32 research outputs found
Optimization of transfection methods for HuhΒ7 and Vero cells: a comparative study
Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for Huh-7 and Vero cells. Different cell confluencies, DNA/reagent ratios and total transfection volumes were optimized for two chemical reagents including jetPEIβ’ and Lipofectamineβ’ 2000. Besides, the effects of electric field strength and pulse length were investigated to improve electroporation efficiency. Transfection of cells by pEGFP-N1 vector and tracking the expression of GFP by FACS and Fluorescence Microscopy analysis were the employed methods to evaluate transfection efficiencies. Optimized electroporation protocols yielded 63.73 Β± Β± 2.36 and 73.9 Β± 1.6 % of transfection in Huh-7 and Vero cells respectively, while maximum achieved level of transfection by jetPEIβ’ was respectively 14.2 Β± 0.69 and 28 Β± 1.11 % for the same cells. Post transfectional chilling of the cells did not improve electrotransfection efficiency of Huh-7 cells. Compared to chemical based reagents, electroporation showed the superior levels of transfection in both cell lines. The presented protocols should satisfy most of the experimental applications requiring high transfection efficiencies of these two cell lines.ΠΠ°Π»ΠΈΡΠΈΠ΅ ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎΠ³ΠΎ ΠΏΡΠΎΡΠΎΠΊΠΎΠ»Π° ΡΡΠ°Π½ΡΡΠ΅ΠΊΡΠΈΠΈ ΡΠ²Π»ΡΠ΅ΡΡΡ ΠΏΠ΅ΡΠ²ΡΠΌ ΡΡΠ»ΠΎΠ²ΠΈΠ΅ΠΌ ΡΡΠΏΠ΅ΡΠ½ΡΡ
ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΠΉ ΠΏΠΎ ΠΏΠ΅ΡΠ΅Π½ΠΎΡΡ Π³Π΅Π½ΠΎΠ² Π² ΠΊΠ»Π΅ΡΠΊΠΈ ΠΌΠ»Π΅ΠΊΠΎΠΏΠΈΡΠ°ΡΡΠΈΡ
, ΡΡΠΎ Π΄ΠΎΡΡΠΈΠ³Π°Π΅ΡΡΡ ΡΠΊΡΠΏΠ΅ΡΠΈΠΌΠ΅Π½ΡΠ°Π»ΡΠ½ΠΎ Π΄Π»Ρ ΠΊΠ°ΠΆΠ΄ΠΎΠ³ΠΎ ΠΊΠΎΠ½ΠΊΡΠ΅ΡΠ½ΠΎΠ³ΠΎ ΡΠΈΠΏΠ° ΠΊΠ»Π΅ΡΠΎΠΊ. ΠΠ΄Π΅ΡΡ ΠΌΡ ΠΏΡΠΈΠ²ΠΎΠ΄ΠΈΠΌ Π΄Π°Π½Π½ΡΠ΅ ΡΡΠ°Π²Π½ΠΈΡΠ΅Π»ΡΠ½ΠΎΠ³ΠΎ ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΡ ΠΏΠΎ ΠΎΠΏΡΠΈΠΌΠΈΠ·Π°ΡΠΈΠΈ ΡΡΠ»ΠΎΠ²ΠΈΠΉ ΡΡΠ°Π½ΡΡΠ΅ΠΊΡΠΈΠΈ ΠΊΠ»Π΅ΡΠΎΠΊ Huh-7 ΠΈ Vero Ρ ΠΏΠΎΠΌΠΎΡΡΡ ΡΠ»Π΅ΠΊΡΡΠΎΠΏΠΎΡΠ°ΡΠΈΠΈ ΠΈ Ρ
