scholarly journals Ex vivo induction of antitumor DEC-205+ CD11c+ cells in a murine neuroblastoma model by co-stimulation with doxorubicin, lipopolysaccharide and interleukin-4

2015 ◽  
Vol 4 (1) ◽  
pp. 27-32 ◽  
Author(s):  
SEIICHIRO INOUE ◽  
YUMIKO SETOYAMA ◽  
YOSHIFUMI BECK ◽  
DAIKI KITAGAWA ◽  
AKIO ODAKA
Keyword(s):  
Ex Vivo ◽  
Interleukin 4 ◽  
Murine Neuroblastoma

scholarly journals Transcutaneous penetration of a single-chain variable fragment (scFv) compared to a full-size antibody: potential tool for atopic dermatitis (AD) treatment

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Audrey Baylet ◽  
Raoul Vyumvuhore ◽  
Marine Laclaverie ◽  
Laëtitia Marchand ◽  
Carine Mainzer ◽  
...  
Keyword(s):  
Atopic Dermatitis ◽  
Ex Vivo ◽  
Topical Therapy ◽  
Human Keratinocytes ◽  
Raman Microspectroscopy ◽  
Interleukin 4 ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Administration Routes

AbstractCurrently, several biologics are used for the treatment of cutaneous pathologies such as atopic dermatitis (AD), psoriasis or skin cancers. The main administration routes are subcutaneous and intravenous injections. However, little is known about antibody penetration through the skin. The aim was to study the transcutaneous penetration of a reduced-size antibody as a single-chain variable fragment (scFv) compared to a whole antibody (Ab) and to determine its capacity to neutralize an inflammatory cytokine involved in AD such as human interleukin-4 (hIL-4). Transcutaneous penetration was evaluated by ex vivo studies on tape-stripped pig ear skin. ScFv and Ab visualization through the skin was measured by Raman microspectroscopy. In addition, hIL-4 neutralization was studied in vitro using HEK-Blue™ IL-4/IL-13 cells and normal human keratinocytes (NHKs). After 24 h of application, analysis by Raman microspectroscopy showed that scFv penetrated into the upper dermis while Ab remained on the stratum corneum. In addition, the anti-hIL4 scFv showed very efficient and dose-dependent hIL-4 neutralization. Thus, scFv penetrates through to the upper papillary dermis while Ab mostly remains on the surface, the anti-hIL4 scFv also neutralizes its target effectively suggesting its potential use as topical therapy for AD.

scholarly journals Transcutaneous penetration of a single-chain variable fragment (scFv) compared to a full-size antibody: potential tool for Atopic Dermatitis (AD) treatment

2021 ◽  
Author(s):  
Audrey Baylet ◽  
Raoul Vyumvuhore ◽  
Marine Laclaverie ◽  
Laëtitia Marchand ◽  
Carine Mainzer ◽  
...  
Keyword(s):  
Atopic Dermatitis ◽  
Ex Vivo ◽  
Topical Therapy ◽  
Human Keratinocytes ◽  
Raman Microspectroscopy ◽  
Poly I:C ◽  
Interleukin 4 ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Polycytidylic Acid

