Journal articles on the topic 'Auto-induction medium'

Abstract Objective In this article, we explore the feasibility of dendritic cells (DCs) induced from chronic myeloid leukemia (CML) with different cytokine cocktails and the mechanisms of immunological responses by specific T lymphocytes primed by this kind of DC vaccine. Methods Monocytes (MNCs) for the generation of DC were collected from patients with high numbers of circulating peripheral blood CML cells, then cultured with different cytokine cocktails in different cultural medium (Fetal calf serum or Human serum albumin medium) to induce DCs. DCs were studied for morphology and immunofluorescent staining. RT-PCR was used to analyze the specific fusion genes and chromosome banding technique were used to detect Philadelphia chromosome (ph’). In vitro, DCs were used to induce auto-lymphocytes to be activated CTL, killing rates and T subgroups were assayed, IL-12 contents in the supernents were assayed also. Results After induction, all these CML-derived MNCs developed typical DC morphology and the DC-associated surface molecules upregulated. The contents of IL-12 in the co-culture system of CML-DC and lymphocytes increased and the activated lymphocyte skewed from Th1 to Th2. At and effector: target ratio of 20:1, 80.67±6.43%, 62.33±12.01%, 23.92±5.62% cytotoxicity was noted with CML-DC/AL against autologous leukemia cells, K562, HL60, exhibiting more killing activities to auto-CML cells than K562 and HL60 cell. Conclusion The results of this research will supply new approach for specific immunotherapy of CML and supply the application value for the CML derived DC vaccine against CML.

The presence of extracellular polymeric substances (EPS) and their relevance for biofilm formation on the mineral surface for a variety of microbial species play a fundamental role in the degradation of sulfide ores. EPS production is associated with induction or auto induction mechanisms as a response of bacteria to environmental conditions. In this study, we tested galactose as an inducer of EPS production in planktonic cells of Acidithiobacillus thiooxidans DSM 14887T and their adherence to polymetallic mineral surfaces. Cells of At. thiooxidans were first adapted to grow at different concentrations of galactose (0.15, 0.25, 0.35%) using a modified 9K liquid medium and elemental sulfur as the energy source. In order to determine EPS production, the microorganisms were grown for 24 hours at different concentrations of galactose. Our results showed a cell adherence of 84% cells within 4 hours in presence of 0.15% galactose compared to 70% without galactose. The optimal concentration of galactose for maximal EPS production was 0.25% and for the attachment of cells it was 0.15%. Higher galactose concentrations inhibited microbial growth and decreased the number of cells attached to the mineral. While with a small amount of galactose in the culture media can shift the balance between sessile cells and planktonic cells, generating an increase in adhesion and therefore a possible increase of the bioleaching rate.

The decline of mammary epithelial cell (MEC) number during mammary gland involution in the cow is due to inhibition of proliferation and induction of apoptosis. Transforming growth factor-beta 1 (TGF-β1) belongs to a group of intramammary auto/paracrine inhibitors of bovine MEC growth and inducers of apoptosis. However, the mechanism responsible for the regulation of TGF-β1 expression in MEC is not known. The present study examined the effect of the hormones, growth hormone (GH), somatostatin (STS), 17-β oestradiol (E2), progesterone (P4), as well as the growth factors, insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF), on TGF-β1 expression in the bovine MEC lines, BME-UV1 and MAC-T. The model of apoptosis in bovine mammary gland in vitro was applied by reduction of fetal bovine serum (FBS) (from 10% to 2% or 0·5% FBS) in the cell environment to show the relationship between TGF-β1 expression and apoptosis in bovine MEC. RT-PCR, Western blot and laser scanning cytometry (LSC) were used for analysis of TGF-β1 transcript and protein level as well as apoptosis and cell cycle in examined MEC. In this model of apoptosis, FBS deficiency (mimicking the naturally occurring decline in the access of bioactive compounds and nutrients at the end of lactation and dry period) was associated with increased TGF-β1 expression at the level of transcript and protein, induction of apoptosis and inhibition of cell cycle. Exogenous TGF-β1, IGF-I, EGF and GH inhibited FBS-deficiency-stimulated TGF-β1 expression. The suppressive effect of GH was reversed when cells were maintained longer in FBS-deficient medium. In general, STS, E2 and P4 increased TGF-β1 expression. However, this effect was dependent on hormone concentration and cell line. BME-UV1 cells were much more responsive to the peptides, GH, STS, IGF-I and EGF, whereas MAC-T cells were more responsive to the steroid sex hormones: E2 and P4.

