scholarly journals In Utero Transplantation of FVIII-Expressing Human Placental Cells Upregulates Gene Pathways Associated with Immune Tolerance, Altering the Response to Postnatal FVIII Infusion

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1863-1863
Author(s):  
Martin Rodriguez ◽  
Brady Trevisan ◽  
Sunil George ◽  
Jordan E Shields ◽  
Jorge Figueroa ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Signaling ◽  
Immune Tolerance ◽  
In Utero ◽  
Publicly Traded ◽  
The Past ◽  
In Utero Transplantation ◽  
Placental Cells ◽  
Inhibitory Antibodies ◽  
Human Placental Cells

Abstract We have previously reported that in utero transplantation (IUTx) of sheep fetuses (n=14) with human placental cells (PLC) transduced with a lentiviral vector encoding mcoET3, an expression/secretion-optimized, bioengineered fVIII transgene (PLC-mcoET3) increased plasma FVIII activity levels by 57%, 42%, and 35% at 1, 2, and 3 years post-IUTx, respectively, without the development of FVIII/ET3 inhibitors. We also demonstrated that immune tolerance to the cell/gene product was maintained after postnatal administration of PLC-mcoET3 (cells producing 20 IU/kg/24h were administered i.v. for 3 consecutive weeks). However, when IUTx-treated animals received weekly i.v. infusions of purified ET3 protein (20IU/kg) for 5 weeks, all recipients developed a robust ET3-specific IgG response that appeared at week 3 of infusion at titers ranging from 1:70 to 1:857 and inhibitory antibodies that ranged from 3-36 BU. Here, we investigated differences in the immune responses of animals that received IUTx with PLC-mcoET3 and were boosted postnatally with PLC-mcoET3 (IUTx-PLC-mcoET3) vs. ET3 protein (IUTx-ET3) to define the pathways by which the immune system differentially responds to protein vs. cell-secreted ET3. A sheep-specific multiplex gene expression analysis with 165 genes involved in immune cell signaling pathways (NanoString) was used to evaluate mRNA isolated from peripheral blood mononuclear cells collected at Weeks (W) 0, 1, and 5 of postnatal infusions. Significant fold-change expression in these mRNA targets was determined using NanoString nSolver 4.0 software. Animals in the IUTx-PLC-mcoET3 group (known to be devoid of inhibitors to ET3 post-boosting) showed that immunoregulation and immune tolerance gene clusters were among the top three clusters that increased expression from W0 to W5 (adj. p-value<0.01). Differential expression of genes in pathways involved in Th1, Th2, and Th17 responses was also found, at differing levels, in the IUTx-PLC group, suggesting a balance between immunity and tolerance was maintained. Surprisingly, the IUTx-ET3 group, which developed inhibitory antibodies after ET3 boosting, also showed significantly increased expression of immune tolerance genes, and downregulation of Th1 and Th17 cell signaling, when evaluated by direct global significance score. Nevertheless, 66% of these animals had a significant upregulation of Th2 cell signaling by W1 vs W0. To determine if the increase in expression of immune tolerance genes was due to the IUTx treatment, we also evaluated a group of aged-matched, non-transplanted sheep that received ET3 protein under the same dose and schedule. Results from Gene Set Analysis (GSA) demonstrated significant upregulation of genes involved in interferon signaling, class I MHC antigen processing, and Th17 signaling in these animals, suggesting the potential involvement of Th17 cells in the immune response in this group. In conclusion, IUTx with PLC-mcoET3 induces the upregulation of genes associated with immune tolerance, providing an explanation for the long-lasting elevation in plasma FVIII levels in these animals in the absence of inhibitors. Nevertheless, despite the continued expression of tolerogenic genes, administration of purified ET3 protein to these IUTx recipients induced upregulation of Th2 signaling, a pathway that was not observed in animals that only received ET3 protein, demonstrating that the mechanism by which tolerance is broken in IUTx recipients differs from that by which an immune response to ET3 occurs in animals with no prior exposure. Of note is that animals that develop inhibitors by the Th17 pathway had considerably higher inhibitor titers than the IUTx recipients that responded to ET3 infusion by the Th2 pathway. These studies underscore the need for a more complete understanding of the mechanisms by which immune tolerance to FVIII develops during ontogeny. Disclosures Doering: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months. Spencer: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months.

