Recombinant Tumor Necrosis Factor Research Articles

Trained Macrophage Bioreactor for Penetrating Delivery of Fused Antitumor Protein.

Macromolecular protein drugs are promising anti-neoplastic agents based on their precise tumor affinity and innocuousness to normal tissues. Although direct delivery of protein drugs remains impractical due to its short half-life in circulation, inefficiency in tumor accumulation, and poor penetrability in intratumoral distribution. Recently, biogenetic cell-based drug vectors have been widely reported for antitumor drug delivery. Macrophage is naturally independent with endogenous proteolysis, elimination of reticuloendothelial system, and immune surveillance. Meanwhile, its innate recruitment behaviors responsive to chronic inflammation signals make it a potential cellular vector for tumor targeting drug delivery. In this study, we develop a trained macrophage bioreactor for tumor homing and an in situ expression of fused antitumor protein. The recombinant tumor necrosis factor related apoptosis-inducing ligand is coded on a plasmid vector with penetrating domain on the C terminus, which improves the intratumoral distribution by facilitating protein dispersion in tumor tissue after in situ secretion. The combination of tumor-infiltrating macrophage bioreactor and multifunctional fused protein drug embodies a new effective tumor homing system for antitumor protein delivery.

The Effect of Human Recombinant Tumor Necrosis Factor Receptor-2 on Reducing Inflammatory of Collagen -Induced Arthritis in Balb/c Mice.

BackgroundThe tumor necrosis factor alpha (TNFα) is a cytokine that produced principally by monocyte/macrophages and T lymphocytes, respectively. TNFα is recognized as the primary mediator of immunity in inflammation reaction. One important application of Tumor Necrosis Factor Receptor 2 (TNFR2) is for the treatment of autoimmune diseases like rheumatoid arthritis (RA).ObjectivesThe aim of this study is to examine the therapeutic trace of the recombinant humanTNFR2 on collagen-induced arthritis (CIA) in mice.Materials and MethodsCIA was created in 20 mice by immunization with bovine type II collagen (CII). After the mice were boosted on day 21, they were injected with the recombinant protein in test group (1 mg.kg-1) and assessed edema in paws and knee joints after two weeks. The quantities of inflammatory cytokines such as TNF-α, interleukin-1 beta (IL-β1), interleukin-6 (IL-6), and interleukin-10(IL-10) in serum were evaluated through enzyme-linked immunosorbent assay (ELISA) kit. In addition, the histopathology of joints sections was analyzed.ResultsThe cytokines TNF-α, IL-1β, and IL-6 values in serum markedly decreased in groups treated with TNFR2 (P < 0.01–0.001). The results showed that treatment with TNFR2 significantly reduced edema in paws and joints (P < 0.01–0.001).ConclusionsPathological investigations proved that administration of recombinant TNF receptor has blocked or protected joints from progressive damage. This study suggests that the anti-arthritic effectiveness of TNFR2 will repress the symptoms of rheumatoid arthritis. Moreover, it seems that TNFR2 is a strong candidate for the treatment of the RA disease.

Tubulin couples death receptor 5 to regulate apoptosis.

Activation of death receptor 5 (DR5) to induce apoptosis in cancer cells is an attractive strategy for cancer therapy. However, many tumor cell lines and primary tumors are resistant to DR5 targeted agents including recombinant tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and anti-DR5 agonistic antibodies. Here we identify tubulin proteins - primarily consisting of α and β subunits folded into microtubule polymers - as a crucial modulator of DR5 mediated apoptosis. Using affinity purification coupled with mass spectrometry, we found that DR5 interacts with both α- and β-tubulin proteins in cancer cells. Pharmacological disruption of microtubules increased DR5 protein expression and subsequently sensitized the cells to TRAIL-induced apoptosis. Similar results were observed by selectively silencing tubulin transcript using small RNA interference. We also demonstrate that tubulin/microtubule blockade augments TRAIL induced apoptosis by stabilizing DR5 protein. Together, our results link the tubulin/microtubule network to the stringent regulation of DR5 mediated apoptosis, which could lead to potential therapeutic strategies to enhance cancer therapy efficacy.