ΠΈΠΌΠΈΡΠ΅ΡΠΊΠΈΠΌΠΈ ΠΌΠ΅ΡΠΎΠ΄Π°ΠΌΠΈ. ΠΠ»Ρ Π΄Π²ΡΡ
Ρ
ΠΈΠΌΠΈΡΠ΅ΡΠΊΠΈΡ
ΡΠΎΠ΅Π΄ΠΈΠ½Π΅Π½ΠΈΠΉ, jetPEIβ’ ΠΈ Lipofectamineβ’ 2000, Π±ΡΠ»ΠΈ ΠΎΠΏΡΠΈΠΌΠΈΠ·ΠΈΡΠΎΠ²Π°Π½Ρ ΡΠΎΡΠ΅ΡΠ°Π½ΠΈΡ ΡΠ°Π·Π»ΠΈΡΠ½ΡΡ
ΠΊΠ»Π΅ΡΠΎΠΊ, ΡΠΎΠΎΡΠ½ΠΎΡΠ΅Π½ΠΈΡ ΠΠΠ/ΡΠ΅Π°Π³Π΅Π½Ρ ΠΈ ΠΎΠ±ΡΠΈΠ΅ ΠΎΠ±ΡΠ΅ΠΌΡ ΡΡΠ°Π½ΡΡΠ΅ΠΊΡΠΈΠΈ. ΠΡΠΎΠΌΠ΅ ΡΠΎΠ³ΠΎ, Π΄Π»Ρ ΡΠ»ΡΡΡΠ΅Π½ΠΈΡ ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ ΡΠ»Π΅ΠΊΡΡΠΎΠΏΠΎΡΠ°ΡΠΈΠΈ Π±ΡΠ»ΠΎ ΠΈΠ·ΡΡΠ΅Π½ΠΎ Π²Π»ΠΈΡΠ½ΠΈΠ΅ ΡΠΈΠ»Ρ ΡΠ»Π΅ΠΊΡΡΠΈΡΠ΅ΡΠΊΠΎΠ³ΠΎ ΠΏΠΎΠ»Ρ ΠΈ Π΄Π»ΠΈΠ½Ρ ΠΈΠΌΠΏΡΠ»ΡΡΠ°. Π’ΡΠ°Π½ΡΡΠ΅ΠΊΡΠΈΡ ΠΊΠ»Π΅ΡΠΎΠΊ Ρ ΠΏΠΎΠΌΠΎΡΡΡ Π²Π΅ΠΊΡΠΎΡΠ° pEGFP-N1, ΠΎΠΏΡΠ΅Π΄Π΅Π»Π΅Π½ΠΈΠ΅ ΡΠΊΡΠΏΡΠ΅ΡΡΠΈΠΈ GFP Ρ ΠΏΠΎΠΌΠΎΡΡΡ FACS ΠΈ ΡΠ»ΡΠΎΡΠ΅ΡΡΠ΅Π½ΡΠ½Π°Ρ ΠΌΠΈΠΊΡΠΎΡΠΊΠΎΠΏΠΈΡ Π±ΡΠ»ΠΈ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π½Ρ Π΄Π»Ρ ΠΎΡΠ΅Π½ΠΊΠΈ ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ ΡΡΠ°Π½ΡΡΠ΅ΠΊΡΠΈΠΈ. Π ΠΎΠΏΡΠΈΠΌΠΈΠ·ΠΈΡΠΎΠ²Π°Π½Π½ΡΡ
ΠΏΡΠΎΡΠΎΠΊΠΎΠ»Π°Ρ
Π΄ΠΎΡΡΠΈΠ³Π°Π»Π°ΡΡ ΡΡΠ°Π½ΡΡΠ΅ΠΊΡΠΈΡ Π½Π° ΡΡΠΎΠ²Π½Π΅ 63.73 Β± 2.36 ΠΈ 73.9 Β± 1.6 % Π² ΠΊΠ»Π΅ΡΠΊΠ°Ρ
Huh-7 ΠΈ Vero ΡΠΎΠΎΡΠ²Π΅ΡΡΡΠ²Π΅Π½Π½ΠΎ, Π² ΡΠΎ Π²ΡΠ΅ΠΌΡ ΠΊΠ°ΠΊ ΠΌΠ°ΠΊΡΠΈΠΌΠ°Π»ΡΠ½ΡΠΉ ΡΡΠΎΠ²Π΅Π½Ρ ΡΡΠ°Π½ΡΡΠ΅ΠΊΡΠΈΠΈ Ρ ΠΏΠΎΠΌΠΎΡΡΡ jetPEIβ’ ΡΠΎΡΡΠ°Π²Π»ΡΠ» 14.2 Β± 0.69 ΠΈ 28 Β± 1.11 % Π΄Π»Ρ ΡΠ΅Ρ
ΠΆΠ΅ ΠΊΠ»Π΅ΡΠΎΠΊ. ΠΡ
Π»Π°ΠΆΠ΄Π΅Π½ΠΈΠ΅ ΠΊΠ»Π΅ΡΠΎΠΊ ΠΏΠΎΡΠ»Π΅ ΡΡΠ°Π½ΡΡΠ΅ΠΊΡΠΈΠΈ Π½Π΅ ΡΠ»ΡΡΡΠ°Π»ΠΎ ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎΡΡΡ ΡΠ»Π΅ΠΊΡΡΠΎΡΡΠ°Π½ΡΡΠ΅ΠΊΡΠΈΠΈ ΠΊΠ»Π΅ΡΠΎΠΊ Huh-7. Π ΠΎΠ±Π΅ΠΈΡ
ΠΊΠ»Π΅ΡΠΎΡΠ½ΡΡ
Π»ΠΈΠ½ΠΈΡΡ
ΡΠ»Π΅ΠΊΡΡΠΎΠΏΠΎΡΠ°ΡΠΈΡ ΠΏΠΎΠ·Π²ΠΎΠ»ΠΈΠ»Π° Π΄ΠΎΡΡΠΈΡΡ Π±ΠΎΠ»Π΅Π΅ Π²ΡΡΠΎΠΊΠΎΠ³ΠΎ ΡΡΠΎΠ²Π½Ρ ΡΡΠ°Π½ΡΡΠ΅ΠΊΡΠΈΠΈ ΠΏΠΎ ΡΡΠ°Π²Π½Π΅Π½ΠΈΡ Ρ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π½ΠΈΠ΅ΠΌ Ρ
ΠΈΠΌΠΈΡΠ΅ΡΠΊΠΈΡ
ΡΠ΅Π°Π³Π΅Π½ΡΠΎΠ². ΠΡΠ΅Π΄ΡΡΠ°Π²Π»Π΅Π½Π½ΡΠΉ ΠΏΡΠΎΡΠΎΠΊΠΎΠ» ΠΌΠΎΠΆΠ΅Ρ Π±ΡΡΡ ΠΏΡΠΈΠ³ΠΎΠ΄Π½ΡΠΌ Π΄Π»Ρ Π±ΠΎΠ»ΡΡΠΈΠ½ΡΡΠ²Π° ΡΠΊΡΠΏΠ΅ΡΠΈΠΌΠ΅Π½ΡΠ°Π»ΡΠ½ΡΡ
