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Medicina ◽  
2022 ◽  
Vol 58 (1) ◽  
pp. 84
Author(s):  
Sergii Tkach ◽  
Andrii Dorofeyev ◽  
Iurii Kuzenko ◽  
Nadiya Boyko ◽  
Tetyana Falalyeyeva ◽  
...  

The intestinal microbiota plays an important role in maintaining human health, and its alteration is now associated with the development of various gastrointestinal (ulcerative colitis, irritable bowel syndrome, constipation, etc.) and extraintestinal diseases, such as cancer, metabolic syndrome, neuropsychiatric diseases. In this context, it is not surprising that gut microbiota modification methods may constitute a therapy whose potential has not yet been fully investigated. In this regard, the most interesting method is thought to be fecal microbiota transplantation, which consists of the simultaneous replacement of the intestinal microbiota of a sick recipient with fecal material from a healthy donor. This review summarizes the most interesting findings on the application of fecal microbiota transplantation in gastrointestinal and extraintestinal pathologies.


2022 ◽  
Vol 22 (1) ◽  
pp. 19
Author(s):  
Carolina Romero ◽  
José-María Díez ◽  
Rodrigo Gajardo
Keyword(s):  

2021 ◽  
pp. 102648
Author(s):  
S.A. Chechetkina ◽  
A.A. Khabarova ◽  
A.S. Chvileva ◽  
O.M. Kurchenko ◽  
A.V. Smirnov ◽  
...  

2021 ◽  
pp. 102613
Author(s):  
Shuxing Cao ◽  
Jun Ma ◽  
Jinyu Zhang ◽  
Ruiyun Guo ◽  
Xin Liu ◽  
...  

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Victoria Ann Remley ◽  
Jianjian Jin ◽  
Sarmila Sarkar ◽  
Larry Moses ◽  
Michaela Prochazkova ◽  
...  

Abstract Background Gene transfer is an important tool for cellular therapies. Lentiviral vectors are most effectively transferred into lymphocytes or hematopoietic progenitor cells using spinoculation. To enable cGMP (current Good Manufacturing Practice)-compliant cell therapy production, we developed and compared a closed-system spinoculation method that uses cell culture bags, and an automated closed system spinoculation method to decrease technician hands on time and reduce the likelihood for microbial contamination. Methods Sepax spinoculation, bag spinoculation, and static bag transduction without spinoculation were compared for lentiviral gene transfer in lymphocytes collected by apheresis. The lymphocytes were transduced once and cultured for 9 days. The lentiviral vectors tested encoded a CD19/CD22 Bispecific Chimeric Antigen Receptor (CAR), a FGFR4-CAR, or a CD22-CAR. Sepax spinoculation times were evaluated by testing against bag spinoculation and static transduction to optimize the Sepax spin time. The Sepax spinoculation was then used to test the transduction of different CAR vectors. The performance of the process using healthy donor and a patient sample was evaluated. Functional assessment was performed of the CD19/22 and CD22 CAR T-cells using killing assays against the NALM6 tumor cell line and cytokine secretion analysis. Finally, gene expression of the transduced T-cells was examined to determine if there were any major changes that may have occurred as a result of the spinoculation process. Results The process of spinoculation lead to significant enhancement in gene transfer. Sepax spinoculation using a 1-h spin time showed comparable transduction efficiency to the bag spinoculation, and much greater than the static bag transduction method (83.4%, 72.8%, 35.7% n = 3). The performance of three different methods were consistent for all lentiviral vectors tested and no significant difference was observed when using starting cells from healthy donor versus a patient sample. Sepax spinoculation does not affect the function of the CAR T-cells against tumor cells, as these cells appeared to kill target cells equally well. Spinoculation also does not appear to affect gene expression patterns that are necessary for imparting function on the cell. Conclusions Closed system-bag spinoculation resulted in more efficient lymphocyte gene transfer than standard bag transductions without spinoculation. This method is effective for both retroviral and lentiviral vector gene transfer in lymphocytes and may be a feasible approach for gene transfer into other cell types including hematopoietic and myeloid progenitors. Sepax spinoculation further improved upon the process by offering an automated, closed system approach that significantly decreased hands-on time while also decreasing the risk of culture bag tears and microbial contamination.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2694-2694
Author(s):  
Ladan Kobari ◽  
Martine Auclair ◽  
Olivier Piau ◽  
Nathalie Ferrand ◽  
Maurice Zaoui ◽  
...  