Nerve growth factor continuously elevates in a rat rotator cuff tear model

Nerve growth factor (NGF) plays a key role in osteoarthritic pain and low back pain. Rotator cuff tear (RCT) is often associated with severe shoulder pain. However, the role of NGF in RCT remains to be fully understood. Rats were divided into sham and RCT groups. The rotator cuff was harvested from the sham and RCT groups on various days for reverse transcription-polymerase chain reaction analysis of Tnfa, Ngf, Il1b, and Cox2 expression. Rotator cuffs from the sham and RCT groups were also harvested at 1 and 14 days for enzyme-linked immunosorbent assay and immunohistochemistry to assess NGF protein levels and localization. Rotator cuff-derived cells were stimulated with rat recombinant tumor necrosis factor (TNF)-α to investigate the involvement of TNF-α in the regulation of NGF expression. Tnfa and Ngf messenger RNA levels increased within 1 day in the RCT group. Notably, Tnfa and Ngf upregulation persisted for up to 56 days after the RCT surgery, while Il1b and Cox2 expression was significantly reduced. NGF levels in the RCT group were significantly higher than those in the sham operation group on days 1 and 14. Certain inflammatory cells and synovial-like cells lining the surface of the laminated tears were NGF-positive on days 1 and 14, respectively. Ngf messenger RNA levels increased significantly in rotator cuff-derived cells after TNF-α stimulation. NGF levels are continuously elevated in RCT, which is mainly regulated by TNF-α. NGF may thus represent a potential target for therapies that modulate RCT pain.

Effect of neoadjuvant chemotherapy and recombinant tumor necrosis factor -thymosin – α 1 (TNF-T) on overall survival in patients with triple negative breast cancer (TNBC).

e12625Background: The data on TNF - drugs used in neoadjuvant treatment in breast cancer patients especially on its effect on survival is very few. Meanwhile some trials show that immunotherapy cou...

Baicalein inhibits acinar-to-ductal metaplasia of pancreatic acinal cell AR42J via improving the inflammatory microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers. Recent research has demonstrated that chronic pancreatitis (CP) is associated with an increased risk of PDAC, partly due to acinar-to-ductal metaplasia (ADM). Baicalein has been shown to exert anti-inflammatory and anti-tumor effects for CP or PDAC, respectively. The aim of our study was to investigate the effect of baicalein, and the putative underlying mechanism, on inflammatory cytokines-induced ADM of rat pancreatic acinar cell line AR42J. To investigate ADM and baicalein effects in vitro, AR42J were treated with recombinant rat Tumor Necrosis Factor alpha (rTNFα) with or without baicalein for 5 days. Results showed that rTNFα-induced AR42J cells switched their phenotype from dominantly amylase-positive acinar cells to dominantly cytokeratin 19-positive ductal cells. Moreover, expression of the transcripts for TNFα or Hes-1, a Notch target, was up-regulated in these cells. Interestingly, baicalein reduced the population of ADM as well as cytokines gene expression but not Hes-1. Baicalein inhibited NF-κB activation induced by rTNFα in AR42J, but no effect on Notch 1activation. Moreover, baicalein suppressed the secretion of TNFα and Nitric Oxide (NO) in macrophages stimulated with LPS and further inhibited ADM of conditional medium-treated AR42J cells. Baicalein also suppressed the inflammatory response of LPS-activated macrophages, thereby inhibited ADM of AR42J by altering their microenvironment. Taken together, our study indicates that baicalein reduces rTNFα-induced ADM of AR42J cells by inhibiting NF-κB activation. It also sheds new light on Chinese material medica therapy of pancreatitis and thereby prevention of PDAC.

Plasmodium circumsporozoite protein suppresses the growth of A549 cells via inhibiting nuclear transcription factor κB

Blocking the activation of nuclear factor κB (NF-κB) is a promising strategy for the treatment of non-small cell lung cancer. The circumsporozoite protein (CSP), a key component of the sporozoite stage of the malaria parasite, was previously reported to block NF-κB activation in hepatocytes. Therefore, in the present study, the effect of CSP on the growth of the human lung cancer cell line, A549, was investigated. It was demonstrated that transfection with a recombinant plasmid expressing CSP was able to inhibit the proliferation of A549 cells in a dose-dependent manner and induce the apoptosis of A549 cells. A NF-κB gene reporter assay indicated that CSP and its nuclear localization signal (NLS) motif were able to equally suppress the activation of NF-κB following stimulation with human recombinant tumor necrosis factor (TNF)-α in A549 cells. Furthermore, western blot analysis indicated that NLS did not affect the phosphorylation and degradation of IκB, but was able to markedly inhibit the nuclear translocation of NF-κB in TNF-α stimulated A549 cells. Therefore, the data suggest that CSP may be investigated as a potential novel NF-κB inhibitor for the treatment of lung cancer.