scholarly journals Administration of FVIII-Expressing Human Placental Cells to Juvenile Sheep Yields Multi-Organ Engraftment, Therapeutic Plasma FVIII Levels and Alter Immune Signaling Pathways to Evade FVIII Inhibitor Induction

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3966-3966
Author(s):  
Brady Trevisan ◽  
Martin Rodriguez ◽  
Jacqueline Dizon ◽  
Sunil George ◽  
Jordan E Shields ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Differentiation ◽  
T Cell ◽  
Lymph Nodes ◽  
Signaling Pathways ◽  
Proinflammatory Cytokine ◽  
T Cell Differentiation ◽  
Publicly Traded ◽  
The Past ◽  
Inhibitory Antibodies

Abstract We have previously reported that normal juvenile sheep that received weekly intravenous (IV) infusions of human (n=3) or an expression/secretion-optimized, bioengineered human/porcine hybrid (ET3) FVIII protein (n=3) for 5 weeks (20 IU/kg) developed anti-FVIII inhibitory antibodies (10-116 BU, and IgG titers of 1:20-1:245) by week 3 of infusion. By contrast, the IV infusion, or IP administration, of human placental mesenchymal cells (PLC) transduced with a lentiviral vector encoding a myeloid codon-optimized ET3 transgene (PLC-mcoET3) to produce high levels of ET3 protein (4.9-6IU/10^6 cells/24h) enabled the delivery of FVIII without eliciting antibodies, despite using PLC-mcoET3 doses that provided ~20-60 IU/kg ET3 each 24h to mirror the amount of FVIII protein infused. In addition, we showed that the route of PLC-mcoET3 administration (IP vs IV) did not impact the resultant plasma FVIII levels, with animals in these two groups exhibiting mean increases in FVIII activity (quantified by aPTT) of 30.9% and 34.2%, respectively, at week 15 post-treatment. Here, we investigated whether the sites and levels of PLC-mcoET3 engraftment were dependent upon the route of administration and performed s sheep-specific multiplexed transcriptomic analysis (NanoString) to define the immune signaling pathways that thwarted FVIII/ET3 protein immune response when ET3 was delivered through PLC. Tissue samples were collected from various organs at euthanasia and RT-qPCR performed using primers specific to the mcoET3 transgene, to the human housekeeping transcript GAPDH, and to sheep GAPDH, to quantify PLC-mcoET3 tissue engraftment, and normalize the results. RT-qPCR demonstrated PLC-mcoET3 engrafted, in both IP and IV groups, in all the organs evaluated (liver, lung, lymph nodes, thymus, and spleen). Animals that received PLC-mcoET3 via the IP route displayed higher overall levels of engraftment than their IV counterparts. The spleen was the preferential organ of engraftment for both IP and IV groups (IP:2.41±1.97%; IV: 0.64±0.54%). The IP group exhibited significantly higher engraftment in the left lobe of the liver (IP: 1.36±0.35%; IV: 0.041±0.022%), which was confirmed by immunohisto-chemistry (IHC) with an antibody to the human nuclear antigen Ku80 and ImageJ analysis (IP:5.24±3.36%; IV: 0±0). Of note is that the IP route resulted in higher levels of engraftment in the thymus, while IV infusion yielded higher levels of PLC-mcoET3 in lymph nodes. Analysis of H&E-stained tissues demonstrated they were devoid of any abnormal histologic changes and exhibited no evidence of hyperplasia or neoplasia, supporting the safety of the cell platform, irrespective of the route of administration. To date, NanoString analysis of PBMC collected at day 0, week 1, and week 5 post-infusion demonstrated that animals who received FVIII protein had upregulation of UBA5 and BATF, genes involved in antigen processing and Th17 signaling pathways, respectively. Although both IV and IP recipients of PLC-mcoET3 also had an increase in BATF, the IV group exhibited upregulation of BTLA, a gene involved in immune-tolerance, and downregulation of NOTCH and DDL1, involved in T cell differentiation, as well as MAPK12 and PLCG1, genes involved in proinflammatory cytokine regulation and T signaling within the Th17 signature. In IP recipients, BTLA, NOTCH, and DLL1 were all downregulated. Since ET3-reactive Th 1 cells were not present in any of the treated animals, it is possible that the Th17 cells are responsible for the inhibitory antibodies seen in the juvenile sheep treated with FVIII/ET3 protein, while in animals receiving PLC-mcoET3, downregulation of genes involved in T cell differentiation and proinflammatory cytokine signaling keeps the immune system in check to avoid an immune response. Disclosures Doering: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months. Spencer: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months.

