The impact of Zataria multiflora Boiss extract on in vitro and in vivo Th1/Th2 cytokine (IFN-γ/IL4) balance

Abstract Maintenance of hematopoietic stem cells (HSCs) and regulation of their quiescence and self-renewal is critical for maintaining a lifelong supply of blood cells. The ability of HSCs to stay quiescent is thought to depend on their specific niche in the bone marrow (BM). Mesenchymal stromal cells (MSC) in the BM are multipotent stem cells that form part of the vascular HSC niche and provide micro-environmental support to HSCs both in vivo and upon expansion ex vivo. Culture-expanded MSCs also exhibit immunomodulatory properties that can be enhanced by pre-treatment with interferon-gamma (IFN-γ). BM MSC are thus attractive candidates for cellular therapy after hematopoietic stem cell transplantation, for promoting rapid hematopoietic recovery and reducing the incidence or severity of graft versus host disease. Although IFN-γ pre-treatment can improve the immunomodulatory properties of MSCs, elevated IFN-γ levels have also been associated with anemia and BM failure in multiple chronic inflammatory diseases. While the impact of IFN-γ on HSC has been elucidated in recent years, it remains largely unknown whether IFN-γ can also influence hematopoietic support by BM stromal cells. In this study, we aim to elucidate the impact of IFN-γ on hematopoietic support of BM MSC. We show that in vitro expansion of primary BM MSC cultures from healthy donors was significantly reduced in the presence of IFN-γ, and this effect could be reproduced in the BM stromal cell line MS-5. Concurrently, IFN-γ diminished the clonal capacity of BM MSC, as measured by CFU-F assays. In addition, BM MSC that were pre-stimulated with IFN-γ produced significantly lower levels of CXCL12, suggesting a loss of hematopoietic support potential. Indeed, support of CD34+ hematopoietic stem and progenitor cells (HSPC) in a co-culture assay was greatly reduced in when MSC were pre-treated with IFN-γ. To determine the impact of IFN-γ on BM MSC in vivo, we investigated the BM stromal compartment of IFN-γ AU-rich element deleted (ARE-Del) mice, which constitutively express IFN-γ in steady state conditions. FACS analysis revealed a remodeling of the BM stromal compartment in ARE-Del mice compared to littermate controls, with significantly fewer MSCs, identified as CD45-Ter119-CD31-CD51+PDGFRa+ cells. Numbers of other stromal cell subsets, such as osteoblasts and fibroblasts, were not altered. The reduction of BM MSC in ARE-Del mice coincided with a loss of quiescence in HSCs; only 35% of long term HSC (LT-HSC) in ARE-Del mice were quiescent, compared to 70% in WT mice, as determined by Ki-67 staining. Loss of quiescence in LT-HSC did not lead to increased self-renewal, but rather induced increased differentiation towards short-term HSC and multi-potent progenitors. We then sorted LT-HSC from WT and ARE-Del mice and performed in vitro HSC culture assays in the absence of IFN-γ. Absolute numbers of LT-HSC were rapidly decreased in ARE-Del compared to WT cultures after 3 and 7 days of HSC culture, while numbers of more differentiated progenitors were increased. These data indicate that an IFN-γ-mediated loss of BM MSC in ARE-Del mice leads to loss of quiescent LT-HSCs and induces a tendency towards HSC differentiation over self-renewal. In conclusion, we have shown that IFN-γ has a negative impact on expansion and hematopoietic support of BM MSC in vitro and in vivo across species. Although IFN-γ treatment enhances the immunomodulatory function of MSCs in a clinical setting, it is obvious from our data that IFN-γ impairs their HSC supporting function. These data also provide more insight in the underlying mechanism by which IFN-γ contributes to the pathogenesis of anemia and BM failure. Disclosures No relevant conflicts of interest to declare.

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Trastuzumab versus MYL-1401O (a proposed trastuzumab biosimilar) in a phase I bioequivalence study: In vivo and in vitro immunomodulation.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.7_suppl.10 ◽

2017 ◽

Vol 35 (7_suppl) ◽

pp. 10-10

Author(s):

RÃƒÂ©gine Audran ◽

Haithem Chtioui ◽

Anne-Christine Thierry ◽

Carole Mayor ◽

Laure Vallotton ◽

...

