Therapies to reduce liver fibrosis and stimulate organ regeneration are urgently needed. We conducted a first-in-human, phase 1 dose-escalation trial of autologous macrophage therapy in nine adults ...with cirrhosis and a Model for End-Stage Liver Disease (MELD) score of 10-16 (ISRCTN 10368050). Groups of three participants received a single peripheral infusion of 10
, 10
or up to 10
cells. Leukapheresis and macrophage infusion were well tolerated with no transfusion reactions, dose-limiting toxicities or macrophage activation syndrome. All participants were alive and transplant-free at one year, with only one clinical event recorded, the occurrence of minimal ascites. The primary outcomes of safety and feasibility were met. This study informs and provides a rationale for efficacy studies in cirrhosis and other fibrotic diseases.
MicroRNA (miRNA) species (miR) regulate mRNA translation and are implicated as mediators of disease pathology via coordinated regulation of molecular effector pathways. Unraveling miR disease-related ...activities will facilitate future therapeutic interventions. miR-155 recently has been identified with critical immune regulatory functions. Although detected in articular tissues, the functional role of miR-155 in inflammatory arthritis has not been defined. We report here that miR-155 is up-regulated in synovial membrane and synovial fluid (SF) macrophages from patients with rheumatoid arthritis (RA). The increased expression of miR-155 in SF CD14⺠cells was associated with lower expression of the miR-155 target, Src homology 2-containing inositol phosphatase-1 (SHIP-1), an inhibitor of inflammation. Similarly, SHIP-1 expression was decreased in CD68⺠cells in the synovial lining layer in RA patients as compared with osteoarthritis patients. Overexpression of miR-155 in PB CD14⺠cells led to down-regulation of SHIP-1 and an increase in the production of proinflammatory cytokines. Conversely, inhibition of miR-155 in RA synovial CD14⺠cells reduced TNF-α production. Finally, miR-155-deficient mice are resistant to collagen-induced arthritis, with profound suppression of antigen-specific Th17 cell and autoantibody responses and markedly reduced articular inflammation. Our data therefore identify a role of miR-155 in clinical and experimental arthritis and suggest that miR-155 may be an intriguing therapeutic target.
To test the hypothesis that miR-155 regulates monocyte migratory potential via modulation of chemokine and chemokine receptor expression in RA, and thereby is associated with disease activity.
The ...miR-155 copy-numbers in monocytes from peripheral blood (PB) of healthy (n = 22), RA (n = 24) and RA SF (n = 11) were assessed by real time-PCR using synthetic miR-155 as a quantitative standard. To evaluate the functional impact of miR-155, human monocytes were transfected with control or miR-155 mimic, and the effect on transcript levels, and production of chemokines was evaluated by Taqman low-density arrays and multiplex assays. A comparative study evaluated constitutive chemokine receptor expression in miR-155
and wild-type murine (CD115
Ly6C
Ly6G
) monocytes.
Compared with healthy monocytes, the miR-155 copy-number was higher in RA, peripheral blood (PB) and SF monocytes (PB P < 0.01, and SF P < 0.0001). The miR-155 copy-number in RA PB monocytes was higher in ACPA-positive compared with ACPA-negative patients (P = 0.033) and correlated (95% CI) with DAS28 (ESR), R = 0.728 (0.460, 0.874), and with tender, R = 0.631 (0.306, 0.824) and swollen, R = 0.503 (0.125, 0.753) joint counts. Enforced-expression of miR-155 in RA monocytes stimulated the production of CCL3, CCL4, CCL5 and CCL8; upregulated CCR7 expression; and downregulated CCR2. Conversely, miR155
monocytes showed downregulated CCR7 and upregulated CCR2 expression.
Given the observed correlations with disease activity, these data provide strong evidence that miR-155 can contribute to RA pathogenesis by regulating chemokine production and pro-inflammatory chemokine receptor expression, thereby promoting inflammatory cell recruitment and retention in the RA synovium.
