector has cytoprotective action on islets cultured in vitro and discuss this method of gene therapy for clinical islet transplantation. Cadaveric pancreatic islets were isolated and purified in vitro. Transduction efficiency of islets was determined by infecting islets with adenovirus vector containing the enhanced green fluorescent protein gene (Ad-EGFP) at multiplicities of infection (MOI) of 2, 5, 10, or 20. Newly isolated islets were divided into three groups: EGFP group, islets transduced with Ad-EGFP using MOI = 20; HO-1 group, transduced with adenovirus vectors containing the human HO-1 gene using MOI = 20; and control group, mock transduced islets. Insulin release after glucose stimulation of the cell lines was determined by a radioimmunoassay kit and the stimulation index was calculated. Flow cytometry was used to detect apoptotic cells in the HO-1 group and in the control group after induction by recombinant human tumor necrosis factor-alpha (rTNFalpha) and cycloheximide (CHX) for 48 hours. Adenovirus vectors have a high efficiency of gene transduction into adult islet cells. Transduction of islets with the Ad-EGFP was most successful at MOI 20, at which MOI fluorescence was very intense on day 7 after transduction and EGFP was expressed in cultured islet cells for more than four weeks in vitro. The insulin release in the control group was (182.36 +/- 58.96) mIU/L after stimulation by high glucose media (16.7 mmol/L), while insulin release from the HO-1 group and the EGFP group were (270.09 +/- 89.37) mIU/L and (175.95 +/- 75.05) mIU/L respectively. Compared to the control group and the EGFP group, insulin release in the HO-1 group increased significantly (P < 0.05). After treatment with rTNFalpha and CHX the apoptotic ratio of islet cells was (63.09 +/- 10.86)% in the HO-1 group, significantly lower than (90.86 +/- 11.25)% in the control group (P < 0.05). Transduction of human islets with Ad-HO-1 can protect against TNF-alpha and CHX mediated cytotoxicity. The HO-1 gene also appears to facilitate insulin release from human islets. Transduction of donor islets with the adenovirus vector containing an HO-1 gene might have potential value in clinical islet transplantation.

Effect of MePEG molecular weight and particle size on in vitro release of tumor necrosis factor-alpha-loaded nanoparticles1

To study the in vitro release of recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) encapsulated in poly (methoxypolyethyleneglycol cyanoacrylate-co-n-hexadecyl cyanoacrylate) (PEG-PHDCA) nanoparticles, and investigate the influence of methoxypolyethyleneglycol (MePEG) molecular weight and particle size. Three sizes (approximately 80, 170, and 240 nm) of PEG-PHDCA nanoparticles loading rHuTNF-alpha were prepared at different MePEG molecular weights (M(r) =2000, 5000, and 10,000) using the double emulsion method. The in vitro rHuTNF-alpha release was studied in PBS and rat plasma. A higher burst-release and cumulative-release rate were observed for nanoparticles with higher MePEG molecular weight or smaller particle size. A decreased cumulative release of rHuTNF-alpha following the initial burst effect was found in PBS, while the particle sizes remained constant and MePEG liberated. In contrast, in rat plasma, slowly increased cumulative-release profiles were obtained after the burst effect. During a 5-h incubation in rat plasma, more than 50% of the PEG-PHDCA nanoparticles degraded. The MePEG molecular weight and particle size had an obvious influence on rHuTNF-alpha release. rHuTNF-alpha released from PEG-PHDCA nanoparticles in a diffusion-based pattern in PBS, but in a diffusion and erosion-controlled manner in rat plasma.

Expression of a functional human tumor necrosis factor-α (hTNF-α) in yeastSaccharomyces cerevisiae

The recombinant soluble human tumor necrosis factor-alpha (hTNF-α) was expressed in a yeastSaccharomyces cerevisiae and its cytotoxicity was evaluated. A cDNA encoding hTNF-α was placed under the control of two different promoters: a glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and a yeast hybridADH2-GPD promoter, consisting of alcohol dehydrogenase II (ADH2) and theGPD promoter. A Northern blot analysis revealed that, although variation in the expression level of hTNF-α existed among transformants, the higher expression was obtained with theGPD promoter. Expressed hTNF-α protein (rhTNF-α) was successfully secreted into the culture medium, producing 2.5 mg per liter of culture filtrate, with no changes in cell growth. The bioassay for observing the cytotoxicity to the murine L929 fibroblast cell line, with serial dilution of rhTNF-α, indicated that the secreted rhTNF-α was bioactive and its doseresponse was improved eight to ten times over that of theE. coli-derived rhTNF-α.

502 Repopulation of the moderately well differentiated GL human squamous cell carcinoma growing in nude mice

Purpose: To evaluate the antitumor activity of recombinant human tumor necrosis factor-alpha (rHuTNF-α) on a human glioblastoma multiforme (U87) xenograft in nude mice, and to study the effect of combining rHuTNF-α with local radiation on the tumor control probability of this tumor model.Methods and Materials: U87 xenograft was transplanted SC into the right hindleg of NCr/Sed nude mice (7–8 weeks old, male). When tumors reached a volume of about 110 mm3, mice were randomly assigned to treatment: rHuTNF-α alone compared with normal saline control; or local radiation plus rHuTNF-α vs. local radiation plus normal saline. Parameters of growth delay, volume doubling time, percentage of necrosis, and cell loss factor were used to assess teh antitumor effects of rHuTNF-α on this tumor. The TCD50 (tumor control dose 50%) was used as an endoint to determine the effect of combining rHuTNF-α with local radiation.Results: Tumor growth in mice treated with a dose of 150 μg/kg body weight rHuTNF-α, IP injection daily for 7 consecutive days, was delayed about 8 days comapred to that in controls. Tumors in the treatment group had a significantly longer volume doubling time, and were smaller in volume and more necrotic than matched tumors in control group. rHuTNF-α also induced a 2.3 times increase of cell loss factor. The administration ofthe above-mentioned dose of rHuTNF-α starting 24 h after single doses of localized irradiation under hypoxic condition, resulted in a significant reduction in TCD50 from the control value of 60.9 Gy to 5035 Gy (p < 0.01).Conclusion: rHuTNF-α exhibits an antitumor effect against U87 xnograft in nude mice, as evidence by an increased delay in tumor growth as well as cell loss factor. Also, there was an augmentation of tumor curability when given in combination with radiotherapy, resulting in a significantly lower TCD50 value in the treatment vs. the control groups.