Abstract Introduction: Myeloma is characterized by bone lesions, which are related to both an increased osteoclast activity and a defect in the differentiation of medullary mesenchymal stem cells (MSCs) into osteoblasts. Outside the medullary environment, adipocyte-derived MSCs (ASCs) could represent a source of functional osteoblasts. However, we recently found a defect in the osteoblastic differentiation of ASCs from myeloma patients (MM-ASCs). We therefore examined the effects of plasma from myeloma patients at diagnosis (MM-plasmas) and in complete remission (CR-plasmas) and from healthy donors on the osteoblastic differentiation of healthy donor-derived ASCs (HD-ASCs) and healthy donor-bone marrow derived MSCs (HD-BM-MSCs). Materials and Methods: We studied 11 MM-ASCs, 5 HD-ASCs and 3 HD-BM-MSCs. The plasmas were from myeloma patients with bone lesions at diagnosis (n=12), in complete remission (n=8) and from 5 pools of 100 healthy donors (HD-plasmas). HD-ASCs were differentiated into osteoblasts and adipocytes and HD-BM-MSCs into osteoblasts with the three types of plasmas as well as newly discovered cytokines. Results: Osteoblastogenesis in HD-ASCs was suppressed by MM-plasmas. Alizarin red coloration and alkaline phosphatase activity were strongly decreased along with a decreased RUNX2 and osteocalcin expression. However, adipocyte differentiation was unaltered. The osteoblastic differentiation deficiency was reversible once the plasma-derived factors were removed. Using cytokine array and comparing MM-plasmas with HD-plasmas, we identified seven cytokines (ANG1, ENA-78, EGF, PDGF-AA/AB/BB and TARC), besides DKK1, highly increased in MM-plasmas which was confirmed by ELISA (Figure). They separately inhibited the osteoblastic differentiation of HD-ASCs. In contrast, myeloma patients in remission had a cytokine plasma level almost normal with barely no osteoblastic differentiation inhibition. In addition, the mixture of the 7 cytokines with and without DKK1 inhibited not only the HD-ASCs but also the HD-BM-MSCs. Concomittantly, we observed that MM-plasmas enhanced adipogenesis-related gene expression. Comparison of MM-ASCs and HD-ASCs by RNA sequencing showed that two master genes characterizing adipocyte differentiation, CD36 and PPARγ, were upregulated in MM-ASCs as compared to HD-ASCs. Moreover, we demonstrated a significant increase in CD36 and PPARγ expression in HD-ASCs in the presence of MM-plasmas or the seven cytokines individually, similarly as in MM-ASCs. Finally, we tried to identify the origin of these cytokines. When myeloma patients were in remission, the cytokines levels were strongly decreased suggesting a malignant plasmocyte secretion. This was reinforced by the detection of the 7 cytokines in three different myeloma cell lines with an especially high secretion of PDGF-AA. We conclude that specific cytokines in MM-plasmas, besides the well-known DKK1, inhibit the osteoblastic differentiation of MM- and HD-ASCs with a skewing towards adipocyte differentiation. Of note, this inhibition by the cytokines were also observed on HD-BM-MSCs suggesting that this could also be the case on myeloma-BM-MSCs. Legend to figure: Cytokine expression in MM, CR and HD-plasmas (A) Representative images of cytokine array blots probed with the plasma samples. The red boxes identify the cytokines significantly dysregulated in MM- as compared to HD- or CR-plasmas and further analyzed by ELISA. The blue boxes identified the cytokines similarly expressed in MM-/CR-/HD-plasmas. (B) Cytokine concentrations in the HD-plasmas (n=5), MM-plasmas (n=11) and CR-plasmas (n=8) were measured by ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001, ns (not significant). Figure 1 Figure 1. Disclosures Delhommeau: Novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy. Garderet: Celgene: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Sanofi: Consultancy; Takeda: Consultancy.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2767-2767
Author(s):  
Henry Barreras ◽  
Sabrina N. Copsel ◽  
Ying Ding ◽  
Charles J. Cash ◽  
Cameron S. Bader ◽  
...  

