IntroductionLiver cirrhosis is a growing global healthcare challenge. Cirrhosis is characterised by severe liver fibrosis, organ dysfunction and complications related to portal hypertension. There ...are no licensed antifibrotic or proregenerative medicines and liver transplantation is a scarce resource. Hepatic macrophages can promote both liver fibrogenesis and fibrosis regression. The safety and feasibility of peripheral infusion of ex vivo matured autologous monocyte-derived macrophages in patients with compensated cirrhosis has been demonstrated.Methods and analysisThe efficacy of autologous macrophage therapy, compared with standard medical care, will be investigated in a cohort of adult patients with compensated cirrhosis in a multicentre, open-label, parallel-group, phase 2, randomised controlled trial. The primary outcome is the change in Model for End-Stage Liver Disease score at 90 days. The trial will provide the first high-quality examination of the efficacy of autologous macrophage therapy in improving liver function, non-invasive fibrosis markers and other clinical outcomes in patients with compensated cirrhosis.Ethics and disseminationThe trial will be conducted according to the ethical principles of the Declaration of Helsinki 2013 and has been approved by Scotland A Research Ethics Committee (reference 15/SS/0121), National Health Service Lothian Research and Development department and the Medicine and Health Care Regulatory Agency-UK. Final results will be presented in peer-reviewed journals and at relevant conferences.Trial registration numbersISRCTN10368050 and EudraCT; reference 2015-000963-15
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.
Microfluidic devices are useful tools for a wide range of biomedical, industrial, and environmental applications. Hybrid microfluidic devices utilising more than two materials are increasingly being ...used for their capacity to produce unique structures and perform novel functions. However, an analysis of publications across the field shows that whilst hybrid microfluidic devices have been reported, there remains no system of classifying hybrid devices which could help future researchers in optimising material selection. To resolve this issue, we propose a system of classifying hybrid microfluidic devices primarily as containing either hybrid structural, chemical, or electrical components. This is expanded upon and developed into a hierarchy, with combinations of different primary components categorised into secondary or tertiary hybrid device groupings. This classification approach is useful as it describes materials that can be combined to create novel hybrid microfluidic devices.
Chemokines have been shown to be essential players in a range of cancer contexts. In this study, we demonstrate that mice deficient in the atypical chemokine receptor Ackr2 display impaired ...development of metastasis in vivo in both cell line and spontaneous models. Further analysis reveals that this relates to increased expression of the chemokine receptor CCR2, specifically by KLRG1
NK cells from the Ackr2
mice. This leads to increased recruitment of KLRG1
NK cells to CCL2-expressing tumors and enhanced tumor killing. Together, these data indicate that Ackr2 limits the expression of CCR2 on NK cells and restricts their tumoricidal activity. Our data have important implications for our understanding of the roles for chemokines in the metastatic process and highlight Ackr2 and CCR2 as potentially manipulable therapeutic targets in metastasis.
Dendritic cells (DCs) comprise heterogeneous subsets of professional antigen-presenting cells, linking innate and adaptive immunity. Analysis of DC subsets has been hampered by a lack of specific DC ...markers and reliable quantitation assays. We characterised the immunophenotype and functional characteristics of psoriatic arthritis (PsA)-derived and rheumatoid arthritis (RA)-derived myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to evaluate their potential role in arthritis. Circulating peripheral blood (PB) pDC numbers were significantly reduced in PsA patients (P = 0.0098) and RA patients (P = 0.0194), and mDCs were significantly reduced in RA patients (P = 0.0086) compared with healthy controls. The number of circulating mDCs in RA PB was significantly inversely correlated to C-reactive protein (P = 0.021). The phenotype of both DC subsets in PsA PB and RA PB was immature as compared with healthy controls. Moreover, CD62L expression was significantly decreased on both mDCs (PsA, P = 0.0122; RA, P = 0.0371) and pDCs (PsA, P = 0.0373; RA, P = 0.0367) in PB. Both mDCs and pDCs were present in PsA synovial fluid (SF) and RA SF, with the mDC:pDC ratio significantly exceeding that in matched PB (PsA SF, P = 0.0453; RA SF, P = 0.0082). pDCs isolated from RA SF and PsA SF displayed an immature phenotype comparable with PB pDCs. RA and PsA SF mDCs, however, displayed a more mature phenotype (increased expression of CD80, CD83 and CD86) compared with PB mDCs. Functional analysis revealed that both SF DC subsets matured following toll-like receptor stimulation. pDCs from PB and SF produced interferon alpha and tumour necrosis factor alpha on TLR9 stimulation, but only SF pDCs produced IL-10. Similarly, mDCs from PB and SF produced similar tumour necrosis factor alpha levels to TLR2 agonism, whereas SF mDCs produced more IL-10 than PB controls. Circulating DC subset numbers are reduced in RA PB and PsA PB with reduced CD62L expression. Maturation is incomplete in the inflamed synovial compartment. Immature DCs in SF may contribute to the perpetuation of inflammation via sampling of the inflamed synovial environment, and in situ presentation of arthritogenic antigen.