Effects of TNF receptor blockade on in vitro cell survival and response to negative energy balance in dairy cattle

BackgroundAssociative data and some controlled studies suggest that the inflammatory cytokine tumor necrosis factor (TNF) α can induce fatty liver in dairy cattle. However, research demonstrating that TNFα is a necessary component in the etiology of bovine fatty liver is lacking. The aim of this work was to evaluate whether blocking TNFα signaling with a synthetic cyclic peptide (TNF receptor loop peptide; TRLP) would improve liver metabolic function and reduce triglyceride accumulation during feed restriction.ResultsCapability of TRLP to inhibit TNFα signaling was confirmed on primary bovine hepatocytes treated with recombinant bovine TNFα and 4 doses of TRLP (0, 1, 10, 50 μmol/L) over 24 h. Next, 4 lactating Holstein cows (parity 1.4 ± 0.5, 433 ± 131 d in milk) in an incomplete Latin rectangle design (3 × 2) were subcutaneously administered with different TRLP doses (0, 1.5, 3.0 mg/kg BW) every 4 h for 24 h, followed by an intravenous injection of TNFα (5 μg/kg BW). Before and for 2 h after TNFα injection, TRLP decreased plasma non-esterified fatty acid (NEFA) concentration (P ≤ 0.05), suggesting an altered metabolic response to inflammation. Finally, 10 non-pregnant, non-lactating Holstein cows (3.9 ± 1.1 yr of age) were randomly assigned to treatments: control (carrier: 57% DMSO in PBS) or TRLP (1.75 mg TRLP /kg BW per day). Treatments were administrated every 4 h for 7 d by subcutaneous injection to feed-restricted cows fed 30% of maintenance energy requirements. Daily blood samples were analyzed for glucose, insulin, β-hydroxybutyrate, NEFA, and haptoglobin concentrations, with no treatment effects detected. On d 7, cows completed a glucose tolerance test (GTT) by i.v. administration of a dextrose bolus (300 mg glucose/kg BW). Glucose, insulin, and NEFA responses failed to demonstrate any significant effect of treatment during the GTT. However, plasma and liver analyses were not indicative of dramatic lipolysis or hepatic lipidosis, suggesting that the feed restriction protocol failed to induce the metabolic state of interest. Injection site inflammation, assessed by a scorer blinded to treatment, was enhanced by TRLP compared to control.ConclusionsAlthough the TRLP inhibited bovine TNFα signaling and altered responses to i.v. administration of TNFα, repeated use over 7 d caused apparent local allergic responses and it failed to alter metabolism during a feed restriction-induced negative energy balance. Although responses to feed restriction seemed atypical in this study, side effects of TRLP argue against its future use as a tool for investigating the role of inflammation in metabolic impacts of negative energy balance.

Differential Characterization of Temozolomide-Resistant Human Glioma Cells.

Glioblastoma multiforme (GBM) is the most common type of primary and malignant tumor occurring in the adult central nervous system. Temozolomide (TMZ) has been considered to be one of the most effective chemotherapeutic agents to prolong the survival of patients with glioblastoma. Many glioma cells develop drug-resistance against TMZ that is mediated by increasing O-6-methylguanine-DNA methyltransferase (MGMT) levels. The expression of connexin 43 was increased in the resistant U251 subline compared with the parental U251 cells. The expression of epithelial–mesenchymal transition (EMT)-associated regulators, including vimentin, N-cadherin, and β-catenin, was reduced in the resistant U251 subline. In addition, the resistant U251 subline exhibited decreased cell migratory activity and monocyte adhesion ability compared to the parental U251 cells. Furthermore, the resistant U251 subline also expressed lower levels of vascular cell adhesion molecule (VCAM)-1 after treatment with recombinant tumor necrosis factor (TNF)-α. These findings suggest differential characteristics in the drug-resistant GBM from the parental glioma cells.