IL-33, a cytokine of the IL-1 family, is closely associated with type II T cell responses. Here, we report an unexpected proinflammatory role of IL-33 in inflammatory arthritis. IL-33 was expressed ...in synovial fibroblasts from patients with rheumatoid arthritis (RA). Expression was markedly elevated in vitro by inflammatory cytokines. Mice lacking ST2, the IL-33 receptor α-chain, developed attenuated collagen-induced arthritis (CIA) and reduced ex vivo collagen-specific induction of proinflammatory cytokines (IL-17, TNFα, and IFNγ), and antibody production. Conversely, treatment of wild-type (WT) but not ST2⁻/⁻ mice with IL-33 exacerbated CIA and elevated production of both proinflammatory cytokines and anti-collagen antibodies. Mast cells expressed high levels of ST2 and responded directly to IL-33 to produce a spectrum of inflammatory cytokines and chemokines in vitro. In vivo, IL-33 treatment exacerbated CIA in ST2⁻/⁻ mice engrafted with mast cells from WT but not from ST2⁻/⁻ mice. Disease exacerbation was accompanied by elevated expression levels of proinflammatory cytokines. Our results demonstrate that IL-33 is a critical proinflammatory cytokine for inflammatory joint disease that integrates fibroblast activation with downstream immune activation mainly via an IL-33-driven, mast-cell-dependent pathway. Thus, this IL-1 superfamily member represents a therapeutic target for RA.
The manufacturing of clinical cellular therapies is a complex process frequently requiring manipulation of cells, exchange of buffers and volume reduction. Current manufacturing processes rely on ...either low throughput open centrifugation-based devices, or expensive closed-process alternatives. Inertial focusing (IF) microfluidic devices offer the potential for high-throughput, inexpensive equipment which can be integrated into a closed system, but to date no IF devices have been approved for use in cell therapy manufacturing, and there is limited evidence for the effects that IF processing has on human cells. The IF device described in this study was designed to simultaneously separate leucocytes, perform buffer exchange and provide a volume reduction to the cell suspension, using high flow rates with high Reynolds numbers. The performance and effects of the IF device were characterized using peripheral blood mononuclear cells and isolated monocytes. Post-processing cell effects were investigated using multi-parameter flow cytometry to track cell viability, functional changes and fate. The IF device was highly efficient at separating CD14+ monocytes (approx. 97% to one outlet, approx. 60% buffer exchange, 15 ml min
) and leucocyte processing was well tolerated with no significant differences in downstream viability, immunophenotype or metabolic activity when compared with leucocytes processed with conventional processing techniques. This detailed approach provides robust evidence that IF devices could offer significant benefits to clinical cell therapy manufacture.
COVID-19 disease caused by the SARS-CoV-2 virus is characterized by dysregulation of effector T cells and accumulation of exhausted T cells. T cell responses to viruses can be corrected by adoptive ...cellular therapy using donor-derived virus-specific T cells. One approach is the establishment of banks of HLA-typed virus-specific T cells for rapid deployment to patients. Here we show that SARS-CoV-2-exposed blood donations contain CD4 and CD8 memory T cells which recognize SARS-CoV-2 spike, nucleocapsid and membrane antigens. Peptides of these antigens can be used to isolate virus-specific T cells in a GMP-compliant process. The isolated T cells can be rapidly expanded using GMP-compliant reagents for use as an allogeneic therapy. Memory and effector phenotypes are present in the selected virus-specific T cells, but our method rapidly expands the desirable central memory phenotype. A manufacturing yield ranging from 10
to 10
T cells can be obtained within 21 days culture. Thus, multiple therapeutic doses of virus-specific T cells can be rapidly generated from convalescent donors for potential treatment of COVID-19 patients.
Background
Cryopreserved platelets are generally produced and stored in plasma. With the advent of additive solutions being routinely used it would be prudent to examine producing and storing these ...units in a platelet additive solution (SSP+).
Study design and methods
Platelet concentrates were prepared from twenty overnight held whole blood units with 10 being re‐suspended in 100% plasma and 10 in approximately 70% SSP + and 30% plasma. All had 6% v/v DMSO added prior to storage at −80°C. On thawing plasma stored platelets were re‐constituted in fresh plasma with additive prepared platelets being subsequently suspended in 100% SSP+. Sample analysis was assessed pre cryopreservation, post thaw and 4 h.
Results
We noted a significant increase in our annexin V levels along with a decrease in GP1bα Von Willebrand binding sites post thaw. The platelets ability to change shape was also significantly reduced as observed with our HSR and ESC values. However, despite this there was still sufficient material within the platelet to allow them to be viable as observed with our thromboelasticity results which, were still observed to be within normal parameters post thaw We also observed an increase in Extracellular vesicles post thaw, suggesting platelet damage which was supported by the reduction in platelet counts. Although there were still sufficient numbers to meet the minimum requirements of the UK guidelines.
