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1.
Sampali Banerjee Shardul S. Salunkhe Anjali D. Apte-Deshpande Naganath S. Mandi Goutam Mandal Sriram Padmanabhan 《Biotechnology letters》2009,31(7):1031-1036
A modified pBAD24 vector (pBAD24M) was constructed with the araBAD promoter of the arabinose operon along with T7g10 sequence
elements and a modified Shine–Dalgarno sequence. While both green fluorescent protein and granulocyte colony stimulating factor
showed negligible expression under the original pBAD24 vector, they were expressed at >35% of total cellular protein with
the modified vector. Similar results were obtained for staphylokinase wherein the pBAD24-SAK construct yielded 8 ng/106 c.f.u. of E. coli induced cells while the pBAD24M-SAK vector showed nearly 55 ng/106 c.f.u. induced bacterial cells as tested by ELISA. Interestingly, the expression levels using modified pBAD24 vector matched
that achieved with T7 promoter based vector system. The modified pBAD24 vector therefore represents a simple and a useful
prokaryotic expression system for efficient repression, modulation and elevated protein expression levels.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
2.
Yamaji K Nabeshima S Murata M Chong Y Furusyo N Ikematsu H Hayashi J 《Cancer immunology, immunotherapy : CII》2006,55(4):394-403
Type I interferon (IFN) possesses antiviral and antitumor activities and also having an immune regulatory effect, activating
cellular immune response and upregulating several cytokines. Recent study has shown that type I IFN upregurates the dendritic
cell production of IL-15 capable of activating natural killer cells and CD8+ memory T lymphocytes. However, it is still unknown if type I IFN induces IL-15 production in non-immune cells and if type
I IFN affects IL-15 production in vivo. The present study investigated the effect of type I IFNs on IL-15 expression in hepatocellular
carcinoma (HCC) cell lines in vitro and in patients with chronic hepatitis C in vivo. When three HCC cell lines, Huh7, HepG2,
and JHH4 were cultured in vitro, IFN upregulation of IL-15 expression was observed at both the mRNA and protein levels. In
experiments using Huh7 cells, upregulation of IL-15 expression occurred within 24 h of the start of IFN stimulation, and both
IFN-α and -β dose-dependently increased IL-15 production in the range from 100 U/ml to 10,000 U/ml of concentration. IFN-β
showed stronger activity in IL-15 production induction in vitro than IFN-α. For in vivo examination, sera were obtained from
21 chronic hepatitis C patients treated with IFN and 29 healthy individuals, and the serum IL-15 level was quantified by ELISA.
The serum IL-15 level of chronic hepatitis C patients before IFN treatment was similar to that of the healthy controls and
significantly increased only during the IFN administration period. These results confirm that IFN-α/β induce IL-15 production
and also suggest that IL-15 may be associated with type I IFN-induced immune response. 相似文献
3.
A mammalian gene expression vector based on cytomegalovirus (CMV)enhancer/promoter (CMVe/p) for the regulation of gene expression
was further optimized by adding oriP elements derived from Epstein-Barr virus (EBV) and the Tat/TAR transactivation axisfrom human immunodeficiency virus type
1 (HIV-1). Using the Tat/TAR-oriP expression vector, a transient transfection system was optimized for an extended culture period to produce large amounts
of secreted IL-2SA (an IL-2 mutein) in HKB11 cells. We observed a 4-fold increase in IL-2SA expression in cells transfected
with vectors containing the HIV-1 transactivation axis (Tat/TAR) or oriP elements alone when compared to cells transfected with the control vector having a CMVe/p. Cells transfected with expression
vectors equipped with both oriP and Tat/TAR showed an 18-fold increase in IL-2SA expression. This transient transfection system maintained high secretion
of IL-2SA for a period of 10-day with no appreciable loss in expression. We demonstrate that during this 10-day culture period,
it was possible to produce 1–100 mg of proteins using 500 μg of plasmid DNA.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
4.
5.