Response to isolated limb perfusion and chemotherapy with epirubicin plus ifosfamide in a metastatic malignant ossifying fibromyxoid tumor

BackgroundOssifying fibromyxoid tumor (OFMT) is a rare soft tissue neoplasm of uncertain lineage and intermediate biological potential. It is more common in middle-aged men, usually arising from the deep tissues of the extremities. It is now established that it is a translocation related tumor, most often marked by translocation of PHF1 gene. Surgery is the mainstay of treatment and proves usually curative, although, in rarer cases the disease shows malignant features and tendency to recur both locally and at distant sites. In such cases, no standard treatment exists.Case presentationWe report on a case of malignant advanced OFMT of the hand with lung metastases responding to isolated limb perfusion with human recombinant tumor necrosis factor and melphalan and chemotherapy with epirubicin and ifosfamide.ConclusionsTo our knowledge, this is the first report of activity of soft tissue sarcoma-oriented chemotherapy in advanced OFMT.

MiR-133a Promotes TRAIL Resistance in Glioblastoma via Suppressing Death Receptor 5 and Activating NF-κB Signaling.

Recombinant tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), as a novel cancer therapeutic, is being tested in phase II and III clinical trials; however, TRAIL resistance remains a big obstacle preventing its clinical application. Considering that TRAIL-induced apoptosis through death receptors DR4 and DR5, their activation may be an alternative pathway to suppress TRAIL resistance. In this study, a negative correlation between DR5 expression and TRAIL resistance was observed, and miR-133a was predicted to be the most promising candidate to suppress DR5 expression. Further investigation demonstrated that miR-133a knockdown dramatically suppressed TRAIL resistance in glioblastoma in vitro and in vivo. An NF-κB family member, phosphorylated IκBα (P-IκBα), was shown to be stimulated by miR-133a, leading to the activation of this signaling. Finally, miR-133a was found to be inversely correlated with DR5 expression in human clinical specimens. In conclusion, our data demonstrate that miR-133a promotes TRAIL resistance in glioblastoma by suppressing DR5 expression and activating NF-κB signaling.

Three-year survival after combined treatment with induction chemoimmunotherapy in patients (pts) with non-small cell lung cancer (NSCLC).

e20064 Background: Induction chemotherapy (iCT) for NSCLC has potential advantages over adjuvant CT, high therapeutic compliance, the ability to influence the primary tumor and metastatic disease. Standard approaches to iCT improving are largely exhausted. However, the development of iCIT techniques using recombinant cytokines and assessment of the effectiveness of its clinical application remains poorly understood. Methods: Clinical trial included 65 pts. with stage IIB, IIIA (N2), IIIB (T4 ipsi. nod.) NSCLC, randomized in main (n = 33) and control (n = 32) groups with comparable clinical and pathological characteristics: m 23 (69.7) vs. 23 (71.8%), W 10 (30,3%) vs. 9 (28.2%); stage IIB - 8 (24.4%) vs. 9 (28.6%), IIIA - 22 (66.6%) vs. 19 (59.4%), IIIB - 3 (9%) vs. 4 (12.5%); 40-49 years 5 (15,5%) vs. 5 (15,6%), 50-59 14 (42,4%) vs.15 (46,8%), 60 years and older 14 (42,4%) vs. 12 (37,8%). Pts. of the control group received 2 iCT cycles: cisplatin 75 mg / m2 i.v. 1 day and gemcitabine 1000 mg / m2 i. v. 1 and 8 days; pts. of the main group additionally received recombinant tumor necrosis factor coupled with thymosin (TNF-T Farmaclon Russia) 50. 000 ED / m2i.v. 3, 5 and 7 days of the cycle. The direct results were evaluated 3 weeks after completion of induction. Pts. who underwent radical surgery received 2 cycles of platinum-based adjuvant CT. DFS was studied by the method of Kaplan-Meier. Results: 3-year DFS in the main group was 72% vs. 48% in the control group (p = 0.10603). Conclusions: Clinical trial of the effectiveness of iCIT with using immune drug TNF-T demonstrated a tendency to improve 3-year DFS by 24%, which favors further studies on a larger number of pts.

Interrelationships between Infliximab and Recombinant Tumor Necrosis Factor-α in Plasma Using Minimal Physiologically Based Pharmacokinetic Models.