Targeting FVIII Expression to Platelets Induces Immune Tolerance in Hemophilia A Mice with or without Pre-Existing Anti-FVIII Immunity,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4170-4170
Author(s):  
Yingyu Chen ◽  
Erin L. Kuether ◽  
Jocelyn A. Schroeder ◽  
Robert R. Montgomery ◽  
David W. Scott ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Immune Response ◽  
Immune Tolerance ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
High Titer ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Therapy Strategy ◽  
Inhibitory Antibodies

Abstract Abstract 4170 Our previous studies have shown that targeting FVIII expression to platelets (2bF8) can correct murine hemophilia A phenotype even in the presence of inhibitory antibodies. In the present study, we wanted to explore 1) whether platelets containing FVIII can act as an immunogen; and 2) whether platelet-derived FVIII can induce immune tolerance in a hemophilia A mouse model. To investigate whether platelets containing FVIII can act as an immunogen in hemophilia A mice, we infused transgenic mouse platelets with a level of platelet-FVIII of 6 mU/108 platelets to naïve FVIIInull mice weekly for 8 weeks. These platelets were between 30 to 50% of total platelets upon infusion and the levels of platelet-FVIII in the infused animals were 0.11 ± 0.01 mU/108 platelets (n = 6) one week after infusion. No anti-FVIII inhibitory antibodies were detected in the infused mice during the study course. All animals developed inhibitors following further challenged with recombinant human FVIII (rhFVIII) at a dose of 50 U/kg by intravenous injection weekly for 4 weeks, indicating that infusion of platelets containing FVIII does not trigger immune response in hemophilia A mice. To explore whether platelet-derived FVIII will act as an immunogen in the presence of primed spleen cells (from mice already producing inhibitory antibody), we co-transplanted splenocytes from highly immunized FVIIInull mice and bone marrow (BM) cells from 2bF8 transgenic mice into 400 cGy sub-lethal irradiated FVIIInull recipients. We monitored the levels of inhibitory antibodies in recipients for up to 8 weeks and found that inhibitor titers declined with time after transplantation. We then challenged co-transplantation recipients with rhFVIII and found that inhibitor titers in the control group co-transplantat of FVIIInull BM cells increased 103.55 ± 64.83 fold (n = 4), which was significantly more than the group receiving 2bF8 transgenic BM cells (14.34 ± 18.48, n = 5) (P <.05). The inhibitor titers decreased to undetectable in 40% of 2bF8 transgenic BM cells co-transplantation recipients even after rhFVIII challenge, indicating immune tolerance was induced in these recipients. To further explore the immune response in the lentivirus-mediated platelet-derived FVIII gene therapy of hemophilia A mice, we transduced hematopoietic stem cells from pre-immunized FVIIInull mice with 2bF8 lentivirus (LV) followed by syngeneic transplantation into pre-immunized lethally irradiated FVIIInull recipients and monitored the levels of inhibitor titers in recipients. After full BM reconstitution, platelet-FVIII expression was sustained (1.56 ± 0.56 mU/108platelets, n = 10), but inhibitor titers declined with time, indicating that platelet-derived FVIII does not provoke a memory response in FVIIInullmice that had previously mounted an immune response to rhFVIII. The t1/2 of inhibitor disappearance in 2bF8 LV-transduced recipients (33.65 ± 11.12 days, n = 10) was significantly shorter than in untransduced controls (66.43 ± 22.24 days, n = 4) (P <.01). We also transplanted 2bF8 LV-transduced pre-immunized HSCs into 660 cGy sub-lethal irradiated naïve FVIIInull mice. After BM reconstituted, recipients were assessed by platelet lysate FVIII:C assay and tail clip survival test to confirm the success of genetic therapy. Animals were then challenged with rhFVIII. Only 2 of 7 2bF8 LV-transduced recipients developed inhibitory antibodies (55 and 87 BU/ml), while all untransduced control developed high titer of inhibitors (735.50 ± 94.65 BU/ml, n = 4). In conclusion, our results demonstrate that 1) platelets containing FVIII are not immunogenic in hemophilia A mice; and 2) platelet-derived FVIII may induce immune tolerance in hemophilia A mice with or without pre-existing inhibitory antibodies. This tolerance induction would add an additional significant benefit to patients with platelet-derived FVIII gene therapy strategy because protein infusion could be administered in some special situations (e.g. surgery in which a greater levels of FVIII may be required) with minimized risk of inhibitor development. Disclosures: No relevant conflicts of interest to declare.