Abstract Mobilized hematopoietic stem / progenitor cells have become the primary option used for adult allogeneic hematopoietic stem cell transplants (aHSCTs) although GVHD remains a major immunologic complication (CIBMTR, 2017). The present studies are directed to developing a translational model for the application of donor regulatory T cells (Tregs) together with the use of mobilized peripheral blood (mPB) for more widespread application of these aHSCTs. We have previously reported that two-pathway Treg expansion strategy markedly elevates Treg numbers and function (Wolf, 2017; Copsel 2018). Additionally, this approach could be effectively applied in a concomitant strategy to amplify Treg suppressor activity in mPB to diminish pre-clinical GVHD (Barreras H, ASTCT Meeting S292 ,2019). Here, we present new findings using mPB transplants examining GVL reactivity in an MHC-matched minor antigen mismatched pre-clinical model and test the notion that human donor Tregs can be employed with mobilized healthy donor PB to effectively regulate xenogeneic GVHD. Donor C3H.SW (H2b) mice were treated with filgrastim for 4 days prior to use for transplants and PB analysis demonstrated significant elevations in c-kit + cells. Some PB donors were concurrently administered the two-pathway Treg expansion strategy (D-6 through D-1) consisting of TL1A-Ig, targeting TNFRSF25, and low dose huIL-2. A significant increase (up to 40%) in the frequency of CD4 +FoxP3 + Tregs occurred during the mobilization process. Pooled C3H.SW PB was collected from both mobilized and Treg unexpanded ("TrUM") or mobilized and Treg expanded ("TrEM") donors and transplanted into lethally irradiated, MHC-matched B6 (H2b) recipients. Recipients of TrEM donors exhibited significantly reduced weight loss and clinical GVHD scores compared to recipients of TrUM (Fig.1A). GVL responses were tested in animals administered B6-MLL-AF9 GFP leukemia cells administered at the time of transplant. Notably, recipients of TrEM exhibited comparable GVL activity to TrUM recipients evidenced by B6-MLL-AF9 GFP levels in bone marrow and spleen (Fig.1B). To begin to translate these findings, we tested the use of ex-vivo expanded human donor Tregs (huTregs) to ameliorate xenogeneic GVHD (xGVHD). First, sorted huTregs (CD4 +CD25 +CD127 lo) from a healthy donor were expanded ex-vivo using anti-CD3/anti-CD28 beads 1 week prior to transplant (Fig 2A). next, these expanded huTregs were combined with human mPB from the same healthy donor (6x10 6 PBMC) and transplanted into NSG/ NOD-scid IL2Rgamma null mice. We found that treatment with ex-vivo expanded huTregs resulted in significant reduction of mortality rate and clinical xGVHD (Fig 2B,C). Notably, 1 week post-transplant PB huTregs levels were still elevated and frequency of huCD4 +Tconv cells was diminished supporting xGVHD outcomes (Fig 2D). In total, these findings demonstrated that the use of mPB containing elevated Treg levels significantly reduced pre-clinical GVHD without loss of GVL activity in an MHC-matched allogeneic model. Moreover, utilizing ex-vivo expanded huTregs from a mPB donor and added back to the same donor at the time of transplant ameliorated xGVHD. The observations indicate that during the donor stem / progenitor cell mobilization process, manipulation of donor Tregs using our two-pathway strategy can be successfully accomplished in PB resulting in an effective and translational approach to ameliorate GVHD following aHSCT. In total, the present studies support the notion that in vivo or ex-vivo manipulation of donor tregs together with mobilized peripheral blood could provide therapeutic approaches to improve aHSCT outcomes. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 515-515
Author(s):  
Claire E. Pillsbury ◽  
Jairo A. Fonseca ◽  
Jodi Dougan ◽  
Hasan Abukharma ◽  
Gloria Gonzalez-Flamenco ◽  
...  