The soluble cytokine tumor necrosis factor-α (TNF-α) is an important target for many therapeutic proteins used in the treatment of rheumatoid arthritis. Biologics targeting TNF-α exert their pharmacologic effects through binding and neutralizing this cytokine and preventing it from binding to its cell surface receptors. The magnitude of their pharmacologic effects directly corresponds to the extent and duration of free TNF-α suppression. However, endogenous TNF-α is of low abundance, so it is quite challenging to assess the free TNF-α suppression experimentally. Here we have applied an experimental approach to bypass this difficulty by giving recombinant human TNF-α (rhTNF-α) to rats by s.c. infusion. This boosted TNF-α concentration enabled quantification of TNF-α in plasma. Free rhTNF-α concentrations were measured after separation from the infliximab-rhTNF-α complex using Dynabeads Protein A. The interrelationship of infliximab and TNF-α was assessed with minimal physiologically based pharmacokinetic models for TNF-α and infliximab with a target-mediated drug disposition component. Knowledge of TNF-α pharmacokinetics allows reliable prediction of the free TNF-α suppression with either free or total TNF-α concentration profiles. The experimental and modeling approaches in our study may aid in the development of next-generation TNF-α inhibitors with improved therapeutic effects.

Reduced [14C]-methionine uptake and fecundity in Schistosoma mansoni females treated with recombinant tumor necrosis factor α in vitro

Recombinant human TNFα (rhTNFα) has previously been shown to reduce fecundity in Schistosoma mansoni adult females maintained in vitro without males, and adversely affect the uptake of [14C]-tyrosine, an amino acid required for schistosome vitellogenesis. Here we report on the effect of rhTNFα on [14C]-methionine uptake in both separated and paired females, and the effect of three different preparations of rTNFα on schistosome oviposition in vitro. In the absence of rhTNFa, separated females incorporated only 30% of the [14C]-methionine incorporated by paired females in a dose and time-dependent manner, suggesting low metabolic activity of females in the absence of males. Separated females and worm-pairs were treated with increasing doses of rhTNFα for 2 or 4 hr and then incubated in RPMI 1640 containing 10% fetal calf serum (FCS) and 5 µCi ml-1[14C]-methionine for 1 hr. Separated females treated with rhTNFa for 4 hr incorporated less methionine than those treated for 2 hr. In contrast, paired females treated with rhTNFa incorporated significantly smaller amounts of [14C]-methionine in a TNFa dose-dependent but time-independent manner [2 hr (P = 0.001) or 4 hr (P = 0.027) One-Way ANOVA]. Worm-pairs maintained in RPMI 1640 containing 10% FCS and 100 ng ml-1 of any of the three rTNFa preparations laid significantly fewer eggs than the worms cultured without rTNFα(P = 0.001; Kruskal-Wallis Test). We also observed that among rTNFα-treated worm-pairs, females were sluggish and tended to separate from their male partners. These observations suggest that TNFa inhibits [14C]-methionine uptake and reduces fecundity in females paired with males. Since paired females incorporate substantially greater amounts of [14C]-methionine, the role of males in stimulating metabolic activity in females is affirmed. Reduced amino acid uptake, and possibly other nutrients, may contribute to the diminished fecundity observed in TNFa-treated females.

NF-κB promotes leaky expression of adenovirus genes in a replication-incompetent adenovirus vector.

The replication-incompetent adenovirus (Ad) vector is one of the most promising vectors for gene therapy; however, systemic administration of Ad vectors results in severe hepatotoxicities, partly due to the leaky expression of Ad genes in the liver. Here we show that nuclear factor-kappa B (NF-κB) mediates the leaky expression of Ad genes from the Ad vector genome, and that the inhibition of NF-κB leads to the suppression of Ad gene expression and hepatotoxicities following transduction with Ad vectors. Activation of NF-κB by recombinant tumor necrosis factor (TNF)-α significantly enhanced the leaky expression of Ad genes. More than 50% suppression of the Ad gene expression was found by inhibitors of NF-κB signaling and siRNA-mediated knockdown of NF-κB. Similar results were found when cells were infected with wild-type Ad. Compared with a conventional Ad vector, an Ad vector expressing a dominant-negative IκBα (Adv-CADNIκBα), which is a negative regulator of NF-κB, mediated approximately 70% suppression of the leaky expression of Ad genes in the liver. Adv-CADNIκBα did not induce apparent hepatotoxicities. These results indicate that inhibition of NF-κB leads to suppression of Ad vector-mediated tissue damages via not only suppression of inflammatory responses but also reduction in the leaky expression of Ad genes.

CFTR Knockdown induces proinflammatory changes in intestinal epithelial cells.