Background aimsInterleukin (IL)-15 and fms-like tyrosine kinase-3 (FLT-3) are crucial factors for the development of human and murine natural killer (NK) cells. Previously, we have demonstrated significant ex vivo expansion and activation of unrelated cord blood (UCB) NK cells with an antibody/cytokine cocktail consisting of anti-CD3 + IL-2 + IL-12 + IL-7 and anti-CD3 + IL-2 + IL-12 + IL-18.MethodsIn the current experiments, we investigated the effects of short-term culture with anti-CD3 + IL-2 + FLT-3 + IL-15 on cord blood (CB) NK cell and NK-cell subset expansion and function. CB mononuclear cells were cultured for 48 h in AIM-V media or AIM-V + IL-2 (5 ng/mL) + anti-CD3 (50 ng/mL) + FLT-3 (50 ng/mL) ± escalating doses of IL-15 (1, 10 or 100 ng/mL). Flow cytometric analysis was performed using various fluorescent-conjugated monoclonal antibodies. In vitro cytotoxicity was determined with a standard europium assay against K562 and Daudi cells.ResultsThere was a 4.8-fold significant increase in NK-cell population (CD3?/16+/56+; P < 0.03), 21-fold significant increase in CD3?/56+/158a+ (KIR2DL1/S1; P < 0.002), 46-fold significant increase in CD3?/56+/158b+ (KIR2DL1/S2; P < 0.002) and 11.5-fold significant increase in CD3?/56+/NKB1+ (KIR3DL1; P < 0.01). We also noted a significant increase in both NK and lymphokine-activated killer (LAK) cytotoxicity with IL-2 + anti-CD3 + FLT-3 + IL-15 (100 ng/mL) compared with IL-2 + anti-CD3 + FLT-3 and media alone against K562 (P < 0.01) and Daudi (P < 0.001), respectively.ConclusionsWe have demonstrated a significant increase in UCB NK cells and NK cells expressing a variety of killer immunoglobulin-like receptor (KIR) receptors after short-term culture with anti-CD3, IL-2, FLT-3 and IL-15. Furthermore, there was a significant increase in in vitro NK/LAK cell cytotoxicity. 相似文献
6.
M Negrini P Rimessi S Sabbioni A Caputo P G Balboni R Gualandri R Manservigi M P Grossi G Barbanti-Brodano 《BioTechniques》1991,10(3):344-353
A eukaryotic vector-host cell system is described where the additive transactivating effects of HIV-1 tat and adenovirus E1A on HIV-1 long terminal repeat (LTR) are exploited to increase expression of exogenous cDNAs. Human 143B and 293 cells, the latter constitutively producing E1A, were used as host cell lines. The bacterial gene chloramphenicol acetyltransferase (CAT) and the hepatitis B surface antigen (HBs-Ag) gene were employed as reporter genes inserted in pRPneoU3R, an episomal vector containing BK virus replication origin and early region, where cDNAs are expressed under control of HIV-1 LTR. The 293 cells were transformed by tat expression vectors to constitutively express tat. Stable cell clones of 293tat cells, constitutively expressing CAT after transformation with pRPneoU3R-CAT, show a CAT activity 600-fold higher than normal 293 transformed cells. CAT expression obtained in normal 293 cells can be transiently increased 10-fold by transfection by vectors expressing tat. The 293tat cells transformed by pRPneoU3R-HBs, an episomal vector expressing HBs-Ag from HIV LTR, yielded stable cell clones secreting HBs-Ag in the culture medium at a concentration up to 744 ng/ml or 44 ng/10(6) cells/24 h, 48-fold more than normal 293 cells. The use of this system for constitutive or inducible expression of sequences under control of HIV-1 LTR is discussed in view of possible applications for diagnostic, vaccinal and therapeutic purposes. 相似文献
7.