ΠΌΠ°Π½ΠΈΠΏΡΠ»ΡΡΠΈΠΉ, ΠΊΠΎΡΠΎΡΡΠ΅ ΡΡΠ΅Π±ΡΡΡ Π²ΡΡΠΎΠΊΠΎΠ³ΠΎ ΡΡΠΎΠ²Π½Ρ ΡΡΠ°Π½ΡΡΠ΅ΠΊΡΠΈΠΈ ΠΈΡΡΠ»Π΅Π΄ΡΠ΅ΠΌΡΡ
ΠΊΠ»Π΅ΡΠΎΡΠ½ΡΡ
Π»ΠΈΠ½ΠΈΠΉ
Prophylactic DNA vaccine targeting Foxp3+regulatory T cells depletes myeloid-derived suppressor cells and improves anti-melanoma immune responses in a murine model
Abstract
Regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) are the two important and interactive immunosuppressive components of the tumor microenvironment that hamper anti-tumor immune responses. Therefore, targeting these two populations together might be beneficial for overcoming immune suppression in the tumor microenvironment. We have recently shown that prophylactic Foxp3 DNA/recombinant protein vaccine (Foxp3 vaccine) promotes immunity against Treg in tumor-free conditions. In the present study, we investigated the immune modulatory effects of a prophylactic regimen of the redesigned Foxp3 vaccine in the B16F10 melanoma model. Our results indicate that Foxp3 vaccination continuously reduces Treg population in both the tumor site and the spleen. Surprisingly, Treg reduction was associated with a significant decrease in the frequency of MDSC, both in the spleen and in the tumor environment. Furthermore, Foxp3 vaccination resulted in a significant reduction of arginase-1(Arg-1)-induced nitric oxide synthase (iNOS), reactive oxygen species (ROS) and suppressed MDSC activity. Moreover, this concurrent depletion restored production of inflammatory cytokine IFN-Ξ³ and enhanced tumor-specific CTL response, which subsequently resulted in the reduction of tumor growth and the improved survival rate of vaccinated mice. In conclusion, our results revealed that Foxp3 vaccine promotes an immune response against tumor by targeting both Treg and MDSC, which could be exploited as a potential immunotherapy approach.