BackgroundHyperinflammation is a hallmark feature of cystic fibrosis (CF) airways. However, inflammation has also been documented systemically and, more recently, in extrapulmonary CF-affected tissues such as the pancreas and intestine. The pathogenesis of CF-related inflammation and more specifically the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in that respect are not entirely understood. We have tested the hypothesis that genetic depletion of CFTR will affect the inflammatory status of human intestinal epithelial cell lines.MethodsCFTR expression was genetically depleted from Caco-2/15 and HT-29 cells using short hairpin RNA interference (shRNAi). Inflammatory conditions were induced by the addition of human recombinant tumor necrosis factor (TNF) or Interleukin-1β (IL-1β) for various periods of time. Gene expression, mRNA stability and secreted levels of interleukin (IL)-6, −8 and 10 were assessed. Analysis of pro- and anti-inflammatory signaling pathways including mitogen-activated protein kinases (p38, ERK 1/2 and JNK), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), and nuclear factor-kappa B (NF-κB) was also performed. Eosinophils were counted in the jejunal mucosa of Cftr−/− and Cftr+/+ mice.ResultsCFTR gene and protein knockdown caused a significant increase in basal secretion of IL-8 as well as in IL-1β-induced secretion of IL-6 and −8. Release of the anti-inflammatory cytokine, IL-10, remained unaffected by CFTR depletion. The enhanced secretion of IL-8 stems in part from increased IL8 mRNA levels and greater activation of ERK1/2 MAPK, IκBα and NF-κB in the CFTR knockdown cells. By contrast, phosphorylation levels of p38 and JNK MAPK did not differ between control and knockdown cells. We also found a higher number of infiltrating eosinophils in the jejunal mucosa of Cftr −/− females, but not males, compared to Cftr +/+ mice, thus providing in vivo support to our in vitro findings.ConclusionCollectively, these data underscore the role played by CFTR in regulating the intestinal inflammatory responses. Such findings lend support to the theory that CFTR exerts functions that may go beyond its role as a chloride channel whereby its disruption may prevent cells to optimally respond to exogenous or endogenous challenges. These observations are of particular interest to CF patients who were found to display alterations in their intestinal microbiota, thus predisposing them to pathogens that may elicit exaggerated inflammatory responses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12950-015-0107-y) contains supplementary material, which is available to authorized users.

Metabolomics investigation of recombinant mTNFα production in Streptomyces lividans.

BackgroundWhilst undergoing differentiation, Streptomyces produce a large quantity of hydrolytic enzymes and secondary metabolites, and it is this very ability that has focussed increasing interest on the use of these bacteria as hosts for the production of various heterologous proteins. However, within this genus, the exploration and understanding of the metabolic burden associated with such bio-products has only just begun. In this study our overall aim was to apply metabolomics approaches as tools to get a glimpse of the metabolic alterations within S.lividans TK24 when this industrially relevant microbe is producing recombinant murine tumour necrosis factor alpha (mTNFα), in comparison to wild type and empty (non-recombinant protein containing) plasmid-carrying strains as controls.ResultsWhilst growth profiles of all strains demonstrated comparable trends, principal component-discriminant function analysis of Fourier transform infrared (FT-IR) spectral data, showed clear separation of wild type from empty plasmid and mTNFα-producing strains, throughout the time course of incubation. Analysis of intra- and extra-cellular metabolic profiles using gas chromatography–mass spectrometry (GC–MS) displayed similar trends to the FT-IR data. Although the strain carrying the empty plasmid demonstrated metabolic changes due to the maintenance of the plasmid, the metabolic behaviour of the recombinant mTNFα-producing strain appeared to be the most significantly affected. GC–MS results also demonstrated a significant overflow of several organic acids (pyruvate, 2-ketoglutarate and propanoate) and sugars (xylitol, mannose and fructose) in the mTNFα-producing strain.ConclusionThe results obtained in this study have clearly demonstrated the metabolic impacts of producing mTNFα in S. lividans TK24, while displaying profound metabolic effects of harbouring the empty PIJ486 plasmid. In addition, the level of mTNFα produced in this study, further highlights the key role of media composition towards the efficiency of a bioprocess and metabolic behaviour of the host cells, which directly influences the yield of the recombinant product.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0350-1) contains supplementary material, which is available to authorized users.