Migration of Donor Cells from the Yolk Sac to Fetal Tissues after In Utero Transplantation of Early to Mid-Gestation Canine Fetuses.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1201-1201
Author(s):  
Andrea K. Vaags ◽  
Cathy Gartley ◽  
Yanzhen Zheng ◽  
Howard Dobson ◽  
Krista B. Halling ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fluorescence Microscopy ◽  
Immune Tolerance ◽  
Donor Cell ◽  
In Utero ◽  
Yolk Sac ◽  
Iron Particles ◽  
In Utero Transplantation ◽  
Donor Cells

Abstract Transplantation of stem and progenitor cells, under permissive conditions, can result in the long-term engraftment of allogeneic donor cells. In utero transplantation is of particular interest in that mixed chimerism may allow for the amelioration of disorders before birth and the development of immune tolerance towards donor cells prior to the maturation of the immune system. In order to more fully establish the feasibility of in utero cell transplantation, we are developing a canine model through the injection of allogeneic cells to the yolk sacs of day 25 or day 35 fetuses. Cell tracking was facilitated by labeling transplanted male canine cells with micron-sized superparamagnetic, fluorescent, polystyrene beads. Total bone marrow (BMMC) and mesenchymal stromal cells (MSC) were labeled for 16 hours with fluorescent superparamagnetic beads, prior to transplantation. Five pregnancies were studied, wherein 1–2 × 106 MSC or 0.1–1 × 107 BMMC were delivered to individual yolk sacs of day 25 (n=13) or day 35 (n=14) fetuses under ultrasound guidance. Each pregnancy included 1–2 fetuses that received an equal volume saline injection (n=7). Fetuses developed in utero for an additional seven to fourteen days at which time ovariohysterectomy and fetal retrieval were performed. Ex vivo whole body fluorescence imaging of fetuses verified cell migration from the yolk sac injection site to the fetus proper based on increased levels of green fluorescence in injected versus non-injected controls. The signal was predominantly localized to the thoracic and abdominal regions, with no fluorescence visible in the yolk sac. Fluorescence microscopy for detection of the fluorophore and light microscopy of Prussian Blue stained sections for detection of superparamagnetic iron particles was performed to assess donor cell localization. The co-localization of iron particles and fluorescence label was detected on images taken from sequential sections. These analyses indicated that labeled BMMC and MSC migrated from the yolk sac to the fetal liver and to a lesser extent to the developing bone marrow cavity. In some cases, the use of superparamagnetic particles has been confounded by free particles being scavenged by macrophage. To determine if cell labeling was restricted to macrophage, sections were stained with an anti-macrophage antibody and analyzed by fluorescence microscopy. Co-localization of the anti-macrophage stain and the fluorescence of the particle was not detected. Furthermore, molecular confirmation of male donor cell engraftment in the livers of female fetuses was obtained via canine Y chromosome specific Q-PCR. Y chromosome positive cells were detected in female fetuses receiving either male MSC or BMMC, but not in saline injected controls. Our studies demonstrate that injection of cells into the yolk sac during early to mid gestation is an effective strategy to deliver cells to the developing fetus, and in particular to sites of fetal hematopoiesis. We are currently following in utero transplant recipients to determine whether long-term engraftment and immune tolerance of donor cells during the neonatal period can be achieved.