Systemic sclerosis (SSc) is characterised by vasculopathy, an aberrantly activated immune system and excessive extracellular matrix deposition. Inflammatory chemokines control migration of cells to ...sites of tissue damage; their removal from inflamed sites is essential for resolution of the inflammatory response. The atypical chemokine receptor D6 has a critical role in this physiological balance. To explore potential deregulation of this system in SSc, inflammatory chemokine and D6 expression were compared with that in healthy controls (HC).
Serum levels of inflammatory mediators were assessed by luminex analysis. Peripheral blood mononuclear cells (PBMCs) were used in molecular and immunocytochemical analysis. Platelet-rich plasma was collected and assessed by western blotting for D6 expression levels. Sex-matched HC were used for comparison.
72 patients with SSc and 30 HC were enrolled in the study. The chemokines MCP-1/CCL2, MIP-1α/CCL3, MIP-1β/CCL4 and IL-8/CXCL8 were significantly increased in patients with SSc, regardless of disease subtype and phase. Quantitative PCR analysis revealed a significant 10-fold upregulation of D6 transcripts in patients with SSc compared with controls, and this was paralleled by increased D6 protein expression in the PBMCs of patients with SSc. Platelet lysates also showed strong D6 expression in patients with SSc but not in controls. Importantly, high levels of D6 expression correlated with reduced levels of its ligands in serum.
Inflammatory chemokines and the regulatory receptor D6 are significantly upregulated in SSc and high D6 levels are associated with lower systemic chemokine levels, indicating that some patients control systemic chemokine levels using D6. These results suggest that chemokines may represent a therapeutic target in SSc.
Limbal stem cell deficiency (LSCD) is a disease resulting from the loss or dysfunction of epithelial stem cells, which seriously impairs sight. Autologous limbal stem cell transplantation is ...effective in unilateral or partial bilateral disease but not applicable in total bilateral disease. An allogeneic source of transplantable cells for use in total bilateral disease can be obtained from culture of donated cadaveric corneal tissue. We performed a controlled multicenter study to examine the feasibility, safety, and efficacy of allogeneic corneal epithelial stem cells in the treatment of bilateral LSCD. Patients were randomized to receive corneal epithelial stem cells cultured on amniotic membrane (AM): investigational medicinal product (IMP) or control AM only. Patients received systemic immunosuppression. Primary endpoints were safety and visual acuity, secondary endpoint was change in composite ocular surface score (OSS). Sixteen patients were treated and 13 patients completed all assessments. Safety was demonstrated and 9/13 patients had improved visual acuity scores at the end of the trial, with no significant differences between IMP and control groups. Patients in the IMP arm demonstrated significant, sustained improvement in OSS, whereas those in the control arm did not. Serum cytokine levels were measured during and after the period of immune suppression and we identified strongly elevated levels of CXCL8 in the serum of patients with aniridia, which persisted throughout the trial. This first randomized control trial of allogeneic corneal epithelial stem cells in severe bilateral LSCD demonstrates the feasibility and safety of this approach. Stem Cells Translational Medicine 2019;8:323–331
Patients with severe ocular surface disorder received transplants of amniotic membrane with (black bars) or without (gray bars) cadaveric‐donor‐derived cultured limbal stem cells. All patients received immune suppression. Only patients who received transplants containing limbal stem cells showed sustained significant improvements (reductions) in combined ocular surface scores (5 factors scored 0–3 where 0 is a normal eye score).