Keyword(s):

Phase I ◽

Mononuclear Cell ◽

Ex Vivo ◽

Reference Drug ◽

Gm Csf ◽

Significant Difference ◽

Ifn Γ ◽

The Impact

10 Background: Trastuzumab is a humanized monoclonal antibody targeting breast cancer cells overexpressing the HER2-oncoprotein. During a Phase-I single centre, single dose, randomized, double-blind, cross-over study assessing the bioequivalence of a proposed trastuzumab biosimilar (MYL-1401O) versus the initially marketed drug (Herceptin), we investigated in addition a large panel of pharmacodynamics parameters comparing the immunomodulatory activity of both drugs. Methods: 22 healthy males were included, 19 subjects receiving randomly a single intravenous infusion of MYL-1401O and 22 of Herceptin, separated by 16 to 22 week wash-out. Blood samples drawn pre- and post- infusion were assessed for in vivo serum cytokines induction (IL-1β, IL-2, IL-6, IL-10, IL-12, TNF-α, GM-CSF and IFN-γ) whereas the impact of treatment on mononuclear cell subsets and their level of activation was tested ex vivo. Volunteers’ PBMC (peripheral blood monocnuclear cells) were stimulated in vitro with recall antigens and mitogen for cytokine production. At baseline, we performed in addition a cytokine release assay on PBMC upon stimulation with trastuzumab as a preclinical safety test. Results: Trastuzumab infusion induced a transient and weak peak of serum IL-6 at 6h, and a modulation of mononuclear cell subset profile and level of activation. Notably CD16+ cells frequency decreased at 3h and peaked at 48h. Except for CD8+ T cells, there were no significant differences between Herceptin and its proposed biosimilar ex vivo. PBMC stimulated in vitro with trastuzumab secreted IL-6, TNF-a, IL-1β, GM-CSF, IFN-γ, and IL-10, but no IL-2. There was no significant difference between the two mAbs. Conclusions: Based on these in vivo, ex vivo and in vitro experiments, there is a strong assumption that MYL-1401O is biosimilar to the reference drug Herceptin for its immunomodulation properties as already proven for its bioequivalence. Clinical trial information: 2011-001406-94.

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The In Vivo Impact of Human Regulatory T Cells on the Graft Versus Tumor Effect.

Blood ◽

10.1182/blood.v110.11.349.349 ◽

2007 ◽

Vol 110 (11) ◽

pp. 349-349 ◽

Cited By ~ 1

Author(s):

Tuna Mutis ◽

Henk Rozemuller ◽

Maarten E. Emmelot ◽

Tineke Aarts-Riemens ◽

Vivienne Verweij ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Tumor Cells ◽

Cell Reactivity ◽

Human Pbmc ◽

Ifn Γ ◽

T Cell Reactivity ◽

The Impact

Abstract The curative Graft-vs-Tumor effect (GvT) of allogeneic Stem cell transplantation (SCT) is frequently complicated with life threatening Graft-vs-Host Disease (GvHD). In mice, prevention of GvHD, without abrogation of GvT is possible by co-transplantation of naturally occurring regulatory T cells (Tregs) with SC grafts. Consistent with these murine studies, we recently demonstrated that also human Tregs possess potent GvHD-downregulatory capacities in a xenogeneic(x) model, where x-GvHD is induced by infusion of autologous human T cells in RAG2−/−γc−/− mice (Mutis et al. Clin. Cancer Res.2006, 12: 5520–5525). Towards clinical application of Tregs, we now explored the impact of human Treg-administration on GvT in a bioluminescence imaging (BLI) based human-GvT model in the RAG2−/−γc−/− mice. In this model, mice inoculated with luciferase (LUC)-transduced human myeloma (MM) cell lines developed BLI-detectable, progressive, MM-like multifocal tumors exclusively in the bone marrow (BM). Full blown tumors were effectively eliminated by infusion of allogeneic human PBMC. This treatment also caused lethal x-GvHD as expected. In this setting, co-infusion of human PBMC with autologous, in vitro cultured Tregs at a 1:1 Treg: T effector cell ratio had no adverse effects on the development of GvT while significantly reducing the lethality of x-GvHD. In vitro analyses of sacrificed mice at day 21 revealed that administered Tregs homed to BM and spleen, significantly downregulated the total numbers of IFN-γ-producing CD4+ and CD8+ T cells responding to CD3 mediated signals, but had no downregulatory effect on the frequencies of IFN-γ-producing T cells responding to tumor cells. There was also no downregulation of cytotoxic activity against tumor cells in Treg-treated mice. Conclusively, these results showed that Tregs, at doses which are inhibitory for x-GvHD-inducing T cells, could maintain the GvT effect by allowing T cell reactivity against tumor cells. Human Tregs thus still hold promise as attractive cellular tools for separating GvT from GvHD.