Young Il Kim Jin-Woo Lee Mu-Hyoung Lee Seung-Won Park Byung-Nam Cho Ha Kyu Lee 《Molecular biology reports》2011,38(5):3207-3212
15-deoxy-∆12,14-prostaglandin J2 (15d-PGJ2) is an anti-inflammatory prostaglandin that plays a role in promoting the resolution of inflammation. We investigated the
effects of 15d-PGJ2 on the production of IL-8 and on the expression of Toll-like receptors (TLRs) 2 in human primary keratinocytes stimulated
with lipopolysaccharide (LPS). Cell proliferation was analyzed using the MTT assay, TLR2 and -4 mRNA expression was detected
by RT–PCR, and IL-8 production and NF-κB p65 activities were determined by ELISA. LPS and 15d-PGJ2 did not influence the proliferation rate at low concentrations (0.5 and 2.0 μM) in keratinocytes, and showed toxicity at
high concentrations (5.0 μM). LPS, compared with control, induced the expression of TLR2 mRNA, increased IL-8 production,
and enhanced NF-κB activity. 15d-PGJ2 decreased TLR2 mRNA, increased IL-8 production, and suppressed NF-κB activity. Costimulation with LPS and 15d-PGJ2, compared with LPS stimulation alone, decreased TLR2 mRNA (1.8-fold), increased IL-8 production (1.8-fold at 0.5 μM and 3.7-fold
at 2.0 μM), and inhibited NF-κB activity (3.3-fold at 0.5 μM and 5.1-fold at 2.0 μM). TLR4 mRNA was not expressed in primary
keratinocytes. These results suggest that 15d-PGJ2 suppresses TLR2 expression and that it up-regulates the production of IL-8 by inhibiting the NF-κB signaling pathway in primary
keratinocytes. Thus, 15d-PGJ2 can have both anti- and pro-inflammatory effects, and 15d-PGJ2-mediated IL-8 up-regulation is related to the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways. 相似文献
8.
Arnaud Bianchi David Moulin Sylvie Sebillaud Meriem Koufany Marie-Madeleine Galteau Patrick Netter Bernard Terlain Jean-Yves Jouzeau 《Arthritis research & therapy》2005,7(6):R1325
Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with
a key function in prostaglandin (PG)E2 synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production
of 6-keto-PGF1α and PGE2 in rat chondrocytes stimulated with 10 ng/ml IL-1β, and compared their modulation by peroxisome-proliferator-activated receptor
(PPAR)γ agonists. Real-time PCR analysis showed that IL-1β induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1
expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF1α and PGE2 peaked 24 hours after stimulation with IL-1β; the induction of PGE2 was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Δ12,14prostaglandin J2 (15d-PGJ2) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 μM), with more potency on PGE2 level than on 6-keto-PGF1α level (-90% versus -66% at 10 μM). A high dose of 15d-PGJ2 partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels.
Rosiglitazone was poorly effective on these parameters even at 10 μM. Inhibitory effects of 10 μM 15d-PGJ2 were neither reduced by PPARγ blockade with GW-9662 nor enhanced by PPARγ overexpression, supporting a PPARγ-independent
mechanism. EMSA and TransAM? analyses demonstrated that mutated IκBα almost completely suppressed the stimulating effect of IL-1β on mPGES-1 expression
and PGE2 production, whereas 15d-PGJ2 inhibited NF-κB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1
is rate limiting for PGE2 synthesis; second, activation of the prostaglandin cascade requires NF-κB activation; third, 15d-PGJ2 strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ2 occurs independently of PPARγ through inhibition of the NF-κB pathway; fifth, mPGES-1 is the main target of 15d-PGJ2. 相似文献
9.