Islet transplantation is an efficacious therapy for type 1 diabetes; however, islets from multiple donor pancreata are required, and a gradual attrition in transplant function is seen. Here, we ...manufactured human umbilical cord perivascular mesenchymal stromal cells (HUCPVCs) to Good Manufacturing Practice (GMP) standards. HUCPVCs showed a stable phenotype while undergoing rapid ex vivo expansion at passage 2 (p2) to passage 4 (p4) and produced proregenerative factors, strongly suppressing T cell responses in the resting state and in response to inflammation. Transplanting an islet equivalent (IEQ):HUCPVC ratio of 1:30 under the kidney capsule in diabetic NSG mice demonstrated the fastest return to normoglycemia by 3 days after transplant: Superior glycemic control was seen at both early (2.7 weeks) and later stages (7, 12, and 16 weeks) versus ratios of 1:0, 1:10, and 1:50, respectively. Syngeneic islet transplantation in immunocompetent mice using the clinically relevant hepatic portal route with a marginal islet mass showed that mice transplanted with an IEQ:HUCPVC ratio of 1:150 had superior glycemic control versus ratios of 1:0, 1:90, and 1:210 up to 6 weeks after transplant. Immunodeficient mice transplanted with human islets (IEQ:HUCPVC ratio of 1:150) exhibited better glycemic control for 7 weeks after transplant versus islet transplant alone, and islets transplanted via the hepatic portal vein in an allogeneic mouse model using a curative islet mass demonstrated delayed rejection of islets when cotransplanted with HUCPVCs (IEQ:HUCPVC ratio of 1:150). The immunosuppressive and proregenerative properties of HUCPVCs demonstrated long-term positive effects on graft function in vivo, indicating that they may improve long-term human islet allotransplantation outcomes.
Adoptive immunotherapy using Epstein–Barr Virus (EBV)‐specific T cells is a potentially curative treatment for patients with EBV‐related malignancies where other clinical options have proved ...ineffective. We describe improved good manufacturing practice (GMP)‐compliant culture and analysis processes for conventional lymphoblastoid cell line (LCL)‐driven EBV‐specific T cell manufacture, and describe an improved phenotyping approach for analysing T cell products. We optimized the current LCL‐mediated clinical manufacture of EBV‐specific T cells to establish an improved process using xenoprotein‐free GMP‐compliant reagents throughout, and compared resulting products with our previous banked T cell clinical therapy. We assessed effects of changes to LCL:T cell ratio in T cell expansion, and developed a robust flow cytometric marker panel covering T cell memory, activation, differentiation and intracellular cytokine release to characterize T cells more effectively. These data were analysed using a t‐stochastic neighbour embedding (t‐SNE) algorithm. The optimized GMP‐compliant process resulted in reduced cell processing time and improved retention and expansion of central memory T cells. Multi‐parameter flow cytometry determined the optimal protocol for LCL stimulation and expansion of T cells and demonstrated that cytokine profiling using interleukin (IL)‐2, tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ was able to determine the differentiation status of T cells throughout culture and in the final product. We show that fully GMP‐compliant closed‐process culture of LCL‐mediated EBV‐specific T cells is feasible, and profiling of T cells through cytokine expression gives improved characterization of start material, in‐process culture conditions and final product. Visualization of the complex multi‐parameter flow cytometric data can be simplified using t‐SNE analysis.
Adoptive immunotherapy using Epstein‐Barr Virus (EBV)‐specific T cells is a potentially curative treatment for patients with EBV‐related malignancies where other clinical options have proved ineffective. Manufacture of these cells requires robust analytical approaches to ensure quality and efficacy of the cells in therapy. We demonstrate that cytokine profiling can define T cell differentiation status and produce improved characterization of start material, in‐process culture conditions and final product using multi‐parameter flow cytometry and t‐SNE visualisation.
Biliary diseases can cause inflammation, fibrosis, bile duct destruction, and eventually liver failure. There are no curative treatments for biliary disease except for liver transplantation. New ...therapies are urgently required. We have therefore purified human biliary epithelial cells (hBECs) from human livers that were not used for liver transplantation. hBECs were tested as a cell therapy in a mouse model of biliary disease in which the conditional deletion of Mdm2 in cholangiocytes causes senescence, biliary strictures, and fibrosis. hBECs are expandable and phenotypically stable and help restore biliary structure and function, highlighting their regenerative capacity and a potential alternative to liver transplantation for biliary disease.
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•Human biliary epithelial cells (hBECs) can be isolated from discarded human livers•hBECs show regenerative properties when grafted into a biliary disease mouse model•Mice transplanted with hBECs regenerate bile ducts and show improved liver function•hBECs can be cultured in good manufacturing process conditions for clinical use
In this manuscript, Forbes and colleagues isolate and expand human biliary epithelial cells (hBECs) from discarded livers. Upon transplantation into a mouse model of biliary disease, hBECs regenerate and repair damaged bile ducts, offering a potential therapy for biliary disease.
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