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Reduced Th1, but Not Th2, Cytokine Production by Lymphocytes after In Vivo Exposure of Healthy Subjects to Endotoxin

Infection and Immunity ◽

10.1128/iai.68.3.1014-1018.2000 ◽

2000 ◽

Vol 68 (3) ◽

pp. 1014-1018 ◽

Cited By ~ 39

Author(s):

Fanny N. Lauw ◽

Tessa ten Hove ◽

Pascale E. P. Dekkers ◽

Evert de Jonge ◽

Sander J. H. van Deventer ◽

...

Keyword(s):

Whole Blood ◽

Healthy Subjects ◽

Cytokine Production ◽

Interleukin 2 ◽

Normal Serum ◽

T Helper 1 ◽

Th2 Cytokine ◽

Ifn Γ

ABSTRACT Endotoxin (lipopolysaccharide [LPS]) tolerance is characterized by a reduced capacity of monocytes to produce proinflammatory cytokines upon restimulation in vitro. To determine whether LPS exposure induces a change in lymphocyte cytokine production and whether this results in a shift in the T-helper 1 (Th1)/Th2 balance, whole blood obtained from seven healthy subjects before and after an intravenous injection of LPS (4 ng/kg) was stimulated in vitro with the T-cell stimulus anti-CD3/CD28 or staphylococcal enterotoxin B. Whole-blood production of the Th1 cytokines gamma interferon (IFN-γ) and interleukin-2 (IL-2) was markedly reduced at 3 and 6 h, while the production of the Th2 cytokines IL-4 and IL-5 was not influenced or was slightly increased. The IFN-γ/IL-4 ratio was strongly decreased at 6 h. Serum obtained after LPS exposure could slightly inhibit the release of IFN-γ but increased IL-4 production during stimulation of blood drawn from subjects not previously exposed to LPS. Normal serum also inhibited IFN-γ production, albeit to a lesser extent. LPS exposure influences lymphocyte cytokine production, resulting in a shift toward a Th2 cytokine response, an effect that may be mediated in part by soluble factors present in serum after LPS administration in vivo.

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The impact of FGF19/FGFR4 signaling inhibition in antitumor activity of multi-kinase inhibitors in hepatocellular carcinoma

Scientific Reports ◽

10.1038/s41598-021-84117-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hiroaki Kanzaki ◽

Tetsuhiro Chiba ◽

Junjie Ao ◽

Keisuke Koroki ◽

Kengo Kanayama ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Molecular Mechanisms ◽

Kinase Inhibitors ◽

Progression Free Survival ◽

Erk Signaling ◽

Enzyme Linked Immunosorbent Assay ◽

Antitumor Effects ◽

The Impact

AbstractFGF19/FGFR4 autocrine signaling is one of the main targets for multi-kinase inhibitors (MKIs). However, the molecular mechanisms underlying FGF19/FGFR4 signaling in the antitumor effects to MKIs in hepatocellular carcinoma (HCC) remain unclear. In this study, the impact of FGFR4/ERK signaling inhibition on HCC following MKI treatment was analyzed in vitro and in vivo assays. Serum FGF19 in HCC patients treated using MKIs, such as sorafenib (n = 173) and lenvatinib (n = 40), was measured by enzyme-linked immunosorbent assay. Lenvatinib strongly inhibited the phosphorylation of FRS2 and ERK, the downstream signaling molecules of FGFR4, compared with sorafenib and regorafenib. Additional use of a selective FGFR4 inhibitor with sorafenib further suppressed FGFR4/ERK signaling and synergistically inhibited HCC cell growth in culture and xenograft subcutaneous tumors. Although serum FGF19high (n = 68) patients treated using sorafenib exhibited a significantly shorter progression-free survival and overall survival than FGF19low (n = 105) patients, there were no significant differences between FGF19high (n = 21) and FGF19low (n = 19) patients treated using lenvatinib. In conclusion, robust inhibition of FGF19/FGFR4 is of importance for the exertion of antitumor effects of MKIs. Serum FGF19 levels may function as a predictive marker for drug response and survival in HCC patients treated using sorafenib.