Keywords
Regulatory T cells Myeloid-derived suppressor cells Foxp3 Melanom
Prophylactic DNA vaccine targeting Foxp3 + regulatory T cells depletes myeloid-derived suppressor cells and improves anti-melanoma immune responses in a murine model
Regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) are the two important and interactive immunosuppressive components of the tumor microenvironment that hamper anti-tumor immune responses. Therefore, targeting these two populations together might be beneficial for overcoming immune suppression in the tumor microenvironment. We have recently shown that prophylactic Foxp3 DNA/recombinant protein vaccine (Foxp3 vaccine) promotes immunity against Treg in tumor-free conditions. In the present study, we investigated the immune modulatory effects of a prophylactic regimen of the redesigned Foxp3 vaccine in the B16F10 melanoma model. Our results indicate that Foxp3 vaccination continuously reduces Treg population in both the tumor site and the spleen. Surprisingly, Treg reduction was associated with a significant decrease in the frequency of MDSC, both in the spleen and in the tumor environment. Furthermore, Foxp3 vaccination resulted in a significant reduction of arginase-1(Arg-1)-induced nitric oxide synthase (iNOS), reactive oxygen species (ROS) and suppressed MDSC activity. Moreover, this concurrent depletion restored production of inflammatory cytokine IFN-ΓΒ³ and enhanced tumor-specific CTL response, which subsequently resulted in the reduction of tumor growth and the improved survival rate of vaccinated mice. In conclusion, our results revealed that Foxp3 vaccine promotes an immune response against tumor by targeting both Treg and MDSC, which could be exploited as a potential immunotherapy approach.. ΓΒ© 2017, Springer-Verlag GmbH Germany, part of Springer Nature
MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression
Superantigens (SAgs) are potent exotoxins secreted by Staphylococcus aureus and Streptococcus pyogenes. They target a large fraction of T cell pools to set in motion a "cytokine storm" with severe and sometimes life-threatening consequences typically encountered in toxic shock syndrome (TSS). Given the rapidity with which TSS develops, designing timely and truly targeted therapies for this syndrome requires identification of key mediators of the cytokine storm's initial wave. Equally important, early host responses to SAgs can be accompanied or followed by a state of immunosuppression, which in turn jeopardizes the host's ability to combat and clear infections. Unlike in mouse models, the mechanisms underlying SAg-associated immunosuppression in humans are ill-defined. In this work, we have identified a population of innate-like T cells, called mucosa-associated invariant T (MAIT) cells, as the most powerful source of pro-inflammatory cytokines after exposure to SAgs. We have utilized primary human peripheral blood and hepatic mononuclear cells, mouse MAIT hybridoma lines, HLA-DR4-transgenic mice, MAIThighHLA-DR4+ bone marrow chimeras, and humanized NOD-scid IL-2RΞ³null mice to demonstrate for the first time that: i) mouse and human MAIT cells are hyperresponsive to SAgs, typified by staphylococcal enterotoxin B (SEB); ii) the human MAIT cell response to SEB is rapid and far greater in magnitude than that launched by unfractionated conventional T, invariant natural killer T (iNKT) or Ξ³Ξ΄ T cells, and is characterized by production of interferon (IFN)-Ξ³, tumor necrosis factor (TNF)-Ξ± and interleukin (IL)-2, but not IL-17A; iii) high-affinity MHC class II interaction with SAgs, but not MHC-related protein 1 (MR1) participation, is required for MAIT cell activation; iv) MAIT cell responses to SEB can occur in a T cell receptor (TCR) VΞ²-specific manner but are largely contributed by IL-12 and IL-18; v) as MAIT cells are primed by SAgs, they also begin to develop a molecular signature consistent with exhaustion and failure to participate in antimicrobial defense. Accordingly, they upregulate lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-3 (TIM-3), and/or programmed cell death-1 (PD-1), and acquire an anergic phenotype that interferes with their cognate function against Klebsiella pneumoniae and Escherichia coli; vi) MAIT cell hyperactivation and anergy co-utilize a signaling pathway that is governed by p38 and MEK1/2. Collectively, our findings demonstrate a pathogenic, rather than protective, role for MAIT cells during infection. Furthermore, we propose a novel mechanism of SAg-associated immunosuppression in humans. MAIT cells may therefore provide an attractive therapeutic target for the management of both early and late phases of severe SAg-mediated illnesses
Designing & producing polytope DNA vaccine containing HBsAg gene for the induction of protective immunity against hepatitis C
Background: Considering the immunosuppressive effects and prevalent mutations in some HCV antigens, induction of CD8+ T cell responses is focused on conserved and critical epitopes which as a multi-epitope vaccine can prevent the chronic nature of the disease.