Christopher A. Lazarski Anushree A. Datar Emily K. Reynolds Michael D. Keller Catherine M. Bollard Patrick J. Hanley 《Cytotherapy》2021,23(1):65-76
Infusion of viral-specific T cells (VSTs) is an effective treatment for viral infection after stem cell transplant. Current manufacturing approaches are rapid, but growth conditions can still be further improved. To optimize VST cell products, the authors designed a high-throughput flow cytometry-based assay using 40 cytokine combinations in a 96-well plate to fully characterize T-cell viability, function, growth and differentiation. Peripheral blood mononuclear cells (PBMCs) from six consenting donors were seeded at 100 000 cells per well with pools of cytomegalovirus peptides from IE1 and pp65 and combinations of IL-15, IL-6, IL-21, interferon alpha, IL-12, IL-18, IL-4 and IL-7. Ten-day cultures were tested by 13-color flow cytometry to evaluate viable cell count, lymphocyte phenotype, memory markers and interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) expression. Combinations of IL-15/IL-6 and IL-4/IL-7 were optimal for the expansion of viral-specific CD3+ T cells, (18-fold and 14-fold, respectively, compared with unstimulated controls). CD8+ T cells expanded 24-fold in IL-15/IL-6 and 9-fold in IL-4/IL-7 cultures (P < 0.0001). CD4+ T cells expanded 27-fold in IL-4/IL-7 and 15-fold in IL-15/IL-6 (P < 0.0001). CD45RO+ CCR7– effector memory (CD45RO+ CCR7– CD3+), central memory (CD45RO+ CCR7+ CD3+), terminal effector (CD45RO– CCR7– CD3+), and naive (CD45RO– CCR7+ CD3+). T cells were the preponderant cells (76.8% and 72.3% in IL-15/IL-6 and IL-15/IL-7 cultures, respectively). Cells cultured in both cytokine conditions were potent, with 19.4% of CD3+ cells cultured in IL-15/IL-6 producing IFNγ (7.6% producing both TNFα and IFNγ) and 18.5% of CD3+ cells grown in IL-4/IL-7 producing IFNγ (9% producing both TNFα and IFNγ). This study shows the utility of this single-plate assay to rapidly identify optimal growth conditions for VST manufacture using only 107 PBMCs. 相似文献
10.
Kentaro Yamada Naoya Morishita Tomohisa Katsuda Shuji Kubo Akinobu Gotoh Hideki Yamaji 《Cytotechnology》2009,59(3):153-160
The use of low-multiplicity infection of 293 cells in static culture with regular medium replacement was investigated for
efficient large-scale production of adenovirus vectors for gene therapy applications. An adenovirus vector carrying the enhanced
green fluorescent protein gene (Ad EGFP) was used to infect 293-F cells at a low multiplicity of infection (MOI) of 0.00001–0.1
transductional unit (TU) cell−1. The cells, which have the ability to grow in suspension, were incubated in T-flasks and the serum-free culture medium was
replaced with fresh medium via centrifugation every 2 days. Because only a small proportion of cells were initially infected
at low MOIs (<1 TU cell−1), uninfected cells continued to grow until they were infected by progeny adenoviruses released from previously infected cells.
When 293-F cells at a relatively low density of 1 × 105 cells cm−3 were infected with Ad EGFP at a low MOI of 0.001 TU cell−1, the vector yield was 2.7-fold higher than the maximum yield obtained with high-multiplicity infection (MOI = 10 TU cell−1) in batch culture. These results indicate that efficient adenovirus vector production using low MOIs is achieved by minimization
of either nutrient depletion and/or accumulation of inhibitory metabolites in the culture medium. 相似文献
11.