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Decitabine Promotes Modulation in Phenotype and Function of Monocytes and Macrophages That Drive Immune Response Regulation

Cells ◽

10.3390/cells10040868 ◽

2021 ◽

Vol 10 (4) ◽

pp. 868

Author(s):

Fabiana Albani Zambuzi ◽

Priscilla Mariane Cardoso-Silva ◽

Ricardo Cardoso Castro ◽

Caroline Fontanari ◽

Flavio da Silva Emery ◽

...

Keyword(s):

Immune Response ◽

Hematological Malignancies ◽

M2 Macrophage ◽

M2 Polarization ◽

Tnf Α ◽

Monocytes And Macrophages ◽

Ifn Γ ◽

And Function ◽

The Impact

Decitabine is an approved hypomethylating agent used for treating hematological malignancies. Although decitabine targets altered cells, epidrugs can trigger immunomodulatory effects, reinforcing the hypothesis of immunoregulation in treated patients. We therefore aimed to evaluate the impact of decitabine treatment on the phenotype and functions of monocytes and macrophages, which are pivotal cells of the innate immunity system. In vitro decitabine administration increased bacterial phagocytosis and IL-8 release, but impaired microbicidal activity of monocytes. In addition, during monocyte-to-macrophage differentiation, treatment promoted the M2-like profile, with increased expression of CD206 and ALOX15. Macrophages also demonstrated reduced infection control when exposed to Mycobacterium tuberculosis in vitro. However, cytokine production remained unchanged, indicating an atypical M2 macrophage. Furthermore, when macrophages were cocultured with lymphocytes, decitabine induced a reduction in the release of inflammatory cytokines such as IL-1β, TNF-α, and IFN-γ, maintaining IL-10 production, suggesting that decitabine could potentialize M2 polarization and might be considered as a therapeutic against the exacerbated immune response.

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Oxidative Stress Compromises Lymphocyte Function in Neonatal Dairy Calves

Antioxidants ◽

10.3390/antiox10020255 ◽

2021 ◽

Vol 10 (2) ◽

pp. 255

Author(s):

Wilmer Cuervo ◽

Lorraine M. Sordillo ◽

Angel Abuelo

Keyword(s):

Oxidative Stress ◽

Immune Responses ◽

Immune Cell ◽

Lymphocyte Activation ◽

Dairy Calves ◽

Lymphocyte Function ◽

Newborn Calves ◽

Ifn Γ ◽

The Impact

Dairy calves are unable to mount an effective immune response during their first weeks of life, which contributes to increased disease susceptibility during this period. Oxidative stress (OS) diminishes the immune cell capabilities of humans and adult cows, and dairy calves also experience OS during their first month of life. However, the impact that OS may have on neonatal calf immunity remains unexplored. Thus, we aimed to evaluate the impact of OS on newborn calf lymphocyte functions. For this, we conducted two experiments. First, we assessed the association of OS status throughout the first month of age and the circulating concentrations of the cytokines interferon-gamma (IFN-γ) and interleukin (IL) 4, as well as the expression of cytokine-encoding genes IFNG, IL2, IL4, and IL10 in peripheral mononuclear blood cells (PBMCs) of 12 calves. Subsequently, we isolated PBMCs from another 6 neonatal calves to investigate in vitro the effect of OS on immune responses in terms of activation of lymphocytes, cytokine expression, and antibody production following stimulation with phorbol 12-myristate 13-acetate or bovine herpesvirus-1. The results were compared statistically through mixed models. Calves exposed to high OS status in their first month of age showed higher concentrations of IL-4 and expression of IL4 and IL10 and lower concentrations of IFN-γ and expression of IFNG and IL2 than calves exposed to lower OS. In vitro, OS reduced lymphocyte activation, production of antibodies, and protein and gene expression of key cytokines. Collectively, our results demonstrate that OS can compromise some immune responses of newborn calves. Hence, further studies are needed to explore the mechanisms of how OS affects the different lymphocyte subsets and the potential of ameliorating OS in newborn calves as a strategy to augment the functional capacity of calf immune cells, as well as enhance calves’ resistance to infections.