Background: Diffuse large B cell lymphoma (DLBCL) is one of the most common malignancy hematologic disease in China. At present, the NCCN recommended first-line therapy to DLBCL is: rituximab combined with cyclophosphamide, doxorubicin, vincristine, prednisolone (R-CHOP) or etoposide, cyclophosphamide, doxorubicin, vincristine, prednisolone (R-EPOCH). For the medium-risk and high-risk adult patients, autologous hematopoietic stem cell transplantation (auto-HSCT) is recommended as consolidation therapy. However, no maintenance therapy is recommended. A number of clinical trials of rituximab or lenadomide for maintenance therapy in DLBCL have been carried out, but the exact effect has not yet been confirmed. Traditional Chinese medicine (TCM) is a unique treatment in China. For economic or other reasons, many Chinese patients with DLBCL choose TCM as maintenance therapy after induction chemotherapy or auto-HSCT. Here we will introduce the application of TCM in maintenance therapy of DLBCL. Methods: We conducted a retrospective analysis of 87 (34 males and 53 females; age ranged from 16 to 86 years old) consecutive patients diagnosed with de novo DLBCL treated at Zhongda Hospital Affiliated to Southeast University between January 2013 and March 2019. All patients were diagnosed by pathology and graded by international prognostic index (IPI) score. All patients enrolled in our cohort were received at least 4 cycles of R-CHOP or R-EPOCH regimen. Then we compared the outcome of maintenance therapy with TCM or rituximab in DLBCL patients who were received standard induction chemotherapy. Results: Survival analysis showed that the overall survival (OS) in maintenance group (including TCM and rituximab) was significantly different from that in non-maintenance group (P=0.036). Among them, the patients received maintenance treatment with rituximab or TCM did not relapse and were still in survival state, but there was no significant difference in OS between the two groups (P>0.05). Because of the short follow-up period of this group, only 5 patients chose TCM and 7 patients chose rituximab for maintenance. Therefore, we will focus on two successful cases of TCM maintenance therapy. Case 1. A 57-year-old woman was diagnosed with DLBCL (stage Ⅳ, group B) in December 2012. After 3 cycles of R-EPOCH regimen chemotherapy, she was assessed as complete remission (CR) by PET/CT. Then auto-HSCT was followed in in May 2013. However, the patient suffered with fatigue and loss of appetite in July 2013. Also, decreased hematopoietic cells were found. Although G-CSF, cyclosporin and androgen were used to stimulate hematopoiesis, the hematopoietic function was still not fully restored. Thinking about the toxic and side effects of chemotherapy, we treated the patient with TCM as maintenance therapy and physical conditioning. After 3 months, the hematopoietic function returned to normal gradually. The symptoms of fatigue and loss of appetite were significantly improved. Then she continued the treatment of TCM for 2 years. The patient reviewd every year and was still in CR state. Case 2. A 46-year-old man was diagnosed with DLBCL (stageⅢ, group B) in June 2015. After 4 cycles of R-CHOP regimen chemotherapy, he was assessed as CR by PET/CT. Then auto-HSCT was followed in November 2015. Because of 2 months' bone marrow depression and skin rashes, the patient chose TCM as maintenance therapy from February 2016 to the present. Up to now, he was still alive and without disease progress. Conclusion: Even after first-line chemotherapy, about 30% high-risk DLBCL patients will be relapse. At present, no maintenance therapy is recommended yet, but more and more clinical trials on cancer-targeting drugs such as rituximab have been carried out these years. TCM is a therapeutic method with Chinese characteristics, which is reported to be used as adjuvant drugs for cancer chemotherapy. Maintenance therapy of TCM is a new option that is expected to be a more economical and effective alternative to rituximab. Due to the small sample size and short follow-up time, there is a certain bias. In the future, we will expand the sample size and extend the follow-up time to obtain more accurate clinical data to verify the effectiveness of TCM maintenance therapy. Disclosures No relevant conflicts of interest to declare.