SummaryBackgroundCurrently, several biologics are used for the treatment of cutaneous pathologies such as atopic dermatitis (AD), psoriasis (PSO) or skin cancers. The main administration routes are subcutaneous and intravenous injections. However, little is known about antibody penetration through the skin.ObjectivesThe aim was to study the transcutaneous penetration of a reduced-size antibody as a single-chain variable fragment (scFv) compared to a whole antibody (Ab) and to determine its capacity to neutralize an inflammatory cytokine involved in AD such as human interleukin-4 (hIL-4).MethodsTranscutaneous penetration was evaluated by ex vivo studies on tape-stripped pig ear skin. Antibody visualization through the skin was measured by Raman microspectroscopy. In addition, hIL-4 neutralization was studied using two 2D models. First, embryonic alkaline phosphatase (SEAP) secretion by HEK-Blue™ IL-4/IL-13 cells, proportional to hIL-4 cells stimulation, was quantified by OD 620 nm measurement in presence or absence of an anti-hIL4 scFv or Ab. Then, normal human keratinocytes (NHKs) were stimulated with polyinosinic-polycytidylic acid (poly I:C) +/− hIL-4 and treated with anti-hIL4 scFv. Human Interleukin-8 (hIL-8) concentrations were determined in culture supernatants by ELISA.ResultsAfter 24h of application, analysis by Raman microspectroscopy showed that scFv penetrated into the upper dermis while Ab remained on the stratum corneum. In addition, the anti-hIL4 scFv showed better efficiency compared to Ab, with a neutralization percentage at 200 nM of 68% and 47%, respectively, in the HEK-Blue™ IL-4/IL-13 model. hIL-8 dosage in stimulated NHKs supernatants revealed that addition of scFv induced a dose-dependent hIL-4 neutralization.ConclusionsscFv penetrates through to the upper papillary dermis while Ab remains on the surface. The anti-hIL4 scFv neutralizes its target effectively in two 2D models suggesting its potential use as topical therapy for AD.

scholarly journals An Ex Vivo Study of T Lymphocytes Recovered from the Lungs of I/St Mice Infected with and Susceptible toMycobacterium tuberculosis

1998 ◽  
Vol 66 (10) ◽  
pp. 4981-4988 ◽  
Author(s):  
Irina Lyadova ◽  
Vladimir Yeremeev ◽  
Konstantin Majorov ◽  
Boris Nikonenko ◽  
Sergei Khaidukov ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Antimycobacterial Activity ◽  
Enzymatic Digestion ◽  
Interleukin 4 ◽  
T Cell Clones ◽  
Cell Clones ◽  
Ifn Γ

ABSTRACT I/St mice, previously characterized as susceptible toMycobacterium tuberculosis H37Rv, were given 103 or 105 CFU intravenously. At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs were enumerated and further characterized phenotypically and functionally. Regarding the T-cell populations recovered at 2 and 5 weeks postinfection, two main results were obtained: (i) the population of CD44− CD45RB+cells disappeared within 2 weeks postinfection, while the number of CD44+ CD45RB−/low cells slowly increased between weeks 2 and 5; (ii) when cocultured with irradiated syngeneic splenocytes, these lung T cells proliferated in the presence of H37Rv sonicate. Using H37Rv sonicate and irradiated syngeneic splenocytes to reactivate lung T cells, we selected five CD3+CD4+ CD8− T-cell clones. In addition to the H37Rv sonicate, the five clones react to both a short-term culture filtrate and an affinity-purified 15- to 18-kDa mycobacterial molecule as assessed by the proliferative assay. However, there was a clear difference between T-cell clones with respect to cytokine (gamma interferon [IFN-γ] and interleukin-4 [IL-4] and IL-10) profiles: besides one Th1-like (IFN-γ+ IL-4−) clone and one Th0-like (IFN-γ+ IL-4+IL-10+) clone, three clones produced predominantly IL-10, with only marginal or no IL-4 and IFN-γ responses. Inhibition of mycobacterial growth by macrophages in the presence of T cells was studied in a coculture in vitro system. It was found that the capacity to enhance antimycobacterial activity of macrophages fully correlated with INF-γ production by individual T-cell clones following genetically restricted recognition of infected macrophages. The possible functional significance of cytokine diversity among T-cell clones is discussed.