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Transcriptional Profiling of Porcine Blastocysts Produced In Vitro in a Chemically Defined Culture Medium

Animals ◽

10.3390/ani11051414 ◽

2021 ◽

Vol 11 (5) ◽

pp. 1414

Author(s):

Josep M. Cambra ◽

Emilio A. Martinez ◽

Heriberto Rodriguez-Martinez ◽

Maria A. Gil ◽

Cristina Cuello

Keyword(s):

Culture Medium ◽

Embryo Quality ◽

Transcriptional Profiling ◽

Defined Medium ◽

Platelet Factor ◽

Defined Media ◽

Defined Culture ◽

The Impact

The development of chemically defined media is a growing trend in in vitro embryo production (IVP). Recently, traditional undefined culture medium with bovine serum albumin (BSA) has been successfully replaced by a chemically defined medium using substances with embryotrophic properties such as platelet factor 4 (PF4). Although the use of this medium sustains IVP, the impact of defined media on the embryonic transcriptome has not been fully elucidated. This study analyzed the transcriptome of porcine IVP blastocysts, cultured in defined (PF4 group) and undefined media (BSA group) by microarrays. In vivo-derived blastocysts (IVV group) were used as a standard of maximum embryo quality. The results showed no differentially expressed genes (DEG) between the PF4 and BSA groups. However, a total of 2780 and 2577 DEGs were detected when comparing the PF4 or the BSA group with the IVV group, respectively. Most of these genes were common in both in vitro groups (2132) and present in some enriched pathways, such as cell cycle, lysosome and/or metabolic pathways. These results show that IVP conditions strongly affect embryo transcriptome and that the defined culture medium with PF4 is a guaranteed replacement for traditional culture with BSA.

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Sarcoma IL-12 overexpression facilitates NK cell immunomodulation

Scientific Reports ◽

10.1038/s41598-021-87700-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Mary Jo Rademacher ◽

Anahi Cruz ◽

Mary Faber ◽

Robyn A. A. Oldham ◽

Dandan Wang ◽

...

Keyword(s):

Immune Response ◽

Cell Lines ◽

Nk Cell ◽

Autologous Tumor ◽

Murine Models ◽

Lentiviral Transduction ◽

Ifn Γ ◽

Human Sarcoma

AbstractInterleukin-12 (IL-12) is an inflammatory cytokine that has demonstrated efficacy for cancer immunotherapy, but systemic administration has detrimental toxicities. Lentiviral transduction eliciting IL-12-producing human sarcoma for autologous reintroduction provides localized delivery for both innate and adaptive immune response augmentation. Sarcoma cell lines and primary human sarcoma samples were transduced with recombinant lentivirus engineering expression of human IL-12 (hu-IL-12). IL-12 expressing sarcomas were assessed in vitro and in vivo following implantation into humanized NSG and transgenic human IL-15 expressing (NSG.Tg(Hu-IL-15)) murine models. Lentiviral transduction (LV/hu-IL-12) of human osteosarcoma, Ewing sarcoma and rhabdomyosarcoma cell lines, as well as low-passage primary human sarcomas, engendered high-level expression of hu-IL-12. Hu-IL-12 demonstrated functional viability, eliciting specific NK cell-mediated interferon-γ (IFN-γ) release and cytotoxic growth restriction of spheroids in vitro. In orthotopic xenograft murine models, the LV/hu-IL-12 transduced human sarcoma produced detectable IL-12 and elicited an IFN-γ inflammatory immune response specific to mature human NK reconstitution in the NSG.Tg(Hu-IL-15) model while restricting tumor growth. We conclude that LV/hu-IL-12 transduction of sarcoma elicits a specific immune reaction and the humanized NSG.Tg(Hu-IL-15) xenograft, with mature human NK cells, can define in vivo anti-tumor effects and systemic toxicities. IL-12 immunomodulation through autologous tumor transduction and reintroduction merits exploration for sarcoma treatment.

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Impact of RARα and miR-138 on retinoblastoma etoposide resistance

Tumor Biology ◽

10.3233/tub-200072 ◽

2021 ◽

Vol 43 (1) ◽

pp. 11-26

Author(s):

Maike Busch ◽

Natalia Miroschnikov ◽

Jaroslaw Thomas Dankert ◽

Marc Wiesehöfer ◽

Klaus Metz ◽

...

Keyword(s):

Cell Viability ◽

Cell Lines ◽

Cancer Progression ◽

Treated Patient ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Gene Assay ◽

The Impact

BACKGROUND: Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. OBJECTIVE: This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. METHODS: RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. RESULTS: A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. CONCLUSIONS: Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

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