Activin A (a homodimer of two activin betaA subunits) has been shown to induce the neuronal differentiation of rat pheochromocytoma PC12 cells. We studied activin A and its receptor gene expression in human pheochromocytomas in vivo and in vitro to clarify the potential involvement of activin A in the pathophysiology of these tumors. We first screened 20 pheochromocytomas and nine normal adrenal tissues for activin betaA mRNA expression. Northern blots hybridized with specific oligonucleotide probes detected weak signals for activin betaA transcripts in pheochromocytomas. Both type I and type II activin receptor (ActR-I, ActR-IB and ActR-II) mRNA expression was also detectable in the pheochromocytoma tissues. In primary cultures of pheochromocytoma cells, expression of activin betaA mRNA was readily detectable by Northern blotting, and secretion of activin A into the conditioned medium was confirmed by an enzyme-linked immunosorbent assay. The expression of activin betaA mRNA and secretion of activin A were induced by (Bu)(2)cAMP after 1 and 3 days of treatment (all P<0.05). A protein kinase inhibitor, staurosporine, inhibited the basal and (Bu)(2)cAMP-induced accumulation of activin betaA mRNA (P<0.05). In addition, induction of chromaffin phenotype by dexamethasone also inhibited the basal and (Bu)(2)cAMP-induced expression of activin A at both mRNA and protein levels (all P<0.05). In contrast, the expression of ActR-I and ActR-IB mRNAs was not affected by these agents in cultured pheochromocytoma cells. In summary, activin betaA subunit and activin receptors are expressed in human pheochromocytomas. Production of activin A in cultured pheochromocytoma cells is induced through the protein kinase A pathway, but reduced during chromaffin differentiation. Therefore, activin A may function as a local neurotrophic factor via an auto/paracrine manner in human pheochromocytomas.

Abstract3,3′-Thiodipropionic acid (TDP) is an antioxidant, which can be used as precursor carbon source to synthesize polythioesters. The bacterium Variovorax paradoxus TBEA6 strain can use TDP as a single source of carbon and energy. In the present study, experiments were carried out to identify proteins involved in the transport of TDP into the cells of strain TBEA6. Hence, eight putative tctC genes, which encode for the TctC proteins, were amplified from genomic DNA of TBEA6 strain using polymerase chain reaction and expressed in E. coli BL21 cells. Cells were grown in auto-induction medium, and protein purification was done using His Spin Trap affinity columns. Purity and molecular weight of each protein were confirmed by SDS-PAGE analysis. Protein-ligand interactions were monitored in thermoshift assays using the real-time PCR system. Two TctC proteins (locus tags VPARA-44430 and VPARA-01760) out of eight proteins showed a significant shift in their melting temperatures when they interact with the ligand (TDP or gluconate). The responsible genes were deleted in the genome of TBEA6 using suicide plasmid pJQ200mp18Tc, and single deletion mutants of the two candidate genes were subsequently generated. Finally, growth of the wild-type strain (TBEA6) and the two mutant strains (ΔVPARA-44430 and ΔVPARA-01760) were monitored and compared using TDP or gluconate as carbon sources. Wild type strains were successfully grown with TDP or gluconate. From the two mutant strains, one (ΔVPARA-44430) was unable to grow with TDP indicating that the tctC gene with locus tag VPARA-44430 is involved in the uptake of TDP.Key Points• Putative tctC genes from V. paradoxus TBEA6 were heterologously expressed in E. coli.• Protein-ligand interactions monitored in thermoshift assays using the real-time PCR.• tctC gene with locus tag VPARA-44430 is involved in the uptake of TDP.