Dendritic Cells Derived In Vitro From Acute Myelogenous Leukemia Cells Stimulate Autologous, Antileukemic T-Cell Responses

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 780-786 ◽  
Author(s):  
A. Choudhury ◽  
J.C. Liang ◽  
E.K. Thomas ◽  
L. Flores-Romo ◽  
Q.S. Xie ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
T Cell ◽  
Acute Myelogenous Leukemia ◽  
Chromosomal Abnormalities ◽  
Fusion Gene ◽  
Ex Vivo ◽  
Myelogenous Leukemia ◽  
Interleukin 4 ◽  
Acute Myelogenous

Abstract We have previously reported that leukemic dendritic cells (DC) can be generated ex vivo from myelomonocytic precursors in chronic myelogenous leukemia. In this study we report the generation of DC from acute myelogenous leukemia (AML) cells and their potent ability to stimulate leukemia-specific cytolytic activity in autologous lymphocytes. DC were generated in vitro using granulocyte-macrophage colony-stimulating factor +interleukin-4 in combination with either tumor necrosis factor- or CD40 ligand (CD40L). Cells from 19 AML patients with a variety of chromosomal abnormalities were studied for their ability to generate DC. In all but 1 case, cells with the morphology, phenotypic characteristics, and T-cell stimulatory properties of DC could be generated. These cells expressed high levels of major histocompatibility complex class I and class II antigens as well as the costimulatory molecules B7-2 and ICAM-1. In three cases these cells were determined to be of leukemic origin by fluorescence in situ hybridization for chromosomal abnormalities or Western blotting for the inv(16) fusion gene product. Autologous lymphocytes cocultured with AML-derived DC (DC-AL) were able to lyse autologous leukemia targets, whereas little cytotoxicity was noted against autologous, normal cells obtained from the patients during remission. We conclude that leukemia derived DC may be useful for immunotherapy of many AML patients.

scholarly journals Interleukin-4 Signaling Plays a Major Role in Urogenital Schistosomiasis-Associated Bladder Pathogenesis

2019 ◽  
Vol 88 (3) ◽  
Author(s):  
Evaristus C. Mbanefo ◽  
Chi-Ling Fu ◽  
Christina P. Ho ◽  
Loc Le ◽  
Kenji Ishida ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Signaling Pathway ◽  
Ex Vivo ◽  
Bladder Wall ◽  
Interleukin 4 ◽  
Wild Type ◽  
Urogenital Schistosomiasis ◽  
Content Type ◽  
Helminth Infections

ABSTRACT Interleukin-4 (IL-4) is crucial in many helminth infections, but its role in urogenital schistosomiasis, infection with Schistosoma haematobium worms, remains poorly understood due to a historical lack of animal models. The bladder pathology of urogenital schistosomiasis is caused by immune responses to eggs deposited in the bladder wall. A range of pathology occurs, including urothelial hyperplasia and cancer, but associated mechanisms and links to IL-4 are largely unknown. We modeled urogenital schistosomiasis by injecting the bladder walls of IL-4 receptor-alpha knockout (Il4ra−/−) and wild-type mice with S. haematobium eggs. Readouts included bladder histology and ex vivo assessments of urothelial proliferation, cell cycle, and ploidy status. We also quantified the effects of exogenous IL-4 on urothelial cell proliferation in vitro, including cell cycle status and phosphorylation patterns of major downstream regulators in the IL-4 signaling pathway. There was a significant decrease in the intensity of granulomatous responses to bladder-wall-injected S. haematobium eggs in Il4ra−/− versus wild-type mice. S. haematobium egg injection triggered significant urothelial proliferation, including evidence of urothelial hyper-diploidy and cell cycle skewing in wild-type but not Il4ra−/− mice. Urothelial exposure to IL-4 in vitro led to cell cycle polarization and increased phosphorylation of AKT. Our results show that IL-4 signaling is required for key pathogenic features of urogenital schistosomiasis and that particular aspects of this signaling pathway may exert these effects directly on the urothelium. These findings point to potential mechanisms by which urogenital schistosomiasis promotes bladder carcinogenesis.