Abstract Immunotherapies have recently shown efficacy in treatment of aggressive, refractory pediatric B cell acute lymphoblastic leukemia (B-ALL), which remains one of the leading causes of cancer-related death in children. The immune evasion mechanisms of B-ALL are still being explored to discover new therapeutic targets and improve patient outcomes. Recent reports have implicated a role for the molecule Siglec-15 (Sig15) in regulating immune response in solid tumor-infiltrating macrophages. Our lab has found higher expression of SIGLEC15 at the RNA level in primary pediatric B-ALL as compared to healthy donor controls, as well as at the RNA and protein levels across a panel of B-ALL, T cell acute lymphoblastic leukemia (T-ALL), and diffuse large B cell lymphoma (DLBCL) cell lines compared to healthy donor PBMCs. Higher expression of SIGLEC15 in pediatric B-ALL samples from the TARGET database correlates with markers of PKC and NFκB activation known to drive B-ALL leukemogenesis, which we have demonstrated to regulate Sig15 RNA and protein expression in vitro. Knockout of Siglec15 expression in a BCR-ABL1 + murine model of B-ALL engrafted in immunocompetent and Rag1 -/- immunodeficient recipients resulted in leukemia clearance in immunocompetent, but not immunodeficient, recipients and 100% survival (Figure A, p=0.01 Sig15 KO into WT vs. Rag1 -/-). Further study indicates that Siglec15 expression on these leukemia cells suppresses T cell effector and memory population expansion at 7 days post-engraftment (Figure B) and correlates with higher levels of IL-10 and lower levels of CCL17 present in the bone marrow, representing a more immunosuppressive bone marrow milieu. These data suggest a prominent role for Sig15 in the suppression of adaptive immune response to B-ALL as well as other hematological malignancies. We have also reported for the first time the release of a soluble form of Sig15 (sSig15), which we have demonstrated to circulate at higher levels in the plasma of pediatric B-ALL patients compared to healthy donors (Figure C, ****P≤0.0001). Detection of this sSig15 negatively correlated with circulating levels of IL-12 and IL-1α/β (Figure D, depicting correlations of cytokines using Pearson's r), suggesting sSig15 levels correspond to a systemically immunosuppressive phenotype. Flow cytometry of fresh pediatric B ALL cells demonstrates expression of surface Sig15 in a subset of cases. Thus, Sig15 has the capacity to promote immunosuppressive effects at both marrow-localized and systemic levels. Together, these results suggest Siglec-15 is a novel, potent immunosuppressive molecule active in leukemia progression that may be targeted therapeutically to activate T lymphocytes against leukemia cells. Figure 1 Figure 1. Disclosures Abukharma: NextCure Inc.: Current Employment. Liu: NextCure: Current Employment, Current holder of stock options in a privately-held company.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 859-859
Author(s):  
Senthil Velan Bhoopalan ◽  
Jonathan Yen ◽  
Thiyagaraj Mayuranathan ◽  
Yu Yao ◽  
Kalin Mayberry ◽  
...  