The effect of ephrin-A1 on resistance to Photofrin-mediated photodynamic therapy in esophageal squamous cell carcinoma cells.

Esophageal squamous cell carcinoma (ESCC), the most prevalent cell type of esophageal cancer, remains a dismal disease with poor prognosis. Photodynamic therapy (PDT) is a minimally invasive treatment option for early esophageal cancer. To explore possible factors involved in resistance to PDT in esophageal cancer cells, we selected PDT-resistant subcell lines by repeated treatment of CE48T/VGH (CE48T) ESCC cells with Photofrin-PDT and then analyzed the global gene modulations in the PDT-resistant cells by whole-genome microarray. More than 700 genes reached a fold change greater than 1.5 in each of the PDT-resistant cells compared to parental cells. Among these genes, both tumor necrosis factor (TNF) and EFNA1 genes were significantly upregulated in resistant cell lines. However, they were significantly downregulated in Photofrin-PDT-treated cells compared to untreated cells. The observations made in the microarray analysis were further confirmed by quantitative PCR. We observed that recombinant tumor necrosis factor alpha (TNF-α) activated the gene expression of EFNA1 at both the messenger RNA (mRNA) level and the protein level in CE48T cells. Functional analysis showed that when incubated with oligomeric and monomeric ephrin-A1 simultaneously, ESCC cells became significantly resistant to Photofrin-PDT. Functional analysis further suggested that transmembrane and soluble ephrin-A1 may cooperate to enhance resistance to Photofrin-PDT in ESCC cells.

Circulating TNF and mitochondrial DNA are major determinants of neutrophil phenotype in the advanced-age, frail elderly

Tumor necrosis factor (TNF), a potent inflammatory cytokine, and mitochondrial DNA (mtDNA), a product of inflammation-induced tissue damage, increase with age (“inflammaging”) and many chronic diseases. Peripheral blood neutrophils, a critical component of innate immunity, have also been shown to be altered with age, and are exceptionally sensitive to external stimuli. Herein, we describe that the phenotype of neutrophils from the advanced-age, frail elderly (ELD) is determined by levels of circulating TNF and mtDNA.Neutrophils from ELD donors are morphologically immature, and have higher levels of intracellular reactive oxygen species (ROS) and expression of the activation markers CD11b and HLA-DR. The frequency of CD11b++ neutrophils correlated with plasma TNF, and recombinant TNF elevated neutrophil CD11b ex vivo and in vivo. Furthermore, neutrophils from aged TNF-deficient mice expressed CD11b similar to young counterparts. The frequency of HLA-DR+ neutrophils, on the other hand, positively correlated with circulating mtDNA, which increased neutrophil HLA-DR expression in a dose-dependent manner ex vivo. Cell-surface TLR-9 expression, however, was unaltered on neutrophils from ELD donors.In summary, we provide novel evidence that products of age-related inflammation modulate neutrophil phenotype in vivo. Given this, anti-inflammatory therapies may prove beneficial in improving neutrophil functionality in the elderly.

Tetraspanin CD9 is involved in pancreatic damage during caerulein-induced acute pancreatitis in mice.

Pancreatic acinar cell necrosis and subsequent inflammatory response aggravate acute pancreatitis (AP). Tetraspanin CD9 has been reported to mediate inflammatory signaling by regulating molecular organization at the cell surface. This study aimed to investigate the role of CD9 in caerulein-induced AP (CIP) in mice. The expression of CD9 was detected in CIP in mice in vivo and cholecystokinin (CCK)/recombinant mouse tumor necrosis factor (rmTNF)-α induced pancreatic acinar cell death in vitro by quantitative real-time polymerase chain reaction, Western blot and immunofluorescence. The roles of CD9 in pancreatic acinar cell death and inflammatory response were further studied through the deletion of CD9 expression using small interfering RNA (siRNA). CD9 was markedly upregulated in pancreatic tissues in mice during the early onset of CIP and was located mainly at the pancreatic acinar cell surface, which was associated with pancreatic damage. Additionally, incubation with CCK or rmTNF-α directly increased the expression of CD9 in isolated mice pancreatic acinar cells in vitro. The deletion of CD9 expression partially reversed both pancreatic acinar cell death induced by CCK and mRNA levels of proinflammatory cytokines produced by damaged acinar cells. These results indicate that increased CD9 expression may be involved in pancreatic injury, possibly via the promotion of cytokine expressions in CIP in mice.