Generation of Dendritic Cells after One-Hit Lentiviral Transduction of Hematopoietic Precursor Cells: Proof of Concept for the Human and Mouse Systems.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3448-3448
Author(s):  
Richard C. Koya ◽  
Nori Kasahara ◽  
Takahiro Kimura ◽  
Antoni Ribas ◽  
Renata Stripecke
Keyword(s):  
Tumor Antigens ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
High Titer ◽  
Lentiviral Vectors ◽  
Genetically Engineered ◽  
Interleukin 4 ◽  
Lentiviral Transduction ◽  
Gm Csf

Abstract Conventional, ex vivo culture of monocytes with recombinant proteins for their differentiation into DCs involves considerable manipulation under “Good Manufacturing Practices” conditions, and is not only more labor intensive but importantly, after ex vivo produced DCs are administered, they lack the stimulatory signals to keep them alive and functional and therefore are short lived. Because of these problems, we have evaluated an one-hit lentiviral transduction approach for genetically modifying monocytes in order to promote autocrine and paracrine production of factors required for their differentiation into immature DCs. High-titer third generation self-inactivating lentiviral vectors expressing granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) efficiently achieved simultaneous and persistent co-delivery of the transgenes into purified human CD14+ monocytes. Co-expression of GM-CSF and IL-4 in monocytes was sufficient to induce their differentiation into lentivirus-modified DCs (“DC/LVs”), as evidenced by their morphology, immunophenotype and immune-function*. Mixed lymphocyte reactions showed that the T-cell stimulating activity of DC/LVs was superior to that of DCs grown by conventional methods. DC/LVs displayed efficient antigen-specific, MHC Class-I restricted stimulation of autologous CD8+ T-cells, as shown by IFN-G production and CTL assays. Importantly, DC/LVs could be maintained metabolically active and viable in culture for 2–3 weeks in the absence of exogenously added growth factors, unlike conventional DCs *. We are now evaluating whether DC/LVs can be re-infused immediately after gene transfer to achieve stable and long-lasting differentiation in vivo. Additionally, the genetic engineering of monocytes is anticipated to generate DCs after one hit of lentiviral transduction, instead of the three consecutive steps for development of DCs (differentiation, maturation, gene delivery of tumor antigens). We have thus established a mouse model for testing DC/LVs in vivo for the treatment of melanoma. Bone marrow cells from C57BL/6 mice transduced with lentiviral vectors expressing GM-CSF and IL-4 recapitulated the same DC/LV morphology and immunophenotype obtained in the human system. Mouse DC/LVs were also more viable in vitro and outperformed conventional mouse DCs in pilot immunization assays as followed by CTL assays and IFN-G ELISPOT. We are currently evaluating the immunotherapeutic efficacy of DC/LVs injected into mice developing B16 melanoma tumors. Co-delivery of a gene for DC maturation (CD40L) and of gene encoding a tumor-associated antigens (MART-1) is being performed. Our goal is to evaluate the implications of simultaneous co-expression of GM-CSF/ IL-4/ CD40L/ MART-1 in DC/LV differentiation and migration to lymph nodes in vivo, immunopotency and safety. Once these pre-clinical considerations are addressed, we foresee a broad clinical application of genetically engineered DCs for vaccination purposes against cancer and chronic infectious diseases.

Interleukin-4 Regulates Eomesodermin In CD8+ T Cell Development and Differentiation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2284-2284
Author(s):  
Shannon A. Carty ◽  
Martha S Jordan
Keyword(s):  
T Cell ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
T Cell Development ◽  
Cd8 T Cell ◽  
Cell Development ◽  
Interleukin 4 ◽  
Transfer Model ◽  
Potent Inducer