Cellular therapeutics are a fast‐growing, highly innovative area of medicine. This field encompasses well‐established immune therapies for infection and cancer, as well as newer cell therapies aimed ...at regenerating diseased tissue. Flow cytometry is arguably the most important tool in the development of advanced cellular therapeutics and plays a role in many aspects of manufacturing. Quality control of raw materials, assessing rates of cellular growth during complex in vitro culture processes, differentiation status, as well as final product characterization, viability, and product stability are all essential data to be collected and documented. This requires robust and validated flow cytometric analysis. In this review, we discuss flow cytometer set up; design and use of assays to maintain quality control in cell processing, and the building of a robust set of release criteria. The use of flow cytometric assays as surrogates of functional assays to allow comparison and release of cellular products for clinical use are reviewed with reference to monocyte‐derived and T cell products.
Autologous macrophage therapy represents a potentially significant therapeutic advance for the treatment of severe progressive liver cirrhosis. Administration of macrophages has been shown to reduce ...inflammation and drive fibrotic scar breakdown and tissue repair in relevant models. This therapeutic approach is being assessed for safety and feasibility in a first-in-human trial (MAcrophages Therapy for liver CirrHosis MATCH trial).
We outline the development and validation phases of GMP production. This includes use of the CliniMACS Prodigy cell sorting system to isolate CD14+ cells; optimizing macrophage culture conditions, assessing cellular identity, product purity, functional capability and determining the stability of the final cell product.
The GMP-compliant macrophage products have a high level of purity and viability, and have a consistent phenotypic profile, expressing high levels of mature macrophage markers 25F9 and CD206 and low levels of CCR2. The macrophages demonstrate effective phagocytic capacity, are constitutively oriented to an anti-inflammatory profile and remain responsive to cytokine and TLR stimulation. The process validation shows that the cell product in excipient is remarkably robust, consistently passing the viability and phenotypic release criteria up to 48 hours after harvest.
This is the first report of validation of a large-scale, fully Good Manufacturing Practice–compliant, autologous macrophage cell therapy product for the potential treatment of cirrhosis. Phenotypic and functional assays confirm that these cells remain functionally viable for up to 48 h, allowing significant flexibility in administration to patients.
Abstract Background aims Macrophages have complex roles in the liver. The aim of this study was to compare profiles of human monocyte-derived macrophages between controls and cirrhotic patients, to ...determine whether chronic inflammation affects precursor number or the phenotype, with the eventual aim to develop a cell therapy for cirrhosis. Methods Infusion of human macrophages in a murine liver fibrosis model demonstrated a decrease in markers of liver injury (alanine transaminase, bilirubin, aspartate transaminase) and fibrosis (transforming growth factor-β, α-smooth muscle actin, phosphatidylserine receptor) and an increase in markers of liver regeneration (matrix metalloproteinases MMP-9, MMP-12 and TNF-related weak inducer of apoptosis). CD14+ monocytes were then isolated from controls. Monocytes were matured into macrophages for 7 days using a Good Manufacturing Practice–compatible technique. Results There was no significant difference between the mean number of CD14+ monocytes isolated from cirrhotic patients (n = 9) and controls (n = 10); 2.8 ± SEM 0.54 × 108 and 2.5 ± 0.56 × 108 , respectively. The mean yield of mature macrophages cultured was also not significantly different between cirrhotic patients and controls (0.9 × 108 ± 0.38 × 108 , with more than 90% viability and 0.65 × 108 ± 0.16 × 108 , respectively. Maturation to macrophages resulted in up-regulation of a number of genes (MMP-9, CCL2, interleukin IL-10 and TNF-related weak inducer of apoptosis). A cytokine and chemokine polymerase chain reaction array, comparing the control and cirrhotic macrophages, revealed no statistically significant differences. Conclusions Macrophages can be differentiated from cirrhotic patients' apheresis-derived CD14 monocytes and develop the same pro-resolution phenotype as control macrophages, indicating their suitability for clinical therapy.
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