Abstract As the most potent antigen-presenting cells, Dendritic cells (DCs), capable of inducing immune responses from naive T cells, are operative tools for tumor immunotherapy. Derived DCs are extremely effective in capturing and presentation of antigens to T cells and play a key role in the induction of cytotoxic T lymphocytes (CTLs). In vitro culture system containing the combination of GM-CSF, IL-4 and TNF-α cytokine can affect CD14 + progenitor cells from mononuclear cells (MNCs) of peripheral blood (PB) developing into functional DCs, which have enough quantities for application in vitro researches and clinical practices. Multiple myeloma cells(MM)are able to secrete a great quantity of immunoglobulin (Ig) expressing idiotypic antigen called idiotype (Id) in its mutational hotspot. This kind of idiotypic structure regions also expressing on the surface of MM cells are high specific autologous tumor associated antigen (TAA). The combination use of DCs and tumor specific antigen can improve the immunogenicity of MM cells and stimulate specific anti-tumor immunological response effectively, so by using this new kind of DC tumor vaccine, following high dose chemical therapy, the tiny residual pathological changes might be cleared totally in the future. To investigate the specific antitumor immune response induced by Id-pulsed dendritic cells(DCs) in vitro. DCs were generated from peripheral blood monocytes of the multiple myeloma(MM) patients using GM-CSF, IL-4, and TNF-α. pulsed with idiotype protein at the immature stage, DCs could activate T cells to become tumor specific cytotoxic T lymphocytes (CTLs). The morphologic characteristics of those cells were observed with light and electron microscopes. The phenotypic figures were analyzed with FACS analysis. Methy-thiazoly-Tetrazolium (MTT) assay was employed to evaluate the effect of proliferation of autologous T cells and the inhibition rate of CTL on MM cells. DCs precursors in peripheral blood could be induced to typical mature DCs in medium containing GM-CSF, IL-4 and TNF-α. Mature DCs with Id could operatively increase proliferation of the autologous T cells and active naive T cells to become tumor specialized CTLs. Any doses of CTLs had significant inhibition or killing ability on autologous MM cells. These results suggest in suitable cytokine environment, the precursors in peripheral blood of MM patients could be induced to functional DCs, and vaccination with Id-pulsed DCs could induce active antitumor immune response. Multiple cycles of immunization using DC as APC in vitro can be beneficial in generating antigen- specific T cells from normal PBMC, and Id an auto-specific tumor antigen, can be got with ammonium sulfate four-step precipitated method, By digestion of pepsin and affinity chromatography so as to stimulate MM specific immunological responce, and Id-pulsed mature DCs from MM patients can stimulate not only the proliferation of autologous T cells, but also the specific CTL immune response against autologous MM cells. In addition, in vitro immunization may provide an alternative approach to in vivo immunization of MM. We believe that DCs vaccine can bring the breakthrough of therapy to MM in the near future.

Clonal proliferation of megakaryoblasts, called transient abnormal myelopoiesis (TAM), is a rare disease of newborns triggered by trisomy 21 (constitutional or somatic) together with acquired mutations of GATA1 resulting in the exclusive production of its short variant - GATA1s. No other TAM drivers have been described so far. We have diagnosed a unique TAM case with a typical clinical and laboratory manifestation but without the gain (or any other aberration) of chromosome 21. Thorough genomic profiling revealed 4 somatic mutations: GATA1 D65_C228del, JAK1 F636del, FN1 R2420C and SPIRE2 R471W. With respect to the generally accepted 2-hit theory, we hypothesized that this TAM arose from a collaboration of the atypical GATA1 mutation (not inducing GATA1s) with (at least) one of the other identified mutations. Unlike SPIRE2 and FN1 aberrations, various mutations of the JAK1 kinase have been previously described as leukemia drivers, suggesting JAK1 F636del as a top candidate for the second hit. Moreover, JAK1 mutations have been associated with the transformation of TAM into acute megakaryoblastic leukemia (Nikolaev et al., Blood, 2013). The aim of our project was to functionally characterize this novel JAK1 mutation. Phenylalanine 636 belongs to a phylogenetically conserved triad of amino acids suggested to control catalytic activity of JAK1 via mediating a switch between the supposedly active and inactive conformations (Toms et al., Nat Struct Mol Biol, 2013). Hence, F636 seems to be essential for JAK1 function. Surprisingly, homology modeling showed that loss of F636 is compatible with both functionally opposite conformations. Indeed, Western blot analysis of JAK/STAT signaling in transiently transformed HEK293T cells showed that catalytic activity is preserved in JAK1 F636del. However, we observed lower levels of auto- and STATs- phosphorylation compared to wild-type (wt) JAK1 suggesting decreased kinase activity of JAK1 F636del. Subsequently, we tested the oncogenic potential of JAK1 F636del in the Ba/F3 cell assay; unlike the known oncogenic JAK1 variant (JAK1 V658I), JAK1 F636del did not induce IL3-independent growth. To further assess phenotypic impact of F636del, we introduced JAK1 F636del into murine bone marrow and fetal liver hematopoietic stem and progenitor cells (HSPCs) using lentiviruses and performed colony forming assays. The number and morphology of colonies did not differ in JAK1 F636del compared to wt JAK1. Furthermore, we assessed the impact of JAK1 F636del in the context of mutated GATA1. We utilized the in-vitro model recently described by Labuhn et al. (Cancer Cell, 2019), in which the CRISPR/Cas9-mediated induction of Gata1s expression leads to the expansion and sustained proliferation of fetal liver HSPCs from embryonic day 13.5 ROSA26:Cas9-EGFPki/wt mice. Similar to wt JAK1, lentivirally introduced JAK1 F636del had no impact on the proliferation and maturation status of such Gata1-edited HPSCs irrespective of the timing of its introduction (simultaneously with Gata1 editing versus into fully established Gata1-edited culture) or of culturing conditions (fully cytokine-supplemented growth-supportive versus cytokine-depleted growth-restrictive medium). Altogether, we show that unlike known oncogenic variants, F636del identified in the first case of trisomy 21-independent TAM attenuates JAK1 kinase activity. The results of our phenotypic studies question the potential contribution of this mutation to TAM development. Interestingly, Labuhn et al. (2019) recently showed that non-activating JAK mutations occur at higher than random frequency in trisomy 21-dependent TAM. This tempts us to speculate that JAK1 mutations may still play a role in TAM. Yet, this role may significantly differ from that of known oncogenic mutations; it may result from attenuation/modulation instead of activation of downstream signaling and it may remain unrevealed utilizing the currently available sophisticated, yet still imperfect experimental models. Support: GAUK 86218, EHA Research Mobility Grant Disclosures No relevant conflicts of interest to declare.