Abstract Cytokines regulate T cell development and function. Interleukin (IL)-4 is a cytokine classically associated with CD4+ T helper (TH) differentiation, specifically TH2 differentiation. However, IL-4 has recently been shown to drive the development of CD8+ innate-like lymphocytes (ILLs). CD8+ ILLs are non-conventional thymocytes that develop with characteristics typically associated with innate and/or memory immune cells, including surface expression of the activation/memory markers CD44 and CD122, expression of the T-box transcription factor Eomesodermin (Eomes) and the rapid production of interferon (IFN)-γ after ex vivo stimulation. Here we show that IL-4 is sufficient to promote Eomes expression in CD8 single-positive (SP) thymocytes in short-term in vitro culture and direct CD8+ ILL development in fetal thymic organ culture. Using genetic deficiency and pharmacologic inhibitors, we demonstrate that IL-4 up-regulation of Eomes in CD8SP thymocytes requires STAT6 and Akt signaling pathways. Next, we investigated the possibility that in addition to directing the fate of developing thymocytes, IL-4 might impact the function of mature CD8+ cells undergoing activation. We found that that in naïve peripheral CD8+ T the combination of IL-4 and low dose T cell receptor (TCR) stimulation is a potent inducer of Eomes. Futhermore, when combined, these stimuli promote the persistence of CD8+ T cells in an adoptive transfer model. Understanding how IL-4 directs CD8+ T cell differentiation may provide a novel way to enhance CD8+ T cell function in adoptive T cell therapies. Disclosures: No relevant conflicts of interest to declare.

Dendritic Cells Derived In Vitro From Acute Myelogenous Leukemia Cells Stimulate Autologous, Antileukemic T-Cell Responses

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 780-786 ◽  
Author(s):  
A. Choudhury ◽  
J.C. Liang ◽  
E.K. Thomas ◽  
L. Flores-Romo ◽  
Q.S. Xie ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
T Cell ◽  
Acute Myelogenous Leukemia ◽  
Chromosomal Abnormalities ◽  
Fusion Gene ◽  
Ex Vivo ◽  
Myelogenous Leukemia ◽  
Interleukin 4 ◽  
Acute Myelogenous

We have previously reported that leukemic dendritic cells (DC) can be generated ex vivo from myelomonocytic precursors in chronic myelogenous leukemia. In this study we report the generation of DC from acute myelogenous leukemia (AML) cells and their potent ability to stimulate leukemia-specific cytolytic activity in autologous lymphocytes. DC were generated in vitro using granulocyte-macrophage colony-stimulating factor +interleukin-4 in combination with either tumor necrosis factor- or CD40 ligand (CD40L). Cells from 19 AML patients with a variety of chromosomal abnormalities were studied for their ability to generate DC. In all but 1 case, cells with the morphology, phenotypic characteristics, and T-cell stimulatory properties of DC could be generated. These cells expressed high levels of major histocompatibility complex class I and class II antigens as well as the costimulatory molecules B7-2 and ICAM-1. In three cases these cells were determined to be of leukemic origin by fluorescence in situ hybridization for chromosomal abnormalities or Western blotting for the inv(16) fusion gene product. Autologous lymphocytes cocultured with AML-derived DC (DC-AL) were able to lyse autologous leukemia targets, whereas little cytotoxicity was noted against autologous, normal cells obtained from the patients during remission. We conclude that leukemia derived DC may be useful for immunotherapy of many AML patients.

scholarly journals The Immunotoxicity of Chronic Exposure to High Levels of Lead: An Ex Vivo Investigation

Toxics ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 56
Author(s):  
Kawinsaya Pukanha ◽  
Supabhorn Yimthiang ◽  
Wiyada Kwanhian
Keyword(s):  
Mononuclear Cells ◽  
Ex Vivo ◽  
Inverse Correlation ◽  
Treg Cells ◽  
Interleukin 4 ◽  
Smoking History ◽  
Control Group ◽  
Peripheral Blood Mononuclear ◽  
Exposed Workers ◽  
Pb Exposure