Peroni CN Cecchi CR Damiani R Soares CR Ribela MT do Rocio Arkaten R Bartolini P 《Molecular biotechnology》2006,34(2):239-245
A gene therapy clinical trial for treatment of growth hormone (GH) deficiency has not been reached yet, but several strategies
using different gene transfer methodologies and animal models have been developed and showed successful results. We have set
up an ex vivo gene therapy protocol using primary human keratinocytes transduced with an efficient retroviral vector (LXSN)
encoding the human (hGH) or mouse GH (mGH) genes. These stably modified cells presented high in vitro expression levels of
hGH (7 μg/106 cells/d) and mGH (11 μg/106 cells/d) after selection with geneticin. When the hGH-secreting keratinocytes were grafted onto immunodeficient dwarf mice
(lit/scid), hGH levels in the circulation were about 0.2–0.3 ng/mL during a 12-d assay and these animals presented a significant body
weight increase (p<0.01) compared to the control. Substitution of conventional grafting methodologies with organotypic raft cultures revealed
a peak value of up to 20 ng mGH/mL in the circulation of grafted lit/scid mice at 1 h postimplantation, followed by a rapid decline to baseline (≈2 ng/mL) within 24 h. One week after grafting, however,
the cultured excised implants still presented approx 45% of their original in vitro secretion efficiency. Further studies
are being carrier out to identify the main factor(s) that still constitute one of the major impediments to the success of
this promising model of cutaneous gene therapy. 相似文献
12.
13.
Production of human factor VIII-FL in 293T cells using the bicistronic MGMT(P140K)-retroviral vector
Fontes AM Melo FU Greene LJ Faça VM Lin Y Gerson SL Covas DT 《Genetics and molecular research : GMR》2012,11(1):775-789
Hemophilia A is the most common X-linked bleeding disorder; it is caused by deficiency of coagulation factor VIII (FVIII). Replacement therapy with rFVIII produced from human cell line is a major goal for treating hemophilia patients. We prepared a full-length recombinant FVIII (FVIII-FL), using the pMFG-P140K retroviral vector. The IRES DNA fragment was cloned upstream to the P140K gene, providing a 9.34-kb bicistronic vector. FVIII-FL cDNA was then cloned upstream to IRES, resulting in a 16.6-kb construct. In parallel, an eGFP control vector was generated, resulting in a 10.1- kb construct. The 293T cells were transfected with these constructs, generating the 293T-FVIII-FL/P140K and 293T-eGFP/P140K cell lines. In 293T-FVIII-FL/P140K cells, FVIII and P140K mRNAs levels were 4,410 (±931.7)- and 295,400 (±75,769)-fold higher than in virgin cells. In 293T-eGFP/P140K cells, the eGFP and P140K mRNAs levels were 1,501,000 (±493,700)- and 308,000 (±139,300)-fold higher than in virgin cells. The amount of FVIII-FL was 0.2 IU/mL and 45 ng/mL FVIII cells or 4.4 IU/μg protein. These data demonstrate the efficacy of the bicistronic retroviral vector expressing FVIII-FL and MGMT(P140K), showing that it could be used for producing the FVIII-FL protein in a human cell line. 相似文献
14.
Viral vectors are valuable tools to deliver genetic materials into cells. Vectors derived from human immunodeficiency virus type 1 are being widely used for gene delivery, mainly because they are able to transduce both dividing and non-dividing cells which leads to stable and long term gene expression. In addition, these types of vectors are safe, with low toxicity, high stability and cell type specificity. Therefore, this work was aimed to produce lentivirus-based vector using a three-plasmid system. To produce this system, the eGFP marker gene was cloned into the plasmid pWPXLd. Subsequently, this vector plasmid, along with packaging plasmids, psPAX2 and envelope plasmid, pMD2.G, was co-transfected into packaging cell line (293T) using calcium phosphate method. 48 h post transfection, the constructed viral vector was harvested, purified and concentrated and stored at −80 °C for next experiments. The titration of the vector was carried out, using ELISA, flowcytometry, and fluorescent microscopy. Finally, transduction of HEK-293T, CHO, HepG2, MCF-7, MEFs and Jurkat cell lines was carried out with indicated cell numbers and multiplicities of infections of the vector in the presence of polybrene. Using this system, high titer lentivirus at titers of up to 2 × 108 transducing units/ml (TU/ml) was successfully generated and its transduction efficacy was improved by seven to over 20-fold in various cell types. We demonstrate the applicability of this vector for the efficient transduction of dividing and non-dividing cells, including HEK-293T, CHO, HepG2, MCF-7, MEFs and Jurkat cell line. Transduction efficiency yielded titers of (6.3 ± 1.2) 105 TU/ml. Furthermore, lentivirus transferred transgene was expressed at high level in the target cells and expression was followed until 90 days after transduction. Thus, the vector generated in this work, might be able to deliver the transgene into a wide range of mammalian cells.