Background: Auto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems. Auto-induction can occur unintentionally using a complex culture medium prepared by mixing culture substrates. The differences in culture substrates sometimes lead to variations in the induction level. Objectives: In this study, we investigated the feasibility of using glucose and lactose as boosters of auto-induction with a complex culture medium. Method: First, auto-induction levels were assessed by quantifying recombinant GFPuv expression under the control of the T7lac promoter. Effectiveness of the additive-containing medium was examined using ovine angiotensinogen (tac promoter-based expression) and Thermus thermophilus manganese-catalase (T7lac promoter-based expression). Results: Auto-induced GFPuv expression was observed with the enzymatic protein digest Polypepton, but not with another digest tryptone. Regardless of the type of protein digest, supplementing Terrific Broth medium with glucose (at a final concentration of 2.9 g/L) and lactose (at a final concentration of 7.6 g/L) was successful in obtaining an induction level similar to that achieved with a commercially available auto-induction medium. The two recombinant proteins were produced in milligram quantity of purified protein per liter of culture. Conclusion: The medium composition shown in this study would be practically useful for attaining reliable auto-induction for E. coli-based recombinant protein production.

Fossil fuels are exhausting day by day at a very faster rate due to excessive demand for energy. Diesel engines are important prime movers used in different industries. When liquid petroleum fuels are burnt in diesel engines they emit harmful exhaust emissions which pollute the environment and may cause severe chronic diseases. Hence to mitigate over-dependency of crude oil and to protect the environment from harmful emissions, different engine experts and scientists have proposed dual fuel combustion technology to utilize low emissions renewable gaseous fuels without compromising its performance. Most of the work in the literature concentrate on utilizing gaseous fuels such as CNG, LPG, biogas, and hydrogen whereas very little quantum of work has been done to utilize acetylene in the IC engine. The higher flame velocity, high auto-ignition temperature, and high calorific value are the important combustion properties of acetylene which makes it more advantageous in CI engine than the available feedstock. The acetylene can be easily produced from calcium carbonate and water. Hence, the author has considered acetylene as a primary fuel in the present study and diesel as a pilot fuel in the modified CI engine. In this experimental investigation, the author has optimized the flow rate of acetylene by analyzing the performance and emission characteristics of the acetylene fuelled diesel engine at different loads and finally, the obtained results were compared with the neat diesel. The acetylene was inducted at a different gas flow rate of 2 LPM, 3 LPM, and 5 LPM. The results show that when acetylene induction takes place at 2 LPM, the brake thermal efficiency (BTE) increases by 1.4 % at full load during dual fuel mode compared to neat diesel. Brake specific energy consumption (BSEC) increases during acetylene induction whereas carbon monoxide, hydrocarbon, and smoke decrease particularly at medium to high engine loads this may be due to homogenous charge mixture formation, leading to stable combustion. However, there is a slight increase in oxides of nitrogen emissions, which may be due to higher flame speed causing uncontrolled combustion at peak loads relative to baseline diesel.