Lead (Pb) is a toxic metal known for its wide-ranging adverse health effects. However, a compound of Pb is still used in the caulking process to repair wooden fishing boats. The present study aimed to measure Pb exposure and its immunologic effects in boatyard workers in Nakhon Si Thammarat province, Thailand, in comparison with an age-matched control group of farmers. The age, body mass index, and smoking history in workers (n = 14) and controls (n = 16) did not differ. The median blood Pb concentration was 8.7-fold higher in workers than controls (37.1 versus 4.3 µg/dL, p < 0.001). Workers had 8.4% lower phagocytic active cells than controls (89.9% versus 98.1%, p = 0.019). In response to a mitogen stimulation, the peripheral blood mononuclear cells (PBMCs) from workers produced 2-fold higher ratios of interleukin-4 (IL-4) to interferon-γ than the PBMCs from controls (p = 0.026). Furthermore, Pb-exposed workers had 33.9% lower cytotoxic T (Tc) cells than controls (24.3% versus 36.8%, p = 0.004). In stark contrast, the percentage of regulatory T (Treg) cells in workers was 2.7-fold higher than controls (6.1% versus 2.3%, p < 0.001). In all subjects, blood Pb showed positive correlations with the percentages of Treg cells (r = 0.843, p < 0.001) and IL-4 (r = 0.473, p = 0.041) while showing an inverse correlation with the percentages of Tc cells (r = −0.563, p = 0.015). These findings indicate that chronic high Pb exposure may cause a shift towards humoral immune response, together with a suppression of cellular immunity, thereby suggesting an elevation in cancer risk in Pb-exposed workers.

scholarly journals Flow Cytometric Determination of Cellular Sources and Frequencies of Key Cytokine-Producing Lymphocytes Directed against Recombinant LACK and Soluble Leishmania Antigen in Human Cutaneous Leishmaniasis

2001 ◽  
Vol 69 (5) ◽  
pp. 3232-3239 ◽  
Author(s):  
R. L. A. Bottrel ◽  
W. O. Dutra ◽  
F. A. Martins ◽  
B. Gontijo ◽  
E. Carvalho ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Cutaneous Leishmaniasis ◽  
Ex Vivo ◽  
Protozoan Parasite ◽  
Recombinant Antigen ◽  
Interleukin 4 ◽  
Leishmania Braziliensis ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Ifn Γ

ABSTRACT Leishmaniasis, caused by infection with the protozoan parasiteLeishmania, affects millions of individuals worldwide, causing serious morbidity and mortality. This study directly determined the frequency of cells producing key immunoregulatory cytokines in response to the recombinant antigen Leishmania homolog of receptors for activated kinase C (LACK) and soluble leishmania antigen (SLA), and it determined relative contributions of these antigens to the overall cytokine profile in individuals infected for the first time with Leishmania braziliensis. All individuals presented with the cutaneous clinical form of leishmaniasis and were analyzed for proliferative responses to LACK antigen and SLA, frequency of lymphocyte subpopulations (analyzed ex vivo), and antigen-induced (LACK and SLA) cytokine production at the single-cell level (determined by flow cytometry). The following were determined. (i) The Th1-type response previously seen in patients with cutaneous leishmaniasis is due to gamma interferon (IFN-γ) production by several different sources, listed in order of contribution: CD4+ T lymphocytes, CD4−, CD8− lymphocytes, and CD8+ T lymphocytes. (ii) SLA induced a higher frequency of lymphocytes producing IFN-γ and tumor necrosis factor alpha (TNF-α) than did LACK. (iii) LACK induced an activation of monocyte populations as reflected by an increased percentage of CD14-positive cells. (iv) Neither SLA nor LACK induced detectable frequencies of cells producing interleukin-4 (IL-4) or IL-5. These data demonstrated a multifaceted immune response to SLA in human leishmaniasis involving Th1 CD4+ T lymphocytes (IFN-γ+ and IL-10−/IL-4−), Tc1 CD8+ T cells (IFN-γ+, and IL-10−/IL-4−), and a high frequency of TNF-α-producing lymphocytes. Moreover, it was determined that the recombinant antigen LACK acts as a weak inducer of Th1-type lymphocyte responses compared to SLA.

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