Electronic supplementary material
The online version of this article (doi:10.1007/s10616-013-9652-5) contains supplementary material, which is available to authorized users. 相似文献15.
Xue G Liu RY Li Y Cheng Y Liang ZH Wu JX Zeng MS Tian FZ Huang W 《Cancer immunology, immunotherapy : CII》2007,56(11):1831-1843
Backgroud and objective Dendritic cells play an important role in initiation and regulation of immune responses. Previous studies demonstrated that
intratumoral administration of 6Ckine-modified DCs enhanced local and systemic antitumor effects. Herein we report the investigation
of the specific CTL responses elicited by adenoviral 6Ckine/IFNγ fusion gene-modified DCs in vitro.
Methods Human monocyte-derived DCs were modified with an adenoviral vector encoding 6Ckine/IFNγ fusion protein (Ad-6Ckine/IFNγ), and
then investigated the effect of 6Ckine/IFNγ fusion protein on the maturation, cytokine and chemokine secretion of DCs, and
their activities of recruiting and activating T cells in vitro were investigated.
Results 6Ckine/IFNγ fusion protein induced DC maturation characterized with the upregulation of CD83 and CCR7. And it up-regulated
the expression of RANTES and IL-12p70, down-regulated that of IL-10 in DCs. Additionally, 6Ckine/IFNγ markedly increased DC’s
recruiting ability for naive T cells, benefiting from the enhanced expression of chemokines 6Ckine and RANTES in DCs. Fusion
gene-modified DCs significantly promoted the proliferation of autologous T cells, induced Th1 differentiation by upregulating
the expression of IL-2 and T-bet in T cells, and increased specific cytotoxicity of CTLs against specific tumor cells, HepG2
or LoVo cells, respectively.
Conclusion Combining the effects of 6Ckine and IFNγ, Ad-6Ckine/IFNγ modified DCs induced enhanced CTL responses in vitro, which indicated
that Ad-6Ckine/IFNγ modified DCs might be used as an adjuvant to trigger an effective antitumor immune response.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Gang Xue, Ran-yi Liu, Yan Li contributed equally to this work. 相似文献
16.
Yunier Rodríguez-Álvarez Klaudia Martínez-Cordovez Alexey Llopiz-Arzuaga Yassel Ramos-Gómez Vladimir Besada-Pérez Dayana García-Lines 《Preparative biochemistry & biotechnology》2017,47(9):889-900
Recombinant simian IL-15 (siIL-15) was obtained for the preclinical assessment of an anti-human IL-15 vaccine. For this purpose, the cDNA from peripheral blood mononuclear cells of a Macaca fascicularis monkey was cloned into a pIL-2 vector. The siIL-15 was expressed in Escherichia coli strain W3110 as an insoluble protein which accounted for 13% of the total cellular proteins. Inclusion bodies were solubilized in an 8?M urea solution, which was purified by ion exchange and reverse phase chromatography up to 92% purity. The protein identity was validated by electrospray ionization-mass spectrometry, confirming the presence of the amino acids which distinguish the siIL-15 from human IL-15. The purified siIL-15 stimulates the proliferation of cytotoxic T-lymphocytes line (CTLL)-2 and Kit 225 cells with EC50 values of 3.1 and 32.5?ng/mL, respectively. Antisera from modified human IL-15-immunized macaques were reactive to human and simian IL-15 in enzyme-linked immunosorbent assays. Moreover, the anti-human IL-15 antibodies from immune sera inhibited siIL-15 activity in CTLL-2 and Kit 225 cells, supporting the activity and purity of recombinant siIL-15. These results indicate that the recombinant siIL-15 is biologically active in two IL-15-dependent cell lines, and it is also suitable for the preclinical evaluation of an IL-15-based therapeutic vaccine. 相似文献
17.