Atopic dermatitis: new horizons of therapy

2019 ◽  
Vol 1 (7) ◽  
pp. 29-32 ◽  
Author(s):  
L. S. Kruglova ◽  
E. M. Gensler
Keyword(s):  
Blood Pressure ◽  
Immune Response ◽  
Allergic Rhinitis ◽  
Atopic Dermatitis ◽  
Targeted Therapy ◽  
Inflammatory Response ◽  
Bronchial Asthma ◽  
New Horizons ◽  
The Past

Over the past decades, the first breakthrough milestone in the treatment of severe forms of atopic dermatitis (AD) has been targeted therapy aimed at inhibiting IL-4 and IL-13. This was made possible thanks to advances in the understanding of the pathogenesis of AD, the driver of which is the Th2-type immune response, which also underlies such manifestations of atopy as bronchial asthma, allergic rhinitis, and polynosis. In the case of the Th2-type immune response, cytokines IL-4 and IL-13 are secreted, which are the main promoters of the inflammatory response in AD. Inhibition of IL-4 and IL-13 leads to the prevention of inflammation and is an effective approach to therapy. The use of therapy aimed at inhibition of cytokines allows you to effectively cope with the manifestations of severe and moderately severe blood pressure.

Vitamin A: dietologist’s position

2020 ◽  
pp. 49-57
Author(s):  
S. V. Orlova ◽  
E. A. Nikitina ◽  
L. I. Karushina ◽  
Yu. A. Pigaryova ◽  
O. E. Pronina
Keyword(s):  
Immune Response ◽  
Urinary Tract ◽  
Gastrointestinal Tract ◽  
Vitamin A ◽  
Developed Countries ◽  
Therapeutic Practice ◽  
The Past ◽  
Increased Risk ◽  
Mucous Membranes

Vitamin A (retinol) is one of the key elements for regulating the immune response and controls the division and differentiation of epithelial cells of the mucous membranes of the bronchopulmonary system, gastrointestinal tract, urinary tract, eyes, etc. Its significance in the context of the COVID‑19 pandemic is difficult to overestimate. However, a number of studies conducted in the past have associated the additional intake of vitamin A with an increased risk of developing cancer, as a result of which vitamin A was practically excluded from therapeutic practice in developed countries. Our review highlights the role of vitamin A in maintaining human health and the latest data on its effect on the development mechanisms of somatic pathology.

scholarly journals MicroRNAs: Biological Regulators in Pathogen–Host Interactions

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 113 ◽  
Author(s):  
Stephanie Maia Acuña ◽  
Lucile Maria Floeter-Winter ◽  
Sandra Marcia Muxel
Keyword(s):  
Immune Response ◽  
Infectious Diseases ◽  
Small Rnas ◽  
Immune Cell ◽  
Fine Tuning ◽  
Biological Processes ◽  
Host Interactions ◽  
The Past ◽  
Biological Aspects

An inflammatory response is essential for combating invading pathogens. Several effector components, as well as immune cell populations, are involved in mounting an immune response, thereby destroying pathogenic organisms such as bacteria, fungi, viruses, and parasites. In the past decade, microRNAs (miRNAs), a group of noncoding small RNAs, have emerged as functionally significant regulatory molecules with the significant capability of fine-tuning biological processes. The important role of miRNAs in inflammation and immune responses is highlighted by studies in which the regulation of miRNAs in the host was shown to be related to infectious diseases and associated with the eradication or susceptibility of the infection. Here, we review the biological aspects of microRNAs, focusing on their roles as regulators of gene expression during pathogen–host interactions and their implications in the immune response against Leishmania, Trypanosoma, Toxoplasma, and Plasmodium infectious diseases.