Materials and Methods: Two immunodominant HLA-A2-restricted human epitopes (E2614-622 and NS31406-1415) and two H-2d-restricted mouse epitopes (core132-142 and E2405-414) were designed in a sequential tandem, predicted by immunoinformatic analyses. Following the synthesis, related nucleotide sequence was cloned into the pcDNA3.1 vector with and without the fusion of hepatitis B surface antigen (HBsAg). Two constructed plasmids (pcDNA3.1.HPOL and pcDNA3.1.POL, respectively) were evaluated for the protein expression and secretion in Cos-7 cell line. After the vaccination of BALB/c mice (n=6 in each group) with different DNA and peptide immunization regimens, CD8+ T cell activity as well as the type and protective potency of the induced responses were evaluated.
Results: Despite the induction of epitope-specific responses in pcDNA3.1.POL injected mice, the group immunized with pcDNA3.1.HPOL indicated a significant increase in the number and activity of CD8+ T cells (P<0.05). Peptide boosting of this group (formulated in two human-compatible adjuvant) still led to the more activation of CD8+ cells, induction of Th1 response and the inhibition of tumor model growth (P<0.05).
Conclusion: Fusion of HBsAg as a particle-forming sequence and the source of helper epitopes along the DNA-prime/peptide-boosting immunization regimen are proposed as two promising strategies to improve the CTL multi-epitope vaccines against HCV
Cloning, Expression and Characterization of Zebra Fish Ferroportin in Hek 293T Cell Line
Background: Ferroportin (Fpn), a regulator of iron homeostasis is a conserved membrane protein that exports iron across the enterocytes, macrophages and hepatocytes into the blood circulation. Fpn has also critical influence on survival of microorganisms whose growth is dependent upon iron, thus preparation of Fpn is needed to study the role of iron in immunity and pathogenesis of micoorganisms.Methods: To prepare and characterize a recombinant ferroportin, total RNA was extracted from Indian zebrafish duodenum, and used to synthesize cDNA by RT-PCR. PCR product was first cloned in Topo TA vector and then subcloned into the GFP expression vector pEGFP-N1. The final resulted plasmid (pEGFP-ZFpn) was used for expression of Fpn-EGFP protein in Hek 293T cells.Results: The expression was confirmed by appearance of fluorescence in Hek 293 T cells. Recombinant Fpn was further characterized by submission of its predicted amino acid sequences to the TMHMM V2.0 prediction server (hidden Markov model), NetOGlyc 3.1 and NetNGlyc 3.1 servers. The obtained Fpn from indian zebrafish also contained eight transmembrane domains with N- and C-termini inside the cytoplasm and harboured 78 O-glycosylated amino acids.Conclusion: The recombinant Fpn from Indian zebra fish was successfully expressed in Hek 293 cell line. Although the discrepancy in two amino acids was observed in our produced Fpn and resulted in an additional O-glycosylation site, but had no effect on the topology of the protein compared to other Fpn described by other researchers. Therefore this construct can be used in future iron studies