Moustaki A Argyropoulos KV Baxevanis CN Papamichail M Perez SA 《Cancer immunology, immunotherapy : CII》2011,60(12):1683-1695
We have previously reported a synergistic effect between hydrocortisone (HC) and IL-15 on promoting natural killer (NK) cell
expansion and function. In the present study, we extend our findings to methylprednisolone (MeP) and dexamethasone (Dex),
thus ascribing to glucocorticoids (GCs) a general feature as positive regulators of IL-15-mediated effects on NK cells. We
demonstrate that each GC when combined with IL-15 in cultures of peripheral blood (PB)-derived CD56+ cells induces increased expansion of CD56+CD3− cells displaying high cytolytic activity, IFN-γ production potential and activating receptor expression, including NKp30,
NKp44, NKp46, 2B4, NKG2D and DNAM-1. Furthermore, GCs protected NK cells from IL-15-induced cell death. The combination of
IL-15 with GCs favored the expansion of a relatively more immature CD16low/neg NK cell population, with high expression of NKG2A and CD94, and significantly lower expression of KIR (CD158a and CD158b)
and CD57, compared to IL-15 alone. IL-15-expanded NK cells, in the presence or absence of GCs, did not express CD62L, CXCR1
or CCR7. However, the presence of GCs significantly increased the density of CXCR3 and induced strong CXCR4 expression on
the surface of NK cells. Our data indicate that IL-15/GC-expanded NK cells, apart from their increased proliferation rate,
retain their functional integrity and exhibit a migratory potential rendering them useful for adoptive transfer in NK cell-based
cancer immunotherapy. 相似文献
18.
Hirofumi Shoda Keishi Fujio Yumi Yamaguchi Akiko Okamoto Tetsuji Sawada Yuta Kochi Kazuhiko Yamamoto 《Arthritis research & therapy》2007,8(6):R166
IL-32 is a newly described cytokine in the human found to be an in vitro inducer of tumor necrosis factor alpha (TNFα). We examined the in vivo relationship between IL-32 and TNFα, and the pathologic role of IL-32 in the TNFα-related diseases – arthritis and colitis.
We demonstrated by quantitative PCR assay that IL-32 mRNA was expressed in the lymphoid tissues, and in stimulated peripheral
T cells, monocytes, and B cells. Activated T cells were important for IL-32 mRNA expression in monocytes and B cells. Interestingly,
TNFα reciprocally induced IL-32 mRNA expression in T cells, monocyte-derived dendritic cells, and synovial fibroblasts. Moreover,
IL-32 mRNA expression was prominent in the synovial tissues of rheumatoid arthritis patients, especially in synovial-infiltrated
lymphocytes by in situ hybridization. To examine the in vivo relationship of IL-32 and TNFα, we prepared an overexpression model mouse of human IL-32β (BM-hIL-32) by bone marrow transplantation.
Splenocytes of BM-hIL-32 mice showed increased expression and secretion of TNFα, IL-1β, and IL-6 especially in response to
lipopolysaccharide stimulation. Moreover, serum TNFα concentration showed a clear increase in BM-hIL-32 mice. Cell-sorting
analysis of splenocytes showed that the expression of TNFα was increased in resting F4/80+ macrophages, and the expression of TNFα, IL-1β and IL-6 was increased in lipopolysaccharide-stimulated F4/80+ macrophages and CD11c+ dendritic cells. In fact, BM-hIL-32 mice showed exacerbation of collagen-antibody-induced arthritis and trinitrobenzen sulfonic
acid-induced colitis. In addition, the transfer of hIL-32β-producing CD4+ T cells significantly exacerbated collagen-induced arthritis, and a TNFα blockade cancelled the exacerbating effects of hIL-32β.