In Utero Transplantation of Human Cord Blood Cells into Rabbits

2005 ◽  
Vol 80 (2) ◽  
pp. 282-283 ◽  
Author(s):  
Georg S. Wengler ◽  
Guerino Lombardi ◽  
Tiziana Frusca ◽  
Daniele Alberti ◽  
Alberto Albertini ◽  
...  
Keyword(s):  
Cord Blood ◽  
Blood Cells ◽  
In Utero ◽  
Human Cord Blood ◽  
In Utero Transplantation ◽  
Cord Blood Cells

9 Mesenchymal features of a novel 27-gene algorithm associate with canonical tumor promoting signaling pathways which may identify therapeutic options for immunotherapy resistant patients

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A9-A9
Author(s):  
Tyler Nielsen ◽  
Rob Seitz ◽  
Douglas Ross ◽  
David Hout ◽  
Brock Schweitzer
Keyword(s):  
Immune Response ◽  
Cell Signaling ◽  
Signaling Pathways ◽  
Pathway Analysis ◽  
Stromal Cell ◽  
Checkpoint Inhibitors ◽  
Targeted Therapeutics ◽  
Primary Resistance ◽  
Durable Response ◽  
Xenograft Models

BackgroundImmune checkpoint inhibitors have emerged as a front-line treatment for cancer in multiple indications. Unfortunately, a majority of patients do not realize durable response as a result of primary resistance to the immunotherapy. We have previously described a novel 27-gene immuno-oncology assay and algorithm which demonstrated significant predictive value in both NSCLC and TNBC. This algorithm utilizes gene expression to assess the tumor immune microenvironment (TIME) by combining aspects of the immune response, surrounding stromal cell signaling, and tumor physiology. We hypothesized that features of this algorithm may not only identify responders to immunotherapy (immunomodulatory, IO subtype) but may better enrich for patients who would benefit from other targeted therapeutics that alter the tumor microenvironment such as VEGF or FAK inhibitors (mesenchymal, M subtype).MethodsPathway analysis was used on TNBC specimens representing both the IO and M subtypes as determined by the 27-gene immuno-oncology algorithm. Expression reads were scaled within each sample and the difference of the mean of expression of each gene within IO and M subtypes was determined to quantify relative expression within each pathway. Finally, the mesenchymal score obtained from the 27-gene immuno-oncology algorithm was used to stratify RNAseq expression data from xenograft models that were either sensitive or resistant to a FAK inhibitor (FAKi).ResultsPathway analysis identified stratification between the 27-gene immuno-oncology algorithm subtypes finding with the mesenchymal subtype is associated with higher WNT, TGF-B, and RAS pathways whereas the IO subtype was more highly associated with the JAK/STAT pathway. Additionally, the mesenchymal score from the 27-gene immuno-oncology algorithm was higher in the FAK inhibitor sensitive (0.36) xenograft models than the FAKi resistant (0.076) models (p = 0.025).ConclusionsThe 27-gene immuno-oncology algorithm assesses the TIME to account for the immune response, surrounding stromal cell signaling, and tumor physiology to provide both an immuno-oncology subtype and mesenchymal subtype. We have previously demonstrated improved ability of the IO subtype to predict response to ICIs over current gold standard biomarkers. These data suggest that the M subtype is a distinct feature of the IO subtype which may enrich for patients more likely to respond to targeted therapeutics that act upon the canonical tumor promoting signaling pathways.

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