Abstract Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia that typically manifests in infancy as macrocytic anemia with reticulocytopenia. About 80% of DBA cases are caused by heterozygous loss-of-function mutations or deletions in one of 23 ribosomal proteins (RP) genes, with RPS19 being affected in ~25% of patients. Current therapies are suboptimal, and it is difficult to obtain DBA patient hematopoietic stem and progenitor cells (HSPCs) in sufficient quantity for preclinical development of new therapies. To address this gap, we used CRISPR/Cas9-edited healthy donor CD34 + HSPCs to create a novel model of RPS19-mutated DBA and used this model to develop an efficient RPS19-encoding lentiviral vector (LV) for gene therapy. Healthy donor CD34 + HSPCs were electroporated with ribonucleoprotein (RNP) complex consisting of Cas9 and guide RNAs (gRNAs) targeting RPS19 or the AAVS1 locus as a negative control, then grown in medium to support erythroid differentiation. All gRNAs analyzed generated high-frequency on-target insertion-deletion (indels) mutations (Fig. A). RPS19 indels specifically declined over time, suggesting that RPS19 disruption impairs cell proliferation and/or survival. To rescue the defect, we constructed a third-generation, self-inactivating LV expressing RPS19+GFP (RPS19/GFP LV). Transduction was optimized using poloxamer and prostaglandin E2 and linear transduction efficiency was noted even at high multiplicity of infection (MOI) (Fig. B). An MOI of 20 was used for subsequent experiments. In methylcellulose medium, RPS19 RNP-treated cells generated 72% fewer burst forming unit-erythroid (BFU-E) colonies compared to AAVS1 RNP-treated control cells. RPS19/GFP LVs with three different promoters (EF1α short, EF1α long and MND) partially restored BFU-E formation similarly (Fig. C); the EF1α short promoter was chosen for subsequent experiments due its track record for clinical use. We down-titrated the RNP concentration to generate a total indel frequency of ~25%, which resulted in approximately equal frequencies of RPS19 +/+ and RPS19 +/- BFU-E colonies. RPS19 -/- colonies were detected only after edited HSPCs were rescued by RPS19/GFP LV, due to lethality of this genotype (Fig. D). Transfection of CD34 + HSPCs with RPS19 RNP caused a 49% reduction in cell number after 14 days of liquid culture in erythroid differentiation medium compared to control HSPCs; this was corrected by treatment with RPS19/GFP LV (Fig. E). RPS19 RNP treatment of CD34 + HSPCs had no effect on the expansion of cells grown under myeloid differentiation conditions (Fig. F). We analyzed RNP-treated CD34 + cells further by transducing them with RPS19/GFP LV or control LV encoding GFPalone, transplanting them into immunodeficient NSGW mice and analyzing human donor cell progeny in mouse bone marrow after 16 weeks. In cells treated with AAVS1 RNP and GFP LV, the indel frequency dropped from 27.2±1.5% (SD) at 72 hours after editing (input) to 15.5±4.4% at 16 weeks post-transplant (43% reduction) (Fig. G). In HSPCs treated with RPS19 RNP and GFP LV the indel frequency dropped from 20.9±3.1% in input cells to 1.8±0.9% after 16 weeks (Fig. G) (92% reduction). In contrast, the indel frequency of donor HSPCs treated with RPS19 RNP and RPS19/GFP LV dropped from 23.6±2.7% in input cells to 8.4±1.6% (64% reduction), which represents a 5-fold increase in indel frequency compared to treatment with control GFP LV (p< 0.01)(Fig. G). In flow cytometry-purified, donor HSPC-derived myeloid, B-lymphocyte, HSPC and erythroid lineages at 16 weeks after xenotransplantation, the mean indel rates were 1.3% to 2.5% in cells derived from HSPCs treated with RPS19 RNP and GFP LV. Indel rates ranged from 6.9% to 9.2% in the progeny of input HSPCs that were rescued by RPS19/GFP LV, representing a 4-6-fold increase compared to transduction with control GFP LV (p<0.01) (Fig. H). In summary, our studies show that Cas9-mediated disruption of RPS19 in CD34 + HSPCs causes a selective erythroid defect in RPS19 +/- cells, recapitulating the canonical DBA defect. Additionally, deficient bone marrow repopulation by RPS19 +/- cells suggests an HSC defect, consistent with pancytopenia that is observed in many older DBA patients. The optimized RPS19 LV transduces HSPCs at high efficiency and alleviates both defects, supporting its potential utility for DBA therapy. Figure 1 Figure 1. Disclosures Yen: Beam Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Weiss: Beam Therapeutics: Current holder of stock options in a privately-held company; Forma Therapeutics: Consultancy; Novartis: Consultancy; Cellarity Inc.: Consultancy.


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