We therefore conclude that IL-32 is closely associated with TNFα, and contributes to the exacerbation of TNFα-related inflammatory
arthritis and colitis. 相似文献
19.
This study compared the effects of the human 70-kDa stress protein (Hsp70) peptide, TKDNNLLGRFELSG (TKD), proinflammatory
cytokines, or a combination of both on the repertoire of receptors expressed by human natural killer (NK) cells and their
capacity to kill human CX colon carcinoma cells, K562 erythroleukemic cells, and leukemic blasts from two patients with acute
myelogenous leukemia. Low-dose interleukin (IL) 2/IL-15 and TKD increase the expression density of activatory (NKG2D, NKp30,
NKp44, NKp46, CD94/NKG2C) and inhibitory (CD94/NKG2A) receptors on NK cells. Concomitantly, IL-2/TKD treatment enhances the
cytotoxicity of NK cells (as reflected by their secretion of granzyme B) against Hsp70 membrane-positive and human leukocyte
antigen (HLA)-E membrane-negative (Hsp70+/HLA-E−) CX+ and K562 cells. However, it had no effect on the responsiveness to Hsp70−/HLA-E− CX− cells over that induced by IL-2 alone. The cytotoxicity of IL-2/TKD-activated, purified NK cells and peripheral blood mononuclear
cells against Hsp70+/HLA-E+ leukemic blasts was weaker than that against Hsp70+/HLA-E− K562 cells. Hsp70-blocking and HLA-E transfection experiments confirmed membrane-bound Hsp70 as being a recognition/activatory
ligand for NK cells, as cytotoxicity was reduced by the presence of the anti-Hsp70 monoclonal antibody cmHsp70.2 and by inhibiting
Hsp70 synthesis using short interference ribonucleic acid. HLA-E was confirmed as an inhibitory ligand, as the extent of NK
cell-mediated lysis of K562 cell populations that had been transfected with HLA-ER or HLA-EG alleles was dependent on the proportion of HLA-E-expressing cells. These findings indicate that Hsp70 (as an activatory molecule)
and HLA-E (as an inhibitory ligand) expression influence the susceptibility of leukemic cells to the cytolytic activities
of cytokine/TKD-activated NK cells. 相似文献
20.
Alison Ward Malcolm Anderson Robert I. Craggs Justine Maltby Caroline Grahames Rick A. Davies Donna Finch Debbie Pattison Heather Oakes Philip R. Mallinder 《Protein expression and purification》2009,68(1):42-48
The physiological activities of Interleukin-15 (IL-15) suggest that it could be useful as an immunomodulator to activate the innate immune system, however, the expression and purification yields of recombinant mature IL-15 have typically been low. In this report, a method was optimised to generate milligram quantities of this cytokine. Human IL-15 with an N-terminal (His)6-tag was expressed in Escherichia coli as an insoluble protein. The IL-15 material was purified from other cellular proteins by dissolution in 6 M guanidine HCl, followed by Ni-NTA chromatography in a buffer containing 8 M urea. Use of a multi-component screen identified the optimal conditions for folding (His)6-tagged human IL-15 and the method was scaled up to produce milligram quantities of folded material in its native conformation, with two intra-molecular disulphides as determined by electrospray mass spectrometry. Mature IL-15 was generated by cleavage with recombinant enterokinase, which was subsequently removed by Ni-NTA chromatography. Identical methods were used to produce mature cynomolgus monkey (Macaca fascicularis) IL-15 in similar quantities. Human and cynomolgus IL-15 were both active in two IL-15 dependent assays; mouse CTLL2 cell proliferation and human and cynomolgus CD69 upregulation on CD3− CD8+ lymphocytes in whole blood. Despite being 96% identical at the amino acid level the human IL-15 was 10-fold more potent than the cynomolgus IL-15 in both assays. The methods described here are useful for producing both mature IL-15 proteins in sufficient quantity for in vivo and in vitro studies, including X-ray crystallography. 相似文献